CZ295445B6 - Porphyrin derivatives - 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

Porphyrin derivatives

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 to cells results from their ability to form sDNA relatively stably complexes and from their lipophilic character by which they can cross the lipoprotein membranes of the cells. The formation of complexes 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., Král, V., Ehrenberg, J. Chem. Soc., Perkin Trans., 1, 933-941 (2000)). Also, some expanded porphyrins, particularly safyrins, exhibit a strong but reversible interaction with DNA (King, V., Furuta, H., Shreder, K., Lynch, V., Sessler, JL, J. Am. Chem. Soc., 118 Sesler, JL Samsom, PI, King, V., O'Connor, D. and Iverson, BLJ Am Chem Soc., 118, 12322-12330 (1996), Iverson, BL Shreder, K., King, V. Sansom, P., Lynch, V., Sesler, JLJ Am Chem Soc., 118, 1608-1616 (1996)). However, some porphyrin derivatives are toxic to cells.

Currently, cationic lipids (CF Bennett, MY Chiang, H. Khan, JE Shoemaker, CK Mirabelli. Mol. Pharmacol., 41, 1023-1033 (1992); Lewis, JG, Lin, KY are currently used to transfer oligonucleotides to cells. Kothavale, A., Flanagan, WM, Matteucci, MD, DePrince, RB, Mook, RA, Hender, RW and Wagner, RW Proc Natl Acad Sci USA; 93: 3176-3181 (1996); Bhat B. Hebeett, N., Marcusson, EG, Dean, NM, Bennett, CF, and Manoharan, M. Nucleosides Nucleotides, 18, 1727-1728 (1999)), liposomes (Clarenc, JP, Degols, G., Leonetti, JP, Milhaud, P. and Leblue, B. Anticancer Drug Des., 8, 81-94 (1993), Arima, H., Aramaki, Y., Tsuchia, S., J. Pharm. Sci., 86, 438-442 (1997), Willis, M., Forssen, E., Adv. Drug, Deliv. Rev., 29, 249-271 (1998), Bennett, CF, (1995) In Akhatar, S. (ed.), Delivery Strategies for Antisens Oligonucleotide 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., Mary, J., Lantredou, N., Divita, G., Heitz, F. Biochem. Biophys. Res. Commun. 243: 601-608 (1998); Antopolsky, M. Azhayeva, E., Tengvall, U., Auriola, 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, D., Kramer, W. Hepatology, 30, 1257-1268 (1999); Niidome, T., Wakamatsu, M., Wada, A., Hirayama, T., Aoyagi, H.J. Pept. Sci., 6,271-279 (2000); Hamilton, S.E., Simmons, C.G., Kathiriay, I.S., Corey, D.R. Chem. Biol., 6: 343-351 (1999); Astriab-Fisher, A., Sergueev, D., Fisher, M., Shaw, B.R., Juliano, R.L. Biochem. Pharmacol., 60, 83-90 (2000)), dendrimers (Poxon, SW, Mitchell, PM, Liang, E., Huges, JA Drug Delivery, 3, 255-265 (1996)), polices (Boussif, O. Lezoualc'h, F., Zanta, MA, Mergny, MD, Scherman, D., Demeneix, B., Behr, JP Proc. Natl. Acad. Sci. USA, 92, 7297-7301 (1995); JP, Degols, G. and Lebleu, B. Bioconjugate Chem., 1, 149-153 (1990)), cationic liposomes (lipoplex) (Islam, A., Handley, SL, Thompsonn, 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, J., Jaaskelainen, I., Syrjanen, SJ Histochem. Cytochem., 45, 265-274 (1997),

Cationic polymers (polyplex) (Brown, MD, Schatzlein, A., Brownlie, A., Jack, V., Wang., W., Tetley, L., Gray, AI, and Uchegbu, IF Bioconjugate Chem. 11, 880-891 (2000);

