DE19720165A1 - Production of polymeric compounds with nucleo-base substituent(s) - Google Patents

Abstract

Production of polymeric compounds (I) containing nucleo-base substituents comprises reacting compounds (II), (III), (IV) and (V) with one another, optionally simultaneously, and optionally removing one or more protecting groups and repeating the reaction m-times, with the product of the immediately preceding step being used in any subsequent step in place of (II). A = a residue of an amine component which is conventional in the Ugi reaction, e.g. H; a substituent; (cyclo)alkyl; (cyclo)alkenyl); (cyclo)alkynyl; (hetero)aroyl; heterocyclyl; or a fluorescence label, intercalator, antibiotic, minor or major groove binder, biotinyl group, intercalating agent, alkylating group, steroid, lipid, polyamine, agent facilitating cell reception, (oligo)saccharide, antisense polymer, peptide, antibody conjugate, synthetic polymer or corresponding modified surface; B = H a group or molecule specified under A; or a group X--NPG of compound (V); R1-G is a grouping of formula (i)-(iv): and is optionally bonded to (IV) through R, R1 or R4 via a molecular spacer; R and R1 = A or a residue of a natural or synthetic nucleo-base (at least one of R and R1 is such a residue); L, M, T and Z = O; S or NR4; R4 = H; F; (cyclo)alkyl; (cyclo(alkenyl); (cyclo)alkynyl; (hetero)aroyl; a heterocyclyl; -O-(cyclo)alkyl; -O-aroyl; -S-(cyclo)alkyl; or -S-aroyl; R2 and R3 = A, a residue of an oxo component which is conventional in the Ugi reaction, e.g. a group or molecule specified under A or a branching site P which is the starting site for a further DNA, RNA, PNA or peptide strand or another oligomer or polymer; X is a group of formula (v): PG = optionally orthogonal protecting groups, e.g. acyl, sulphonyl, alkyl or silyl or a carbamate or one of its salts; R5 = H; optionally substituted (cyclo)alkyl, alkoxyalkyl or aryl; or heterocyclyl; U, W, K and Y = optionally substituted (cyclo)alkyl, alkenyl, alkynyl, alkanoyl, alkoxyalkanoyl, aroyl or heterocyclyl; or NR5; a, b, c, n, o and p = 0-10, preferably 0-5; Q = optionally substituted (cyclo)alkyl, aryl, alkenyl, alkynyl, alkanoyl, alkoxyalkanoyl, cycloalkanoyl, aroyl or heterocyclyl; NR5; P; P(O); P(S); B; BR5; or SO2; F = O; S; Se or NH; J is a group of formula (vi)-(ix): R6 = H; a substituent; (cyclo)alkyl; (cyclo)alkenyl; (cyclo)alkynyl; (hetero)aroyl; or heterocyclyl.

Description

The invention relates to a process for the production of polyme ren, which have nucleobases as side groups, using Multi Component reactions, especially the Ugi reaction.

Regulatory by means of polymeric peptidic nucleic acids (PNA) Influencing gene expression first occurred in the 1960s Years of Svachkin et al. described (R. A. Paégle, M. G. Plata, M. Yu. Lidak, S.A. Giller, Yu. P. Shvachkin, in: Present State of the Chemotherapy of Malignant Tumors [in Russian], Riga (1968), 103 ff; Review: Yu. P. Shavachkin, G. P. Mishin, G. A. Korshunova, Russian Chemical Reviews, 51, 1982, 178-188). In 1978 Zamecnik and Stephenson used the terms antisense and Antigen Introduced: These terms describe mechanisms such as therapeutically in the translation and transcription of genes can be intervened (Proc. Natl. Acad. Sci. USA., 1978, 75, 280 and 285).

In the antisense strategy, an antisense molecule binds to the mRNA and thus prevents their translation into a protein. The antigen strategy uses a triple helix of the antigen Molecule formed with the double-stranded DNA and thus the Transcription modified into the mRNA (E. Uhlmann, A. Peyman, Chem. Rev., 90, 1990, 544-584). Different substances in this context as potential therapeutic agents of viral diseases, cancer etc. in various clinical phases.

A good antisense molecule should a. the following An requirements are sufficient:  

• 1. It should be without the help of so-called transfection reagents or Liposomes have good cell and nucleus mobility;
• 2. It should help you achieve adequate bioavailability Exhibit nuclease and peptidase resistance; and
• 3. The sequence of the natural sense strand should be precise detect.

As a promising antisense and antigen polymer, that of Nielsen et al. PNA described proved (Science 1991, 254, 1497-1500; WO 92/20702), which has also found a variety of uses as a molecular biological tool. This is mainly due to the high affinity of the PNA for the sense strand (DNA, RNA) with very good sequence specificity. PNA can identify mismatches in sequences much better than natural DNA or RNA. Furthermore, pyrimidine-rich PNA strands can unravel the DNA double helix and form a (PNA) ₂ DNA triple helix. Such structures are potential translation and replication complex mimetics and one could be able to switch genes on and off in a targeted manner. For an in vivo application, however, various properties of the PNA have to be improved and optimized. For example, the PNA is not common in cells. The mentioned (PNA) ₂ DNA triple helix formation only takes place with pyrimidine-rich PNA strands and only under unphysiological salt concentrations. PNA's aggregate and are poorly water-soluble. PNA - itself a polar polymer - binds both parallel and antiparallel with similar binding constants to the target structure DNA or RNA. Strong cytotoxic effects of PNA are also reported (EP 0 672 677 A2).

New improved PNA's have been shown to be most effective found by screening a large number of variants  (S. Jordan et al., Bioorganic & Medicinal Chemistry Letters, 1997, 7, 681 and 687). For systematic and quick Manufacture of many analogues appear the previous ones described methods of PNA synthesis based on sequential Two-component reactions of monomers are based on which of them partly over many steps from suitable commercial preliminary stages must be synthesized, not optimally suitable (M. Egholm et al., Journal of the American Chemical Society, (1992) 114, 1895).

It is therefore an object of the present invention to provide a method with the complex mono, oligo and Polymers which have nucleobases as side groups, and in particular PNA's with a large variability can be produced.

According to the invention, a process for the preparation of compounds of the formula (I) is disclosed

characterized in that compounds of the formulas

A-NH ₂ II

R ₁ -G III

R ₂ -C (O) -R ₃ IV and

C = NX-NPG V

in a first step, if necessary, implemented simultaneously with one another,
optionally one or more protective groups are removed
and the reaction is repeated m times
the product of the preceding step being used after the first step instead of the compound having the formula II,
in which
m is 0 or an integer from 1 to 1000, preferably 1 to 300,
A is a residue of the amine component customary in the Ugi reaction, such as a hydrogen atom, a substituent, (cyclo) alkyl, (cyclo) alkenyl, (cyclo) alkynyl, aroyl, heteroaroyl, a heterocycle, a fluorescence marker, an intercalator, an antibiotic , a minor groove binder, a major groove binder, a biotinyl residue, an intercalating residue, an alkylating residue, a steroid, a lipid, a polyamine, an agent that facilitates cell uptake, a saccharide or oligosaccharide, an antisense polymer, a peptide, an antibody Conjugate, a synthetic polymer or a correspondingly modified surface,
B is a hydrogen atom, a substituent, (cyclo) alkyl, (cyclo) alkenyl, (cyclo) alkynyl, aroyl, heteroaroyl, a heterocycle, a fluorescent label, an intercalator, an antibiotic, a minor groove binder, a major groove binder , a biotinyl residue, an intercalating residue, an alkylating residue, a steroid, a lipid, a polyamine, an agent which facilitates cell uptake, a saccharide or oligosaccharide, an antisense polymer, a peptide, an antibody conjugate, a synthetic polymer or one appropriately modified surface or a residue X - NPG of compound V,
R1-G is selected from structures of the following formulas:

where the group R ₁ -G can be linked via a molecular spacer via R ₁ or R or R ₄ to the compound of the formula IV;
R ₁ and R independently of one another are a residue of the acid component customary in the Ugi reaction, such as H, a substituent, (cyclo) alkyl, (cyclo) alkenyl, (cyclo) alkynyl, aroyl, heteroaroyl, a heterocycle, a fluorescent marker Intercalator, an antibiotic, a minor groove binder, a major groove binder, a biotinyl residue, an intercalating residue, an alkylating residue, a steroid, a lipid, a polyamine, a saccharide or oligosaccharide, an antisense polymer, a peptide, an antibody conjugate , are a synthetic polymer or a correspondingly modified surface, or residues derived from the natural nucleobases or from synthetic nucleobases;
L, M, T and Z independently of one another are O, S or NR ₄ , where R _{4 is} H, fluorine, (cyclo-) alkyl, (cyclo-) alkenyl, (cyclo-) alkynyl, aroyl, heteroaroyl, heterocycle or -O Are (cyclo) alkyl, -Oaroyl, -S (cyclo) alkyl, -Saroyl;
R ₂ and R ₃ independently of one another are a radical of the oxo component which is customary in the Ugi reaction, such as H, a substituent, (cyclo) alkyl, (cyclo) alkenyl, (cyclo) alkynyl, aroyl, heteroaroyl, a heterocycle, a fluorescent marker, an intercalator, an antibiotic, a minor groove binder, a major groove binder, a biotinyl residue, an intercalating residue, an alkylating residue, a steroid, a lipid, a polyamine, a saccharide or oligosaccharide linked via an amine spacer, an antisense molecule, a peptide an antibody conjugate, a synthetic polymer, a modified surface or a branching point P, which in turn is the starting point for a further strand of DNA, RNA, PNA or peptide or another oligomer or polymer,
X has the following structure:

