CZ20013138A3 - Organic compounds based on porphyrin - Google Patents

Abstract

In the present invention, there are claimed novel derivatives of 5,15-bis(p-tolyl)porphyrin and 5,10.15,20-tetrakis(p-tolyl)porphyrin of the general formula I, in which the substituents Re1 through Re8 are identical or different and are selected from the group consisting of hydrogen, alkyl having one to eight carbon atoms; the substituents Re9 and Re10 are identical or different and are selected from the group consisting of hydrogen, alkyl, phenyl, substituted phenyl wherein the substituent is represented by methyl, amino, nitro, cyano, halo, methoxy, or hydroxy group, carboxyamido guanidinium; the substituents Re11 and Re12 are identical or different substituted phenyl groups in positions 2 or 3, 5, 6, and the substituents are selected from the group consisting of hydrogen, aryl, binaphthoyl, a halogen, a cyano group, a nitro group, carboxyl and methyl through octyl esters thereof; the substituent Re13 represents oxygen O or two hydrogen atoms Hi2; the substituents X and Y, which can be identical or different denote trialkylammonium group (N(Re14)i3), in which Re14 represents alkyl, a hydroxyalkyl group, a polyhydroxyalkyl group, an aminoalkyl group, a polyaminoalkyl group; further, X and Y represent an N-substituted pyridine group wherein the substituent is represented by methyl, amino, nitro, cyano, halo, methoxy or hydroxy group, or X and Y represent a guanidinium group NH(CH=NHi2)NHRe15, aminoguanidinium group NHNH(CH=NHi2)NHRe15, methylguanidinium group CHi2NH(CH=NHi2)NHRe15, and hydroxyethyl guanidinium group OCHi2CHi2NH(CH=NHi2)NHRe15 in which Re15 denotes hydrogen, alkyl, or X and Y denote a dialkylsulfonium group S(Re16)i2, in which Re16 represents alkyl, a or X and Y represent a trialkylphosphonium group P(Re17)i3, in which Re17 denotes alkyl. Further claimed is a process for preparing the above-described derivatives as well as their use for DNA and oligonucleotide transfer into primary cells of vertebrates to influence expression of selected genes.

Description

Technical field

The present invention relates to novel porphyrin-based organic compounds and their use

for the transfer of DNA and oligonucleotides

BACKGROUND OF THE INVENTION

Chemical compounds that could effectively introduce DNA and oligonucleotides into cells must have the following properties: (a) they must form solid but dissociable complexes with DNA so that the bound DNA can be released inside the cells; (b) they must be readily transportable across cell membranes; and (c) they must not be toxic; and (d) they must not (in themselves) interfere with the basic regulatory mechanisms of cells. The use of porphyrins to transfer DNA into cells results from their ability to form relatively stable complexes with DNA and from their lipophilic character, through which they can cross the lipoprotein membranes of cells. Complex formation between DNA and water-soluble cationic porphyrins has been studied in a number of studies (see review article Kubát, P., Lang, K., Anzenbacher, P.Jr., Jursíková, K., Klál, V., Ehrenberg, BJ Chem. Soc. Perkin Trans.,

I, 933-941 (2000)). Also, some expanded porphyrins, particularly safyrins, show a strong but reversible interaction with DNA (King, V., Furuta, H., Shreder, K., Lynch, V., Sessler,

Am. Chem. Soc., 118, 1595-1607 (1996); Sessler, J. L., Sansom, P.I., King, V., O'Connor, D. and Iverson, B.L. J. Am. Chem. Soc., 118, 12322-12330 (1996); Iverson, B. L., Shreder, K., King, V., Sansom, P., Lynch, V., Sessler, J.L. J. Am. Chem. Soc., 118, 1608-1616 (1996)). However, some porphyrin derivatives are toxic to cells.

Currently, cationic lipids are used to transfer oligonucleotides to cells (CF Bennetb MY Chiang, H. Khan, JE Shoemaker, CK Miiabelli. Mol. Pharmacol., 41, 10231033 (1992); Lewis, JG, Lin, KY, Kothavale, Flanagan, WM, Matteucci, MD, DePrince,

R. B., Mook, R. A., Hendren, R. W. and Wagner, R.W. Why. Nati. Acad. Sci. USA 93: 3176-3181 (1996); Bhat, B., Hebett, N., Marcusson, E.G., Dean, N.M., Bennett, C.F. and Manoharan, M. Nucleosides Nucleotides, 18, 1727-1728 (1999)), liposomes (Clarenc, JP, Degols, G., Leonetti, JP., Milhaud, P., and Lebleu, B. Anticancer Drug Des., 8, 81 -94 (1993); Arima, H., Aramaki, Y., Tsuchiya,

