PL222376B1 - Morpholino, and morpholino nitro-imidazole phthalocyanine derivatives, process for their preparation and the use - Google Patents

Abstract

The subject of the invention are new morpholino and morpholino-nitroimidazole derivatives of phthalocyanines of the general formula 1, the method of their preparation and use for the production of drugs used in photodynamic therapy, in which - M is 2H, Mg(II), Zn(II), Mn(II) , Mn(III)Cl, Fe(II), Fe(III)Cl, Co(II), Cu(II) and the substituents R1, R2, R3, R4, R5, R6, R7 and R8 are H or groups of the formula 2 or formula 3. The invention also relates to salts of general formula 1 where R1 R2 R3, R4, R5, R6, R7 and R8 are H or a group of formula 4 and a method for their preparation. The invention also includes the use of compounds for the production of drugs used in anticancer therapies, in particular PDT.

Description

(12) PATENT DESCRIPTION (19) PL (11) 222376 (13) B1 (21) Application number: 402263 ^{(51) Int.Cl.}

C07D 487/22 (2006.01) C09B 47/04 (2006.01) C09B 47/12 (2006.01) ⁽²²⁾ g ^of D ^{ata łoszenia 27} · ¹² · ²⁰¹² C09B 47/32 (2006.01)

A61K 41/00 (2006.01) A61P 35/00 (2006.01)

Morpholine and morpholine-nitroimidazole phthalocyanine derivatives, their preparation and use (73) Patent holder:

MEDICAL UNIVERSITY KAROLA MARCINKOWSKIEGO IN POZNAN, Poznań, PL (43) Application was announced:

07/07/2014 BUP 14/14 (45) The following was announced about the grant of the patent:

29.07.2016 WUP 07/16 (72) Inventor (s):

MARCIN WIERZCHOWSKI, Poznań, PL TOMASZ GOŚLIŃSKI, Poznań, PL WOJCIECH SZCZOTKO, Wilkowo, PL PAULINA SKUPIN-MRUGALSKA, Poznań, PL STANISŁAW SOBIAK, Poznań, PL MAREK MURIAS, Poznań, PL MAŁGORZATA KUCIŃSKA, PLźo, Inowstrocii MATEUSZ ŚCIEPURA, Murowana Goślina, PL MACIEJ KORPUSIŃSKI, Gorzów Wielkopolski, PL (74) Plenipotentiary:

item. stalemate. Wojciech Lisiecki

PL 222 376 B1

Description of the invention

The subject of the invention is new morpholine and morpholine-nitroimidazole phthalocyanine derivatives of the general formula I, the method of their preparation and the use for the preparation of drugs used in photodynamic therapy.

Continuous adaptation of pathogenic microorganisms to the therapy used, as well as the variety of neoplastic diseases, forces the search for new, more effective methods of therapy and remedies.

One of the directions of development of therapeutic methods in oncology is Photodynamic Therapy - PDT, which enables the destruction of neoplastic tissues, without damaging healthy tissues, in a selective manner. This method is based on the use of photochemical reactions sensitized with special dyes. In PDT, the photosensitizing agent is injected into the tumor tissue where it is then activated by light.

Photodynamic therapy is also used in the treatment of diseases caused by bacterial or protozoal infections. Scientific press reports also confirm the possibility of using photodynamic therapy in the case of skin diseases such as solar keratosis, psoriasis, autoimmune diseases, in ophthalmology and dentistry (1).

The role of the photosensitizer is to transfer the excitation energy to molecular oxygen to generate active oxygen species or by the attack of an excited photosensitizer on biomolecules present in the cell. Of the reactive oxygen species produced in the photodynamic process, the most important role is played by the so-called singlet oxygen, i.e. an oxygen molecule excited to the first singlet state (A _g ). Two mechanisms of the photodynamic reaction have been described in the literature. Both can occur simultaneously, but the dominance of one of them is determined by the partial pressure of oxygen at the site of the photochemical reaction (2).

The reaction is type I, when there is a low partial concentration of oxygen molecules in the tissue undergoing photodynamic therapy. Under the influence of light, the photosensitizer molecule is excited from the ground state S ₀ , to the first excited state Si, and then goes to the lowest triplet state T ₁ . The transition to the T ₁ state is crucial because it is this excited form of the molecule that is responsible for all further photodynamic changes. The photosensitizer, excited to the triplet state, reacts directly with acceptor molecules, transferring hydrogen or an electron to substrate molecules, such as various types of biomolecules present in the cell, which damage the organelles of the cells (3). Damage to pathologically changed tissues can also occur through the reaction of water molecules with the photosensitizer in the T ₁ state, generating the formation of reactive oxygen species such as peroxide radicals, hydrogen peroxide, and hydroxyl radicals. These reactive oxygen species attack the biomolecules present in the cell, such as aminolipids and proteins (4).

In the type II reaction, as well as in the type I reaction, the photosensitizer molecules in the T ₁ state play a key role. In this case, the partial concentration of oxygen is so high that it is with the oxygen molecule that the photosensitizer reacts in the T ₁ state, which leads to the formation of a reactive form of oxygen, singlet _{oxygen 1 Δ θ} . This active form of oxygen is particularly advantageous in the treatment of bacterial diseases, as no immune mechanisms to this lethal factor have developed in the organisms of bacteria or protozoa.

The reactions caused by both mechanisms of photosensitizing action lead to cell death, most often through apoptosis. Clinical studies have shown that in vivo the advantage is the type II mechanism, however its induction depends on various environmental parameters, such as pH, oxygen concentration and others. The type II mechanism, by consuming oxygen molecules in the reaction, causes that the state of tissue hypoxia gradually increases and more and more photodynamic changes occur according to the I mechanism.

In the current practice, the group of compounds used most frequently as photosensitizers in PDT are porphyrinoids, in particular porphyrins and phthalocyanines.

Porphyrins are macrocyclic natural or synthetic aromatic compounds composed of four pyrrole rings connected by methane bridges. An example of natural porphyrins found in nature, such as heme or protoporphyrin IX.

Phthalocyanines are aromatic macrocyclic systems composed of four isoindole (benzopyrrole) rings linked by azamethine bridges. Unlike porphyrins, they are compounds that do not occur in nature.

PL 222 376 B1

Currently, porphyrinoid preparations derived from natural porphyrins such as protoporphyrin IX are most often used in photodynamic therapy. The most commonly used clinically photosensitizer is sodium porfimer, which is a mixture of approx. 60 oligomers obtained by chemical treatment of hematoporphyrin.

Moreover, prometabolites of porphyrin photosensitizers such as 5-aminolevulinic acid and methyl 5-aminolevulinate have found clinical application.

Examples of other clinically used photosensitizers are temoporfin (3,3 ', 3,3' - (2,3-dihydropyrin-5,10,15,20-tetrayl) tetraphenol), talaporfin (mono-L-aspartyl chlorin e6) and photochlor (2- (1'-hexyloxyethyl) -2-devinylpyropheophorbide-a).

Phthalocyanines are aromatic macrocyclic systems composed of four isoindole (benzopyrrole) rings linked by azamethine bridges. Unlike porphyrins, they are compounds that do not occur in nature. Phthalocyanines are a dynamically developing group of compounds with desired properties in photodynamic therapy.

These compounds have a strong absorption band (the so-called Q-band) responsible for the formation of the photodynamic mechanism in the range of 600 to 800 nm, which is in the so-called biological window. This means that these compounds can be safely excited with light in this wavelength range without the risk of radiation being absorbed by the water and biomolecules present in the cells. Compared to phthalocyanines, currently used porphyrins have a weak Q-band and are excited by irradiation most often with light with a wavelength of about 400-410 nm in the so-called the Soret band (from 200 to 400 nm). Radiation of this length is characterized by lower tissue penetration.

An example of a phthalocyanine photosensitizer used in clinical practice is Photosens, which is a mixture of varying degrees of sulfonated aluminum phthalocyanine. This relationship has been applied with good results, incl. in the treatment of malignant neoplasms of the skin and bronchi in 36 patients (5). Research by Hu M. (6) confirmed the antitumor activity of zinc (II) 2-hydroxyphthalocyanine, zinc (II) 2,3-dihydroxyphthalocyanine, zinc (II) 2,9-dihydroxyphthalocyanine and zinc (II) 2,9,16-trihydroxyphthalocyanine in in vitro tests confirmed the possibility of using them in photodynamic therapy of neoplasms.

Fadel M., (7) demonstrated the possibility of using zinc (II) phthalocyanine as a photosensitizer. However, this compound is characterized by low solubility in water, therefore its use in phototherapy requires special techniques. Fadel M., demonstrated the possibility of using zinc phthalocyanine in phototherapy in animals with the use of thin polymer films as carriers. Similar results were obtained by Larroque C. (8), who confirmed the effectiveness of zinc phthalocyanine combinations with bovine serum albumin against the EMT-6 tumor as a photosensitizer. In the case of another phthalocyanine derivative, 1- [4- (hydroxymethyl) phenoxy] zinc (II) phthalocyanine, the photodynamic antifungal activity against Candida albicans was confirmed in vitro (9).

The presence of various substituents in the peripheral and non-peripheral positions of the phthalocyanine rings allows for a significant change in the properties, in particular depending on the types of substituents, it is possible to obtain compounds with different solubilities in an aqueous medium or lipids. This is of great importance for the bioavailability of the compounds and the selective accumulation process. Phthalocyanine molecules can undergo numerous chemical modifications. As shown by Nyokong and Michel (10) (11), phthalocyanines can be modified by substituting metal ions in the coordination center and by introducing various substituents into the peripheral part.

Lv et al. (12) describe the preferred combination of zinc phthalocyanine with four chain ether substituents. The use of this type of substituents has a positive effect on water solubility, and thus bioavailability. Also, the introduction of ionic substituents increases water solubility, which in the case of medicinal substances results in an increase in bioavailability. Higher water solubility and a special affinity to the cell membrane of gram-negative photosensitizers with cationic substituents is particularly desirable in the treatment of bacterial infections (13).

The possibility of modifying the chemical structure of phthalocyanines allows this group of molecules to be linked with other molecules with their own biological activity. According to one of the applied principles of drug design, medical chemical hybridization (MCH Medicinal Chemical Hybridization) aims to combine two pharmacologically active molecules. New particles

According to the MHC method, apart from the features resulting from the presence of both pharmacologically active fragments, they may have additional unique properties (14) (15).

Compounds used as photosensitizers have different selectivity towards diseased tissues, i.e. they also accumulate in normative tissues. Therefore, there is a need to modify the structure of molecules in order to maximize the selectivity of absorption of compounds used in PDT (16).