PG, independently of one another, optionally represent orthogonal protective groups such as amine protective groups from the class of the N-acyl derivatives, the N-sulfonyl derivatives, the N-alkyl derivatives, the N-silyl derivatives, the carbamates or the salts;
the radicals R ₅ independently of one another represent hydrogen atoms, unsubstituted or substituted alkyl, cycloalkyl, alkoxyalkyl or aryl groups or heterocycles;
the radicals U, W, K and Y independently of one another are unsubstituted or substituted alkyl, alkenyl, alkynyl, alkanoyl, alkoxyalkanoyl, cycloalkyl or aryl groups, unsubstituted or substituted heterocycles or the group NR ₅ , where R _{5 is} like is defined above;
a, b, c, n, o and p are independently an integer from 0-10, preferably 0-5;
Q is an unsubstituted or substituted alkyl, aryl, al kenyl, alkynyl, mono- or polyvalent alkanoyl, cycloalkyl, alkoxyalkanoyl, cycloalkanoyl, aroyl group or an unsubstituted or substituted heterocycle, or one of the groups NR ₅ , Represents P, P (O), P (S), B, BR ₅ or SO ₂ , where R _{5 is} as defined above and the indices a, b, o and p each have corresponding values;
F represents an oxo, thio, seleno or imino group, and
J is selected from the following structures, the upper vertical line representing the bond to R ₁ and the lower vertical line representing the bond to the nitrogen atom of the main chain of the polymer in general formula I:

wherein R _{6 is} H, a substituent, (cyclo-) alkyl, (cyclo-) alkenyl, (cyclo-) alkynyl, aroyl, heteroaroyl, heterocycle, with the proviso that at least one of the radicals R or R ₁ in the compound of Formula I is a residue derived from a natural or synthetic nucleobase (claim 1).

In a preferred embodiment, the method is characterized in that: the fluorescent markers are fluorescein, Texas red, lissamine, rhodamine, cyanine or rhodamine, the intercalators are psoralen, acridine, phenanthroline, a phenanthroline metal complex or ellipticin, the antibiotic endiins, β- Lactams, tetracyclines, anthracyclines, polyethers, mitomycin-like, phosphomycin-like, macrolides, bleomycin-like or an aminoglycoside, which is minor groove binder, netropsin or distamycin, which is polyamine, a spermidine-like polyamine, which antisense polymer is a DNA (5 'or 3' linked) or RNA strand (5 'or 3' linked) or a phosphothioate, the peptide linked N- or C-terminal, the antibody conjugate is selected from antibody conjugates which allow cell-specific uptake, address specific carrier systems or cause endocytosis, the synthetic polymer is CPG, Wang, or Tentagel, the natural nucleobases adenine, thymine , Guanine, cytosine or uracil and the synthetic nucleobases are pseudouracil, 5-propynyluracil, 5-hexenyluracil, 5-fluorocytidine, 5-hydroxymethyluracil, 5-methylcytidine, 5-bromocytidine and compounds of the following formulas

where R ₈ and R _{9 are} independently H, (cyclo-) alkyl, (cyclo-) alkenyl, (cyclo-) alkynyl, aroyl, heteroaroyl, heterocycle, chlorine or fluorine, R ₁₀ = fluorine, bromine, iodine, chlorine, Is alkynyl, (cyclo-) alkyl, aroyl, heteroaroyl or H, and n = 1-20, preferably 1-10 and particularly preferably 1-5, the side groups of the bases having protective groups known to the person skilled in the art, such as, for example, B. tert-butylbenzoyl, p-methoxybenzyl, iso-butanoyl can be protected.

All previous and functional mentioned below Units like antibiotics, groove binders, antisense molecules,  Steroids, antibody conjugates, intercalators and Oligosaccharides can have a suitably constructed spacer be bound to the main polymer. Suitable means at least a functional group of the Ugi reaction such as oxo, acid, Containing amine or isocyano function. But can also be suitable mean another functional group through which the Expert connects two molecular units together.

The protective groups PG are, in themselves, easily removable (Temporary) protecting groups for amine groups. Otherwise would have to their separation to be used under drastic conditions, which lead to undesirable side reactions or even to Can lead to decomposition of the reaction products. Prefers represent the protective groups PG N-acyl derivatives, N-alkyl derivatives or azide groups, and particularly N-acyl, N-sulfonyl, N-alkyl, N-silyl protecting groups such as. B. tert-Boc, Alloc, Fmoc, Moz, Z, Tr, MMTr, DMTr, Pixyl, TBDMS protecting groups, Salts of the amine preferred.

The radicals R ₅ preferably independently of one another are hydrogen atoms, or unsubstituted or substituted alkyl, aryl, cycloalkyl groups or heterocycles, in particular sterically less demanding groups such as alkyl, aryl, cycloalkyl, heterocycles and particularly preferably hydrogen atoms, since primary amines are usually more reactive are secondary amines [I. Ugi, Angew. Chem. 74, 9 (1962)].

The radicals U, W, K and Y are preferably independent of one another unsubstituted or substituted alkyl, cycloalkyl or Aryl groups and particularly preferably methylene, ethylene, Propylene, heptylene, octylene, nonylene, oxymethylene, Oxyethylene, cyclohexyl, phenyl groups and heterocycles.

The indices a, b, m, n, o and p are preferably independent integers from 0-50, more preferably from 0-20  or from 0-10, even more preferably from 0-5 and particularly preferred from 0-3 and most preferred are a, b, p and o 0 and are m and n 0, 1 or 2.

The radical Q preferably represents at least one unsubstituted Al kyl, aroyl or aryl group or an unsubstituted or substituted heterocycle and particularly preferably methylene, Ethylene, propylene, oxymethylene, oxyethylene, dioxymethylene, 1,2-dioxyethylene, cyclohexyl, phenyl groups, N and Heterocycles.

Compounds of formula V can be prepared in that a compound of formula VII

with the groups PG as defined above, which may be independent of one another, is protected by conventional methods, whereby a compound of the formula VIII is prepared

wherein the residues and indices are as defined above, and then the compound of formula VIII using conventional methods an isonitrile of the formula V is implemented. Such usual Process for the reaction of compounds of formula VIII Isonitriles of the formula V are e.g. B. in W.P. Weber, G.W. Gokel, Tetrahedron Lett. 1972, 1637; W.P. Weber, G.W. Gokel, I.K. Ugi, Appl. Chem. 84, 587 (1972); Appl. Chem. Int. Ed. Engl. 11, 530 (1972). Compounds of formula VII are commercially available available.

In a further embodiment, the compound of the formula VIII is reacted with conventional formylation reagents to give a compound of the formula IX:

which then, under normal conditions, combine to form For mel V is implemented, the residues and indices as above are defined (claim 4). Formylation with such ex union formylation reagents is such. B. in U. Schöllkopf et al., Liebigs Ann. 89, 351-360 (1977). A common one Process for converting a compound of formula IX into a Compound of formula V is a dehydration, such as. B. from I. Hagedorn, H. Tönjes, Pharmazie 11, 409 (1956); I. Ugi, R. Meyr, Angew. Chem. 70, 702 (1958); HM. Walborsky, G.E. Niznik, J. Org. Chem. 37, 187 (1972); I. Ugi, W. Betz, U. Fetzer, K. Offermann, Chem. Ber. 94, 2814 (1961) I. Ugi, R. Obrecht, R. Herrmann, Synthesis 1985, 400-402; G. Gokel, D. Marquarding, P.  Hoffmann, I. Ugi in Isonitrile Chemistry; I. Ugi (Ed.), Academic Press, New York, London 1971, 9. To be favoured phosphorus oxychloride and a suitable base such as Triethylamine or diisopropylamine used.