S.. J. Pharm. Sci., 86: 438-442 (1997); Willis, M., Forssen, E. Adv. Drug Deliv. Rev., 29, 249-271 (1998); Bennett C.F. (1995) In Akhatar, S. (ed.), Antisens Oligonucleotide Delivery Strategies • 4 • 4 • 4 φ. · 4 4 44 • 4 4 4 4 4

Π 4 4 4 4 4 44

Zr 4444 44 4 44 444

Therapeutics. CRC Press, Boca Raton, FL, pp.223-232), cationic oligopeptides (Dokka, S., Toledo-Velasquez, D., Shi, X., Wang, L., Rojanasakul, Y. Pharm. Res., 14 , 1759-1764 (1997), Chaloin, L. ,, Vidal, P., Lory, P., Mery, J., Lautredou, N., Divita, G., Heitz, F. Biochem., Biophys, Res. 243, 601-608 (1998), Antopolsky, M., Azhayeva, E., Tengvall, U., Autiola, S., Jaaskelainen, I., Ronkko, S., Honkakoski, P., Urtti, A., Lonnberg, H., Azhayev, A. Bioconjugate Chem., 10, 598-606 (1999); Petzinger, E., Wickboldt, A., Pagels, P., Starke, P., Kramer, W. Hepatology, 30, 1257-1268 (1999), Niidome, T., Wakamatsu, M., Wada, A., Hirayama, T., Aoyagi, HJ Pept. Sci., 6, 2Τ1-2Ί9 (2000); Hamilton, SE, Simmons, CG, Kathiriya, IS, Corey, DR Chem Biol., 6, 343-351 (1999), Astriab-Fisher, A., Sergueev, DS, Fisher, M., Shaw, BR, Juliano, RL, Biochem, Pharmacol. 60, 83-90 (2000)), dendrimers (Poxon, SW, Mitchell, PM, Liang, E., Huges, JA Drug Delivery, 3, 255-265 (1996)), swallowed onty (Boussif, O., Lezoualc'h, F., Zanta, et al

M.A., Mergny, M. D., Scherman, D., Demeneix, B., Behr, J.P. Why. Nati. Acad. Sci. USA, 92, 72977301 (1995); Leonetti, JP, Degols, G. and Leblcu, B. Bioconjugate Chem., 1, 149-153 (1990)), cationic liposomes (lipoplex) (Islam, A., Handley, SL, Thompson, KS, Akhtar, SJ Drug Target., 7, 373-382 (2000); Gao, WY, Jaroszewski, JW, Cohen, JS, Cheng, YCJ Biol. Chem., 265, 20172-20178 (1990); Lee, RJ, Huang, L. Crit Rev. Ther Drug Carrier Syst., 14, 173-206 (1997), Choi, JS, Lee, EJ, Yang, HS, Park, JS, Bioconjugate Chem., 12, 108-113 (2001). Lappalainen, K., Miettinen, R., Kellokoski, I, Jaaskelainen, I., Syqanen, SJ Histochem. Cytochem., 45, 265-274 (1997), cationic polymers (polyplex) (Brown, MD, Schatzlein, A. Brownlie, A., Jack, V., Wang, W., Tedey, L., Gray, Al and Uchegbu, IF Bioconjugate Chem., 11, 880-891 (2000); Pouton, CW, Lucas, P. Thomas, BJ, Udueei, AN, Milroy, DA, Moss, SHJ Control, Release, 53, 289-299 (1998); Kwon, GS, Okano, T. Pharm. Res., 16, 597-600 (1999) Gamier, F., Korri-YousoufFi, H., Srivastava, P., Mand Rand, B., Delair, T. Synth. Met., 100, 89-94 (1999); Benns, J.M., Choi, J.-S., Mahato, R.I., Park, J.S., Kim, S.W. Bioconjugate Chem., 11, 637-645 (2000)) or a combination of cationic liposomes and cationic polymers (lipopolyplex) (Hong, K., Zheng, W., Baker, A., Papahadjopoulos, D. FEBS Leti., 400, 233). -237 (1997)), surface modified nanoparticles (Chavany, C., Le Doan, T., Couvreur, P., Puisieux, F., Helene, C. Pharm. Res., 9, 441-449 (1992); Vinogradov, S., Batrakova, E., Kabanov, A. Colloids Surf, BBiointerfaces, 16, 291-304 (1999); Fan, ZH, Mangru, S., Granzow, R., Heaney, P., Ho, W., Dong, Q., Kumar, R. Anal. Chem., 71, 4851-4859 (1999)) and novel synthetic surfactants (niosomes) (Uchegbu, IF, Schatzlein, A., Vanlerberghe, G., Morgatini, N. and. Florence, ATJ Pharm. Pharmacol., 49, 606-610 (1997)).