The aim of the invention was to create new morpholine and morpholine-nitroimidazole phthalocyanine derivatives, to develop a method of their preparation and use for the production of drugs used in photodynamic therapy with antitumor, antibacterial, antiprotozoal and antiviral properties.

The subject of the invention is new morpholine and morpholine-nitroimidazole phthalocyanine derivatives and their isomers of the general formula 1,

wherein

- M means 2H, Mg (II), Zn (II), Mn (II), Mn (III) Cl, Fe (II), Fe (III) Cl, Co (II), Cu (II)

- R ₁ , R ₂ R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and R ₈ form pairs R ₁ and R ₂ , R ₃ and R ₄ , R ₅ and R ₆ , R ₇ and R ₈ and when they are equal in pair, they are H or a group of formula 2

wherein at least one pair of R ₁ and R ₂ or R ₃ and R ₄ or R ₅ and R ₆ , or R ₇ and R ₈ is a group of formula 2.

or

- R _t R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and R ₈ form pairs R ₁ and R ₂ , R ₃ and R ₄ , R ₅ and R ₆ , R ₇ and R ₈ and when they are equal in pair, they are the group of formula II or when they are different one is H and the other is the group of formula III

wherein:

- n takes a value from 0 to 20

- at least one R ₉ , R ₁₀ , R ₁₁ is -NO ₂ and the other two are equal or different and represent -H, -CH ₃ , -NO ₂ , with at least one pair of R ₁ and R ₂ or R ₃ and R ₄ or R ₅ and R ₆ or R ₇ and R ₈ represent a group of formula 2.

PL 222 376 B1

The invention relates to all isomeric forms of the general formula I.

In a second aspect, the invention relates to the salts of the morpholine and morpholine-nitroimidazole phthalocyanine derivatives and their isomers of the general formula 1,

wherein

- M means 2H, Mg (II), Zn (II), Mn (II), Mn (III) Cl, Fe (II), Fe (III) Cl, Co (II), Cu (II)

- R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and R ₈ form pairs R ₁ and R ₂ , R ₃ and R ₄ , R ₅ and R ₆ , R ₇ and R ₈ and when they are equal in pair, represent H or a group of formula 4 wherein

X is Cl, J, Br, F,

- Alk represents an alkyl chain from 1 to 20 chain length, wherein at least one pair of R ₁ and R ₂ or R ₃ and R ₄ or R ₅ and R ₆ or R ₇ and R ₈ is a group of formula 2, or R ₁ , R ₂ R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and R ₈ form pairs R ₁ and R ₂ , R ₃ and R ₄ , R ₅ and R ₆ , R ₇ and R ₈ and when one pair is equal to the group of formula II or when different one is H and the other is the group of formula III

wherein:

- n takes a value from 0 to 20

- at least one R ₉ , R ₁₀ , R ₁₁ is -NO ₂ and the other two are equal or different and represent -H, -CH ₃ , -NO ₂ , with at least one pair of R ₁ and R ₂ or R ₃ and R ₄ or R ₅ and R ₆ or R ₇ and R ₈ represent a group of formula 2.

In a third aspect, the invention relates to a process for the preparation of the morpholine phthalocyanine derivatives and their isomers of the general formula I, wherein

- M means Mg and

- R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and R ₈ form pairs R ₁ and R ₂ , R ₃ and R ₄ , R ₅ and R ₆ , R ₇ and R ₈ and when they are equal in pair, they are H or a group of formula 2

GB 222 376 B1 wherein the at least one pair of substituents R ι and R ₂ or R ₃ and R ₄ or R ₅ and R ₆ or R ₇ and R ₈ is a group of formula 2 consisting of macrocyclization (cyklotetrameryzacji) of the corresponding dinitriles of general formula 5

optionally with an appropriate amount of 1,2-dicyanobenzene, by the Linsted method in the presence of magnesium alkoxide, preferably obtained directly in the reaction medium by reacting the ground magnesium metal with an alcohol. The addition of 1,2-dicyanobenzene is used in the synthesis of compounds of general formula I in which at least one but not more than three pairs of R ₁ and R ₂ or R ₃ and R ₄ or R ₅ and R ₆ or R ₇ and R ₈ are H.

In a fourth aspect, the invention relates to a process for the preparation of morpholine and morpholine-nitroimidazole phthalocyanine derivatives and isomers of the general formula I, wherein

- M means Mg and

- R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and R ₈ form pairs R ₁ and R ₂ , R ₃ and R ₄ , R ₅ and R ₆ , R ₇ and R ₈ and when they are equal, they are a group of formula 2

or

- R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and R ₈ form pairs R ₁ and R ₂ , R ₃ and R ₄ , R ₅ and R ₆ , R ₇ and R ₈ and when they are equal they are the group of formula II or when they are different one is H and the other is the group of formula 3

wherein:

- n takes a value from 0 to 20

- at least one R ₉ , R ₁₀ , R ₁₁ is -NO ₂ and the other two are equal or different and represent -H, -CH 3, -NO 2, with at least one pair of R ₁ and R ₂ or R 2 ₃ and R ₄ or R ₅ and R ₆ or R ₇ and R ₈ is the group of formula 2,

And the amount of dinitriles of formula 6 appropriate to the planned structure of the final product

wherein:

- n takes a value from 0 to 20

- at least one substituent R ₉ , R ₁₀ , R ₁₁ is -NO ₂ , the other two are equal or different and represent -H, -CH ₃ , -NO ₂ , by the Linsted method in the presence of magnesium alkoxide, preferably obtained directly in the medium reaction by reacting the ground magnesium metal with alcohol.

The method using the Linsted method to obtain magnesium phthalocyanine complexes according to both the fourth and fifth aspects consists in dissolving or suspending in a solvent primary magnesium alkoxides having a chain length of C ₁ to C ₁₀ and the corresponding dinitrile or corresponding dinitriles. The reactions are carried out at a temperature higher than 70 ° C, with vigorous agitation for 30 minutes to 30 days, under an inert gas atmosphere, preventing the access of oxygen from the air. The magnesium alkoxides can be used ready-made or generated immediately before adding the dinitrile or dinitriles. The reactions are carried out in a primary alcohol with a chain length of C ₄ to C ₄₀ . The magnesium alcoholate is obtained by known methods, preferably immediately before the cyclization reaction. The macrocyclization process is carried out at a temperature greater than 70 ° C, preferably at a temperature of 110 to 200 ° C. Due to the possibility of decomposition of the resulting macrocyclization products, the process should be carried out with the lowest possible exposure to light.

After completion of the reaction, the reaction mixture is cooled to room temperature, filtered of precipitated by-products and unreacted starting materials, e.g. through diatomaceous earth. After evaporating off the solvent, preferably under reduced pressure, the products are purified and isolated by column chromatography. Solvents and eluents used in particular are mixtures of such solvents as: ethyl acetate, methanol, tetrahydrofuran, dichloromethane, chloroform, triethylamine. The stationary phases used are silica gel and reverse phase silica gel.

Only magnesium phthalocyanines are obtained by this method.

In a fifth aspect, the invention relates to a process for the preparation of morpholine and morpholine-nitroimidazole phthalocyanine derivatives and isomers of the general formula I, wherein

- M means Mg (II), Zn (II), Mn (II), Mn (III) Cl, Fe (II), Fe (III) Cl, Co (II), Cu (II)

- R ₁ , R ₂ , R ₃ R ₄ , R ₅ , R ₆ , R ₇ and R ₈ form pairs R ₁ and R ₂ , R ₃ and R ₄ , R ₅ and R ₆ , R ₇ and R ₈ and when in pair are equal to denote a group of formula 2

or R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and R ₈ form pairs R ₁ and R ₂ , R ₃ and R ₄ , R ₅ and R ₆ , R ₇ and R ₈ , and when they are equal they are the group of formula 2 or when they are different one is H and the other is the group of formula 3

wherein:

- n takes a value from 0 to 20

- at least one R ₉ , R ₁₀ , R ₁₁ is -NO ₂ and the other two are equal or different and represent -H, -CH ₃ , -NO ₂ ,

PL 222 376 B1, wherein at least one pair of R1 and R2 or R3 and R4 or R5 and R6 or R7 and R8 represents a group of formula 2, consisting in macrocyclization (cyclotetramerization) of the corresponding dinitriles of general formula 4 and / or formula 5

wherein:

- n takes a value from 0 to 20

- at least one R ₉ , R ₁₀ , R ₁₁ is -NO ₂ and the other two are equal or different and represent -H, -CH ₃ , -NO ₂ , in a solvent or in the melt (in molten form) in the presence of a heterocyclic base, most preferably 1,8-diazabicyclo [5.4.0] undec-7-ene (DBU) and when M is Mg (II), Zn (II), Mn (II), Fe (II), Mn (III ) Cl, Fe (III) Cl, Cu (II), Co (II), additionally, the starting material is salts of these elements, preferably chlorides or acetates. The solvent used is primary alcohols with a chain length of C ₄ to C ₄₀ , preferably selected from the group: n-pentanol, n-hexanol, dimethylaminoethanol or mixtures thereof, in an alkaline medium. In the case of using salts other than manganese and iron chlorides for the Mn (III) and Fe (III) derivatives in the synthesis, the anion is exchanged by mixing the dissolved phthalocyanine in an organic solvent, immiscible with water, with a saturated sodium chloride solution. In another variant, in the case of the synthesis of phthalocyanine derivatives Fe (III) Cl and Mn (III) Cl, phthalocyanine derivatives Fe (II) and Mn (II) are dissolved phthalocyanine complexes in a water-immiscible organic solvent and then mixed with a saturated aqueous sodium chloride solution with the access of oxygen from the air.

For free phthalocyanines (M is 2H), the process consists of dissolving or suspending in a solvent, dinitrile or dinitrile and a heterocyclic base, and then vigorously stirring at a temperature greater than 70 ° C for 30 minutes to 30 days, in an inert gas that prevents access of oxygen from the air. In the case where the product is to be phthalocyanine metal complexes, the salts of the metal element forming the complex are additionally used in a salt: dinitrile ratio of at least 0.25: 1, preferably in a ratio of 0.5: 1. Suitable solvents are primary alcohols having a chain length of C3 to C40, preferably 1-pentanol and 1-hexanol, or aminoalcohols (amino alcohol derivatives I-row) with a chain length of C ₁ to C _20, preferably dimethylaminoethanol. The reaction is carried out at a temperature greater than 70 ° C, and preferably at a temperature of 110 to 200 ° C. It is preferable to carry out the reaction with the lowest possible exposure to light as this can lead to the degradation of the macrocyclization products formed. As the salts of the metallic element, inorganic and organic salts thereof are used, with the use of carbonates, chlorides and acetates being preferred. After completion of the reaction, isolation and purification of the products are carried out according to the methods described in the Linsted method.