In a further embodiment, starting from compounds of the formula X.

wherein the residues and indices are as defined above next the amine function z. B. after an above Formulated method, then one or more of the functionalities M, T and S, which independently of one another are halogens or hydroxyl functions, be preferably chlorine, bromine and hydroxyl mean after a usual Process converted into one or more azide functions, these Azides into the corresponding amines by a customary process converted and then using the usual procedures with the above Protecting groups provided to ge to compounds of formula IX long. Likewise, the compounds of formula X according to Formy Formation and conversion of groups M, T and S into corresponding azides e.g. B. according to the above Methods first in the corresponding isonitriles are implemented, the azide functions converted to amines by a conventional method and then with o.g. Protecting groups are provided in order to connect the Formula V to arrive.

The following definitions are used in the description and the claims:
Alkyl: chain length C1 to C100, preferably C1 to C20, more preferably C1 to C10, even more preferably C1 to C6 and most preferably C1 to C4, linear or branched, substituted (as defined below) or unsubstituted,
Cycloalkyl: ring size C3 to C20, preferably C3 to C9, more preferably C3 to C7, most preferably C5, C6 or C7, sub-substituted (as defined below) or unsubstituted,
Alkenyl: 1 to 5, preferably 1 or 2 conjugated or non-conjugated double bonds containing alkyl or cycloalkyl,
Alkynyl: 1 to 5, preferably 1 or 2 conjugated or non-conjugated triple bonds containing alkyl or cycloalkyl,
Heterocycle: 3-7-membered, preferably 5 or 6-membered, heterocycles such as. B. substituted (as defined below) or unsubstituted oxirane, thiirane, aziridine, oxaziridine, oxetane, thietane, azetidine, tetrahydrofuran, dihydrofuran, tetrahydrothiophene, dihydrothiophene, pyrrolidine, dihydropyrrole, 1,3-dioxolane, 1,3-ditholidinane, im Oxazolidine, thiazolidine, 2H-pyran, 4H-pyran, tetrahydropyran, 2H-thiopyran, 4H-thiopyran, tetrahydrothiopyran, piperidine, morpholine, 1,4-dioxin, 1,4-dioxane, 1,4-dithiine, 1,4- Dithian, Piperazin, Oxepan, Thiepan, Thiepin, 1H-Azepin, 2H-Azepin, Azepan,
Aroyl: substituted (as defined below) or unsubstituted benzene, naphthalene, anthracene, biphenyl, triphenyl, azulene, ferrocene, cyclopropenylium,
Heteroaroyl: 5-6-membered heterocyclic aromatic heterocycles with 1, 2 or 3 heteroatoms such as B. substituted (as defined below) pyrrole, furan, thiophene, pyrazole, isoxazole, isothiazole, imidazole, oxazole, thiazole, 1,2,4-triazole, 1,2,4-oxadiazole, 1,2,4-thiadiazole, 1,2,5-oxadiazole, 1,2,5-thiadiazole, tetrazole, pyridine, pyrylium, thiapyrylium, pyridazine, pyrimidine, pyrazine, 1,2,3-triazine, 1,2,4-triazine, 1,3, 5-triazine, 1,2,3,4-tetrazine, 1,2,3,5-tetrazine, 1,2,4,5-tetrazine, indole, coumarone, thionaphthene, carbazole, bibenzofuran, dibenzothiophene, 1H-indazole, Indoxazole, benzo [d] isothiazole, anthranil, benzimidazole, benzoxazole, benzothiazole, benzotriazole, quinoline, isoquinoline, benzopyrylium, thiabenzopyrylium, acridine, benzo [g] quinoline, benzo [g] isoquinoline, benzo [c] quinoline, cinnoline, cinnoline Quinazoline, quinoxaline, phenazine, benzo [g] cinnoline, benzo [g] quinazoline, benzo [g] quinoxaline, 1,5-naphthyridine, 1,6-naphthyridine, 1,7-naphthyridine, 1,8-naphthyridine, 2, 6-naphthyridine, 2,7-naphthyridine, 1,7-phenanthroline, 1,8-phenanthroline, 1,9-phenanthroline, 1,10-phenanthroline, indolizine, 4H -Chinolizine, car bolin, ergoline, purine, pteridine, alloxazine, flavin,
Substituent or "substituted with": -H, -OH, -R _a , -O-, (cyclo-) alkyl, -O-aryl, -O-heteroaroyl, -O-heterocycle, -NH ₂ , -NO ₂ , -CN, -N ₃ , -CNR _a NR _b R _c , -NR _a R _b , NR _a R _b R _c ⁺, fluorine, chlorine, bromine, a-, b- to w-amino acid esters, -NR _a COR _b , -NR _a COXR _b (X = -O, -NR, -PO _0.2.3.4 R, -SO _0.1.2.4, R, -NR _a NR _b R _c ), -COR _a , -COOR _a , -OCOOR _a , -CONR _a R _b , -OCONR _a R _b , -NRCCONR _a R _b , -R _a -OR _b , -R _c -NR _a R _b , -R _a -SR _b , -R _a -SO-R _b , -R _a -S (O) ₂ -R _b , -OR _a -OR _b , -NR _a R _b -OR _c , -SO ₂ R _a , -SO _{1.2 , 3,4} R _a -OR _b , -COR _a -OR _b , -COOR _a -OR _b , -OCOR _a -OR _b , -OCOOR _a - OR _b , -NR _b COR _a -OR _b , -CONR _a R _b -OR _c , OCONR _a R _b -OR _c , -NR _c CONR _a R _b - OR _d , -NR _a COR _b -OR _c , -OR _a -SR _b , -NR _a R _b -SR _c , -SO _1,2,3,4 R _a -SR _b , -COR _a -SR _b , -OCOR _a -SR _b , -OCOR _a -SR _b , NR _a COR _b -SR _c , -CONR _a R _b - S- R _c , -NR _a CONR _b R _c -SR _d , -OR _a -NR _b R _c , -NR _a R _b -NR _c R _d , -SO _1,2,3,4 R _b - NR _b R _c , -COR _a -NR _b R _c , -COOR _a -NR _b R _c , -OCOR _a -NR _b R _c , -OCOOR _a -NR _b R _c , -NR _a CONR _b R _c -NR _d , -NR _a COOR _b -NR _c R _d , -OCONR _a R _b -NR _c R _d , -NR _a CONR _b R _c - NHR _d , -NR _a COOR _b -NR _c R _d , -POOR _a OR _b , -NR _c POOR _a OR _b ,
where R _a , R _b , R _c and R _d independently of one another are as defined above (cyclo) alkyl, alkenyl, akinyl, aroyl, heteroaroyl, a heterocycle, aralkyl, aralkenyl or perhaloalkyl and where R _a , R _b , R _c and R _d may be substituted.

The method according to the invention thus relates to the production of mono-, oligo- and polymers which have nucleobases as side groups, in particular peptide nucleic acid (PNA) and their variants by means of multi-component reactions (MCR's), especially isocyanide-based MCR's such as the Ugi -Reaction. In this case, monomers are not used as in conventional processes, but rather smaller building blocks than previously used, so-called submonomers. The method presented here allows an improved synthesis of the previously described PNA variants and, above all, the rapid synthesis of new variants not described so far with potentially improved and / or new properties. Due to the multicomponent nature of this process, the simultaneous incorporation of many different types of residues in a quasi combinatorial manner z. B. in the polyamide backbone of the PNA, and with suitable synthetic building blocks described below, the construction of (PNA) ₂ -, (PNA (DNA) -, (PNA) (RNA) -, (PNA) (peptide) -, ( PNA) ₂ (DNA) -, (PNA) ((oligo) saccharide) -, (PNA) ₂ (DNA) (peptide) chimeras etc., as well as the construction of modified PNA polymer backbones such as thioamides. Shorter oligomers or even monomers produced by the process described are potentially pharmacologically or agrochemically relevant active ingredients.

