Ruthenium (II) polypyridyl complexes as photosensitizers in photodynamic therapy and processes for preparing ruthenium (II) polypyridyl complexes

Abstract

A ruthenium (II) coordination compound: (III) wherein n is 0 or 2 and R is selected from the group consisting of methyl; phenyl; -CH2NH2,-CH2NH2; -CH2OH,-CH2OH; -COOH,-COOH; the present invention relates to a coordination compound comprising an ester group-COOR1, an amide group-CONR2R3, which coordination compound is useful as a photosensitizer in photodynamic therapy for the treatment of tumors. In particular, the light-sensitive drug comprises a compound (III) incorporated into a liquid lipid nanoparticle-layered and / or non-layered crystal, in particular cubic liquid crystal nanoparticles. The process of obtaining the compound (III) involves three reaction steps (IV). # imgabs0 #

Description

[0001] Description

[0002] Scope of the present invention

[0003] The present invention relates to Ru(II) polypyridyl complexes for use as photosensitisers (i.e., photosensitising agents) in photodynamic therapy for treating tumours, to their synthesis process and to pharmaceutical compositions comprising such complexes.

[0004] Prior art - Technical problem

[0005] In recent years, photodynamic therapy has attracted increasing attention in the scientific community as an alternative to chemotherapy and radiotherapy for treating various malignant tumours in order to overcome well-known drawbacks such as severe side effects, drug resistance and poor drug efficacy.

[0006] Photodynamic therapy involves the use of low-energy light-activated photosensitisers that can be activated to generate highly cytotoxic reactive oxygen species (ROS), including singlet oxygen 1 1O2. In addition to the side effects typically associated with non-specific drug delivery, photodynamic therapy has the advantages of allowing spatiotemporal control of the activation of photosensitisers and allowing improved selectivity and efficacy of treatment for selected tumour targets.

[0007] Photosensitisers for photodynamic therapy must have low toxicity under dark conditions and a high ability to generate ROS upon photoexcitation. Most of the photosensitisers known to date contain a tetrapyrrole structure, such as porphyrins, phthalocyanines and chlorins. Although these drugs have a high ability to generate cytotoxic species upon irradiation, they have poor solubility and selectivity in biological media and induce prolonged photosensitivity in patients.

[0008] Some transition metal complexes used as photosensitisers in photodynamic therapy may overcome these drawbacks. In particular, polypyridyl Ru(II) complexes exhibit favourable photophysical and electrochemical properties, including high thermodynamic and kinetic stability, as well as the ability to generate singlet oxygen with high quantum yield. Examples of such compounds can be found in WO2020260424A1, EP3521295, CN113321687A, CN111875643A, CN110857310A, CN112266402A and WO2021032952A1.

[0009] In recent years, several Ru(II) polypyridyl complexes containing a benzo[i]dipyrido[3,2-a:2’,3’-c]phenazine unit (hereinafter referred to as “dppn”) have been synthesized. This ligand includes a particularly extensive aromatic system, which makes it possible to adjust the chemo-physical properties of the obtained compounds within a wide range. In particular, it allows maximizing the yield of singlet oxygen generation, thereby obtaining excellent therapeutic outcomes.

[0010] A very limited number of polypyridyl complexes containing two dppn units have also been synthesized. In this regard, two publications are known and are mentioned below.

[0011] In the paper of Wang and his colleagues 1 the compound of the following formula was mentioned:

[0012] [RuL(dppn)2] 2+ ,

[0013] where L is 3,8-bis(benzothiazolylfluorenyl)-1,10-phenanthroline, which was obtained by first introducing two dppn units and then reacting the ligand L with the intermediate [Ru(dppn)2Cl2] in ethylene glycol. However, details regarding the manner of conducting this intermediate synthesis were omitted, and no basic characterization data such as the 2+ H NMR spectrum of 1 [RuL(dppn)2] was provided. This synthetic route via the intermediate [Ru(dppn)2Cl2] has the following disadvantages: this intermediate has poor solubility in most organic solvents, leading to complications in the inherent synthesis.