C.W. Lucas, P., Thomas, B.J., Udeehi, A. N., Milroy, D.A., Moss, S.H. J. Control. Rev. 53, 289-299 (1998); Kwon, G. S., Okano, T. Pharm. Res., 16: 597-600 (1997); gamier, F., Korriyousouffi, H., Srivastava, P., Mandrand, B., Delair, T., Synth. Met., 100, 89-94 (1999); Benns, J.J., 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, Kl., Zheng, W., Baker, A., Papahadjopoulos, D. FEBS Lett., 400, 233) (1997)), surface modified nanoparticles (Chavany, C., Le Doan, T., Couvreur, P., Puissieux, F., Helene, C. Pharm. Res., 9, 411-449 (1992); Vinogradov, 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., 11, 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, SK, Dean, NM, Fisher, MH, Delong., R., Manoharan, M., Tivel, KL, Juliano, RL Mol. Pharmacol. 50, 808-). 819 (1996)) or oligonucleotide complexation with cell receptor ligand (Williams, M. and Forseen, E. Adv. DrugDeliv. Rev., 29, 249-271 (1998); Wu-Pong, S., Bard, J. Huffman, J., Jimerson, J. Biol. Cell., 89, 257-261 (1997), Citro, G., Perrotti, D., Cucco, C., D'Agnano, I., Sacchi, A., Zupi, G., Calabretta, B. Proc Natl Acad Sci USA, 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, which have the general formula I

(D wherein R ¹ to R ⁸ are the same or different and are selected from the group consisting of hydrogen, alkyl of one to eight carbon atoms R ⁹ and R ¹⁰ are the same or different are selected from the group consisting of hydrogen, alkyl of one to eight eight carbon atoms, phenyl, substituted phenyl, wherein the substituent is methyl, amino, nitro, cyano, halogen, methoxy, hydroxy, carboxyamidoguanidino;

R ¹¹ and R ¹² are the same or different substituted phenyl groups at the 2 or 3,5,6 position, and the substituents are selected from the group consisting of hydrogen, aryl, halogen, cyano, nitro, carboxyl, and methyl to octyl esters thereof. ;

R ¹³ is O or H ₂ ;

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, hydroxymethyl, hydroxyethyl, hydroxypropyl and hydroxybutyl; a polyhydroxyalkyl group of two to six hydroxyl groups of two to ten carbon atoms, an aminoalkyl group of two to six carbon atoms, and a polyaminoalkyl group of three to eight carbon atoms;

further X and Y is an N-substituted pyridinium group wherein the substituent is methyl, amino, nitro, cyano, halogen, methoxy, hydroxy, or X and Y is a guanidinium group NH (C = NH ₂ ) NHR ¹⁵ , an aminoguanidinium group NHNH ( C = NH 2) NHR ¹⁵ , methylguanidinium CH ₂ NH (C = NH ₂ ) NHR ¹⁵ , and hydroxyethylguanidinium OCH ₂ CH ₂ NH (C = 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 atoms carbon.

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 vzorce

wherein X 'and Y' are hydroxyl ^and R and R ¹² are as defined above, act on dicyclohexyl or diisopropylcarbodiimide, and the activated compound is treated with aminoalkylguanidine hydrochloride, wherein the alkyl is selected from methyl, ethyl or propyl, which allows its covalent attachment to the porphyrin backbone via an amide bond.

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 2 hydrogens is to form a compound of formula (I),

wherein R ¹ -R ¹² are as defined above and X and Y are bromine or chlorine, act by a nucleophile selected from the group consisting of alcoholates selected from the group of methoxide, ethylene glycolate or ethanolate, or sodium phenolate, tertiary amines such as trimethylamine, triethylamine, tripropylamine, followed by pyridine, dimethylsulfide or trimethylphosphine, the reaction being carried out under reflux in methanol, acetonitrile or a mixture thereof under an inert nitrogen atmosphere for 3 to 12 h.