The process described here is based on isocyanide-based Multicomponent reactions (MCR's) of the four component type Condensation or Ugi reaction (Isocyanide Chemistry, (I. Ugi, ed.), Wiley, New York, 1971; I. Ugi, R. Karl, in: Comprehensive Organic Synthesis), (B.M. Trost, C.H. Heathcock (ed.), Vol. II, 1083-1109, Pergamon Press, New York 1991). In this reaction react the four reactant components isocyanide, oxo compound (Aldehydes or ketones), amine-like compounds (e.g. ammonia, primary amines, secondary amines, hydrazine and derivatives, hydroxyl  amine and derivatives) and suitable acid components (e.g. carbon acids, carbonic acid monoesters, water, thiosulfate, selenium water substance, hydrochloric acid, cyanic acid, thiocyanic acid) uniform products, whose central basic structure is essential depends on the nature of the acid component. Remarkably can the residues of the individual components without reactivity ver desire to be varied within wide limits. So react sterically sophisticated or small, aromatic, heteroaromatic like also aliphatic or heterocyclic, electron-withdrawing or electron-donating educts equally in the Ugi reaction. Related MCRs based on isocyanides are the Passerini Reaction (I. Ugi in, Isocyanide Chemistry, (I. Ugi, ed.), Wiley, New York, 1971), and a number of heterocycle syntheses (p. Marcaccini, T. Torroba, OPPI, 143.).

With the help of this method, homopolymers and heteropolymers of general formula I as shown in Scheme I.  

The process for the preparation of homopolymers and heteropolymers General formula I is characterized in that four ver different compounds with suitable functional groups if necessary synchronously if necessary several times with each other be implemented. After the first step, i.e. H. of the possibly synchronous implementation of the connections II, III, IV and V, will be the product of the previous one Then step in place of the compound of formula II used. To get to heteropolymers, the Compounds do not have the same residues in all synthetic steps or functional groups.

The syntheses of the classes of compounds shown under Formula I can on surfaces, in the liquid phase or on polymeric substrates like to be done.

The compounds of formula I can also be synthesized on so-called chips:
a substrate is produced on which, in a first loading area, a compound selected from compounds of the formulas II, III, IV and V is applied, which have protective groups which can be removed using activators;
this step may be repeated on other areas of the surface, in each case with a different compound II, III, IV and V having the corresponding protective group (s),
a region of the surface which optionally has one or more of the compounds II, III, IV and V is exposed to an activator in order to remove at least one protective group,
this area is exposed to a compound of formula II, III, IV or V, which in turn has a photosensitive protective group,
and if necessary repeat this step in other areas with any combination of the compounds of formula II, III, IV and V.

The substrate can be a polymerized Langmuir Blodgett film, a functionalized glass, germanium, silicon, polymers, (poly) tetrafluoroethylene, polystyrene, gallium arsenite or combinations thereof;
Ion beams, electron beams, γ-rays, X-rays, ultraviolet rays, light, infrared rays, microwaves, electrical currents, radio waves and combinations thereof can be used as activators.

As light-sensitive protective groups such. B. local Nitrobenzyl derivatives, 6-nitroveratryloxycarbononyl, 2-nitrobenzyloxycarbonyl, zinnamoyl derivatives and mixtures thereof in Consideration.

The substrate to be used will also be used the activators, the photosensitive protective groups and the Conditions in which the individual steps are carried out full reference to the following publications taken: WO 90/15070, WO 91/07087, WO 92/10092, EP 0 728 520.

Advantages of PNA chips over DNA chips are u. a. the much better detection of mutations in the to investigating DNA, as well as the greater stability of PNA chips compared to DNA chips.  

A DNA or RNA strand in A is built up according to the methods known to the person skilled in the art, such as the Triester method, the H-phosphonate method or phosphoamidite method, preferably according to the standard phosphoamidite method according to Caruthers (MH Caruthers et al ., J. Am. Chem. Soc., 103, 3185 (1981)). The following compound can serve as a linker building block for connection to the conventional PNA synthesized in Scheme I:

where x is an optionally substituted alkyl, cycloalkyl, (cyclo-) alkenyl, (cyclo-) alkynyl, aroyl, heteroaroyl, or heterocycle
PG is a protecting group which must be compatible with a synthesized DNA or RNA or other antisense polymer strand, and e.g. B. dimethoxytrityl, Fmoc, Moc, 5-dimethoxybenzyl carbamate, o- or m-nitrophenyl carbamates such as Nvoc or 3,4-dimethoxy-6-nitrobenzyl carbamate or phenyl (o-nitrophenyl) methyl carbamate.

A peptide strand in A is constructed according to the subject known methods, such as the Merrifield method (E. Ather ton, R.C. Sheppard, Solid Phase Peptide Synthesis - A Practical Approach, IRL Press, New York, 1989). As a linker module for Connection to the conventional PNA synthesized in Scheme I can be any natural or synthetic N-protected anzo acid to serve. After the protective group has been split off, the Peptide strand a PNA strand according to the sketch in Scheme I. Methodology made.

The construction of an oligosaccharide strand in A takes place according to the methods known to the person skilled in the art, such as, B. the Schmidt trichloroacetimidate method or the ring opening of epoxides (P. Collins, R. Ferrier, Monosaccharides, Wiley, New York 1996, 415-430). As a linker building block for connection to the conventional PNA synthesized in Scheme I, e.g. B.

where X is an optionally substituted alkyl, cycloalkyl, (cyclo-) alkenyl, (cyclo-) alkynyl, aroyl, heteroaroyl, or heterocycle,
Y is a nucleophile such as S, O, NR with R = an optionally substituted alkyl, cycloalkyl, (cyclo-) alkenyl, (cyclo-) alkynyl, aroyl, heteroaroyl, heterocycle or -CHPL, where P and L independently of one another -CN , -NC, -COOR, -PO (OR) ₂ or -SO ₂ R,
PG is a protecting group that must be compatible with a synthesized strand of oligosaccharide, and e.g. B. Dimethoxytrityl, Fmoc or Pmoc or Nvoc mean.

The group X in formula V can be the starting point for another DNA, RNA, PNA or peptide or another oligomer or Polymer synthesis.

A DNA or RNA strand in X is built up according to the methods known to the person skilled in the art, such as the Triester method, the H-phosphonate method or phosphoamidite method, preferably according to the standard phosphoamidite method according to Caruthers (MH Caruthers et al ., J. Am. Chem. Soc., 103 3185 (1981)). As a linker building block for connection to the conventional PNA synthesized in Scheme I, e.g. B. serve the following compound of the general formula CN-X-NPG:

where the hydroxy group as an anchor for the following RNA / DNA or Phosphothioate antisense synthesis is used.

A peptide strand or another PNA strand in X is built up according to methods known to the person skilled in the art, such as the Merrifield method (E. Atherton, RC Sheppard, Solid Phase Peptide Synthesis - A Practical Approach, IRL Press, New York, 1989) or the method described in Scheme I (PNA). As a linker, any isocyanide with a protected amine function such. B. the following trifunctional isocyanide of the general formula CN-X-NPG are used:

After completion of the Ugi reaction with the isocyanide z. B. the Fmoc protecting group split off and a peptide or PNA strand built up. After the side chain synthesis is completed by Splitting off the Boc protecting group continues the PNA of the main chain synthesized.

The construction of an oligosaccharide strand in X is carried out according to the methods known to the skilled worker, such as, B. the Schmidt trichloroacetimidate method or the ring opening of epoxides (P. Collins, R. Ferrier, Monosaccharides, Wiley, New York 1996, 415-430). As a link module z. B. the following trifunctional isocyanide of the general formula CN-X-NPG are used:

where the glycolysis takes place on the free hydroxy function or the following isocyanide of the general formula CN-X-NPG are used:

where the glycolysis takes place via the nucleophilic Y group,
X substituted alkyl, cycloalkyl, (cyclo-) alkenyl, (cyclo-) alkynyl, aroyl, heteroaroyl, heterocycle,
Y is a nucleophile such as S, O, NR with R = substituted alkyl, cycloalkyl, (cyclo-) alkenyl, (cyclo-) alkynyl, aroyl, heteroaroyl, heterocycle or -CHPL, where P and L independently of one another -CN, - NC, -COOR, -PO (OR) ₂ or -SO ₂ R mean.