Regardless of whether the synthesis is carried out by the Linsted method or by macrocyclization in the presence of a metal salt and a base, a mixture of two or more different dinitriles is used in the synthesis of phthalocyanine derivatives containing different substituted isoindole moieties. Depending on the

In the expected structure of the phthalocyanine derivative, either an equal proportion of individual dinitriles or a molar excess of one dinitrile over the others, e.g. to obtain phthalocyanine type A ₃ B (three substituted isoindole groups come from dinitrile A and one from dinitrile B), is used dinitrile itriles in a molar ratio of at least 3: 1 or more, with an excess of from 7: 1 to 10: 1 being preferred.

In the case of the synthesis of substituted phthalocyanines containing various substituted isoindole groups, from one to several products and their various constitutional isomers are obtained, in which there are a different number of isoindole fragments depending on the composition of the initial mixture of various dinitriles, but not all products are produced in quantities that can be isolated. The amount of individual isomeric products and the configuration of the fragments derived from a given dinitrile depend on the relative reactivity of the dinitrile used in the mixture, the symmetry of the dinitriles, the steric hindrance due to the presence of the dinitrile substituents and the temperature.

In a sixth aspect, the invention relates to a process for the preparation of morpholine salts and morpholine-nitroimidazole phthalocyanine derivatives and their isomers of the general formula 1,

wherein

- M means 2H, Mg (II), Zn (II), Mn (II), Mn (III) Cl, Fe (II), Fe (III) Cl, Co (II), Cu (II)

- R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and R ₈ form pairs R ₁ and R ₂ , R ₃ and R ₄ , R ₅ and R ₆ , R ₇ and R ₈ and when they are equal in pair, they are H or the group of formula 4

wherein

- X is Cl, J, Br, F,

- Alk represents an alkyl chain with a chain length of 1 to 20, wherein at least one pair of R ₁ and R ₂ or R ₃ and R ₄ or R ₅ and R ₆ or R ₇ and R ₈ represent a group of formula 2.

or R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and R ₈ form pairs R ₁ and R ₂ , R ₃ and R ₄ , R ₅ and R ₆ , R ₇ and R ₈ and when they are equal in pair, they represent a group of formula II or when they are different one is H and the other is a group of formula 3

wherein:

- n takes a value from 0 to 20

At least one R ₉ , R ₁₀ , R ₁₁ is -NO ₂ and the other two are equal or different and are -H, -CH ₃ , -NO ₂ with at least one pair of R ₁ and R ₂ or R ₃ and R ₄ or R ₅ and R ₆ or R ₇ and R ₈ represent a group of formula 2, consisting in an alkylation reaction between a compound of general formula 1,

wherein

- M means 2H, Mg (II), Zn (II), Mn (II), Mn (III) Cl, Fe (II), Fe (III) Cl, Co (II), Cu (II)

- R ₁ , R ₂ R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and R ₈ form pairs R ₁ and R ₂ , R ₃ and R ₄ , R ₅ and R ₆ , R ₇ and R ₈ and when they are equal in pair, they are H or a group of formula 2

wherein at least one pair of R ₁ and R ₂ or R ₃ and R ₄ or R ₅ and Re or R ₇ and R ₈ is a group of formula 2.

or R ₁ , R ₂ R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and R ₈ form pairs R ₁ and R ₂ , R ₃ and R ₄ , R ₅ and R ₆ , R ₇ and R ₈ and when they are equal in pair, they are the group of formula II or when they are different one is H and the other is the group of formula III

wherein n and R ₉ , R ₁₀ , R ₁₁ are as defined above, and the corresponding α-alkyl halide with a chain length of 1 to 20 by mixing both components dissolved or suspended in an organic solvent.

The method of obtaining the salt according to the invention consists in the quaternization of morpholine nitrogen atoms. The phthalocyanine or its metal complex is dissolved or suspended in an organic solvent that does not react with the alkylating agent, and then a solution or suspension of the alkylating agent is added. The reaction is carried out in a temperature range of 0 ° C to 200 ° C, preferably 50 to 120 ° C. Reaction times range from 30 minutes to 30 days, with a preferred reaction time of 24 to 72 hours. The solvents used are two or more C ₁ to C ₂₀ halogenated alkanes, in particular: chloroform, dichloromethane, tetrachloromethane, 1,2-dichloroethane; also DMF, DMSO, N-methylpyrrolidone, acetonitrile. N-haloalkanes having a chain length of C1 to C20, in particular methyl iodide and ethyl bromide, are used as alkylating agents.

The amount of the alkylating agent is from two to several hundred times molar excess for each 2- (morpholin-4-yl) ethoxy group present in the phthalocyanine molecule. Preferably, a five to twenty-fold molar excess is used.

PL 222 376 B1

The reaction will be carried out in an inert gas atmosphere preventing the access of oxygen from the air and with the lowest possible access to light to avoid photo-decay processes. After completion of the reaction and cooling the reaction to a temperature in the range from 15 to 25 ° C, the separated product in the form of a precipitate is separated from the remaining components of the reaction mixture by filtration, for example, under reduced pressure. The resulting solid containing the crude product is washed with acetone, then with ethyl or methyl alcohol and then with diethyl ether and dried to yield the final product.

In a further aspect the invention relates to the use of the compounds according to the invention for the preparation of medicaments used in anti-cancer therapy, in particular in the treatment of PDT, also in the treatment of skin diseases.

The compounds according to the invention, in particular, may find application in the PDT treatment of neoplasms and pre-neoplastic conditions, such as: bladder cancer, esophageal cancer, colorectal cancer, skin and mucosa cancer, basal cell carcinoma of the skin, Bowen's disease, squamous cell carcinoma of the skin, mycosis, bronchial granuloma lung cancer, vulva cancer, in particular skin cancers like malignant melanoma, cancers of the female genital organs like cervical cancer, lung cancers and prostate cancers like androgen-dependent prostate adenoma.

The compounds according to the invention may in particular be used in the PDT treatment of skin diseases such as: Paget's disease (cutaneous form), senile keratosis, rosacea, acne resistant to other forms of therapy, alopecia areata, light red cheilitis, condylomata acuminata, psoriasis, lichen. scleroderma vulva, cystitis.

In order to determine the suitability of the compounds according to the invention for medical applications, in particular antitumor activity, a representative of the group, namely 1,4-bis [2- (morpholin-4-yl) ethoxy] phthalocyanine zinc (II) was performed against four tumor cell lines designated: A375 , Calu-1, HeLa and LNCaP. Cytotoxicity was tested both in terms of the so-called dark toxicity, i.e. in the absence of light, and bright after irradiation for 10 minutes and 20 minutes with light with a wavelength of 670 nm, i.e. in terms of the suitability of these compounds in PDT therapy. The biological activity was assessed on the basis of two tests:

• MTT test (Methylthiazolyldiphenyltetrazolium bromide test) - test with 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide • LDH test (LACTATE DEHYDROGENASE) lactate dehydrogenase activity test

Cell lines used in research

The following cell lines were used to assess the degree of cytotoxicity:

• A375 - human malignant melanoma, • Calu-1 - human lung cancer, • HeLa - human cervical adenoma, • LNCaP - androgen dependent prostate adenoma.

The cell lines were sourced from the European Collection of Cell Cultures (ECACC, Salisbury, UK).

Cells were grown in DMEM (Dulbecco's Modified Eagle's Medium) cell medium without phenol red with the addition of 10% (v / v) FBS (fetal bovine serum), 1% antibiotic solution (penicillin / streptomycin) and 1 % L-lutamine solution. Cultures were carried out at 37 ° C, 95% humidity in an atmosphere containing 5% CO ₂ . The culture medium was changed every two days. After reaching 80% confluence, cells were transferred to new culture vessels at a ratio of 1: 4, twice a week.

MTT test

The MTT assay is based on the ability of mitochondrial dehydrogenase to convert the water-soluble yellow salt of 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide to a purple-colored water-insoluble formazan. This transformation is possible only in living cells, and therefore the test allows to assess the degree of survival of the cells. The resulting formazan crystals are collected inside the cells and extracted with DMSO. The intensity of the color obtained is inversely proportional to the cytotoxicity of the compounds used in the test. Cells of the following cell lines: Calu-1, LNCaP, HeLa, and A375 were seeded in 96-well plates at approximately 2x10 ⁴ and incubated for 24 hours. The cells were then washed twice with PBS solution and solutions of 1,4-bis [2- (morpholin-4-yl) ethoxy] zinc (II) phthalocyanine in cell medium containing 2.5% FBS were administered. For the test, solutions of the tested compounds were prepared at the following concentrations: 10; 5; 2.5; 1.25; 0.6; 0.3; 0.15 pM. DMSO with a concentration of 12 was used as a negative control

The reduction did not exceed 0.1% in the culture medium. After 24 hours of culture, cells were washed twice with phosphate buffer solution and cell medium was added. The plates naświe ₂ tlano LED lamp having a wavelength of 670 nm at an intensity of irradiation of 7.9 ± 0.4 mW / cm for a time 10 and 20 minutes from a distance of about 6 cm in order to assess the degree of cytotoxicity in the light phase. In order to assess the degree of dark phase cytotoxicity, one plate in each experiment was not illuminated. Cells were incubated for 24 hours and 170 µl of MTT solution (5 mg / ml PBS) in cell medium were administered after incubation. After 2 hours, the precipitated formazan was dissolved in 200 µl DMSO and shaken for 10 minutes. Absorbance was measured at 570 nm using a plate reader (Biotek Instruments, Elx-800). The results of two independent experiments performed in six times, in the form of mean values ± standard deviation, are presented in the graphs where:

• in figure 1 the results for cells of the LNCaP line, • in figure 2 for cells of the CALU-1 line, • in figure 3 for cells of the HeLa line, • in figure 4 for cells of the line A375.

In Figures 1, 2, 3 and 4, the dark phase toxicity data are represented by circles, the light phase toxicity data after 10 minutes irradiation squares, and the bright phase toxicity data after 20 minutes exposure by triangles.

LDH test

The test is based on the ability to convert lactate to pyruvic acid by the enzyme lactate dehydrogenase, which is located in the cytosol of all cells. As a result of damage to the continuity of the cell membrane under the influence of mechanical, chemical factors or cell death by apoptosis or necrosis, the enzyme is released from the cells into the culture medium. Measurement of the enzyme activity allows the degree of integrity of the cell membrane to be assessed and thus the degree of cytotoxicity of the test compound to be determined. The commercial LDH Cytotoxicity Assay Kit used for the study is based on a two-step reaction. In the first step, the reduction reaction of NAD (nicotinamide adenine dinucleotide) to NADH (reduced form of nicotinamide adenine dinucleotide) + H + takes place. As a result of the oxidation of lactate to pyruvate catalyzed by the released enzyme. In the second stage, the tetrazole salt is reduced to colored formazan under the influence of NADH and H +. The increase in the amount of secreted formazan was observed spectrophotometrically. Absorbance was measured at 490 nm using a plate reader (Biotek Instruments, Elx-800. The increase in absorbance at 490 nm is directly proportional to LDH activity.

