PL212930B1 - New derivatives of chlorophyll, method for obtaining new derivatives of chlorophyll and their application - Google Patents

Description

Description of the invention

The subject of the invention is new chlorophyll derivatives in which the central Mg atom is substituted with Pt (II), the method of preparation of these derivatives and preparations containing them.

A characteristic feature of chlorophylls is their intense green or blue-green color, associated with strong light absorption in the range of blue and red waves. The absorbed light energy excites the dye molecules, which radiate excess energy in the form of fluorescence or transfer the excitation energy to other molecules through photosensitization. Chlorophylls are the most important photosynthetic pigments of plants, cyanobacteria and photosynthetic bacteria. In the photosynthesis process, chlorophylls are responsible for both the absorption of light and the transfer of excitation energy and its conversion into chemical energy, taking place with the participation of their excited singlet states.

As shown in the figure, in formula 1, structurally all chlorophylls are porphyrin derivatives in which an additional fifth V isocyclic ring is attached to the four-pyrrole macrocyclic system.

Depending on the type of chlorophyll, the pyrrole rings have different degrees of oxidation, there are different substituents in the side positions of the macrocyclic system, and the carboxyl group at the C-17 ³ carbon is esterified with a long carbon chain alcohol which gives the chlorophyll a lipid (hydrophobic) character and makes them insoluble in water.

Hydrolysis of the ester bond at carbon C-17 ³ leads to the formation of chlorophyllides having a free carboxyl group and soluble both in organic solvents and in water. In naturally occurring chlorophylls, the four central nitrogen atoms bind to a magnesium atom, or in rare cases a zinc atom. However, the ability of the central atom to bind additional ligands is important.

In an acidic environment, chlorophylls easily undergo pheophytinization reactions, creating derivatives, the so-called pheophytins in which the central magnesium ion is substituted with two protons. In organic solvents in the presence of zinc (II) or copper (II) salts, pheophytin easily complexes with ions of these metals and transforms into metal-substituted chlorophyll. Under similar conditions, the central magnesium atom in chlorophylls also spontaneously exchanges with zinc or copper (II), respectively, this reaction is also observed under natural conditions, for example in plants subjected to stress with salts of these metals. Insertion or substitution reactions with other metal atoms take place under more drastic conditions and require the use of appropriate synthetic methods. The use of special methods also requires the substitution of the central metal in bacterial chlorophylls.

Depending on the nature of the central metal atom, the physicochemical properties of metal-substituted chlorophylls change. The basic features that are determined by the type of the central metal are: (i) absorption spectra; (ii) emission spectra; (iii) lifetime of excited states; (iv) quantum efficiency of triplet state formation; (v) number of coordinated ligands; (vi) chemical stability and redox potential.

It is known that particularly drastic changes in the physicochemical properties of metalloporphyrins and metal-substituted chlorophylls are associated with the presence in the macrocyclic ring of a transition metal atom, for example Cu (II) or Ni (II). They cause a very strong shortening of the lifetime of excited states of metal-substituted derivatives and quenching of fluorescence, they increase their redox potential, as well as cause changes in the parameters of intersystem transitions related to the heavy atom effect. If the central metal, apart from the four nitrogen atoms of the porphyrin system, binds additional ligands, as in the case of Ni (II), after excitation of the dye molecules with light, ultrafast dissociation of these additional ligands is observed (the so-called photo-ionization of ligands) and their possible re-binding (Rodriguez J. and Holten DJ, J. Phys. Chem. 92, 5944, 1990; Musewald et al., J. Am. Chem. Soc. 103, 7055, 1999).

The above-mentioned parameters largely depend on the interaction of chlorophylls with light and the ways of deactivating their excited states (electron excitation and emission), the reactions of the transfer of excitation energy (photosensitization), and the interaction of metalloporphyrins with the environment (Porphyrins and Metalloporphyrins (1975) Smith Ed., Elsevier , Amsterdam).