Conjugation of oligonucleotides to lipophilic molecules such as cholesterol has also been used (Alahari, S.K., Dean, N.M., Fisher, M.H., Delong, R., Manoharan, M., Tivel, K.L.,

Juliano, R.L. Mol. Pharmacol. 50, 808-819 (1996)) or complexing of oligonucleotides with cell receptor ligands (Willis, M. and Forssen, E. Adv. Drug Deliv. Rev., 29, 249-271 (1998); Wu-Pong, S. Bard, J., Huffman, J., Jimerson, J. Biol. Cell., 89, 257-261 (1997);

Perrotti, D., Cucco, C., D'Agnano, I., Sacchi, A., Zupi, G., Calabretta, B. Proc. Nati. Acad. Sci. U SA, 89, 7031-7035 (1992)).

It has been observed that a particular delivery agent efficiently delivers DNA or oligonucleotides mostly only to certain cell types. In addition, the transfer of DNA or oligonucleotides to primary cells, i.e., to a polyclonal population of cells obtained directly from the organism, is shown to be generally inefficient.

SUMMARY OF THE INVENTION

These disadvantages are overcome by the novel compounds derived from 5,15, -bis (p-tolyl) porphyrin and 5,10,15,20-tetrakis (p-tolyl) porphyrin having the general formula (I)

wherein the substituents R ^{1 to} R ⁸ are the same or different and are selected from the group consisting of hydrogen (H), alkyl of one to eight carbon atoms, C _n H _2n + i wherein n is 1 to 8;

R ⁹ and R ¹⁰ are the same or different and are selected from the group consisting of hydrogen, C 1 -C 8 alkyl, phenyl (C 5 H 5), substituted phenyl, wherein the substituent is methyl, amino, nitro, cyano, halogen, methoxy, hydroxy, carboxyamidoguanidinium;

R ¹¹ and R ¹² are the same or different and are in the 2- or 3,5,6-position of the phenyl group, and are selected from the group consisting of hydrogen, aryl, halogen (fluoro, chloro, bromo, iodo), cyano, nitro, carboxyl an acid and its esters of one to eight carbon atoms; ...

R ¹³ is O or two H ₂ atoms;

the substituents X and Y, which may be the same or different, are a trialkylammonium (N (R ¹⁴ ) 3) group wherein R ¹⁴ is alkyl of one to eight carbon atoms, hydroxyalkyl, polyhydroxyalkyl, aminoalkyl, polyaminoalkyl;

further X and Y is an N-substituted pyridinium group C 6 H, wherein the substituent is methyl, amino, nitro, cyano, halogen, methoxy, hydroxy, or X and Y is guanidinium NH (CH = NH ₂ ) NHR ¹⁵ , aminoguanidinium NHNH (CH = NH 2) NHR ¹⁵ , methylguanidinium CH ₂ NH (CH = NH ₂ ) NHR ¹⁵ , and hydroxyethylguanidinium OCH ₂ CH ₂ NH (CH = NH ₂ ) NHR ¹⁵ wherein R ¹⁵ is hydrogen, alkyl of one to eight carbon atoms or X and Y is a dialkylsulfonium group S (R ¹⁶ ) ₂ , wherein R ¹⁶ is an alkyl of one to eight carbon atoms, or X and Y is a trialkylphosphonium group P (R ¹⁷ ) 3, wherein R ¹⁷ is an alkyl of one to eight carbon atoms.

A process for the preparation of a compound of formula I wherein R ¹ to R ¹² are as defined above and wherein R ¹³ is oxygen is to form a compound of formula I wherein X and Y are hydroxy and R ¹³ is oxygen and R ¹ to R ¹² have the aforementioned meaning, act as an amino component which allows covalent attachment to the porphyrin backbone via an amide bond and provides the meanings of X and Y according to claim 1.

A process for the preparation of a compound of formula (I) wherein R ¹ to R ¹² is as defined above and wherein R ¹³ is 2 hydrogens is to form a compound of formula (I) wherein: ·········· ··· Wherein R ¹ to R ¹² are as defined above and wherein R ¹³ are 2 hydrogen and X and Y are bromine or chlorine atoms, act by O, N, S, P by a nucleophile selected from the group consisting of alcoholates, phenolates , amines, pyridines, sulfides or phosphines.

A process for the preparation of 5,15-bis derivatives of a compound of formula I wherein R ¹ to R ¹² are as defined above and wherein R ¹³ are 2 hydrogels with a nucleophile act on a compound of formula I wherein R ¹ to R ⁸ are as defined above and wherein R ¹³ are 2 hydrogen and X and Y are bromine or chlorine atoms and wherein R ⁹ and R ¹⁰ are hydrogen.