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 hydrogens is characterized in that the nucleophile above is treated with a compound of formula I wherein R ¹ to R 12 ⁸ have the aforementioned meanings and X and Y are bromine or chlorine atom and 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 to R ¹² are as defined above and wherein R ¹³ are 2 hydrogens consists in treating the compound of formula I with the nucleophile above R ¹ to R ⁸ are as defined above 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,

(I), wherein the substituents X, Y and R ¹ to R ¹³ are as defined above, are useful for transferring 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:

-4GB 295445 B6

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 substituents at the 13-position are two hydrogen atoms - the preparation method is referred to as route A, or the oxygen atom the preparation method is referred to as route B.

Preparation Method A

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 formula I,

-5GB 295445 B6

wherein X and Y are bromine or chlorine, and R ¹ to R ¹² are as previously defined, have been recently described. The introduction of other substituents in the R-R ⁷ is copied aggregate reports of porphyrin chemistry, which are listed in the literature review, as well as introduction of substituents R ¹¹ and R ^12th The substitution at positions R ⁹ and R ¹⁰ determines whether they are 5,10,15,20-tetrakis derivatives, in which case R ⁹ and R ¹⁰ 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. of formula Γ,

-6CZ 295445 B6 wherein X 'and Y' are halogen or hydroxy and the substituents defined by R ¹ to ^{R! Z} have the aforementioned meanings, are either known compounds or can be prepared by modification of procedures described above.

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 that its other substituents, defined by R ¹⁴ -R ^{17, would} allow the introduction of positive centers into the transport agent molecule, the 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 necessary for the synthesis of the novel compounds of the general formula I

(D, wherein X, Y and R ¹ to R ¹³ are as previously defined, 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. Král, M.C. Hoehmer, K.O.A. Chin, R.M. Gave: Pure Appl. Chem. 68, 1291-1295 (1996); J.L. Sessler, R.M. Dávila. V. Kings: Tetrahedron Lett. 37, 6469-6972 (1996);

J.L. Sessler, P.I. Sansom, V. King. D. O'Connor. B.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. King, B.L. Iverson: Supramolecular Chem., 8, 45-52 (1996); J.L. Sessler, A. Andrievsky, P.I. Sansom, V. King. B.L. Iverson: Bioorg. Copper. Chem. Lett. 7, 1433-1436 (1997); J. L. Sessler, A. Andrivsky, V. King, V. Lynch: J. Am. Chem. Soc. 119, 9385-9392 (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. Essler, J. Porphyrins Phthaloxyanines 2, 315-325 (1998); S.L. Springs, D. Gosztola. M.R. Wasielewski, V. King. A. Andrievsky, J.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. (23), 2367-2368 (1999); M. Dudic, P. Lhotak, V. Kral, K. Lang, I. Stibor: Tetrahedron Lett. 40 (32): 5949-5952 (1999); King V., A. Cattani, A., Sinica, F.P. Schmidtchen: Tetrahedron 55, 7829-7834 (1999); J.L. Sester, N.A. Tvermoes,

Davis J., Anzenbacher P., Jursíková K., Sato W., Seidel D., Lynch V., C.B. Black, A. Try, B. Andrioletti. G. Hemmi, T.D. Mody, D.J. Magda, V. Kral: PURE AND APPLIED CHEMISTCZ 295445 B6

RY 71: (11) 2009-2018 NOV 1999; Lang Lang, Anzanbacher Jr., Kapusta P., Kubát P., Král King,

D.M. Wagner: Photochem. Photobiol. 57 2000 (51-59); Kubat P., Lang K., Anzenbacher Jr., 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. Sanytsya, A. Synytsya, V. Kral., K. Volka. J. Copikova, J. L. Sessler: J. Chem. Soc., Perkin Trans. 2, 1876-1884 (2000); V. Král,