After chlorophyll is extracted from the natural biological environment and the dye is placed, for example, in organic solvents or in an aqueous environment, triplet excited states of the dye and photosensitization reactions become particularly important. They lead to the formation of active oxygen species, including singlet oxygen, under the influence of light in the presence of a photosensitizer, and consequently to the so-called photodynamic effect, widely described in the literature for many dyes, mainly from the group of porphyrins, including chlorophylls. (Spikes and Bommer, Chlorophylls (1991) Scheer H. Ed., Pp. 1181-1202, CRC Press, Boca Raton; Rosenbach-Belkin V., Chen L., Fiedor L., Salomon Y. and Scherz A. Photodynamic Tumor Therapy. 2nd and 3rd generation photosensitizers (1998) Moser JG Ed., Pp. 117-125, Harwood Academic Publishers, Amsterdam).

The photodynamic effect manifests itself in the ability of a photosensitizer such as chlorophyll to kill living organisms when exposed to light, for example bacteria or animal and human cells. Since a focused beam of light can selectively and precisely illuminate diseased human tissues, this effect has been used for some time to fight cancer in a method called photodynamic therapy (PDT).

One example of a substance approved for medical use as a photosensitizer in photodynamic therapy is the synthetic hematoporphyrin (Hp) derivative with the commercial name Photofrin II, which chemically is a mixture of at least some structurally related compounds. The approval of Photofrin II was due to the slight but favorable accumulation of Photofrin II in cancer cells and the absorption spectrum of this compound, typical for porphyrins, with a strong absorption band in the blue part (approx. 400-500 nm) and a much weaker band in the range above 600 nm. For therapy, it is the weaker, long-term band of this drug that is important, because it is in the range of relatively deeper permeability of light waves through the tissues (the so-called therapeutic window), falling between the range of light absorption by water and the range of endogenous porphyrin absorption, i.e. between 600 and 900 nm. A negative feature of Photofrin II is the low intensity of this band, which makes it necessary to administer very high doses of the drug and use intense and expensive light sources, such as lasers.

Another negative feature of Photofrin II is its long retention time of at least several weeks in the body of treated patients, during which the photosensitizer is still highly photosensitizing and may lead to side effects dangerous for the patient. For the above reasons, intensive work is underway to improve and develop new types of photosensitizers with more favorable physicochemical parameters and higher biocompatibility. In this context, chlorophyll derivatives can be an alternative in therapeutic applications.

Due to the very intense light absorption bands shifted to the range of over 600 nm and above, chlorophylls belong to the 2nd generation of photosensitizers. The location of the light absorption bands is primarily determined by the structure of chlorophylls and their interactions with the environment. For example, chlorophyll a (Chla), a dye of green plants, in acetone solution has an intense QY absorption band at 660 nm, which in natural systems is shifted to 700 nm.

Moreover, as shown by recent research on chlorophyllids (Rosenbach-Belkin V., Chen L., Fiedor L., Tregub I., Pavlotsky F., Brumfeld V., Salomon Y. and Scherz A., Photochem. Photobiol. 64, 174) , 1996), they decompose relatively quickly in living organisms and have short retention times in tissues. For the reasons mentioned above, from the point of view of photodynamic therapy, chlorophylls constitute a very attractive and therapeutically promising group of photosensitizers.

For many reasons (including: water insolubility, chemical stability, photosensitization efficiency), direct use of natural chlorophylls in therapy and diagnostics is impossible, and hence the need to subject these dyes to appropriate chemical modifications.

From the descriptions of Polish patents No. 173128 and 173150 we know new chlorophyll and bacteriochlorophyll derivatives and the method of their production by enzymatic transesterification and, respectively, by catalytic condensation.

The patent specification No. 173128 further states that methods of obtaining chlorophyll and bacteriochlorophyll derivatives substituted with metals such as Cu, Ni, Zn, V, Co are known from the literature (Hambright, 1975; Hynninen 1991; Fiedor et al. 1993). However, neither the patent nor the specialist literature discloses chlorophyll derivatives in which the central atom is substituted with platinum (II).

The therapeutic interest in platinum compounds results from the fact that they are widely used in classic cancer chemotherapy, where one of the most commonly used drugs is the platinum (II) derivative, the so-called cis-platinum with the formula Pt (NH3) 2Cl2. In addition to this compound, other platinum derivatives are also used in chemotherapy, for example carboplatin and oxyplatin.