A process for the preparation of 5,10,15,20-tetrakis derivatives of a compound of formula I wherein R ¹ -R ¹² are as defined above and wherein R ¹³ are 2 nucleophile hydrogens act on a compound of formula I wherein R ¹ -R ⁸ have a wherein R ¹³ is 2 hydrogen and X and Y are bromine or chlorine atoms and wherein R ⁹ and R ¹⁰ are bromomethylphenyl or chloromethylphenyl groups.

Said novel compounds derived from 5,15'-bis (p-tolyl) porphyrin and 5,10,15,20-tetrakis (p-tolyl) porphyrin having the general formula (I),

wherein the substituents R ¹ to R ⁸ are the same or different and are selected from the group consisting of hydrogen (H), alkyl of one to eight carbon atoms, C 11 Phen + i wherein n is 1 to 8;

• 99 •. 99

9 9 9 9 9 99 9999

9 9 9 9 9 99

R ⁹ and R ¹⁰ are the same or different and are selected from the group consisting of hydrogen, alkyl of one to eight carbon atoms, phenyl (C 6 H 5), substituted phenyl, wherein the substituent is methyl, amino, nitro, cyano, halogen, methoxy, hydroxy, carboxyamidoguanidinium;

R ¹¹ and R ¹² are the same or different and are in the 2- or 3,5,6-position of the phenyl group, and are selected from the group consisting of hydrogen, aryl, halogen (fluoro, chloro, bromo, iodo), cyano, nitro a carboxylic acid and its esters of one to eight carbon atoms;

R ¹³ O is oxygen or two hydrogens H2;

the substituents X and Y, which may be the same or different, are a trialkylammonium group (N (R ¹⁴ ) 5), wherein R ¹⁴ is alkyl of one to eight carbon atoms, hydroxyalkyl, polyhydroxyalkyl, aminoalkyl, polyaminoalkyl;

further X and Y is N-substituted pyridinium group C6H5, wherein the substituent is methyl, amino, nitro, cyano, halogen, methoxy, hydroxy, or X and Y is a guanidinium group NH (CH-NH2) NHR ¹⁵ aminoguanidiniová group NHNH ( CH = NH 2) NHR ¹⁵ , methylguanidinium CH ₂ NH (CH = NH 2) NHR ¹⁵ , and hydroxyethylguanidinium OCH ₂ CH ₂ NH (CH = NH 2) NHR ¹⁵ wherein R ¹⁵ is hydrogen, alkyl of one to eight carbon atoms, or X and Y is a dialkylsulfonium group S (R ¹⁶ ) 2 wherein R ¹⁶ is alkyl of one to eight carbon atoms or or X and Y is a trialkylphosphonium group P (R ¹⁷ ) 3 wherein R ¹⁷ is alkyl of one to eight carbon atoms are useful for the transfer of DNA and oligonucleotides to vertebrate primary cells in order to influence the expression of selected genes, particularly useful for transferring oligonucleotides (DNA) to primary leukemic cells.

The synthesis leading to the preparation of the novel compounds of formula I can be illustrated by the following scheme:

Process for the preparation of the compounds of formula I.

For the synthesis of these compounds we used two basic procedures, which differ and are directly determined by the substituent at the R13 position. Thus, the preparation process depends on whether the substituent at the 13-position is two hydrogen atoms - the preparation method is referred to as path A, or the oxygen atom - the preparation method is referred to as path B.

Preparation Method A

* * * * * * * · Φ φ φ φ φ φ φ φ

8Φ φφφφφ φφφ φφφ φφφφφφφφφφφ

The general procedure leading to a group of compounds of formula I wherein R ¹³ is two hydrogen atoms is based on an alkylation reaction using a bromomethyl or chloromethyl derivative. These starting materials, which can be characterized according to general formula I, in that R ¹³ are two hydrogen atoms and the substituents X and Y are bromine or chlorine atoms, have recently been described. The introduction of the other substituents at the R 1 -R ^{7 positions} is described in the literature review of porphyrin chemistry as well as the introduction of the R ¹¹ and R ¹² substituents. The substitution at the R ⁹ and R ^{10 positions} determines whether they are 5,10,15,20-tetrakis derivatives, in which case the R ⁹ and R ¹⁰ substituents are bromomethylphenyl or chloromethylphenyl groups.

For the 5,15-bis derivatives, R ⁹ and R ^{10 are} hydrogen atoms.