[0014] The paper of Turro and his colleagues 2 deals with the synthesis and characterization of a compound having the following formula:

[0015] [Ru(NN)(dppn)2] 2+ ,

[0016] where NN is “bpy” = 2,2’-bipyridine. In this case, the preparation of the metal complex was carried out through a preliminary synthesis of the intermediate [Ru(NN)Cl4] and then [Ru(NN)(CH3CN)4] 2+ avoiding the use of [Ru(dppn)2Cl2] and introducing two dppn groups only in subsequent reaction steps. However, a reaction time of 7 to 24 days is required to obtain [Ru(NN)Cl4], which is very time-consuming and thus industrially unacceptable. This method also requires many complex purification steps, resulting in a relatively low overall yield of 28% - 37%. It is noteworthy that in the above-mentioned report of Turro, the compound [Ru(NN)(dppn)2]2+ It has only been studied as a dye (DSSC) for solar cell applications.

[0017] Accordingly, there is a desire to define a route for synthesizing a polypyridyl Ru(II) complex containing two dppn units that does not have the disadvantages of the prior art briefly discussed above, i.e., a route that provides intermediate compounds that are readily soluble in organic solvents and allows the desired complex to be obtained in higher yields and shorter reaction times. SUMMARY OF THE INVENTION

[0018] Accordingly, an object of the present invention is to provide a class of coordination compounds or Ru(II) complexes that are suitable for use as photosensitizers in the photodynamic therapy of tumors.

[0019] Another specific object of the present invention is to provide such a class of Ru(II) complexes that are more selective for tumor cells than the photosensitizers currently used in photodynamic therapy.

[0020] Another specific object of the present invention is to provide such a class of Ru(II) complexes that are capable of maximizing singlet oxygen production and thus exhibit better therapeutic efficiency compared to the photosensitizers currently used in photodynamic therapy.

[0021] Another object of the present invention is to provide an innovative and direct synthesis method for such Ru(II) complexes that allows the above complexes to be obtained in higher yields and shorter reaction times compared to prior art processes.

[0022] Another object of the present invention is to provide a drug containing such a Ru(II) complex in which there are two dppn (benzo[i]dipyrido[3,2-a:2’,3’-c]phenazine) ligands, which is fully soluble in water and thus in biological media in order to improve patient absorption and distribute to target sites in the patient's body without the use of potentially harmful co-solvents.

[0023] The above objects are achieved by the ruthenium(II) complex disclosed in claim 1, by the method for preparing said complex disclosed in claim 8, and by the photosensitizer drug for photodynamic therapy disclosed in claim 4. Advantageous embodiments of the complex and the drug and advantageous modifications of the method are defined in the corresponding dependent claims.

[0024] According to one aspect of the present invention, a class of ruthenium(II) coordination compounds is described, which ruthenium(II) coordination compounds have the following formula:

[0025]

[0026] where n is selected from between 0 and 2,

[0027] where R is a substituent group selected from the group consisting of:

[0028] - a methyl group;

[0029] - a phenyl group;

[0030] -- a CH2NH2 group;

[0031] -- a CH2OH group;

[0032] - a carboxyl group -COOH;

[0033] - an ester group of the formula -COOR1, where R1 can be selected from the group consisting of:

[0034] - a straight-chain or branched alkyl group having a carbon atom number in the range of 1 to 4;

[0035] - an unsubstituted benzyl group;

[0036] - a polyethylene glycol methoxy group having the formula H(OCH2CH2) 1-4 OH;

[0037] - an amide group of the formula -CONR2R3,

[0038] where

[0039] - R2 can be hydrogen and R3 can be a group selected from the group consisting of:

[0040] - a straight-chain or branched alkyl group having a carbon atom number in the range of 1 to 4;

[0041] -- a (CH2)2NH2 group;

[0042] - a phenyl group,

[0043] or

[0044] - R2 and R3 can be straight-chain or branched alkyl groups having a carbon atom number in the range of 1 to 4,

[0045] or

[0046] - NR2R3 can be a group selected from the group consisting of:

[0047] - a cycloalkyl secondary amine having a carbon atom number in the range of 4 to 5;

[0048] - a heterocyclic alkyl secondary amine containing a heteroatom selected from between nitrogen and oxygen,

[0049] This coordination compound is used as a photosensitizer in the photodynamic therapy of tumors.