According to the generally accepted mechanism of action of cis-platinum, the active form of this compound are its aqua derivatives, which are formed as a result of the exchange of chloride ligands with water molecules in the environment of the interior of living cells. Due to the lability of aqua ligands, cis-platinum becomes

It is more reactive and cytotoxic, by strongly selectively adding to nitrogen atoms of two adjacent guanine bases of the DNA chain and causing inhibition of replication of the entire chain.

According to the invention, the new chlorophyll derivatives have the structure represented by the formula 1, where the central atom M is a Pt (II) atom and R is a phyll or a hydrogen atom.

The method of obtaining new chlorophyll derivatives consists in reacting chlorophyll, pheophytin or their derivatives with the platinum (II) complex compound, preferably with K2 [PtCl4], in the environment of a carboxylic acid with 1 to 4 carbon atoms, preferably acetic acid. , in the temperature range 30 to 110 ° C, preferably 40 to 100 ° C.

A therapeutic or diagnostic preparation contains as active substance new chlorophyll derivatives having the structure shown in the formula 1, where the central atom M is a Pt (II) atom and R is a phyll or a hydrogen atom.

Laboratory tests have shown that the chlorophyll derivatives which are the subject of the invention are very efficient photosensitizers and show high photocytotoxic activity. Therefore, they can be effectively used for therapeutic or diagnostic purposes, for example for the generation of active oxygen species, including singlet oxygen, or the light-induced release of active platinum ion species, as well as for the detection of oxygen.

The table below shows the nomenclature of chlorophyll, its natural derivative chlorophyllide, and the chlorophyll derivatives according to the invention shown in formula 1.

Substituent Dye M. R chlorophyll a (Chla) Mg phyll Pt-Chla Fri. phyll chlorophyllide (Chlide) Mg H. Pt-Chlide Fri. H.

Formula 1 shows the chemical structure and numbering of carbon atoms in the molecule of chlorophyll a and its derivatives.

The properties of the chlorophyll derivatives of the invention are illustrated in the drawing, in which Fig. 2 shows the electron absorption spectra of Pt-Chla and Pt-Chlide in methanol;

Fig. 3 - survival of S91 cells incubated in the dark in the presence of Pt-Chlide;

Fig. 4 Comparison of the photodynamic effect of Pt-Chlide on S91 cells with the photodynamic effect of Chlide and the commercial photosensitizer Photofrin II. The logarithmic scale of the ordinate axis was used;

Figure 5 shows the effect of the concentration of the photosensitizer used to incubate the cells on the magnitude of the Pt-Chlide photodynamic effect on S91 cells. The logarithmic scale of the ordinate axis was used.

The following non-limiting examples illustrate the invention.

Example I (Pt-Chla synthesis and purification)

A portion of pure chlorophyll a (1.8 mg) was dissolved in 1.1 ml of glacial acetic acid and, at 80 ° C, mixed with 10 mg of K2 [PtCl4] salt (10-fold stoichiometric excess) dissolved in 0.1 ml of distilled water. Then 55 mg of sodium acetate is added to the reagent solution.

The reaction for the insertion of Pt into the chlorophyll ring is carried out for 20 minutes by heating the reaction mixture in an oil bath at 80-85 ° C. The course of the reaction is monitored by recording the absorption spectra of samples of the reaction mixture in methanol.

After the reaction, the solvent is evaporated under a nitrogen stream and optionally a little acetone is added and evaporation continued.

After drying, the product is dissolved in a small volume of chloroform and filtered through chromatography paper to separate solid impurities. The filtrate is evaporated under nitrogen and dried under vacuum and the final product, Pt-Chla, is chromatographed on DEAE-Sepharose CL6B (Pharmacia, Sweden).

To this end, the 14 mm diameter column is filled to a height of about 5-6 cm with DEAE-Sepharose gel suspended in acetone. A sample dissolved in a mixture of 5% methanol / 95% acetone (v / v) is applied to the column and eluted with a solvent of the same composition. The dark green fraction containing the purified Pt-Chla is collected, the solvent is evaporated off and the product is dried thoroughly under vacuum. The pure product is stored under argon at -30 ° C.