The introduction of the X and Y substituents of the compound of formula I is then based on a synthetic protocol of the alkylation reactions, for example the description of the compound II wherein the bromomethyl group reacts with O (alcoholate, phenolate), N (amine, pyridine), S (sulfide) , P (phosphine) nucleophile. The products of the reaction are the corresponding ammonium, sulfonium and phosphonium salts described by the enumeration of substituents in the general formula I.

Thus, the process for the preparation of the novel compounds of formula (I) by Process A is a one-step reaction resulting in a wide variety of onion salts depending on the reaction component; the process for preparing this group of compounds is analogous to that described in Example 1 for the compound of formula II.

Method of Preparation B.

The process of Preparation B is documented in Example 2 for the preparation of a compound of Formula III.

It is based on the formation of an amide bond between the starting porphyrinic acid (which may be mono- to tetracic acid depending on the substitution at positions 5, 10, 15 and 20) with an amino component defined by substituents X and Y of formula I. wherein X and Y are halogen or hydroxy, and defined substituents R1 ^; to R ¹² are as defined above, are known or can be prepared by modification of the processes described.

Key to the desired properties is the use of an amino component that would not only allow covalent attachment to the porphyrin backbone via an amide bond, but its other substituents defined by R ¹⁴ -R ¹⁷ * · allow the introduction of positive centers into the transport agent molecule, which are then responsible for its properties described below. Similarly, as described in Example 2 for the preparation of a compound of formula III, it is possible to prepare a whole group of other substances according to the present patent.

Preparation of the starting materials required for the synthesis of the novel compounds of the general formula (I)

wherein the substituents P ⁸ to R ⁸ are the same or different and are selected from the group consisting of hydrogen (H), alkyl of one to eight carbon atoms, C _n H ₂ n + i wherein n is 1 to 8;

R ⁹ and R ¹⁰ are the same or different and are selected from the group consisting of hydrogen, C 1 -C 8 alkyl, phenyl (C 6 H 5), substituted phenyl, wherein the substituent is methyl, amino, nitro, cyano, halogen, methoxy, hydroxy carboxyamidoguanidinium;

R ¹¹ and R ¹² are the same or different and are in the 2- or 3,5,6-position of the phenyl group, and are selected from the group consisting of hydrogen, aryl, halogen (fluoro, chloro, bromo, iodo), cyano, nitro, carboxyl an acid and its esters of one to eight carbon atoms;

R ¹³ is O or two H ₂ atoms;

the substituents X and Y, which may be the same or different, are a trialkylammonium group (N (R ¹⁴ ) 3), wherein R ¹⁴ is alkyl of one to eight carbon atoms, hydroxyalkyl, polyhydroxyalkyl, aminoalkyl, polyaminoalkyl;

further X and Y is an N-substituted pyridinium group C 6 H, wherein the substituent is methyl, amino, nitro, cyano, halogen, methoxy, hydroxy, or X and Y is guanidinium NH (CH = NH ₂ ) NHR ¹⁵ , aminoguanidinium NHNH (CH = NH 2) NHR ¹⁵ , methylguanidinium CH ₂ NH (CH = NH ₂ ) NHR ¹⁵ , and hydroxyethylguanidinium OCH ₂ CH ₂ NH (CH = NH ₂ ) NHR ¹⁵ wherein R ¹⁵ is hydrogen, alkyl of one to eight carbon atoms or X and Y is a dialkylsulfonium group S (R ¹⁶ ) ₂ , wherein R ¹⁶ is an alkyl of one to eight carbon atoms, or X and Y is a trialkylphosphonium group P (R ¹⁷ ) 3, wherein R ¹⁷ is an alkyl of one to eight carbon atoms.

is described in the following publications:

The Porphyrin Handbook, Vol. 1, Synthesis and Organic Chemistry

Eds. K. M. Kadish, K.M. Smith, R. Guilard, Academic Press, 2000, San Diego, 92101 United States

The Porphyrins, Vol. I, Ed. D. Dolphin, Academic Press, New York, 1978.

J.L. Sessler, V. Kral, M.C. Hoehner, K.O.A. Chin, R.M. Gave: Pure Appl. Chem. 68, 1291-1295 (1996); J.L. Sessler, R.M. Dávila, V. Král: Tetrahedron Lett. 37,6469-6472 (1996);