A DNA or RNA strand in B is built up according to the methods known to the person skilled in the art, such as the Triester method, the H-phosphonate method or phosphoamidite method, preferably according to the standard phosphoamidite method according to Caruthers (MH Caruthers et al ., J. Am. Chem. Soc., 103 3185 (1981)). As a linker building block for connection to the conventional PNA synthesis synthesized in Scheme I, e.g. B. serve the following compound of the general formula CN-X-NPG:

where X is substituted alkyl, cycloalkyl, (cyclo-) alkenyl, (cyclo-) alkynyl, aroyl, heteroaroyl or heterocycle,
PG is a protective group which must be compatible with a synthesized DNA or RNA or other antisense polymer strand, and z. B. dimethoxytrityl, Fmoc, Moc, 5-dimethoxybenzylcarbamate, or o- and m-nitrophenylcarbamates such as Nvoc or 3,4-dimethoxy-6-nitrobenzylcarbamate or phenyl (o-nitrophenyl) methylcarbamate, the hydroxyl group being the anchor for the following RNA / DNA or phosphothioate antisense synthesis is used.

Liquid phase processes are preferred to build shorter ones large amounts of oligomers used, while Solid phase process for the construction of long and small to large Amounts of oligomers are suitable and surface processes suitable for building up long and small amounts of oligomers are.

Experimental conditions of liquid phase synthesis

In the liquid phase, the amine component is reacted with the oxo component, the acid component and a suitably substituted amine-protected isocyanoamine component in accordance with formula scheme I. One equivalent of each of the four different components is advantageously added to one another and thus brought to reaction. It is also advantageous to precondense the amine and the oxo components to give the Schiff base. Aprotic polar as well as non-polar and protic polar are suitable as solvents. Due to the solubility properties of the nucleobasic acid components, protic and protic polar solvents such as e.g. B. alcohols such as water, methanol, ethanol, propanol, ethylene glycol, glycerol, trifluoroethanol and aprotic polar solvents such as. B. pyridine, N-methylmorpholine, methylene chloride, chloroform, dimethylformamide, dimethyl sulfoxide, acetonitrile, ethylene glycol dimethyl ether or mixtures thereof, such as. B. ethylene glycol dimethyl ether / glycerol or solvents promoting the base formation such as trimethyl orthoformate. Acylation catalysts such as B. pyridine or 4-dimethylaminopyridine prove to promote the reaction in the Ugi reaction. Lewis acids such as ZnCl ₂ , TiCl ₄ , ZrCp ₂ Cl ₂ etc. also prove to promote the reaction in the Ugi reaction. The reaction temperature can be -20 ° C and + 100 ° C, but preferably 10-40 ° C. Depending on the reactivity of the components, the reaction time is seconds to several days. The Ugi reaction is advantageously carried out in a concentrated manner, ie the concentrations of the individual components are 0.1M to 4M.

Experimental conditions of solid-phase synthesis

Polymers for the solid-phase synthesis of nucleobase polymers can e.g. B. polystyrene, polyethylene glycol, polyethylene glycol-Po lystyrene copolymers, polyacrylamide, controlled porous glass (CPG), Teflon, polyethylene, polypropylene, nylon, cellulose. Suitable linkers are e.g. B. aminomethyl, 4-methylbenzhydrylamine (MBHA), 4- (2 ', 4'-dimethyloxyphenyl-Fmoc-aminomethyl) - phenoxyacetainido-norleucylaminomethyl (Rink Amide AM), 4- (2 ', 4'- Dimethyloxyphenyl-Fmoc-aminomethyl) -phenoxyacetamido-norleucyl- MBHA (Rink Amide MBAH), 4- (2 ', 4'-dimethyloxyphenyl Fmoc aminomethyl) phenoxy (Rink amide), 9-Fmoc-aminoxanthen-3-yloxy (Sieber amide), hydrazine-2-chlorotrityl, 4-sulfamylbenzoyl AM, 4-sulfamylbutyryl AM, bis (2-aminoethyl) ether trityl, 1,3-bis (aminomethyl-phenyltrityl, or with N-terminal protected Resins loaded with amino acids.

The starting materials for the synthesis can be present in a wide variety of solvents mentioned in the previous section which are compatible with the swelling properties of the polymers. Based on the loading of the polymer, the starting materials are used in a 2-20-fold excess. Lewis acids such as ZnCl ₂ , TiCl ₄ , ZrCp ₂ Cl ₂ etc. also prove to promote the reaction in the Ugi reaction. The reaction temperature can be -20 ° C and + 100 ° C, but preferably 10-40 ° C. Depending on the reactivity of the components, the reaction time is seconds to several days. The Ugi reaction is advantageously carried out in a concentrated manner, ie the concentrations of the individual components are 0.1M to 4M.

Experimental conditions of synthesis on surfaces

The surfaces must be through linkers for polymer synthesis be modified. Possible surfaces are e.g. B. Glass surfaces, polymer surfaces such as Teflon, polyethylene, Polypropylene, nylon, cellulose. Be advantageous about one Left photo protection groups attached to the surface. By Removal of protecting groups from a selective area of the Surface can be created using the process described in Scheme I Construction of PNAs can be started. Everyone is advantageous Protection groups of the individual components Photo protection groups such as shown in the figure below.

Advantages and possible uses of the described invention

The process for the production of polymers presented here with nucleobases as side groups using Multicomponent reactions outperform in the Synthetic effectiveness and accessibility of new PNAs all previously described method. The PNA described so far Variants have many disadvantages for use as Antisense and antigenic agents. Most effective can be after new improved variants with the presented here Procedures are sought. Since in previous procedures over Monomers to be prepared in several stages polymerized to the PNAs  is the effectiveness of variation compared to this presented methods much lower. In which presented method is a monomer unit from four ver different components in one step. Since the Educts of the Ugi reaction in a large variety commercially are available or simply in one or two stages commercially available compounds are readily available can at the same time with comparable effort significantly more variants are produced.

About the combinatorial procedure presented here Monomer or di- and trimers shown can be potent Antivirals or cancerostats are presented.

Examples

The abbreviations used are listed below.
Alloc: allyloxycarbonyl
Boc: tert-butyloxycarbonyl
Boc ₂ O: di-tert-butyl pyrocarbonate
DCM: dichloromethane
DMF: dimethylformamide
Dmt: 4,4'-dimethoxytriphenylmethyl
Fmoc: 9-fluorenylmethoxycarbonyl
FmocONSu: N- (9-fluorenylmethoxycarbonyloxy) succinimide
Mmt: 4-methoxytriphenylmethyl
Moz: p-methoxybenzyloxycarbonyl
Pixyl: 9- (9-phenyl) xanthenyl
TBDMS: tert-butyldimethylsilyl
TFA: trifluoroacetic acid
Trt: triphenylmethyl
Z: benzyloxycarbonyl

General instructions for the synthesis of the 2-tritylamino-ethyliso-cyanide residues (X) Preparation of the 2-tritylethylenediamines

40 mmol of the respective trityl chloride (triphenylmethyl chloride, 4-methoxytriphenylmethyl chloride, 4,4'-dimethoxytriphenylmethyl chloride), which was dissolved in 500 ml of THF, are slowly added dropwise at RT to 12.20 g (200 mmol) of ethylenediamine in 500 ml of THF. The mixture is then stirred at RT for 12 h, the solvent is removed on a rotary evaporator, the oily, slightly yellow to orange residue is taken up in 500 ml of ethyl acetate and extracted three times with 250 ml of saturated sodium chloride solution. The organic phase is dried with sodium sulfate. After removal of the solvent, the respective amine is obtained as an oil or foam in yields of 50-95%.
N-tritylethylenediamine
C ₂₁ H ₂₂ N ₂ (288.42)
R _F = 0.21 (DCM / MeOH 5: 1, v, v); Spot colors with ninhydrin spray or with HCl steam)
N- (4-Monomethoxytrityl) ethylenediamine
C ₂₂ H ₂₄ N ₂ O (332.45)
R _F = 0.21 (DCM / MeOH 5: 1, v, v); Spot colors with ninhydrin spray or with HCl steam)
N- (4,4'-dimethoxytrityl) ethylenediamine
C ₂₃ H ₂₆ N ₂ O ₂ (362.48)
R _F = 0.22 (DCM / MeOH 5: 1, v, v); Spot colors with ninhydrin spray or with HCl steam)