O. Rusin, F.P. Schmidtchen: Org. Lett., 3 (6), 873-876, (2001); C. Bucher, R.S. Zimmerman, V. Lynch, V. King, J.L. Sessler: J. Am. Chem. Soc., 123 (9), 2099-2100 (2001); Kubat P., Lang Lang, Anzenbacher P. J, V. Král, Wagner D. M.: J. Phys. Chem. in press; A. Synytsya, V. King, P. Pouckova, K. Volka: App. Spectrocs., 55 (2), 175-180 (2001); A. Synytsya, V. King,

P. Matejka, P., Pouckova, K. Volka, J.L. Sisters - submitted by Photochemistry and Photobiology; K. Zaruba, Z. Tomankova, J. Charvatova, I. Kavenova, P. Bouř, 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 (α-trimethylammonium-p-tolyl) -10,20- (ptolyl) porphyrin dibromide) was prepared which contains trimethylammonium groups at positions 5,15 and at positions 10,20-p-tolyl , with structural formula II.

Process for the preparation of the compound of formula II

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 formed by suspending 0.1 mmol of 5. 15Bis (4-bromomethylphenyl) -10,20- (p-tolyl) porphyrin in 10 ml of a 31 to 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- (ptolyl) porphyrin was prepared according to method (48). The yield of the compound of formula II was 96%.

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

-8C 295445 B6 1 H NMR (DMSO-d ₆ ): 8.94 (s, 4H, β-pyrrole), 8.86 (s, 4H, β-pyrrole), 8.39 (m, 4H, p- tolyl), 8.11 (m, 4H, ₂ phenylCH), 7.98 (m, 4H, p-tolyl), 7.65 (m, 4H, ₂ phenylCH), 4.97 (d, 4H, CH ^ ), 3.32 (s, 18H, CH ₃ N ⁺ ), 2.71 (s, 6H, CH ₃ ), -2.98 (bs, 2H, NH-pyrrole).

Elemental analysis: Calculated for C54H ₅ 4 N ₆ Br ₂ (Mw 946.86, C, 68.50; H, 5.75; N, 8.88., Found: 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 M ^-1 cm ^-1 ), 522, 566, 600, 650 nm.

Example 2

Preparation of the substance III.

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

A process for preparing a compound of formula III.

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

Amide bond formation has been accomplished in two ways, either through activation by the use of carbodiimide or another dehydrating agent, for example 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide (EDC), or through primarily formed tetrafenyl-tetracarboxylic acid porphyrin 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 diisopropylcarbodiimide 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 h and then 10 mmol of the aminoguanidine derivative: 2- (2-aminoethyl) amino-1,4,5,6-tetrahydro-pyrimidine iodide was added, the reaction mixture was stirred at room temperature for 56 h. h, then dimethylformamide was distilled off in

The product was obtained by crystallization from methanol-dichloromethane (1: 3). Yield of product III 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 ^ HH ^ CClulOkoClC (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 MALDITOF: calcd for C72H82Cl4N20O4 1430.56 found 1431.

Examples of use

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

To monitor the transfer, the transferred oligonucleotide was labeled with a radioisotope. By way of comparison, the same oligonucleotide has been transferred to leukemic cells by commercial transporters which are currently considered to be one of the most potent. These compounds are indicated in the graph by KL (cationic lipid dimyristoylamidogracyl-Nw-isopropoxycarbonyl-arginine dihydrochloride) and nonLP (mixed non-liposomal reagent). The activity of the compound of formula II is about 20 times higher, the activity of the compound of formula III is 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 compound of formula II is illustrated by the following Figure 2.

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 II 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). The analysis was performed by Western blotting (Bartunek P, Carnation V. Dvorakova M, Zahorova V, Madikova S, Zenke M, Dvorak M. Oncogene 15: 2939-49 (1997)).