Sessler, J.L. Sansom, P.I., King V., O'Connor, D.L. Iverson, J. Am. Chem. Soc. 118, 12322-12330 (1996); V. King, H. Furuta, K. Shreder, V. Lynch, J.L. Sessler, J. Am. Chem. Soc. 118, 1595-1607 (1996); B.L. Iverson, K. Shreder, V. King, P. Sansom, V. Lynch, J.L. Sessler, J. Am. Chem. Soc. 118, 1608-1616 (1996); J.L. Sessler, J.W. Genge, V.K., B.L. Iverson: Supramolecular Chem. 8, 45-52 (1996); J.L. Sessler, A. Andrievsky, P.I. Sansom, V. Král, B.L. Iverson: Bioorg. Copper. Chem. Lett. 7, 1433-1436 (1997); J.L. Sessler, Andrievsky, V. King, Lynch V.: Am. Chem. Soc. 119, 93859392 (1997); P. Kavenova, P. Anzenbacher Jr., V. Kriz, M. Husak, V. Kral: Chem. sheets 91, 1027 (1997); P. Anzenbacher, Jr., V. Kriz, I. Rosenberg, Z. Tocik, I. Kavenova, K. Jursikova, V.Kral:

Chem. sheets 91, 1025 (1997); Springs S.A. Andrievsky, A.V. King, J.L. Sessler: J. Porphyrins Phthalocyanines 2, 315-325 (1998); Springs L., Gosztola D., Wasielewski M., King V., Andrievsky A., L. Sessler, J. Am. Chem. Soc. 1999, 121, 2281-2289; P. Bouř, V. Král: J. Biomol. Structure and Dynamics; O. Rusin, V. Kral: J. Chem. Soc. Chem. Commun.

• a • 9 9 9

998 9 99

99 99 9 9 (23), 2367-2368 (1999); M. Dudic, P. Lhotak, V. Kral, K. Lang, I. Stibor: Tetrahedron Leti., 40 (32), 5949-5952 (1999); King V., A. Cattani, A. Sinica, F. P. Schmidtchen: Tetrahedron 55 7829-7834 (1999); J.L. Sessler, N.A. Tvermoes, J. Davis, P. Anzenbacher, K. Jursikova, W. Sato, D. Seidel, V, Lynch, C.B. Black, A. Tiy, B. Andrioletti, G. Hemmi, T.D. Mody, D.J. Magda, V. Kral: PURE AND APPLEED CHEMISTRY 71: (11) 2009 - 2018 NOV 1999; Lang Lang, P. Anzenbacher Jr., Kapusta P., Kubat P. V. Král, D.M. Wagner: Photochem. Photobiol. 57 2000 (51-59); Kubat P., Lang Lang, Anzenbacher P. J, V. Král, B. Ehrenberg, J. Chem. Soc., Perkin Trans. 1, 933-941 (2000); P. Bouř, V. Král: Collect. Czech. Chem. Commun. 65, 631-643 (2000); A. Synytsya, A. Synytsya, V. Kral, K. Volka, J. Copikova, J. L. Sessler: J. Chem. Soc., Perkin Trans. 2, 1876-1884 (2000); V. Král, V. Rusin, F. P. Schmidtchen: Org. Lett., 3 (6), 873-876, (2001); C. Bucher, R. S. Zimmerman, V. Lynch,

V. Král, J. L. Sessler, J. Am. Chem. Soc., 123 (9), 2099-2100 (2001); Kubat, P. Lang, Anzenbacher, Jr., V. Král, Wagner, D. M.: J. Phys. Chem. in press; A. Synytsya, V. King, P. Pouckova, K. Volka: App. Spectrosc., 55 (2), 175-180 (2001); A. Synytsya, V. Kral, P. Matejka, P. Pouckova, K. Volka, JL Sessler - submitted Photochemistry and Photobiology; K. Zaruba, Z. Tomankova, J. Charvatova, I. Kavenova, P. Storm, P. Matejka, J. Fahnrich, K. Volka, V. Kral: Analytica Chimica Acta, 21192,1-15 (2001).

Examples

Example 1

Preparation of Formula II.

An organic compound ((5,15-Bis (.alpha.-trimethylammonium-p-tolyl) 10,20- (p-tolyl) porphyrin dihalide)) containing trimethylammonium groups at positions 5.15 and hydrogen at positions 10.20 was prepared. with structural formula Π.

e M% N

NM%

Method of preparation of the compound of formula II 44 4 4 44 ft4

444 * 4 44 444 · 4

4 ^ 4444444

1Ζ 4444 44 44 44 444

Compound (5,15-Bis (α-trimethylammonium-p-tolyl) -10,20- (p-tolyl) porphyrin dibromide) was prepared by heating (85 ° C, 8 hours) a solution which was formed by suspending 0.1 mmol of 5. 15-Bis (4-bromomethylphenyl) -10,20- (p-tolyl) porphyrin in ml of a 31-35% ethanol solution of trimethylamine in a sealed tube. The reaction mixture was stirred vigorously during heating. After cooling to room temperature, the precipitated product was collected by filtration, washed with a small amount of ethanol-THF (1: 1), and dried. The starting material 5,15-Bis (4-bromomethylphenyl) -10,20- (p-tolyl) porphyrin was prepared according to method (48). The yield of the compound of formula II was 96%.