Representation of the 2-tritylaminoethylformamides

190 mmol of the respective amine are refluxed in 300 ml of ethyl formate for 12 h (oil bath temperature 75 ° C.). After removing the excess ester on a rotary evaporator, the respective formamide is obtained as a stable or sticky, yellow to orange foam. The yields are between 80 and 95%.
2-tritylaminoethylformamide
C ₂₂ H ₂₂ N ₂ O (330.42)
R _F = 0.87 (DCM / MeOH 5: 1, v, v); Spot colors with ninhydrin spray or with HCl steam)
2- (4-monomethoxytrityl) aminoethylformamide
C ₂₃ H ₂₄ N ₂ O ₂ (360.46)
R _F = 0.86 (DCM / MeOH 5: 1, v, v); Spot colors with ninhydrin spray or with HCl steam)
¹ H NMR (CDCl ₃ , 250.133 MHz): = 1.70 (br s, 1H); 2.32 (tr, 2H, J = 6.0 Hz); 3.37 (m, 2H); 3.76 (s, 3H); 5.99 (br s, 1H); 6.78-7.49 (m, 14H); 8.17 (s, 1H).
¹³ C-NMR (CDCl ₃ , 62.896 MHz):
= 38.7 (CH ₂ ); 43.1 (CH ₂ ); 55.2 (CH ₃ ); 70.2 (C); 113.2 (CH); 126.4 (CH); 127.9 (CH); 128.3 (CH); 129.7 (CH); 137.8 (C); 145.9 (C); 158.0 (C); 164.7 (CHO).
2- (4,4'-Dimethyoxytrityl) aminoethylformamide
C ₂₄ H ₂₆ N ₂ O ₃ (390.49)
R _F = 0.92 (DCM / MeOH 5: 1, v, v); Spot colors with ninhydrin spray or with HCl)

Preparation of the 2-tritylaminoethylisonitrile

180 mmol of the respective formamide are dissolved in 250 ml of methylene chloride. After adding 400 mmol of TEA, the mixture is cooled to 0.degree. C., 180 mmol of phosphorus oxychloride are slowly added dropwise and stirring is continued at this temperature for two hours. Then 340 mmol of sodium carbonate in 150 ml of water are slowly added at 20 ° C. with vigorous stirring and the mixture is stirred for a further 30 min at RT. The aqueous phase is diluted to 400 ml and extracted twice with 150 ml of DCM. After washing the organic phase with saturated NaCl solution, drying over potassium carbonate and stripping off the solvent, the respective Isoni trile result as a foam in 85-95% yield.
2-tritylaminoethyl isocyanide
C ₂₂ H ₂₀ N ₂ (312.42)
R _F = 0.90 (DCM / MeOH 5: 1, v, v); Spot colors with ninhydrin spray or with HCl steam)
2- (4-monomethoxytrityl) aminoethyl isocyanide
C ₂₃ H ₂₂ N ₂ O (342.44)
R _F = 0.89 (DCM / MeOH 5: 1, v, v); Spot colors with ninhydrin spray or with HCl steam)
2- (4,4'-Dimethyoxytrityl) aminoethyl isocyanide
C ₂₄ H ₂₄ N ₂ O ₂ (372.47)
R _F = 0.95 (DCM / MeOH 5: 1, v, v); Spot colors with ninhydrin spray or with HCl steam)

General instructions for the synthesis of the N-acylethyl isocyanides N- (tert-butyloxycarbonyl) ethylenediamine

0.25 mol of Boc ₂ O in 500 ml of THF are added dropwise to a solution of 2.0 mol of ethylenediamine in 700 ml of THF at RT within 12-48 h. The solution is decanted from the precipitated solid and the solvent is stripped off. The residue and the precipitated solid are dissolved in 500 ml of water and the suspension formed is filtered. The aqueous phase is extracted three times with 150 ml of DCM, washed with concentrated NaCl solution, dried and evaporated. 36 g of an oil result (90% based on Boc ₂ O).

N- (tert-butyloxycarbonyl) aminoethylformamide

21.8 mmol of N- (tert-butyloxycarbonyl) ethylenediamine are dissolved in 50 ml of methyl or methyl formate and p-toluenesulfonic acid is added as a catalyst. This mixture is refluxed for 12 hours. After the solution has been spun in, the residue is taken up in 100 ml of DCM or ethyl acetate, washed twice with water, dried and the solvent is stripped off. 3.86 g of an oil are obtained which gradually crystallize out (94% yield).
Mp .: 84-86 ° C

2- (N-tert-butyloxycarbonyl) aminoethylisonitrile

The isonitrile is prepared according to the above. Regulation.

The result is an almost quantitative oil that gradually crystallizes out.
¹ H NMR (250 MHz, CDCl ₃ ): 1.42 (s, 9H, CH ₃ ), 2.92 (m, 2H, CH ₂ ), 3.40 (m, 2H, CH ₂ ) 5.00 (s, 1H, NH).
¹³ C NMR (62 MHz, CDCl ₃ ): 28.1 (CH ₃ ); 38.1 (t, ¹ J = 7.5 Hz, CH ₂ -NC), 39.0 (CH ₂ -NH), 80.0 (C), 155.8 (CO), 156.2 (t, ¹ J = 5.5 Hz, NC).

According to the above regulations, the following connections are in produced analogously:  

3- (N-tert-butyloxycarbonyl) aminopropylisonitrile

¹ H-NMR (250 MHz, CDCl ₃ ): 1.44 (s, 9H, CH ₃ ), 1.89 (m, 2H, CH ₂ ), 3.26 (q, 2H, ³ J = 6.2 Hz, CH ₂ ), 3.46 ( m, 2H, CH ₂ ) 4.92 (s, 1H, NH). ¹³ C NMR (62 MHz, CDCl ₃ ): 28.2 (CH ₃ ); 29.4 (CH ₂ ), 38.1 (t, ¹ J = 7.6 Hz, CH ₂ -NC), 39.0 (CH-NH), 79.4 (C), 155.9 (CO), 156.4 (t, ¹ J = 5.3 Hz , NC).

6- (N-tert-butyloxycarbonyl) aminohexylisonitrile

¹ H NMR (250 MHz, CDCl ₃ ): 1.36 (m, 6H, CH ₂ ), 1.44 (s, 9H, CH ₃ ), 1.89 (m, 2H, CH ₂ ), 3.27 (m, 2H, CH ₂₎ ), 3.47 (m, 2H, CH ₂ ), 5.02, 4.92 (2s, 1H, NHCO rotamers).
¹³ C-NMR (62 MHz, CDCl ₃ ): 28.2 (CH ₃ ), 28.3 (CH ₂ ), 29.5 (CH ₂ ), 30.5 (CH ₂ ), 38.2 (t, ² J = 6.7 Hz, CH ₂ -NC ), 39.0 (CH ₂ -NH), 77.4 (C), 156.2 (CO), 156.4 (t, ² J = 5.3 Hz, NC).

8- (N-tert-Butyloxycarbonyl) aminooctylisonitrile

¹ H-NMR (250 MHz, CDCl ₃ ): 1.31 (m, 10H, CH ₂ ), 1.44 (s, 9H, CH ₃ ), 1.69 (m, 2H, CH ₂ ), 3.11 (m, 2H, CH ₂₎ -NC), 3:37 (m, 2H, CH ₂ -NHCO), 4:57 (s, 1H, NH).
¹³ C-NMR (62 MHz, CDCl ₃ ): 26.5 (CH ₂ ), 26.6 (CH ₂ ), 28.5 (CH ₃ ), 28.9 (CH ₂ ), 29.0 (CH ₂ ), 29.1 (CH ₂ ), 29.9 ( CH ₂ ), 40.5 (CH ₂ -NC), 41.5 (CH ₂ -NH), 77.4 (C), 156.6 (CO), 155.9 (NC).

N-1- (4-methoxybenzyloxycarbonyl) -N'-2-formylethylene diamine

Synthesis of N- (4-methoxybenzyloxycarbonyl) ethylenediainine according to the instructions [LS Richter, RN Zuckermann, Bioorg. Med. Chem. Lett., 5, 1159-1162 (1995)], the organic methylene chloride phase being washed twice with 100 ml of saturated saline. Yield: 7.1 g, 64% (lit .: 6.0 g, 54%).
7.1 g of N- (4-methoxybenzyloxycarbonyl) ethylenediamine are boiled under reflux in 100 ml of ethyl formate for 3 hours and a spatula tip of 4-dimethylaminopyridine. The excess formic acid ethyl ester is removed on a rotary evaporator under vacuum. The remaining solid is taken up in 100 ml CH ₂ Cl ₂ and extracted with 30 ml H ₂ O. The organic phase is dried with Na ₂ SO ₄ and evaporated in vacuo. The remaining oil (6.53 g, 81% yield) crystallizes after some time and is pure enough for the further reaction.
¹ H-NMR (250 MHz, CDCl ₃ ): 3.37 (m, 4H), 3.80 (s, 3H), 5.02 (s, 2H), 5.28 (s, br, 1H), 6.32 (s, br, 1H) , 6.87 (m, 2H), 7.27 (m, 2H), 8.14 (s, br, 1H).
¹³ C-NMR (62 MHz, CDCl ₃ ): 39.0, 40.6, 55.3, 66.8, 113.9, 128.4, 129.9, 159.7, 161.8, 163.4.
R _f (CH ₂ Cl ₂ / MeOH 9/1): 0.6.