Cells of the LNCaP and A375 cell lines were seeded in 96-well plates at approximately 2x10 ⁴ and incubated for 24 hours. The cells were then washed twice with PBS solution and solutions of 1,4-bis [2- (morpholin-4-yl) ethoxy] zinc (II) phthalocyanine in cell medium containing 2.5% PBS were administered. For the test, solutions of the tested compounds were prepared at the following concentrations: 10; 5; 2.5; 1.25; 0.6; 0.3; 0.15 μΜ. As a negative control, DMSO was used, the concentration of which did not exceed 0.1% in the cell medium. After 24 hours of culture, cells were washed twice with phosphate buffer solution and cell medium was added. Subsequently, the plates were irradiated with the LED lamp ₂ with a wavelength of 670 nm, at an irradiation intensity of 7.9 ± 0.4 mW / cm for 10 and 20 minutes from a distance of about 6 cm in order to assess the degree of cytotoxicity in the light phase. In order to assess the degree of dark phase cytotoxicity, one plate in each experiment was not illuminated. The cells were incubated for 24 hours and then the activity of lactate dehydrogenase was measured. For this, the plates were centrifuged (400 xg, 5 minutes) and the supernatant from each plate was transferred to new 96-well plates. Then a series of standard solutions of lactate dehydrogenase was prepared in duplicate. 100 μl of the reaction solution was applied to each well. Plates were placed on a shaker and incubated for 30 minutes at room temperature, then absorbance was read at 490 nm. The results of the experiment performed in duplicate, in the form of mean values ± standard deviation, are presented in the graphs, where in figure 5 the results for LNCaP cells and in figure 6 for A375 cells.

IC ₅₀ values (IC ₅₀ concentration causing inhibition of half-cell growth) from the MTT assays were read from the data in Graphs 1, 2, 3 and 4 and are shown in Table 1.

PL 222 376 B1

T a b e l a 1

Cell line Exposure time [min] 0 10 twenty IC50 [pM] [pM] [pM] A375 > 10 1.10 0.49 Calu-1 > 10 0.30 0.22 HeLa 8.60 0.49 0.26 LNCaP 6.10 0.15 0.09

The model compound 1,4, -bis [2- (morpholin-4-yl) ethoxy] zinc (II) phthalocyanine showed much lower cytotoxicity in the MTT test in the dark phase (without illumination) than in the light phase. The LNCaP cell line turned out to be the most sensitive to the effects of the test compound in the MTT test. The IC ₅₀ value under MTT assay conditions in the dark phase for this cell line was 6.1 pM. In the light phase at a duration of illumination of 20 minutes, the MTT IC ₅₀ assay was 0.09 µM. The irradiation resulted in a 68-fold increase in activity, which confirms the usefulness of 1,4, -bis [2- (morpholin-4-yl) ethoxy] zinc (II) phthalocyanine as a photosensitizer in photodynamic therapy. The data presented in Table 1 for the remaining tumor cell lines support the usefulness of the compounds of the invention in photodynamic therapy. _{The IC 50} values ranged from 0.22 to 0.49 pM under 20 minute irradiation. The increase in activity for A375 cells was twenty-fold, for Calu-1, forty-five-fold and for HeLA, thirty-fold.

The increased antitumor activity observed from several dozen to several dozen times during irradiation of cells confirms the effectiveness of the new compounds according to the invention in photodynamic therapy of neoplasms. The obtained large differences in the activity between the dark and the light phase will allow for a significant reduction in the doses of photosensitizers used in order to achieve a killing effect against irradiated tumor cells with a simultaneous reduction of the risk of exposure to toxicity to non-irradiated normative tissues.

The second LDH test for the two LNCaP and A375 cell lines showed an increase in the activity of the enzyme lactate dehydrogenase in the culture medium. This phenomenon is associated with the breakdown of the cell membrane caused by the action of chemical factors or biological phenomena such as necrosis or cell apoptosis induced by a cytotoxic agent. In the case of the LNCaP line (Figure 5), a rapid increase in activity was observed at 10 minutes irradiation for a concentration of 1 pM, and at 20 minutes irradiation for a concentration of 0.6 pM. The second line of A375 was only tested at 10 min irradiation, a sharp increase in activity was observed at 2.5 pM (figure 6). This proves that at these concentrations, apart from the inhibition of cell growth described by the MTT test, their disintegration occurs additionally.

The LDH test confirms the cytotoxic effects also observed in the MTT test. With regard to the tested cell lines, it was proved that the photodynamic effect leading to the breakdown of neoplastic cells was dependent on the type of tumor. This effect is four times stronger in androgen-dependent prostate adenoma cancer than in human malignant melanoma cells.

The photodynamic killing effect against neoplastic cells and pathogenic microorganisms is caused by the presence of reactive oxygen species (ROS). The most desirable form of RTF is molecular singlet oxygen Δ _θ . This compound is generated under the influence of light and photosensitizer from the basic, triplet oxygen, present in the tissues. In the case of therapeutic applications, photodynamic processes take place with a predominance of the photodynamic mechanism II, in which the emerging singlet oxygen is responsible for the therapeutic effect. The new phthalocyanines according to the invention have the ability to generate singlet oxygen from triplet oxygen dissolved in a solution by irradiating the new phthalocyanine solutions with light in the 600 to 800 nm range.

The compounds of the invention are characterized by the ability to generate singlet oxygen.

₁

As a result of the reaction of singlet oxygen Δ _θ formed by the photosensitizer and light, 1,3-diphenylisobenzofuran (DPBF) is converted into o-dibenzoylbenzene (ODB). Excited to

In the singlet form, molecular oxygen attaches to the DPBF molecule to form a peroxide-like adduct which is further converted to ODB. The evaluation of the singlet oxygen generation capacity was performed in DMSO and DMF. For this purpose, DPBF working solutions with an absorbance of 1.0 w / in solvents and the phthalocyanine solutions according to the invention with an absorbance of 1.0 were prepared under limited light access. Then the working solutions were mixed together in a 1: 1 ratio. The mixture thus obtained was irradiated with a monochromatic light beam with a wavelength corresponding to the absorption maximum of the Q band of the test compound. The radiation intensity was ~ 0.5 mW / cm, measured with an Optel RD 0.2 / 2 radiometer with a TD probe. A set consisting of a 150 W xenon arc lamp (Optel) and a monochromator (Optel) was used as a light source. The solution was irradiated in a cuvette with an optical path length of 1 = 1 cm. During the irradiation, the solution was stirred with a magnetic stirrer, interrupted for the duration of the spectrophotometric measurement. The test was carried out at room temperature. UV-Vis absorption spectra were plotted at second intervals: 0, 15, 30, 45, 60, 90, 120, 150, 180, 240, 300, 360, 420, 480, 540.

The parameter that allows to compare the efficiency of singlet oxygen generation is the quantum efficiency of singlet oxygen generation (Φδ). The higher the value of Φ _Δ, the greater the capacity of a given photosensitizer to generate singlet oxygen.

The singlet oxygen generation capacity was determined as the decrease in the intensity of the DPBF absorption band at the wavelength of 417 nm. The relationship ln (A ₀ / A) = k (t) was _{used in the calculations, where A - absorbance, A 0} - absorbance at time t ₀ , k - DPBF decay rate constant. Then, from the relationship between the DPBF decay rate constant and the directional line ln (A ₀ / A) = k (t) k = a, the quantum yields of singlet oxygen generation (Φδ) were determined by comparing the slope of the kinetic curves of the standard - phthalocyanine (II) with the slope of the tested photosensitizer phthalocyanine determined under the same conditions.

Table 2 shows the quantum yields of singlet oxygen (Φδ) generation under aerobic conditions for a known photosensitizer considered to be the standard zinc (II) phthalocyanine (data taken from Seotsanyan-Mokhosi I., Kuznetsova N., Nyokong T .: Photochemical studies of tetra2,3 -pyridinoporphyrazines. J. Photochem. Photobiol. A. 2001, 140, 215-222) and the quantum yields of selected phthalocyanines according to the invention, 1,4, -bis [2- (morpholin-4-yl) ethoxy] zinc (II) phthalocyanine and magnesium (II) 1,4,8,11,15,18,22,25-octakis [2- (morpholin-4-yl) ethoxy] phthalocyanine.

The lower photodynamic activities of the new compounds according to the invention towards the standard zinc phthalocyanine are compensated for by the increased bioavailability of these derivatives as they are characterized by a much better water solubility.

The standard zinc phthalocyanine is insoluble in water, therefore its clinical application requires the use of special pharmaceutical formulations, e.g. liposomes. This hinders its penetration into tissues in the therapeutic process.

The differences in the observed values of quantum efficiency of singlet oxygen generation are also influenced by the phenomenon of aggregate formation by photosensitizer molecules. The new modified derivatives according to the invention having substituents in the form of morpholine rings undergo less aggregation or opsonization (phagocytosis), which results in greater bioavailability and stability of the compounds in the tissue environment.

T a b e l a 2

Derivative Solvent Excitation light wavelength [nm] Quantum yield of singlet oxygen Φ _Δ 1,4-bis [2- (morpholin-4-yl) ethoxy] zinc (II) phthalocyanine DMF 691 0.4454 ± 0.0123 DMSO 696 0.0224 ± 0.0004 1,4,8,11,15,18,22,25-octakis- [2- (morpholin-4-yl) ethoxy] phthalocyanine magnesium (II) DMF 738 0.2487 ± 0.0054 DMSO 745 0.2143 ± 0.0036 Reference substances zinc (II) phthalocyanine DMF 670 0.67 * DMSO 672 0.56 *

^w) Seotsanyana-Mokhosi I.

PL 222 376 B1

The new phthalocyanine derivatives have the ability to generate singlet oxygen. The compounds can therefore act according to the second mechanism of photodynamic therapy. This is advantageous because the mechanism is more efficient and there are no reports of the emergence of single oxygen resistance in the treatment of infection with pathogenic microorganisms. It is known from the literature that compounds with the ability to generate singlet oxygen generally exhibit antibacterial activity. Based on studies, McCluskey et al. (17) demonstrated the relationship between the generation of singlet oxygen in the photodynamic process by photosensitizers, which were derivatives of C60, C70 and C80 fullerenes, and antibacterial activity against gram-negative and gram-positive bacteria. The key role in the antibacterial activity of singlet oxygen against E. coli was also confirmed by Bagchi and Basu (18). Also, studies on antiprotozoal activity carried out by Barbos (19) confirm the effectiveness of photodynamic therapy against Trypanosoma cruzi, which is explained by the presence of singlet oxygen generated in the photodynamic process.