Compound II was characterized ^! 1 H NMR, elemental analysis, and MS spectrometry.

1 H NMR (DMSO-d 6): 8.94 (s, 4H, β-pyrrole), 8. 86 (s, 4H, β-pyrrole), 8.39 (m, 4H, ptolyl), 8.11 (m, 4H, phenylCH ₂₎ 7.97 (m, 4H, p-tolyl), 7.65 (m, 4H, phenylCH ₂ ), 4.97 (d, 4H, CH ₂ N ⁺ ), 3.32 (s, 18H, CH 3 N ⁺ ), 2.71 (s, 6H) , CH 3), -2.98 (bs, 2H, NH-pyrrole).

Elemental analysis: Calculated for Cs4H54N6Br ₂ (Mw 946.86, C 68.50, H

N, 8.88. Observed: C 68.39, H 5.82, N 8.69.

MS (FAB, MALDI): 947 [MH +], 393 [M-2Br] / 2

UV-Vis (water-methanol 4: 1): 414 (Soret, ε = 1.6 x 10 ⁵ MW), 522, 566, 600, 650 nm.

Example 2

Preparation of the substance III.

Conjugation of tetrafenylporphyrin with an amino-monocyclic guanidine derivative produced the organic compound (5,10,15,20 -Tetrakis ((4-carboxamidoethyl) -2amino-1,4,5,6-tetrahydro-pyrimidinium) porphyrin tetrajodide) with the structural formula

III i

· ♦ '* 4 4 4 4 4 4 4 4 4 4 4 4 4

999 99 99 9 9 9 99 · · · · · · · · · · · · 99 99

Process for preparing a compound of formula ΙΠ.

The compound of formula ΙΠ was prepared by forming an amide bond between the porphyrin tetracarboxylate and the corresponding amino component aminoethyl derivative of monocyclic guanidine: 2- (2-aminoethyl) amino-1,4,5,6-tetrahydro-pyrimidinium iodide.

Amide bond formation was accomplished in two ways, either through activation using a carbodimide or other dehydrating agent (e.g. EDC), or through a primarily formed porphyrin tetracarboxylic acid chloride.

Preparation of Formula III.

Tetrakis (4-carboxyphenyl) porphyrin 79 mg, 1 mmol was dissolved in 10 mL of anhydrous dimethylformamide and 10 mmol of diisopropylcarbodiimimide was added under ice cooling under argon along with a catalytic amount (3 mg) of 4-dimethylaminopyrimidine and 1-hydroxybenzotriazole. The reaction mixture was stirred at 0 ° C for 1 hour and then 10 mmol of the aminoguanidine derivative: 2- (2-aminoethyl) amino-1,4,5,6-tetrahydro-pyrimidinium iodide was added, the reaction mixture was stirred at room temperature for 56 hours, then dimethylformamide was distilled off in vacuo and the product obtained by crystallization from methanol-dichloromethane (1: 3). The yield of ΠΙ is 88 mg (62%).

Reaction with the appropriate chloride gave a yield of 68%, anhydrous 1,2 dimethoxyethane was used as the reaction solvent, along with 1% triethylamine.

Elemental analysis: For C 72 H 82 Cl 14 N 2 O 4 (1433.36): C, 60.33; H, 5.77; Cl, 9.89; N, 19.54. Found: C, 60.01; H, 5.82; Cl, 9.71; N, 19.37.

MS MALDI TOF: calcd for C72H82Cl4N20O4 1430.56 found 1431.

Examples of use

Compounds of formula II and ΙΠ were used to transfer oligonucleotides to chicken primary leukemic monoblasts in which tumor transformation was induced by the v-myb oncogene.

To monitor the transfer, the transferred oligonucleotide was labeled with a radioisotope. By way of comparison, the same oligonucleotide was transferred to leukemic cells by commercial cells as well. · Transport agents that are currently considered to be one of the most effective. These compounds are indicated in the graph by KL (cationic lipid dimyristoylamidoglycyl-Nw-isopropoxycarbonyl-arginine dihydrochloride) and nonLP (mixed non-liposomal reagent). The efficacy of the compound of formula II is about 20 times higher, the efficacy of the compound of formula ΠΙ about 7 times higher than that of commercial preparations, as shown in Figure /

1.

The suitability of the compound of formula II for influencing the expression of selected genes was verified as follows. Primary leukemia cells synthesize the oncogenic protein v-Myb, which is the cause of their transformed state. Tzv. an antisense oligonucleotide directed against the information mRNA from which v-Myb is synthesized can stop the production of this protein, provided that it is efficiently transported to sites where the mRNA and protein are synthesized. The applicability of the substance of formula Π is documented in Figure 2 below.