The purity of the product was determined by analytical thin layer chromatography (TLC) on silica gel (Merck, West Germany). The chromatogram was developed on the eluent composed of: 68% dichloromethane, 25% n-hexane, 5% 2-propanol, 2% methanol (v / v). The molecular weight of the product, determined by mass spectroscopy, is 1065 IU (m / z), calculated from the sum formula of 1064.3 IU.

Example II (Pt-Chlide synthesis and purification)

A portion of pure chlorophyllide a (Chlide) (0.25 mg) is dissolved in 0.2 ml of glacial acetic acid and 1.6 mg of K2 [PtCl4] dissolved in 0.2 ml of water and a portion of sodium acetate ( 13 mg).

The reaction mixture is kept at 90-95 ° C under reflux for about 1.5 hours. The course of the Pt insertion reaction is monitored by recording the absorption spectra of the samples taken from the reaction mixture.

After completion of the reaction, the solvent of the reaction mixture was evaporated under nitrogen, optionally with the addition of a small amount of methanol.

The dry residue is dissolved in a little methanol and centrifuged (5 min, 7000 rpm, 15 ° C), the supernatant is collected and evaporated again under nitrogen.

The final product, Pt-Chlide, is obtained after purification twice by column chromatography on CM-Sepharose (Pharmacia, Sweden). For this purpose, a sample of the crude product dissolved in a mixture of 40% methanol in acetone is applied to a 10 mm diameter column filled to a height of about 3 cm with CM-Sepharose gel previously stabilized in acetone. The pure product fraction is eluted from the column first with a mixture of 40% methanol in acetone and then with a solution of 10% acetic acid in acetone. After evaporating off the solvent, the purification procedure is repeated.

The product is finally dried under vacuum and stored at -30 ° C under an argon atmosphere.

The purity of the Pt-Chlide was determined by reversed-phase high-performance thin layer chromatography (HPTLC RP-18, Merck). After the dye sample was applied to the plate, the chromatogram was developed on the eluent with the composition: 50% methanol / 50% acetonitrile (v / v). The molecular weight of the product, determined by mass spectroscopy, is 786 IU (m / z) and it is calculated from the sum formula of 785.5 IU.

P r x l a d III (Absorption spectra of Pt-Chla and Pt-Chlide)

The absorption spectra of Pt-Chla and Pt-Chlide, determined by the known method, are shown in Fig. 2. They are typical for chlorites, but in shape differ slightly from the spectra of the natural dyes, Chla and Chlide, which are the starting materials for their synthesis.

The main difference is the shift of the position of the QY band maximum from 660 nm to 635 nm, which additionally confirms the presence of Pt (II) as the central atom in these compounds.

Example IV (Determination of Pt-Chlide cytotoxicity in the dark)

Melanoma tumor cells (line S91) in the amount of 1 x 10 ^{5 were} placed on the bottom of the culture dishes (Petri dishes) in RMPI 5 medium containing antibiotics (penicillin and streptomycin) and left in an oven (37 ° C, 5% CO2) to flatten them. . After removing the medium and washing the cells with a buffered saline solution (PBS, without Ca ²⁺ and Mg ²⁺ ions), freshly prepared and sterilized Pt-Chlide solutions in PBS containing 0 to 0.5 μΜ of photosensitizer were applied to the cell dishes. incubated in an incubator (15 min, 37 ° C, 5% CO2). Then, the photosensitizer solution was removed, the cells were washed with a buffered PBS solution, a new aliquot of the culture medium was administered and the cells were left in the incubator. All operations were performed by protecting cell cultures from light. After 48 hours incubation, to obtain a homogeneous suspension, cells were treated with 0.05% trypsin solution containing EDTA. The surviving fraction was determined by counting in a Burker chamber against control unlit cells not incubated with the photosensitizer.

The graph - Fig. 3 shows the dependence of the size of the surviving S91 cell fraction on the Pt-Chlide concentration used. The graph illustrates the fact that in the concentration range up to 0.5 μΜ Pt-Chlide in the dark is not cytotoxic to S91 cells, and in the lowest concentration range it has slight stimulating properties.