Due to the activity of singlet oxygen generated by the compounds according to the invention, they can also be used as bactericides, both for disinfection and as therapeutic agents for combating: bacterial infections, protozoal infections, fungal infections, viral infections, disinfection of the operating field, surgical instruments and medical equipment, as preparations accelerating the healing of surgical wounds.

Water solubility is an important parameter determining bioavailability and selective transport to the tissue undergoing photodynamic therapy. The solubility of the new phthalocyanines in water was determined on the basis of the UV-Vis spectra for various concentrations. UV-Vis spectra for selected concentrations, in water, ranging from 0.80x10 ^-6 to 12.86x10 ^-6 mol / dm ³ for 1,4,8,11,15,18,22,25-octakis [2- Magnesium (II) (morpholin-4-yl) ethoxy] phthalocyanine (II) is shown in figure 7 and figure 8 shows the linear dependence of absorbance on the concentration of photosensitizer in aqueous solution. The standard zinc (II) phthalocyanine is insoluble in the above-mentioned concentration range and the UV-Vis absorption spectra for this compound cannot be plotted. The new phthalocyanines according to the invention have an advantage over the standard because they are soluble in water in the concentration range mentioned above. This allows them to be used in therapy in the form of aqueous solutions, as opposed to zinc (II) phthalocyanine, which must be used in special drug forms, e.g. microemulsions.

The new morpholine and morpholine-nitroimidazole phthalocyanine derivatives have fluorescent properties when excited by external light.

The phenomenon of fluorescence is commonly used in photodynamic diagnostics - PDD (Photodynamic diagnostics). The currently used diagnostics uses the phenomenon of accumulation of endogenous porphyrinoids such as protoporphyrin IX or prodrugs, such as hexyl 5-aminolevulinate or 5-aminolevulinic acid, which are metabolized in the neoplastic tissue to porphyrins. The use of phthalocyanines allows the use of the Soret band and Q band radiation absorption in diagnostics, which is much stronger than in the case of porphyrins. Vasilchenko et al. Confirm the possibility of using aluminum derivatives of phthalocyanines in dentistry and skin autotransplantology (20).

For example, Figure 9 shows the emission spectrum for 1,4, -bis [2- (morpholin-4-yl) ethoxy] zinc (II) phthalocyanine at an excitation wavelength close to the Q band in DMF-670 nm, and Figure 10 for 1,4,8,11,15,18,22,25-octakis [2- (morpholin-4-yl) ethoxy] phthalocyanine of magnesium (II) at the excitation wavelength close to the Q band - 730 nm.

The same compounds show fluorescence upon irradiation with light in the Soret band. This phenomenon is shown in figure 11 for 1,4, -bis [2- (morpholin-4-yl) ethoxy] zinc (II) phthalocyanine and figure 12 for 1,4,8,11,15,18,22,25-octakis Magnesium (II) [2- (morpholin-4-yl) ethoxy] phthalocyanine.

Morpholine and morpholine-nitroimidazole phthalocyanine derivatives exhibit fluorescence in the light wavelengths of the biological window, making them potentially useful as markers in medical diagnostics.

The photosensitizers of the invention can be excited for fluoridation using light sources in the range 300 to 400 nm, i.e. in the wavelength range emitted by most diagnostic devices as well as light sources with a wavelength of 600 to 800 nm, by observing the near infrared fluorescence.

Table 3 shows the values of the quantum fluorescence efficiency (0> _F ) (parameter describing the ratio of photons emitted in the process of fluorescence in relation to the number of absorbed photons).

PL 222 376 B1

For selected phthalocyanines in DMF, 0> _F values ranging from 32% to 42% of the value relative to the standard zinc (II) phthalocyanine were obtained. In the case of fluorescence in DMSO, these values ranged between 15% to 31% 0> _F with respect to the standard zinc (II) phthalocyanine. The phenomenon of fluorescence is used for diagnostic purposes, in particular to assess the accumulation of compounds in tissues. Accumulation depends on the type of tissue (normative or mutant) and on bioavailability, which in turn depends on water solubility. The O> _F values observed are lower than for zinc (II) phthalocyanine, which is compensated for by the good water solubility of the new phthalocyanine derivatives, as demonstrated in this specification.

T a b e l a 3

Derivative Solvent Excitation light wavelength [nm] Quantum fluorescence yield ⁽ 'I> f 1,4,8,11,15,18,22,25-octakis [2- (morpholin-4-yl) ethoxy] phthalocyanine magnesium (II) DMF 738 0.071 DMSO 744 0.030 1,4, -bis [2- (morpholin-4-yl) ethoxy] zinc (II) phthalocyanine DMF 690 0.055 DMSO 696 0.062 Reference substance zinc (II) phthalocyanine DMF 670 0.17 DMSO 672 0.20

Fluorescence studies have shown that the compounds of the invention are capable of fluorescing as a result of the excitation of light with high tissue penetration.

The phthalocyanine and nitroimidazole fragments present in the molecules of the new phthalocyanines have a complementary mechanism of action. During photodynamic therapy, the process of consumption of molecular oxygen present in the tissue takes place, which undergoes changes within the framework of the second mechanism of photodynamic therapy. Therefore, a gradual hypoxia of the treated tissue occurs, which additionally enhances the action of the nitroimidazole fragment present in the molecule. Both the nitroimidazole and phthalocyanine fragments present have a synergistic effect.

Some of the new phthalocyanines obtained by the invention have nitroimidazoles linked by an alkyl linker as substituents. Nitroimidazoles are a group of compounds with their own antitumor and antibacterial activity with a mechanism of action other than phthalocyanine.

Antibacterial, antitumor and antiprotozoal activity and the use of new phthalocyanines containing nitroimidazole groups in diagnostics will be enhanced due to the spontaneous activity of the nitroimidazole group. Therefore, the biological activity of this group of phthalocyanines will be the sum of the activity of two fragments (phthalocyanine and nitroimidazole ring) having their own biological activity.

Compounds from the group of imidazole derivatives are widely used in medical therapy and diagnostics. 5-nitroimidazole derivatives such as metronidazole, ornidazole, tinidazole, secnidazole are most commonly used in the treatment of protozoal and anaerobic infections. Therefore, it is expected that the antiprotozoal and antimicrobial activity of the new phthalocyanines with a nitroimidazole ring will intensify.

Nitroimidazole derivatives are also evaluated in preclinical studies and in phase III clinical trials and show promising anti-tuberculosis activity. These derivatives are highly active in in vitro and rodent studies against strains resistant to other chemotherapeutic agents and show no cross-resistance with other drugs. The mechanism of their action is based on the inhibition of lipid synthesis of the cell wall of Mycobacterium tuberculosis (21), (22). Therefore, the anti-tuberculosis activity of new phthalocyanines with a nitroimidazole ring in their structure should be expected.

Nitroimidazole derivatives also show antiviral activity. For 2-methyl-5-nitroimidazole derivatives, activity of HIV reverse transcriptase inhibitors has been reported (23). This increases the potential therapeutic use of the new phthalocyanines in the treatment of cancer with accompanying viral infections.

PL 222 376 B1

Moreover, the nitroimidazole moiety affects the ability to accumulate in hypoxic tissues.

This property has been confirmed in clinical tests of F-linked nitroimidazole preparations in positron emission tomography, e.g. FMISO and PHASE (24). Therefore, one should expect a selective accumulation in hypoxic tissues of new phthalocyanines with a nitroimidazole ring in their structure.

The invention is illustrated by, but not limited to, examples.

The synthesis reactions in the examples were carried out in reaction vessels that were isolated from light during the reaction, which could degrade the resulting products or starting materials.

In the examples (unless otherwise stated), 50 cm glass columns packed with Merck silica gel 60 H (40-60 µm) as stationary phase were used for the chromatographic separation.

The examples use abbreviations that mean:

• THF-tetrahydrofuran, • TEA -triethylamine

Example 1 ₃

In a dry flask with a capacity of 100 cm placed magnesium turnings (72 mg, 3 mmol), 7 mg jo ₃ large 20 cm and 1-butanol. The reaction mixture was heated to reflux for 6 h with a vigorous nitrogen purge. After cooling to room temperature, 773 mg (2 mmol) of 2,3-dicyano-1,4-bis [2- (morpholin-4-yl) ethoxy] benzene were added. The mixture was refluxed with vigorous stirring under an inert atmosphere for 20 hours. After cooling to room temperature, the dark green solution was filtered through diatomaceous earth and the 1-butanol was evaporated to dryness in vacuo. The dry _{residue 3} was suspended in 2 cm of eluent with the composition CH ₂ Cl ₂ : CH ₃ OH: TEA 10: 1: 0.11 and pre-purified on a 50 cm column with the eluent composition CH ₂ Cl ₂ : CH ₃ OH: TEA 10 : 1: 0.11. The eluate was evaporated to dryness under reduced pressure and the dry residue was suspended in 2 cm ₃ of the composition of the eluent CH ₂ Cl _2: CH ₃ OH: TEA 10: 1: 0.11, and purified on a 50 cm column using an eluent of the composition CH ₂ Cl ₂ : CH ₃ OH: TEA 10: 1: 0.11. 433 mg (55%) obtained

1.4.8.11.15.18.22.25- octakis [2- (morpholin-4-yl) ethoxy] phthalocyanine magnesium (II) in the form of dark green crystals. mp > 300 ° C,

R, (CH ₂ Cl ₂ : CH ₃ OH: TEA 10: 1: 1%) 0.47.

UV-Vis (CH ₂ Cl ₂ ): X _ma x nm (loge) 324 (4.91), 745 (5.04), 800 (4.91).

¹ H NMR (500 MHz; pyridine-d ₅ ): δ, ppm 8.00 (s, 8H), 5.41-5.38 (t), 3.70-3.68 (t), 3.31 -3.29 (t), 2.73 (s).

¹³ C NMR (125 MHz; pyridine-d ₅ ): δ, ppm 153.2, 152.2, 130.1, 119.6, 70.4, 67.2, 58.4, 54.7.

MS (MALDI TOF): m / z 1571 [M + H] +.