[0050] In other words, the present invention relates to various polypyridyl complexes of ruthenium(II), hereinafter also referred to as [Ru(NN)(dppn)2] n+ , wherein

[0051] -dppn is a benzo[i]dipyrido[3,2-a:2’,3’-c]phenazine unit, i.e., a bidentate heterocyclic ligand characterized by extensive aromaticity, and

[0052] -NN is a functionalized 2,2’-bipyridine ligand with its R group as described above.

[0053] The presence of two dppn ligands in the same molecule enables the maximization of singlet oxygen generation, and thus the above ruthenium(II) compounds have been proven to be useful as photosensitizers in photodynamic therapy. More specifically, the dppn ligands exhibit extensive π-π conjugation, which allows them to easily interact with important biological targets such as proteins and / or DNA, and in the latter case, the interaction is a π-π stacking interaction with the DNA double helix. In addition, the extensive π-π conjugation of dppn extends the lifetime of the lowest-energy triplet excited state of the photosensitizer, resulting in improved generation of singlet oxygen 1 O2 upon excitation with appropriate low-energy light radiation, which is a key feature of the effectiveness of the PS potential.

[0054] In fact, as shown below, the obtained Ru(II) compounds exhibit excellent singlet oxygen generation properties upon photoexcitation, effectively interact with DNA as a possible biological target, and show significant phototoxicity against various cancer cell models.

[0055] According to another aspect of the present invention, a method for obtaining a compound of a class of ruthenium(II) coordination compounds having the following formula:

[0056]

[0057] wherein n is selected between 0 and 2,

[0058] wherein R is a substituent group selected from the group consisting of:

[0059] - hydrogen;

[0060] - a methyl group,

[0061] - a phenyl group,

[0062] --CH2NH2 group,

[0063] --CH2OH group

[0064] - carboxyl group -COOH,

[0065] - ester group of formula -COOR1, where R1 can be selected from the group consisting of:

[0066] - straight-chain or branched alkyl group with 1 to 4 carbon atoms;

[0067] - unsubstituted benzyl group;

[0068] - polyethylene glycol methoxy group with formula H(OCH2CH2) 1-4 OH.

[0069] - amide group of formula -CONR2R3,

[0070] where

[0071] - R2 can be hydrogen and R3 can be a group selected from the group consisting of:

[0072] - straight-chain or branched alkyl group with 1 to 4 carbon atoms;

[0073] --(CH2)2NH2 group;

[0074] - phenyl group,

[0075] or

[0076] - R2 and R3 can be straight-chain or branched alkyl groups with 1 to 4 carbon atoms.

[0077] or

[0078] - NR2R3 can be a group selected from the group consisting of:

[0079] - cycloalkyl secondary amine with 4 to 5 carbon atoms;

[0080] - heterocyclic alkyl secondary amine containing a heteroatom selected between nitrogen and oxygen, the method comprising the following consecutive steps:

[0081] - obtaining a reactive polymer precursor [Ru(CO)2Cl2] by reacting ruthenium(III) chloride with paraformaldehyde m ;

[0082] - obtaining a trans-Cl[Ru(NN)Cl2(CO)2] intermediate complex by:

[0083]

[0084] where NN is a functionalized 2,2'-bipyridine ligand, whose R groups are as described above,

[0085] React the reactive polymer precursor [Ru(CO)2Cl2] m with a predetermined symmetrically functionalized 2,2'-bipyridine ligand having two identical R functional groups;

[0086] - By reacting the intermediate complex

[0087]

[0088] with benzo[i]dipyrido[3,2-a:2',3'-c]phenazine to obtain the ruthenium(II) coordination compound.