N-1- (4-methoxybenzyloxycarbonyl) -2-isocyano-1-aminoethane

To 6.53 g of N-1- (4-methoxybenzyloxycarbonyl) -N'-2-formylethylenediamine (25.9 mmol) and 10.85 ml of triethylamine (78 mmol) in 100 ml of CH ₂ Cl ₂ , tempered with an ice bath, are slowly added within one 3.97 g of POCl ₃ (25.91 mmol) were added dropwise for 1/2 h and the mixture was stirred at 0 ° C. for a further 4 h. An aqueous solution of 5.51 g Na ₂ CO ₃ in 40 ml H ₂ O is slowly added dropwise at 0 ° C. Finally, stirring is continued for 1 h at room temperature. It is diluted with a further 100 ml of water, the organic phase is separated off and the aqueous phase is extracted twice with 50 ml of CH ₂ Cl ₂ . The combined organic phases are dried with MgSO ₄ and evaporated in vacuo. 6.0 g (99%) of red solid remain, the purity of which is sufficient for the subsequent reactions. An analytical sample, recrystallized from ethyl acetate, gives a colorless solid.
¹ H NMR (250 MHz, CDCl ₃ ): 3.43 (m, 2H), 3.53 (m, 2H), 3.80 (s, 3H), 5.04 (s, 2H), 5.50 (s, br, 1H), 6.88 (m, 2H), 7.28 (m, 2H).
¹³ C-NMR (62 MHz, CDCl ₃ ): 40.2, 41.8, 55.3, 66.9, 114.0, 128.2, 130.0, 156.4, 159.7.
R _f (CH ₂ Cl ₂ / MeOH 9/1): 0.8.

example 1

1 mmol thymine acetic acid, 1 mmol oxo component, 1 mmol amine component and 1 mmol isocyanide component are stirred in 1 ml methanol for 24 h and the precipitated product is filtered off. The yields can be increased if the filtrate is concentrated in half and subjected to a further filtration.

C ₂₃ H ₃₁ N ₅ O ₆
M _w : 473.53342
¹ H-NMR (360 MHz / d ₆ -DMSO): 1.35 (9H, s), 1.76 (3H, s), 2.95-3.1 (4H, m), 3.77 and 3.93 (2H, 2 × s {rotamers}) , 4.46, 4.58, 4.62, 4.67, 4.76, 4.80 (4H, 6 × s {rotamers}), 6.78 (1H, m), 7.21-7.39 (6H, m), 7.87 and 8.13 (1H, 2 × tr {rotamers }), 11.29 (1H, s, thymine-NH).
¹³ C-NMR (62.9 MHz): 11.7, 28.1, 47.9, 48.8, 49.5, 77.5, 107.9, 127.1, 127.4, 127.6, 128.2, 128.5, 136.1, 136.8, 142.2, 150.9, 155.5, 158.0, 164.3, 167.2.
ES-MS: 474.0 (m + H) ⁺

Example 2

1 mmol thymine acetic acid, 1 mmol oxo component, 1 mmol amine component and 1 mmol isocyanide component are stirred in 1 ml methanol for 24 h and the precipitated product is filtered off. The yields can be increased if the filtrate is concentrated in half and subjected to a further filtration.

Example 3

1 mmol thymine acetic acid, 1 mmol oxo component, 1 mmol Amine component and 1 mmol isocyanide component are in 1 ml Methanol was stirred for 24 hours and the precipitated product was filtered off. The yields can be increased if the filtrate is half is concentrated and subjected to a further filtration.  

Example 4

1 mmol thymine acetic acid, 1 mmol oxo component, 1 mmol amine component and 1 mmol isocyanide component are stirred in 1 ml methanol for 24 h and the precipitated product is filtered off. The yields can be increased if the filtrate is concentrated in half and subjected to a further filtration.

Example 5

A deep well multi-titer plate made of polypropylene with a volume of 1 ml per well is loaded with thymine-1-acetic acid in methanol and 1-isocyano-2-N-tert-butoxycarbonylethane in methanol per well. Rows AH are filled with the amines shown below and columns 1-12 with the oxo components shown below. The sealed MTPs are shaken at room temperature and the precipitated products are filtered off. To increase the yields, the filtrate can be concentrated in half and the products which have precipitated again can be filtered off. The products were characterized by HPLC-ES, TLC and by ¹ H / ¹³ C-NMR.

Equipping the 96-MTP

Example 6 Preparation of a thymine pentamer

C ₆₅ H ₉₃ N ₂₁ O ₂₀
M _w = 1488.56
HPLC-ESI-MS: corresponds.

The following synthesis scheme is used to prepare this compound on Fmoc-Rinkamid resin:

• 1. Remove the Fmoc group of the resin with morpholine.
• 2. shaking the resin treated in this way with acetone, Thyme acetic acid and N-1- (4-methoxybenzyloxycarbonyl) -2- isocyano-1-aminoethane in DMSO / DMF.
• 3. Capping step with acetic anhydride in DMF.
• 4. Remove the Moz protective group with 5% trifluoroacetic acid, 2.5% thioanisole, 2.5% dimercaptoethane in CH ₂ Cl ₂ .
• 5. Neutralize with morpholine.
• 6. Repeat steps 2-5 4 times.
• 7. Elimination from the resin with 95% TFA.

Example 7

1 mmol thymine acetic acid, 1 mmol oxo component, 1 mmol Amine component and 1 mmol isocyanide component are in 1 ml Methanol was stirred for 24 hours and the precipitated product was filtered off. The yields can be increased if the filtrate is half is concentrated and subjected to a further filtration.  

¹ H-NMR (250 MHz / d ₆ -DMSO): 1.23 (6H, s), 1.37 (9H, s), 1.75 (3H, s), 2.95-3.17 (4H, m), 4.58 (2H, s) , 4.73 (2H, s), 6.70 (1H, tr), 7.29-7.50 (6H, m), 11.25 (1H, d, thymine-NH).
¹³ C-NMR (62.9 MHz): 12.2, 24.4, 28.5, 43.0, 47.4, 49.7, 62.9, 78.0, 108.0, 126.9, 127.5, 128.9, 128.2, 138.8, 142.6, 151.4, 156.1, 164.8, 169.9, 174.1.
ES-MS: 502.0 (m + H) ⁺

Example 8

1 mmol thymine acetic acid, 1 mmol oxo component, 1 mmol amine component and 1 mmol isocyanide component are stirred in 1 ml methanol for 24 h and the precipitated product is filtered off. The yields can be increased if the filtrate is concentrated in half and subjected to a further filtration.

¹ H-NMR (360 MHz / d ₆ -DMSO): 0.83-1.54 (8H, m), 1.37 (9H, s), 1.75 (3H, s), 2.24 (2H, m), 2.95-3.17 (4H, m), 4.56 (2H, s), 4.73 (2H, s), 6.67 (1H, tr), 7.27-7.48 (6H, m), 11.28 (1H, s, thymine-NH).
¹³ C-NMR (62.9 MHz): 11.6, 22.0, 24.7, 28.0, 32.2, 46.6, 49.7, 65.2, 77.4, 107.8, 126.5, 126.9, 128.4, 138.1, 136.1, 142.1, 142.4, 151.0, 155.5, 157.1, 164.3 , 167.7, 172.7.
ES-MS: 542.0 (m + H) ⁺

Example 9

1 mmol thymine acetic acid, 1 mmol oxo component, 1 mmol amine component and 1 mmol isocyanide component are stirred in 1 ml methanol for 24 h and the precipitated product is filtered off. The yields can be increased if the filtrate is concentrated in half and subjected to a further filtration.