Example 2 ₃

In a dry flask with a capacity of 100 cm ^{3 was} placed 1546 mg (4 mmol) 2,3-dicyano-1,4-bis [2- (morpholin-4-yl) ethoxy] benzene, 600 µL (4 mmol) 1,8-diazabicyclo [5.4.0 ] undec-7-ene, 734 mg ₃ (4 mmol) zinc acetate and 3 cm ³ 1-pentanol. The mixture was refluxed with vigorous stirring under argon for 24 hours. After completion of the reaction ₃ re joint stock was added 50 cm ³ of toluene was distilled from the reaction mixture the solvents under reduced pressure until the appearance of visible residues. Then to the remaining ₃ portions of solvent and precipitate was again added 50 cm ³ of toluene and evaporated to dryness. The su ₃ residue was suspended in 2 cm of eluent with the composition CH ₂ Cl ₂ : CH ₃ OH: TEA 20: 1: 0.11 and pre-purified on a 50 cm column with the eluent composition CH2Cl2: CH3OH: TEA 20: 1: 0 , 11.

The eluate was evaporated to dryness under reduced pressure, after which the dry residue was suspended in 2 cm ₃ of the composition of the eluent CH ₂ Cl _2: CH ₃ OH: TEA 20: 1: 0.11, and purified on a 50 cm column using an eluent of the composition of CH ₂ Cl ₂ : CH ₃ OH: TEA 20: 1: 0.11. 138 mg (8.6%) was obtained

1.4.8.11.15.18.22.25-octakis- [2- (morpholin-4-yl) ethoxy] zinc (II) phthalocyanine. mp > 300 ° C,

R t 0.61 (CH ₂ Cl ₂ : CH ₃ OH: TEA 10: 1: 1%); V-Vis (CH ₂ Cl ₂ ): X _has x, nm (logs) 324 (4.91), 672 (4.68), 740 (5.28), 803 (4.82).

¹ H NMR (500 MHz; pyridine-d ₅ ): δ, ppm 2.73 (s, 32H), 3.29-3.32 (t, 16H), 3.68-3.69 (t, 32H) 5.38-5.40 (t, 16H), 8.01 (s, 8H).

MS (MALDI TOF): m / z 1523 [M + H] +.

PL 222 376 B1

P r z k ł a d 3

In a dry flask with a capacity of 100 cm3 placed 386 mg (1 mmol), 3-dicyano-1,4-bis [2- (morpholin-4-yl) ethoxy] benzene, 1280 mg (10 mmol) 1,2-dicyanobenzene, 822 µL, (5 , 5 mmol) 1,8-diazabicyclo [5.4.0.] Undec-7-ene and 1001 mg (5.5 mmol) zinc acetate in 3 ml of 1-pentanol. The mixture was refluxed with vigorous stirring under argon for 24 hours. ₃ after completion of the reaction the reaction mixture was added 50 cm ³ of toluene was distilled from the reaction mixture the solvents under reduced pressure until the _three visible precipitate. Then to the remaining portion of the solvent and precipitate was again added 50 cm ^{3 of} toluene _three ene and evaporated to dryness. The dry residue was suspended in 2 cm ^{3 of the} eluent with the composition CH2Cl2: CH3OH: TEA 20: 1: 0.11 and pre-purified on a 50 cm column with the eluent composition CH2Cl2: CH3OH: TEA 20: 1: 0.11. Two eluates were obtained by chromatographic separation: one contained zinc (II) phthalocyanine and the other 1,4-bis [2- (morpholin-4-yl) ethoxy] zinc (II) phthalocyanine. Zinc (II) phthalocyanin was a by-product and was not further purified. An eluate containing

1.4-bis [2- (morpholin-4-yl) ethoxy] phthalocyanine zinc (II) was evaporated to dryness under reduced ₃ pressure. The dry residue was suspended in 2 cm of eluent with the composition MeOH: THF 5: 1 and purified on a 50 cm column chromatography filled with 90 C18 reverse phase gel - Reversed phase from Merck and washed with eluent. The eluate was evaporated to dryness to 128 mg, (15.0%)

1, 4, -bis [2- (morpholin-4-yl) ethoxy] phthalocyanine zinc (II). mp. > 300 ° C.

R, 0.71 (CH ₂ Cl ₂ : CH ₃ OH: TEA 10: 1; 1%) ¹ H NMR (500 MHz; pyridine-d ₅ ): δ, ppm 9.42-9.33 (m, 6H ), 8.26-8.22 (m, 6H), 7.82 (s, 2H), 3.76-3.74 (t, 4H), 3.67-3.65 (t, 4H), 3.29h (4H), 3.16-3.14 (t, 4H);

MS (MALDITOF): m / z 835 [M + H] +.

Example 4 ₃

In a dry flask with a capacity of 100 cm of magnesium shavings were placed 188 mg; (7.73 mmol), ₃ mg of iodine and 20 cm ³ of 1-butanol. The reaction mixture was heated to the reflux temperature of the alcohol for 20 hours with a vigorous nitrogen purge. After cooling the reaction mixture to room temperature, 1.815 g (4.7 mmol) 2,3-dicyano-1,4-bis [2- (morpholin-4-yl) ethoxy] benzene and 140 mg (0.47 mmol) were added. ) 1- [2- (2,3-dicyanophenyloxy) ethyl] -2-methyl-5-nitro-1H-imidazole. The mixture was refluxed with vigorous stirring under an inert atmosphere for 18 hours. After cooling to room temperature, the dark green solution was filtered through diatomaceous earth. After completion of the reaction ₃ reactive tive was added 50 cm ³ of toluene was distilled from the reaction mixture the solvents under reduced pressure until the appearance of visible residues. Then to the remaining portion _{3 of} the solvent and precipitate was again added 50 cm ³ of toluene and evaporated to dryness. The dry after ₃ zostałość suspended in 2 cm eluent with a composition of CH ₂ Cl _2: CH ₃ OH 10: 1 and pre-separated on a 50 cm column using an eluent of the composition of CH ₂ Cl _2: CH ₃ OH 10: 1, which, after washing, the substrate changed to CH ₂ Cl ₂ : CH ₃ OH: TEA 10: 1: 0.11. Fraction containing 1- [2- (2-methyl-5-nitro-1, H-imidazol-1-yl) ethoxy] -8,11,15,18,22,25-hexakis [2- (morpholin-4- yl) ethoxy] phthalocyanine magnesium (II) was evaporated to dryness in vacuo, the same was done for the fraction containing

1,4,8,11,15,18,22,25-octakis [2- (morpholin-4-yl) ethoxy] phthalocyanine magnesium (II). Both ₃ fractions were then separately purified, for this purpose they were dissolved in 2 cm ^{3 of} methanol and purified on 30 cm 3 column chromatography columns filled with Merck 90 C18 Reversed Phase Gel. A mixture of H 2 O: CH 3 OH 7: 3 was used as eluent. Evaporation of the eluates containing both reaction products under reduced pressure gave: 2 mg of 1- [2- (2-methyl-5-nitro-1, H-imidazol-1-yl) ethoxy] -8,11,15,18,22 , 25-hexakis [2- (morpholin-4-yl) ethoxy] phthalocyanine magnesium (II) (0.27% yield);

R f = 0.47 (dichloromethane-methanol-triethylamine, 100: 10: 0.5);

MS (MALDI) m / z 1480.6 [M + H] +;

¹ H NMR (400 MHz, d _5- pyridine): δ 9.67; 8.25; 8.12; 8.01; 7.88; 7.72; 5.72; 5.41; 5.31; 5.26; 5.05; 3.83; 3.69; 3.50; 3.31; 3; 19; 2.92; 2.77; 2.73.

and

265 mg; 1,4,8,11,15,18,22,25-octakis [2- (morpholin-4-yl) ethoxy] phthalocyanine magnesium (II) ed. (3.6%);

R f = 0.41 (dichloromethane-methanol-triethylamine, 100: 10: 0.5);

MS (MALDI) m / z 1569.7 [M + H] +;

GB ¹ 222 376 B1 H NMR (400 MHz, d ₅ -pyridine) δ 8.05; 5.45 (t, 16H, ³ J = 8Hz); 3.76 (t, 16H, ³ J = 6Hz); 3.35 (t, 16H, ³ J = 6Hz);

¹³ C NMR (100 MHz, d 5 -pyridine): 153.4; 152.7; 130.7; 119.8; 70.6; 67.3; 58.5; 54.8.

Example 5 ₃

In a dry flask with a capacity of 100 cm ³ provided, 93 g (5 mmol) of 2,3-dicyano-1,4-bis [2- (morpholin-4-yl) ethoxy] benzene, 148 mg (0.5 mmol) of 1- [2- ( 2,3-dicyanophenyloxy) ethyl] -2-methyl-5-nitro-1Himidazole, 500 mg (2.75 mmol) zinc acetate, 420 mg (2.75 mmol) 1,8-diazabicyclo [5.4.0] undec-7-ene and 3 ml of 1-pentanol. The mixture was kept at 130 ° C with vigorous stirring under argon for 21 hours. _{After completion of the reaction, 3} cm ^{3 of} toluene was added to the reaction mixture, and the solvents were distilled off from the reaction mixture under reduced pressure until visible precipitate appeared. Then, to the remaining portion of the solvent ₃ together with the precipitate, 50 cm ^{3 of} toluene were again added and evaporated to dryness. The reaction products were purified and isolated from the mixture by column chromatography. The dry residue suspension ₃ szono 2 cm eluent with a composition of CH ₂ Cl _2: CH ₃ OH 10: 1 and pre-purified on a 50 cm column using an eluent of the composition of CH ₂ Cl _2: CH ₃ OH 10: 1, which, after washing, the substrate changed to CH ₂ Cl ₂ : CH ₃ OH: TEA 10: 1: 0.11. Fraction containing 1- [2- (2-methyl-5-nitro-1, H-imidazol-1-yl) ethoxy] -8,11,15,18,22,25-hexakis [2- (morpholin-4- yl) ethoxy] zinc (II) phthalocyanine was evaporated to dryness under reduced pressure, the same was done for the fraction containing 1.4.8,

11,15,18,22,25-octakis [2- (morpholin-4-yl) ethoxy] zinc (II) phthalocyanine. Both fractions ₃ were then separately purified, for this purpose they were suspended in 2 cm ^{3 of} methanol and purified on 30 cm 3 column chromatography columns filled with 90 C18 - Reversed phase gel from Merck. A mixture of H 2 O: CH 3 OH 7: 3 was used as eluent. Evaporation of the eluates under reduced pressure containing both reaction products gave:

mg 1- [2- (2-methyl-5-nitro-1, H-imidazol-1-yl) ethoxy] -8,11,15,18,22,25-hexakis [2- (morpholin-4-yl) ) zinc (II) ethoxy] phthalocyanine (0.27% yield);

R _f = 0.54 (dichloromethane-methanol-triethylamine, 100: 10: 0.5);

MS (MALDI) m / z 1520.6 [M + H] +.