[0089] In other words, the present invention provides a direct synthetic route for heteroleptic ruthenium(II) complexes which, according to the following general scheme, contain two extended aromatic bidentate ligands containing nitrogen atoms (these ligands consist of a benzo[i]dipyrido[3,2-a:2',3'-c]phenazine (dppn) unit) plus a symmetrically functionalized 2,2'-bipyridine ligand (the R groups of which are indicated hereinafter),

[0090] wherein the complex of formula III is shown without its charge and wherein three reaction steps are involved, namely:

[0091]

[0092] a) Preparation of the polymer precursor I;

[0093] b) Preparation of the trans-Cl[Ru(NN)Cl2(CO)2] complex of formula II;

[0094] c) Preparation of the complex of formula [Ru(NN)(dppn)2] n+ (where n = 0,

[0095] 2)(III) by reaction with two dppn equivalents.

[0096] This three-step synthesis process allows the desired compound to be obtained in relatively high yields (usually with an overall reaction yield between 22% and 47%) and the reaction time is at most a few hours.

[0097] In the method according to the invention, the NN ligand is introduced before the Ru(II) coordination by the dppn ligand. In the synthesis method of the present invention, the complex of formula III is obtained through the preliminary synthesis of intermediate II, overcoming the problems related to the insufficient solubility of [Ru(dppn)2Cl2] (which is commonly used in the synthesis of bis-heterocoordinated Ru(II) polypyridyl complexes) in most organic solvents, see the reference of Wang, cit. above. Typically, this is also the strategy commonly used to prepare Ru(II) complexes containing only one dppn unit.

[0098] On the other hand, the method of the present invention allows the obtaining of Ru(II) complexes with a reaction time much shorter than that required by the route according to Turro et al. (cit.).

[0099] The present invention provides a simple method for preparing a class of complexes [Ru(NN)(dppn)2] n+ (n = 0, 2) in relatively high yields, which relies on the preliminary preparation of the reactive polymeric compound [Ru(CO)2Cl2] m (I), first reacting this reactive polymeric compound with a functionalized 2,2'-bipyridine unit to obtain the monomer trans-Cl[Ru(NN)Cl2(CO)2] (II), and then allowing this monomer to react with two equivalents of the dppn unit to obtain compound III. This method makes it possible to overcome the solubility problems caused by the use of the intermediate [Ru(dppn)2Cl2] and allows the obtaining of various substituted III compounds in good yields.

[0100] Furthermore, using the synthetic route described herein, the reaction intermediates I and II can be used without purification, which shortens the total method time, reduces the amount of solvent required and increases the yield. Only compound III needs to be purified before it can be processed for characterization and further use.

[0101] The R-functional groups of the remaining bidentate NN ligand can be optimally selected to fine-tune the chemo-physical properties of the resulting Ru(II) complex.

[0102] Preferably, the 2,2'-bipyridine ligand is functionalized with R substituents at the 5,5' and 4,4' positions. In other words, each of the R substituent groups is bonded to a carbon atom at a position between the 5,5' position and the 4,4' position of each pyridine ring selected from the 2,2'-bipyridine group

[0103]

[0104] The above-mentioned positions are indeed those which make it possible to obtain compounds which are stable upon irradiation (since they do not have such unfavourable conditions as steric hindrance) and which at the same time do not cause complications in the synthesis of the complexes.

[0105] In particular, the 2,2'-bipyridine ligand is symmetrically modified with two R-substituents at the 5,5' or 4,4' position. In other words, the substituent groups R are bonded to carbon atoms at the symmetrical 5,5' or 4,4' positions of each pyridine ring of the 2,2'-bipyridine group. This allows the product to be obtained as a mixture of enantiomers, avoiding the formation of structural isomers.

[0106] According to another aspect of the present invention, there is also provided a photosensitizing drug for use in photodynamic therapy for treating tumors, the drug containing a ruthenium (II) coordination compound having the following formula as a photosensitizer:

[0107]

[0108] Where n is selected between 0 and 2,

[0109] wherein R is a substituent group selected from the group consisting of:

[0110] -hydrogen,

[0111] - methyl group,

[0112] -phenyl group,

[0113] --CH2NH2 group,

[0114] --CH2OH group

[0115] -carboxyl group -COOH,

[0116] - an ester group of formula -COOR1, wherein R1 may be selected from the group consisting of:

[0117] - a linear or branched alkyl group having a number of carbon atoms ranging from 1 to 4;

[0118] - unsubstituted benzyl groups;

[0119] -With the formula H(OCH2CH2) 1-4 OH-containing polyethylene glycol methoxy groups.