Legend to Fig. 2: Protein v-Myb formation without affecting (1), in the presence of a compound of formula Π (2), in the presence of an antisense oligonucleotide alone (3), in the presence of a compound of formula Π and an antisense oligonucleotide 1: 5 ( 4), in the presence of a compound of Formula II and an antisense oligonucleotide in a ratio of 1:10 (5), in the presence of a compound of Formula II and an oligonucleotide that is not precisely directed against v-Myb in a ratio of 1:10 (6). Western blot analysis was performed (Bartunek P, Karafiat V, Dvorakova M, Zahorova V, Mandikova S, Zenke M, Dvorak M. Oncogene 15: 2939-49 (1997)).

Claims (6)

Patent claims Organic compounds derived from 5,15, -bis (p-tolyl) porphyrin and 5,10,15,20 tetrakis (p-tolyl) porphyrin having the general formula (I) wherein the substituents R ⁸ to R ⁸ are the same or various and are selected from the group consisting of hydrogen (H), alkyl of one to eight carbon atoms, C _n H 2 _ft + wherein n is 1 to 8; R ⁹ and R ¹⁰ are the same or different and are selected from the group consisting of hydrogen, C 1 -C 8 alkyl, phenyl (C 6 H 5), substituted phenyl, wherein the substituent is methyl, amino, nitro, cyano, halogen, methoxy, hydroxy carboxyamidoguanidinium; R ¹¹ and R ¹² are the same or different and are in the 2- or 3,5,6-position of the phenyl group and are selected from the group consisting of hydrogen, aryl, halogen (fluoro, chloro, bromo, iodo), cyano, nitro, carboxyl an acid and its esters of one to eight carbon atoms; R ¹³ is O or two 1'-hydrogen atoms; X and Y substituents, which may be the same or different, are trialkylammonium (N (R ¹⁴ ) a), where R ¹⁴ is alkyl of one to eight carbon atoms, hydroxyalkyl group , polyhydroxyalkyl, aminoalkyl, polyaminoalkyl; / Further X and Y is N-substituted pyridinium group C 6 H _5> wherein R is methyl, amino, nitro, cyano, halogen, methoxy, hydroxy, or X and Y is a guanidinium group NH (CH-NH ₂₎ NHR ¹⁵ aminoguanidiniová NHNH (CH = NH 2) NHR ¹⁵ , methylguanidinium CH ₂ NH (CH = NH 2) NHR ¹⁵ , and "hydroxyethylguanidinium OCH ₂ CH ₂ NH (CH = NH 2) NHR ¹⁵ , where R ¹⁵ is hydrogen, alkyl of one to eight atoms or X and Y is a dialkylsulfonium group S (R ¹⁶ ) ₂ , wherein R ¹⁶ is an alkyl of one to eight carbon atoms, or X and Y is a trialkylphosphonium group P (R ¹⁷ ) 3, wherein R ¹⁷ is an alkyl of one to eight carbon atoms. eight carbon atoms. A process for the preparation of a compound of formula I according to claim 1, wherein R ¹³ is oxygen, characterized in that to a compound of formula I wherein X and Y are hydroxy and R ¹³ is oxygen and R ¹ to R ^{4 * 12} have: as defined in claim 1, acts by an amino component that allows covalent attachment to the porphyrin backbone via an amide bond and provides the meanings of X and Y according to claim 1. A process for the preparation of a compound of formula (I) according to claim 1, wherein R ¹³ is 2 hydrogen, characterized in that to a compound of formula (I) wherein X and Y are bromine or chlorine, R ¹³ is 2 hydrogen and R ¹ to R ¹² are as defined in claim 1, acting by a Ο, Ν, δ, Ρ nucleophile selected from the group consisting of alcoholates, phenolates, amines, pyridines, sulfides or phosphines. A method according to claim 3, characterized in that for preparing the 5,15-bisderivatives of the compound according to claim 1, a nucleophile is treated with a compound of the formula I according to claim 2, wherein: R ^{9 and} R ¹⁰ are hydrogen. A process according to claim 3, characterized in that for preparing the 5,10,15,20 tetrakisivatives of the compound according to claim 1, a nucleophile is treated with a compound of the formula I according to claim 2, wherein R ⁹ and R ¹⁰ are bromomethylphenyl or chloromethylphenyl groups. ' 6. Use of compounds of the formula I according to claim 1 for the transfer of DNA and oligonucleotides into vertebrate primary cells in order to influence the expression of selected genes.