PL 212 930 B1

Example V (Photodynamic effect of Pt-Chlide)

The following example illustrates the photodynamic effect of Pt-Chlide on the in vitro model cultures of S91 melanoma cells and compares it with the photodynamic effects of two other photosensitizers, i.e. the commercially available Photofrin II, a photosensitizer approved for photodynamic therapy, and chlorophyllide (Chlide ), a hydrophilic derivative of Chla.

A) Comparison of the photodynamic effects of Pt-Chlide, Chlide and Photofrin II

Melanoma cells of the S91 line in an amount of 1 x 10 ^{5 were} placed on the bottom of culture dishes (Petri dishes) in RMPI 5 medium containing antibiotics (penicillin and streptomycin) and left in an incubator (37 ° C, 5% CO 2) to dissolve them. After removing the medium and washing the cells with a buffered saline solution (PBS, without Ca ²⁺ and Mg ²⁺ ions), freshly prepared and sterilized photosensitizer solutions were applied to the cell dishes and incubated in an incubator (15 min, 37 ° C, 5 % CO2). Then, the cells were irradiated with red light with wavelengths above 600 nm with an intensity of 108 mW / cm ² , regulating the amount of light doses through appropriately selected exposure times. The HOP microprocessor halogen illuminator (Optel, Opole) equipped with an optical fiber (diameter 8 mm) with an internal high-pass filter transmitting radiation in the ranges above 600 nm was used as the light source.

After irradiation, the photosensitizer solution was removed, the cells were washed with buffered saline (PBS, without Ca ²⁺ and Mg ²⁺ ions) and incubated for 48 h. in fresh culture medium in an incubator. After the cells were treated with trypsin, they were counted as in Example IV. The surviving fraction was determined against a control culture of the same cells, not treated with either photosensitizer or light.

The following concentrations of photosensitizers were used: Pt-Chlide: 0.5 μΜ (0.38 μg / ml), Chlide: 1 μΜ (0.59 μg / ml), Photofrin II: 10 μg / ml.

The above-described procedure was performed in quadruplicate, and the graph of Fig. 4 shows the averaged results.

In all cases, the graph shows the typical dependence of the photodynamic effect on the delivered light dose. However, modification of the natural plant dye Chlide by substituting the central Mg ion with the Pt ion drastically increases its photocytotoxic properties. The comparison with the commercial photosensitizer Photofrin II shows that a similar photodynamic effect on S91 cells in vitro is achieved already at concentrations of Pt-Chlide 25 times lower than those required for Photofrin II.

The photocytotoxic effect of Pt-Chlide is stronger than might be expected. This seems to be the result of a synergistic mechanism of action consisting in amplifying the photodynamic effect of chlorophyll (or a derivative) by active forms of platinum (II) ions released in the photodissociation reaction.

B) Dependence of the photodynamic effect on the Pt-Chlide concentration.

Red light with wavelengths above 600 nm and power of 108 mW / cm ² was used to illuminate the cells, light dose delivered: 97 J / cm ² . The remaining conditions for the determination of the photodynamic effect were followed in A of this example, using cells of the S91 line. The variable parameter was the concentration of Pt-Chlide used for the incubation of the cells. The obtained results are shown in the diagram - Fig. 5.

This graph illustrates the strong dependence of the photodynamic effect on the concentration of the photosensitizer.

Claims (3)

1. New chlorophyll derivatives with the structure represented by the formula 1, in which M is Pt (II) and R is phyl or a hydrogen atom. 2. The method of obtaining new chlorophyll derivatives with the structure represented by the formula 1, characterized in that chlorophyll, pheophytins or their derivatives are reacted with the platinum (II) complex compound, preferably with K2 [PtCl4], in a carboxylic acid medium with 1 to 4 atoms carbon, preferably acetic acid, in the temperature range 30 to 110 ° C, preferably 40 to 100 ° C. 3. A therapeutic or diagnostic preparation, characterized in that as the active substance it contains new chlorophyll derivatives with the structure represented by the formula 1, in which M is Pt (II) and R is phytate or a hydrogen atom.