[0120] - an amide group of the formula -CONR2R3,

[0121] in

[0122] - R2 may be hydrogen and R3 may be a group selected from the group consisting of:

[0123] - a straight-chain or branched alkyl group having 1 to 4 carbon atoms;

[0124] -- the (CH2)2NH2 group;

[0125] - a phenyl group,

[0126] or

[0127] - R2 and R3 may be a straight-chain or branched alkyl group having 1 to 4 carbon atoms.

[0128] or

[0129] - NR2R3 may be a group selected from the group consisting of:

[0130] - a cycloalkyl secondary amine having 4 to 5 carbon atoms;

[0131] - a heterocyclic alkyl secondary amine containing a heteroatom selected from between nitrogen and oxygen.

[0132] Wherein the coordination compound is incorporated into nanoparticles, in particular, it is incorporated into liquid crystal lamellar lipid nanoparticles and / or non-lamellar liquid crystal lipid nanoparticles. The effect of this incorporation is to improve the water solubility of the coordination compound and thus allow its administration without using any co-solvent.

[0133] In other words, the present invention relates to various polypyridyl ruthenium(II) complexes, hereinafter also referred to as [Ru(NN)(dppn)2] n+ , wherein, in each of said complexes,

[0134] - dppn is a benzo[i]dipyrido[3,2-a:2’,3’-c]phenazine unit, i.e., a bidentate heterocyclic ligand characterized by extensive aromaticity, and

[0135] - NN is a functionalized 2,2’-bipyridine ligand, wherein the R group is as described above.

[0136] This allows one or more of the following therapeutic advantages:

[0137] a) More uniform and / or more selective drug distribution at the tumor site;

[0138] b) Crossing epithelial and organ barriers, depending on the administration route. For example, such barriers can be the stratum corneum of the skin, the blood-brain barrier, the esophageal or intestinal mucosa, etc.;

[0139] c) Regulating the release and half-life in circulation;

[0140] d) Reducing the toxicity at non-target sites.

[0141] Preferably, the liquid crystalline lipid nanoparticles are cubic liquid crystalline nanoparticles (cubosomes).

[0142] Cubic liquid crystalline nanoparticles (commonly referred to as "bicontinuous cubic liquid crystalline nanoparticles") are special nanoparticles for delivering lipophilic drugs and / or hydrophilic drugs. They are characterized by a three-dimensional honeycomb arrangement of lipid bilayers with internal aqueous channels and can encapsulate many bioactive components such as pharmacological reagents, proteins, and amino acids. Generally, cubic liquid crystalline nanoparticles exhibit several benefits over other nanoparticles (especially liposomes, which are regarded as the gold standard in nanomedicine):

[0143] - Stability: From a colloidal perspective, cubic liquid crystalline nanoparticles exhibit greater resistance to rupture, which is a crucial property for drug delivery as it increases the likelihood of the drug reaching the target location without deteriorating or losing its effectiveness;

[0144] - Increased drug loading: Cubic liquid crystalline nanoparticles exhibit a larger bilayer area to particle volume ratio, and they display a large internal surface area that allows for carrying a large pharmacological payload;

[0145] - Improved bioavailability: By overcoming solubility and stability deficiencies, cubic liquid crystalline nanoparticles can increase drug bioavailability, which allows for lower drug doses and thus reduces the likelihood of adverse effects.

[0146] As a result of the above, cubic liquid crystalline nanoparticles show higher encapsulation efficiency and more effective pharmacological effects both in vitro and in vivo compared to, for example, liposomes.

[0147] Similarly, when compared to solid lipid nanoparticles (SLNs) loaded with the same drug, cubic liquid crystalline nanoparticles show higher in vitro cytotoxicity, cell uptake, and tumor growth inhibition. In addition, when applied to the skin, cubic liquid crystalline nanoparticles show higher drug permeability compared to liposomes, transfersomes, and ethosomes.

[0148] As an alternative, the liquid crystalline lipid nanoparticles can be selected from the group consisting of:

[0149] - Hexagonal liquid crystalline nanoparticles (hexosomes);

[0150] - Solid lipid nanoparticles;

[0151] - Nanoemulsions;

[0152] - Combinations of the above.