¹ H NMR (400 MHz, d _5- pyridine): δ 9.66; 8.21; 8.14; 8.04; 7.90; 7.77; 5.72; 5.41; 5.31; 5.26; 5.05; 3.82; 3.68; 3.62; 3.50; 3.31; 3.20; 2.92; 2.76; 2.73.

and mg 1,4,8,11,15,18,22,25-octakis [2- (morpholin-4-yl) ethoxy] zinc (II) phthalocyanine (3.6% yield);

R _f = 0.51 (dichloromethane-methanol-triethylamine, 100: 10: 0.5);

MS (MALDI) m / z 1609.7 [M + H] + ^.

Example 6 ₃

In a dry flask with a capacity of 50 cm ^{3 were} placed 200 mg (0.13 mmol) 1,4,8,11,15,18,22,25-octakis [2- (morpholin-4-yl) ethoxy] phthalocyanine of magnesium (II), prepared according to Example 5 and solution ₃

518 μl (8.32 mmol) of methyl iodide in 12 cm of anhydrous chloroform. The reaction mixture thus obtained was kept at reflux under argon for 5 hours. The green precipitate formed by the reaction was then filtered off and washed with acetone, methanol and diethyl ether. 320 mg of magnesium (II) iodide 1,4,8,11,15,18,22,25-octakis [2- (4-methylmorpholin-4-yl) ethoxy] phthalocyanine were obtained (93% yield). mp > 300 ° C,

UV-Vis (H ₂ O): X _has x, nm (loge) 324 (4.83), 663 (4.70), 736 (5.36).

¹ H NMR (400 MHz; D ₂ O): δ, ppm 7.91 (s, 8H), 5.37 (s), 4.28 (s), 3.93-3.89 (t), 3 , 53-3.50 (d), 3.30-3.26 (t).

¹³ C NMR (100 MHz; D ₂ O): δ, ppm 156.2, 153.0, 131.3, 121.8, 67.85, 64.1.64.3, 64.1.51.5 .

P r z k ł a d 7

The preparation containing the new phthalocyanines is administered from 1 to 100 hours before irradiation, (preferably between 24 and 72 hours) as intravenous injections. This is the time for selective accumulation in neoplastic tissue and elimination from normative tissues. The preparation containing phthalocyanine in a concentration from 0.001 mg / ml to 100 mg / ml is used in the form of an isotonic solution (e.g. saline solution or dextran solution). Then, after the time necessary for selective accumulation has elapsed, the tissue is irradiated with light with a wavelength of 600 to 750 nm, for example: argon pumped dye laser (rhodamine B dye) with a wavelength of 630 nm emitted radiation. The total dose of light radiation used varies with the treatment and the tissues treated, and generally ranges from 50 to 22

1000 J / cm ² , preferably between 100 and 350 J / cm ² .

PL 222 376 B1

P r z k ł a d 8

It is possible to apply photodynamic therapy with the use of new phthalocyanines on the tissue surface in the form of an ointment, emulsion or gel, in which the preparation is at least 0.001% by weight, and its concentration depends on the type of tissue, the location of lesions and the bioavailability of phthalocyanine. In the time interval from immediate application to 100 hours after application of the preparation, the tissue is irradiated with a light source with a wavelength of 600 to 750 nm, e.g. an argon pumped dye laser (rhodamine B dye) with an emitted wavelength of 630 nm. The total dose of light radiation used varies zależno ₂ COMPONENTS of the treatment and the tissues to be treated, and generally it is in the range of 50 to 1000 J / cm ₂ preferably between 100 and 350 J / cm ²

Literature:

1. Konopka K., Goslinski T., J. Dent. Res., 86 (2007) 694-707.

2. Kramarenko G.G., Wilke W.W., Dayal D., Buettner G.R., Schafer F.Q., Free Radical Biology and Medicine, 40 (2006) 1615-1627

3. Agostinis P., Berg K., Cengel KA, Foster TH, Girotti AW, Gollnick SO, Hahn SM, Hamblin MR, Juzeniene A., Kessel D., Korbelik M., Moan J., Mroz P., Nowis D ., Piette J., Wilson BC, Golab J., Ca-Cancer. J. Clin., 61 (2011) 250-281

4. Wainwright M., Journal of Antimicrobial Chemotherapy, 42 (1998) 13-28

5. Allison R.R., Downie G.H., Cuenca R., Hu X.H., Childs C., Sibata C.H., Photodiag. Photodyn. Ther., 1 (2004) 27-42

6. Hu M., Brasseur N., Yildiz S.Z., Van Lier J.E., Leznoff C.C., J. MED. CHEM. 41 (11) (1998) 1789-1802

7. Fadel M., Kassab K., Fadeel D.A., Lasers Med Sci. 25 (2) (2010) 283-72

8. Larroque C., Pelegrin A., Van Lier J. E., Br. J. Cancer., 74 (12) (1996) 1886-1890

9. Cosimelli B., Roncucci G., Dei D., Fantetti L., Ferroni F., Ricci M., Spinelli D., Tetrahedron 59 (2003) 10025-10030

10. Nyokong T., Coord. Chem. Rev., 251 (2007) 1707-1722.

11. Michel S., Hoffman B.M., Baum S., Barrett A.G.M., Prog. Inorg. Chem., Ed. Karlin K.D.,

J. Wiley & Sons, New York, 50 (2001) 473-590

12. Lv F, Cao B, Cui Y, Liu T., Molecules, 17, (2012) 6349-6361

13. Minnock A., Vernon D. L, Schofield J., Griffiths J., Parish J. H., Brown S. B. Antimicrob. Agents Chemother. 44 (3) (2000) 522-527

14. Gasco A., Boschi D., Chegaev K., Cena C., Di Stilo A., Fruttero R., Lazzarato L., Rolando B., Tosco P., PureAppl. Chem :, 80 (8) (2008) 1693-1701

15. Viegas C., Danuello A., Silva Bolzani V., Barreiro E.J., Fraga C.A.M., Curr. Med. Chem., 14 (2007) 1829-1852

16. Haimovici R, Ciulla TA, Miller JW, Hasan T, Flotte TJ, Kenney AG, Schomacker KT, Gragoudas ES., Retina, 22 (2002) 65-74

17. McCluskey D. M., Smith T. N., Madasu P. K., Coumbe C. E., Mackey M. A., Fulmer P. A., Wynne J. H., Stevenson S., Phillips J. P. ACS Appl Mater Interfaces. 1 (4) (2009) 882-887

18. Bagchi, B., Basu, S. Photochemistry and Photobiology, 29, (1979), 403-405)

19. A. F. S. Barbosa, L. G. P. Soares, J. M. S. Aciole, G. T. S. Aciole, I. R. Pitt, S. L. Galdino, A. L. B. Pinheiro AIP Conf. Proc. 1364, pp. 55-59, 5-6 November 2010, Florence, Italy

20. Vasilchenko S. Y., Volkova A. I., Ryabova A. V., Loschenov V. B., Konov V. I., Mamedov A. A., Kuzmin S. G., Lukyanets E. A., J. Biophotonics 3 (5-6) (2010) 336-346.

21. Matsumoto M., Hashizume H., Tomishige T., Kawasaki M., Tsobouchi H., Sasaki H., Shimokawa Y., Komatsu M., PLoSMed., 3 (2006) 2131-2144

22. Walczak K., Gondela A., Suwiński J., Eur. J. Med. Chem., 39 (2004) 849-853.

23. De Martino G., La Regina G., Di Pasquali A., Ragno R., Bergamini A., Ciaprini C., Sinistro A., Maga G., Crespan E., Artico M., Silvestri R., J Med. Chem., 48 (2005) 4378-4388.

24. Abdel-Jalil R.J., Ubele M., Ehrlichmann W., Voelter W., Machulla H.J., J. Radioanal. Nucl. Chem., 267 (2006) 557-560.

PL 222 376 B1

Claims (29)