Claims (6)

PATENT CLAIMS 5 1. Derivatives of 5,15-bis (p-tolyl) porphyrin, and 5,10,15,20-tetrakis (p-tolyl) porphyrin of formula I (D io wherein the substituents R ¹ to R ⁸ are identical or different and are selected from the group consisting of hydrogen and alkyl of one to eight carbon atoms; 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, substituted phenyl, wherein the substituent is methyl, 15 amino, nitro, cyano, halogen, methoxy, hydroxy, carboxyamidoguanidino; R ¹¹ and R ¹² are the same or different substituted phenyl groups at the 2- or 3,5-position, and the substituents are selected from the group consisting of hydrogen, aryl, halogen, cyano, nitro, carboxyl and its methyl to octyl esters; R ¹³ is O or H ₂ ; the substituents X and Y, which may be the same or different, are the trialkylammonium group N (R ¹⁴ ) ₃ , Wherein R ¹⁴ is C 1 -C 8 alkyl, hydroxyalkyl, polyhydroxyalkyl, aminoalkyl, and polyaminoalkyl; further X and Y is an N-substituted pyridinium group wherein the substituent is methyl, amino, nitro, cyano, halogen, methoxy, hydroxy, or X and Y is a guanidinium group NH (C = NH ₂ ) NHR ¹⁵ , an aminoguanidinium group NHNH ( C = NH 2) NHR ¹⁵ , methylguanidinium CH ₂ NH (C = NH ₂ ) NHR ¹⁵ , and hydroxyethylguanidinium OCH ₂ CH ₂ NH (C = NH ₂ ) NHR ¹⁵ , wherein R ¹⁵ is hydrogen or alkyl of one to eight carbon atoms, or X and Y is the dialkylsulfonium group S (R ¹⁶ ) 2, wherein R ¹⁶ is alkyl of one to eight carbon atoms, or X and Y is the trialkylphosphonium group P (R ¹⁷ ) 3, wherein R ¹⁷ is alkyl of one to eight carbon atoms. A process for the preparation of porphyrin derivatives according to claim 1, wherein R ¹³ is oxygen, and X and Y are guanidinium NH (C = NH ₂ ) NHR ¹⁵ , aminoguanidinium NHNH (C = NH 2) NHR ¹⁵ , methylguanidinium CH 2 NH (C = NH ₂ ) NHR ¹⁵ , and the hydroxyethylguanidinium group OCH ₂ CH ₂ NH (C = NH ₂ ) NHR ¹⁵ , wherein R ¹⁵ is hydrogen, alkyl of one to one 45 eight carbon atoms, characterized in that the compound of the general formula Γ, -11 CZ 295445 B6 (I7 _Z wherein X 'and Y' are hydroxy, and R ^{* 1} through R ¹² are as defined in claim 1, treated with 5 dicyclohexyl or diisopropyl carbodiimide, and thus an activated substance with an appropriate guanidine derivative, which allows its covalent attachment to the porphyrin backbone via an amide bond. A process for the preparation of porphyrin derivatives according to claim 1, wherein R ¹³ is 2, wherein the compound of formula I wherein X and Y are bromine or chlorine, and R ¹ to R ¹² are as defined in claim 1, acting by the appropriate nucleophile to introduce residues X and Y. A process according to claim 3, characterized in that for the preparation of the 5,15-bisphosphoryl derivatives of claim 1, bis-bromomethyl derivatives of the formula I wherein X and Y are bromine and R ⁹ and R ¹⁰ are hydrogen , acting by the appropriate nucleophile to introduce 20 rings in the meaning of X and Y. A process according to claim 3, characterized in that for the preparation of the 5,10,15,20-tetrakisderivatives of porphyrins according to claim 1, the tetracycl bromomethyl derivatives of the formula I wherein X and Y are bromine atoms and wherein R ^{9 is} and R ¹⁰ are bromomethylphenyl or chloro- The reaction is carried out under reflux in methanol, acetonitrile or a mixture thereof under an inert nitrogen atmosphere for 3 to 12 hours. Use of porphyrin derivatives according to claim 1 of the formula I for the preparation of a medicament for the transfer of 30 DNA and oligonucleotides to primary cells of vertebrates.