C ₂₃ H ₄₈ N ₆ O ₇
M _w : 520.67501
¹ H-NMR (250 MHz / d ₆ -DMSO):
¹³ C-NMR (62.9 MHz): 11.8, 23.7, 24.0, 28.1, 44.9, 45.6, 47.9, 49.8, 53.0, 54.4, 55.3, 66.1, 77.6, 107.8, 142.3, 150.9, 155.5, 164.3, 166.9, 167.3, 167.9 , 168.0.
ES-MS: 522 (m + H) ⁺

Example 10

Execution analogous to the previous examples.
Isocyanide component: 1-isocyano-3-N (tert-butoxycarbonyl) propane
Oxo component: paraformaldehyde
Amine component: benzylamine
Acid component: N- (p-methoxybenzoyl) -N ₉ -adeninetic acid
C ₃₂ H ₃₈ N ₈ O ₆
M _w = 630.71
ES-MS: 632 (m + H) ⁺

Example 11

1 mmol thymine acetic acid, 1 mmol oxo component, 1 mmol amine component and 1 mmol isocyanide component are stirred in 1 ml methanol for 24 h and the precipitated product is filtered off. The yields can be increased if the filtrate is concentrated in half and subjected to a further filtration.

C ₂₇ H ₂₈ N ₆ O ₁₀
M _w = 596.56

Claims (5)

1. A process for the preparation of compounds of formula (I) is disclosed
characterized in that compounds of the formulas
A-NH ₂ II
R ₁ -G III
R ₂ -C (O) -R ₃ IV and
C = NX-NPG V
in a first step, if necessary, implemented simultaneously with one another,
optionally one or more protective groups are removed and the reaction is repeated m times,
the product of the preceding step being used after the first step instead of the compound having the formula II,
in which
m is 0 or an integer from 1 to 1000, preferably 1 to 300,
A is a residue of the amine component customary in the Ugi reaction, such as a hydrogen atom, a substituent, (cyclo) alkyl, (cyclo) alkenyl, (cyclo) alkynyl, aroyl, heteroaroyl, a heterocycle, a fluorescence marker, an intercalator, an antibiotic , a minor groove binder, a major groove binder, a biotinyl residue, an intercalating residue, an alkylating residue, a steroid, a lipid, a polyamine, an agent that facilitates cell uptake, a saccharide or oligosaccharide, an antisense polymer, a peptide, an antibody Conjugate, a synthetic polymer or a correspondingly modified surface,
B is a hydrogen atom, a substituent, (cyclo) alkyl, (cyclo) alkenyl, (cyclo) alkynyl, aroyl, heteroaroyl, a heterocycle, a fluorescent label, an intercalator, an antibiotic, a minor groove binder, a major groove binder , a biotinyl residue, an intercalating residue, an alkylating residue, a steroid, a lipid, a polyamine, an agent which facilitates cell uptake, a saccharide or oligosaccharide, an antisense polymer, a peptide, an antibody conjugate, a synthetic polymer or one appropriately modified surface or a residue X - NPG of compound V,
R1-G is selected from structures of the following formulas:
where the group R ₁ -G can be linked via a molecular spacer via R ₁ or R or R ₄ to the compound of the formula IV;
R ₁ and R independently of one another are a residue of the acid component customary in the Ugi reaction, such as H, a substituent, (cyclo) alkyl, (cyclo) alkenyl, (cyclo) alkynyl, aroyl, heteroaroyl, a heterocycle, a fluorescent marker Intercalator, an antibiotic, a minor groove binder, a major groove binder, a biotinyl residue, an intercalating residue, an alkylating residue, a steroid, a lipid, a polyamine, a saccharide or oligosaccharide, an antisense polymer, a peptide, an antibody conjugate , are a synthetic polymer or a correspondingly modified surface, or residues derived from the natural nucleobases or from synthetic nucleobases;
L, M, T and Z independently of one another are O, S or NR ₄ , where R _{4 is} H, fluorine, (cyclo-) alkyl, (cyclo-) alkenyl, (cyclo-) alkynyl, aroyl, heteroaroyl, heterocycle or -O Are (cyclo) alkyl, -Oaroyl, -S (cyclo) alkyl, -Saroyl;
R ₂ and R ₃ independently of one another are a radical of the oxo component which is customary in the Ugi reaction, such as H, a substituent, (cyclo) alkyl, (cyclo) alkenyl, (cyclo) alkynyl, aroyl, heteroaroyl, a heterocycle, a fluorescent marker, an intercalator, an antibiotic, a minor groove binder, a major groove binder, a biotinyl residue, an intercalating residue, an alkylating residue, a steroid, a lipid, a polyamine, a saccharide or oligosaccharide linked via an amine spacer, an antisense molecule, a peptide an antibody conjugate, a synthetic polymer, a modified surface or a branching point P, which in turn is the starting point for a further strand of DNA, RNA, PNA or peptide or another oligomer or polymer,
X has the following structure:
PG, independently of one another, optionally represent orthogonal protective groups such as amine protective groups from the class of the N-acyl derivatives, the N-sulfonyl derivatives, the N-alkyl derivatives, the N-silyl derivatives, the carbamates or the salts;
the radicals R ₅ independently of one another represent hydrogen atoms, unsubstituted or substituted alkyl, cycloalkyl, alkoxyalkyl or aryl groups or heterocycles;
the radicals U, W, K and Y independently of one another are unsubstituted or substituted alkyl, alkenyl, alkynyl, alkanoyl, alkoxyalkanoyl, cycloalkyl or aryl groups, unsubstituted or substituted heterocycles or the group NR ₅ , where R _{5 is} like is defined above;
a, b, c, n, o and p are independently an integer from 0-10, preferably 0-5;
Q is an unsubstituted or substituted alkyl, aryl, al kenyl, alkynyl, mono- or polyvalent alkanoyl, cycloalkyl, alkoxyalkanoyl, cycloalkanoyl, aroyl group or an unsubstituted or substituted heterocycle, or one of the groups NR ₅ , Represents P, P (O), P (S), B, BR ₅ or SO ₂ , where R _{5 is} as defined above and the indices a, b, o and p each have corresponding values;
F represents an oxo, thio, seleno or imino group, and
J is selected from the following structures, the upper vertical line representing the bond to R ₁ and the lower vertical line representing the bond to the nitrogen atom of the main chain of the polymer in general formula I:
wherein R _{6 is} H, a substituent, (cyclo-) alkyl, (cyclo-) alkenyl, (cyclo-) alkynyl, aroyl, heteroaroyl, heterocycle, with the proviso that at least one of the radicals R or R ₁ in the compound of Formula I is a residue derived from a natural or synthetic nucleobase. 2. The method according to claim 1, characterized in that the fluorescent marker fluorescein, Texas red, lissamine rhodamine, cyanine or rhodamine, the intercalators are psoralen, acridine, phenanthroline, a phenanthroline-metal complex or ellipticin, the antibiotic endiins, β-lactams, Tetracyclines, anthracyclines, polyethers, mitomycin-like, phosphomycin-like, macrolides, bleomycin-like or an aminoglycoside, the minor groove binder is netropsin or distamycin, the polyamine is a spermidine-like polyamine, the antisense polymer is a DNA (5 ' or 3 'linked) or RNA strand (5' or 3 'linked) or is a phosphothioate, the peptide is N- or C-terminally linked, the antibody conjugate is selected from antibody conjugates that allow cell-specific uptake, address specific carrier systems or Endocytosis, the synthetic polymer is CPG, Wang, or Tentagel, the natural nucleobases adenine, thymine, guanine, cytosine or uracil and the synthetic nucleobases are pseudouracil, 5-propynyluracil, 5-hexenyluracil, 5-fluorocytidine, 5-hydroxymethyluracil, 5-methylcytidine, 5-bromocytidine and compounds of the following formula
where R ₈ and R _{9 are} independently H, alkyl, (cyclo-) alkenyl, (cyclo-) alkynyl, aroyl, heteroaroyl, heterocycle, chlorine or fluorine,
R ₁₀ = fluorine, bromine, iodine, chlorine, alkynyl, alkyl, aroyl, heteroaroyl or H, and n = 1-20, preferably 1-10 and particularly preferably 1-5, where the side groups of the bases can have protective groups. 3. The method according to any one of the preceding claims, characterized ge indicates that m = 1-200, preferably 1-100. 4. The method according to any one of the preceding claims, characterized ge indicates that in each reaction step at least one of the Components III, IV, and / or V is varied. 5. The method according to any one of the preceding claims, characterized ge indicates that it is on a solid phase such as a chip is carried out.