Patent claims 1. New morpholine and morpholine-nitroimidazole derivatives of phthalocyanines and their isomers of the general formula I, in which - M means 2H, Mg (II), Zn (II), Mn (II), Mn (III) Cl, Fe (II), Fe (III) Cl, Co (II), Cu (II) - R ₁ , R ₂ R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and R ₈ form pairs R ₁ and R ₂ , R ₃ and R ₄ , R ₅ and R ₆ , R ₇ and R ₈ and when they are equal in pair, they are H or a group of formula 2, where at least one pair of R ₁ and R ₂ or R ₃ and R ₄ or R ₅ and R ₆ or R ₇ and R ₈ is a group of formula 2, or - R ₁ , R ₂ R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and R ₈ form pairs R ₁ and R ₂ , R ₃ and R ₄ , R ₅ and R ₆ , R ₇ and R ₈ and when they are equal in pair, they are a group of formula II or when they are different one is H and the other is a group of formula III in which: - n takes a value from 0 to 20 - at least one R ₉ , R ₁₀ , R n is -NO ₂ and the other two are equal or different and represent -H, -CH ₃ , -NO ₂ , with at least one pair of R ₁ and R ₂ or R ₃ and R ₄ or R ₅ and R ₆ or R ₇ and R ₈ represent a group of formula 2. 2. Salts of morpholine and morpholine-nitroimidazole derivatives of phthalocyanines and their isomers of general formula 1, PL 222 376 B1 in which - M means 2H, Mg (II), Zn (II), Mn (II), Mn (III) Cl, Fe (II), Fe (III) Cl, Co (II), Cu (II) - R ₁ , R ₂ R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and R ₈ form pairs R ₁ and R ₂ , R ₃ and R ₄ , R ₅ and R ₆ , R ₇ and R ₈ and when they are equal in pair, they are H or the group of formula 4 wherein - X is Cl, J, Br, F, - Alk is an alkyl chain with a chain length of 1 to 20, at least one pair of R ₁ and R ₂ or R ₃ and R ₄ or F ₅ and R ₆ , or R ₇ and R ₈ represent a group of formula 2, or R ₁ , R ₂ R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and R ₈ form pairs R ₁ and R ₂ , R ₃ and R ₄ , R ₅ and R ₆ , R ₇ and R ₈ and when they are equal in pair, they mean a group of formula II or when they are different, one is H and the other is a group of formula III in which - n takes a value from 0 to 20 - at least one R ₉ , R ₁₀ , R ₁₁ is -NO ₂ and the other two are equal or different and represent -H, -CH ₃ , -NO ₂ , with at least one pair of R ₁ and R ₂ or R ₃ and R ₄ or R ₅ and R ₆ or R ₇ and R ₈ represent a group of formula 2. 3. The method of obtaining morpholine phthalocyanine derivatives and their isomers of general formula 1, PL 222 376 B1 in which - M means Mg and - R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and R ₈ form pairs R ₁ and R ₂ , R ₃ and R ₄ , R ₅ and R ₆ , R ₇ and R ₈ and when they are equal in pair, they are H or a group of formula 2, wherein at least one pair of R ₁ and R ₂ or R ₃ and R ₄ or R ₅ and R ₆ or R ₇ and R ₈ is a group of formula 2, characterized by that it consists in macrocyclization of the corresponding dinitriles of the general formula V, optionally with an appropriate amount of 1,2-dicyanobenzene, by the Linsted method in the presence of magnesium alkoxide at a temperature higher than 70 ° C. 4. The method according to p. The process of claim 3, wherein the reaction is carried out at a temperature of 110 to 200 ° C. 5. The method according to p. A process according to claim 3 or 4, characterized in that for the preparation of compounds in which at least one pair of R1 and R2 or R3 and R4 or R5 and R6 or R7 and R8 is H, an appropriate amount of 1,2-dicyano benzene is used. 6. The method according to p. The process of claim 3, 4 or 5, characterized in that the magnesium alkoxide with a chain length of C ₁ to C ₁₀ is obtained directly in the reaction medium. The method according to claim 3 or 4, 5 or 6, characterized in that the reaction is carried out in a primary alcohol with a chain length of C4 to C40. 8. The method of obtaining morpholine and morpholine-nitroimidazole derivatives of phthalocyanines and their isomers of the general formula 1, PL 222 376 B1 in which - M means Mg and - R ₁ , R ₂ R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and R ₈ form pairs R ₁ and R ₂ , R ₃ and R ₄ , R ₅ and R ₆ , R ₇ and R ₈ and when are equal signify a group of formula 2 or - R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and R ₈ form pairs R ₁ and R ₂ , R ₃ and R ₄ , R ₅ and R ₆ , R ₇ and R ₈ and when they are equal they are the group of formula II or when they are different one is H and the other is the group of formula III wherein: - n takes a value from 0 to 20 - at least one R ₉ , R ₁₀ , R ₁₁ is -NO ₂ and the other two are equal or different and represent -H, -CH ₃ , -NO ₂ , with at least one pair of R ₁ and R ₂ or R ₃ and R ₄ or R ₅ and R ₆ or R ₇ and R ₈ is a group of formula 2, characterized in that it consists in macrocyclization of appropriate dinitriles of general formula 5 and the amount of dinitriles of formula 6 appropriate for the planned structure of the final product PL 222 376 B1 in which: - n takes a value from 0 to 20 - at least one of R ₉ , R ₁₀ , R ₁₁ is -NO ₂ and the other two are equal or different and are -H, -CH ₃ , -NO ₂ , by the Linsted method in the presence of magnesium alkoxide at a temperature higher than 70 ° C. 9. The method according to p. The process of claim 8, wherein the reaction is carried out at a temperature of 110 to 200 ° C. 10. The method according to p. The process of claim 8 or 9, characterized in that the magnesium alkoxide with a chain length of C ₁ to C ₁₀ is obtained directly in the reaction medium. 11. The method according to p. 8. The process according to claim 8, 9 or 10, characterized in that the reaction is carried out in a primary alcohol with a chain length of C ₄ to C ₄₀ . 12. Method for the preparation of morpholine and morpholine-nitroimidazole phthalocyanine derivatives and their isomers of the general formula I, in which - M means Mg (II), Zn (II), Mn (II), Mn (III) Cl, Fe (II), Fe (III) Cl, Co (II), Cu (II) - R1, R2, R3, R4, R5, Re, R7 and R8 form pairs R and R2, R3 and R4, R5 and Re, R7 and R9, and when they are equal in pair, they represent a group of formula 2 or R ₁ , R groups ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and R ₈ form pairs R ₁ and R ₂ , R ₃ and R ₄ , R ₅ and R ₆ , R ₇ and R ₈ and when they are equal, they are a group of Formula 2 or, when different, one is H and the other is a group of Formula PL 222 376 B1 in which - n takes a value from 0 to 20 - at least one R ₉ , R ₁₀ , R ₁₁ is -NO ₂ and the other two are equal or different and represent -H, -CH ₃ , -NO ₂ , with at least one pair of R ₁ and R ₂ or R ₃ and R ₄ or R ₅ and R ₆ or R ₇ and R ₈ represent a group of formula 2, characterized in that it consists in macrocyclization of the corresponding dinitriles of general formula 4 and / or formula 5 in which - n takes a value from 0 to 20 - at least one R ₉ , R ₁₀ , R ₁₁ is -NO ₂ and the other two are equal or different and represent -H, -CH ₃ , -NO ₂ , in a solvent or in the melt in the presence of a heterocyclic base, and in the case of when M is Mg (II), Zn (II), Mn (II), Fe (II), Mn (III) Cl, Fe (III) Cl, Cu (II), Co (II), additional salts are used as substrate inorganic and organic salts of these elements, the reaction being carried out in a solvent or melt at a temperature greater than 70 ° C. 13. The method according to p. The process of claim 12, wherein the reaction is carried out at a temperature of 110 to 200 ° C. 14. The method according to p. A process as claimed in claim 12 or 13, characterized in that 1,8-diazabicyclo [5.4.0] undec-7-ene is used as the heterocyclic base. 15. The method according to p. Primary alcohols of C ₄ to C ₁₀ chain length, preferably selected from the group: n-pentanol, n-hexanol, dimethylaminoethanol or mixtures thereof; or amino alcohols having a chain length of C ₁ to C ₂₀ , preferably dimethylaminoethanol. 16. The method according to p. A process as claimed in any preceding claim in which the metal salts are carbonates, chlorides or acetates. 17. The method according to p. The method of claim 12 or 13 or 14 or 15 or 16, characterized in that the metal salt to dinitrile ratio is at least 0.25: 1. 18. The method according to p. The process of claim 17, wherein the metal salt to dinitrile ratio is at least 0.5: 1. 19. The method of preparation is the salts of morpholine and morpholine-nitroimidazole phthalocyanine derivatives and their isomers of the general formula 1, PL 222 376 B1 in which - M means 2H, Mg (II), Zn (II), Mn (II), Mn (III) Cl, Fe (II), Fe (III) Cl, Co (II), Cu (II) - R1, R2, R3, R4, R5, R6, R7 and R8 form pairs R1 and R2, R3 and R4, R5 and R6, R7 and R8, and when they are equal in pair, they are H or a group of formula 4 in which - X is Cl, J, Br, F, - Alk is an alkyl chain with a chain length of 1 to 20, at least one pair of R1 and R2 or R3 and R4 or R5 and R6 or R7 and R8 is a group of formula 2, or R1, R2 R3, R4, R5, R6, R7 and R8 form pairs R1 and R2, R3 and R4, R5 and R6, R7 and R8, and when they are equal in pair, they represent a group of formula 2 or when they are different, one is H and the other of formula 3 in which - n takes a value from 0 to 20 - at least one R9, R10, R11 is -NO2 and the other two are equal or different and represent -H, -CH3, -NO2, with at least one pair of R1 and R2 or R3 and R4 or R5 and R6 or R7 and R8 is a group of formula 2, characterized in that it consists of an alkylation reaction between a compound of general formula 1, PL 222 376 B1 in which - M means 2H, Mg (II), Zn (II), Mn (II), Mn (III) Cl, Fe (II), Fe (III) Cl, Co (II), Cu (II) - R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ i, R ₇ and R ₈ form pairs R ₁ and R ₂ , R ₃ and R ₄ , R ₅ and R ₆ , R ₇ and R ₈ and when the pair are equal to H or grupęo formula 2 wherein at least one pair of substituents R ₁ and R ₂ or R ₃ and R ₄ or R ₅ and R ₆ or R ₇ and R ₈ is a group of formula 2, or R ₁ , R ₂ R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and R ₈ form pairs R ₁ and R ₂ , R ₃ and R ₄ , R ₅ and R ₆ , R ₇ and R ₈ and when in a pair they are equal means a group of formula 2 or when they are different one is H and the other is a group of formula 3 in which n and the substituents R ₉ , R ₁₀ , R ₁₁ are as defined above, and the corresponding α-alkyl halide of chain length from 1 to 20 by mixing both components dissolved or suspended in an organic solvent. 20. The method according to p. A process as claimed in 19, characterized in that n-haloalkanes having a chain length of C ₁ to C _20, in particular methyl iodide, ethyl bromide, are used as the alkylating agents. 21. The method according to p. A process as claimed in claim 19 or 20, characterized in that the amount of the alkylating agents is from two to several hundred times excess for each 2- (morpholin-4-yl) ethoxy group present in the phthalocyanine molecule. 22. The method according to p. The process of claim 21, wherein the amount of the alkylating agents is 5 to 20 fold excess for each 2- (morpholin-4-yl) ethoxy group present in the phthalocyanine molecule. 23. The method according to p. 19 or 20 or 21 or 22, characterized in that the solvent is DMSO, DMF, N-methylpyrrolidone, acetonitrile two or more halogenated alkanes of C ₁ to C _20, in particular chloroform, dichloromethane, tetrachloromethane, 1,2-dichloroethane . 24. Use of morpholine and morpholine-nitroimidazole derivatives of phthalocyanines and their isomers and their salts of general formula 1, in which M, R ₁ and R ₂ are as defined in claim 1, and their salts of general formula 1, in which M, R ₁ and R ₂ are as defined in claim 2, characterized in that the compounds used for the preparation in therapy. 25. Use according to claim 1 24. A method according to claim 24, characterized in that the compounds are used in the preparation of preparations used in antitumor therapy. 26. Use according to claim 1 25, characterized in that the compounds are used for the preparation of preparations used in the treatment of neoplasms and pre-neoplastic conditions such as: bladder cancer, esophageal cancer, colon cancer, vulvar cancer, cancer of the skin and mucous membranes, basal cell carcinoma of the skin, Bowen's disease, squamous cell carcinoma of the skin, head and neck, mycosis fungoides, bronchial lung cancer, vulvar cancer. 27. The use of claim 27 26. A method according to claim 26, characterized in that the compounds are used in the preparation of preparations for the treatment of oral squamous cell carcinomas. 28. Use according to claim 28 24, characterized in that the compounds are used in the preparation of preparations used in the PDT therapy of neoplasms and pre-neoplastic conditions such as: bladder cancer, esophageal cancer, colorectal cancer, vulvar cancer, cancer of the skin and mucous membranes, basal cell carcinoma of the skin, Bowen's disease, squamous cell carcinoma of the skin, head and neck, mycosis fungoides, bronchial lung cancer, vulvar cancer. 29. Use according to claim 1 24, characterized in that the compounds are used for the preparation of preparations used in the PDT treatment of skin diseases such as: Paget's disease, cutaneous form, senile keratosis, rosacea, acne resistant to other forms of therapy, alopecia areata, luminous red cheilitis, condylomas acuminata, psoriasis, vulvar lichen sclerosus, bladder inflammation.