[0153] As another alternative, the liquid crystalline lipid nanoparticles can also be liposomes.

[0154] Also falling within the scope of the present invention is a photodynamic therapy method for treating tumors, the method comprising the steps of administering to a patient a medicament containing the above-described nanoformulated coordination compound as a photosensitizer, and applying light to a tumor region to initiate singlet oxygen generation and induce site-specific cytotoxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0155] With reference to the accompanying drawings, further features and advantages of the present invention will be better understood from the following description of its variations and embodiments, given by way of example and not limitation, in which

[0156] - Figure 1 shows the 1 1H-NMR spectrum of complex IIIa in (CD3)2CO);

[0157] - Figure 2 shows the 1 1H 1 H-COSY spectrum of complex IIIa in (CD3)2CO);

[0158] - Figure 3 shows the 13 13C-NMR spectrum of complex IIIa in (CD3)2CO;

[0159] - Figure 4 shows the HSQC spectrum of complex IIIa in (CD3)2CO);

[0160] - Figure 5 shows the HR MS spectrum of complex IIIa;

[0161] - Figure 6 shows the 1 1H-NMR spectrum of complex IIIb in (CD3)2SO;

[0162] - Figure 7 shows the 1 1H 1 H-COSY spectrum of complex IIIb in (CD3)2SO;

[0163] - Figure 8 shows the HR MS spectrum of complex IIIb;

[0164] - Figure 9 shows the 1 1H-NMR spectrum of complex IIIc in (CD3)2CO;

[0165] - Figure 10 shows the 1 1H 1H-COSY spectrum;

[0166] - Figure 11 shows the 13 13C NMR spectrum of complex IIIc in (CD3)2CO;

[0167] - Figure 12 shows the HSQC spectrum of complex IIIc in (CD3)2CO;

[0168] - Figure 13 shows the HR MS spectrum of complex IIIc;

[0169] - Figure 14 shows the absorption spectra of complexes IIIa, IIIb and IIIc in CH3CN;

[0170] - Figure 15 shows the absorption spectra of solutions containing DHN (330 μM) and complexes IIIa, IIIb and IIIc (10 μM) in acetonitrile irradiated with a 30 W LED lamp with an emission spectral width in the range of 400 nm to 500 nm for different irradiation times (up to 200 s);

[0171] - Figure 16 shows the semi-logarithmic plots of In(A t / A0) versus irradiation time for complexes IIIa, IIIb and IIIc and [Ru(phen)3]Cl2, the latter being used as a reference ([DHN] = 330 μM, [Ru] = 10 μM);

[0172] - Figure 17 shows the absorption spectra of complexes IIIa, IIIb and IIIc (10 μM) in a buffered 10 mM Tris-HCl, 50 mM NaCl solution (pH 7.2), the spectra being detected in the presence of increasing concentrations of double-stranded DNA;

[0173] - Figure 18 shows the plot of [DNA] / |ε a -ε b | values versus DNA molar concentration for complexes IIIa, IIIb and IIIc (Ru = 10 μM, Tris-HCl buffer 10 mM, NaCl 50 mM, pH 7.2);

[0174] - Figure 19 shows the results of the cytotoxicity tests of different concentrations of complex IIIa on epidermoid carcinoma (A431) and glioblastoma (U87MG) models after irradiation with LED light for 30 minutes and in the dark;

[0175] -Figure 20 Show the results of cytotoxicity tests of complex IIIb at different concentrations on an epidermal squamous cell carcinoma model (A431) after 30 minutes of irradiation with LED light and in the dark;

[0176] - Figure 21 Show the results of cytotoxicity tests of complex IIIb encapsulated in cubic liquid crystal nanoparticles at different concentrations on an epidermal squamous cell carcinoma model (A431) after 30 minutes of irradiation with LED light and in the dark;

[0177] - Figure 22 Show the results of cytotoxicity tests of complex IIIc at different concentrations on a metastatic lung adenocarcinoma (Calu-3) after 30 minutes of irradiation with LED light and in the dark;

[0178] - Figure 23 Show the results of cytotoxicity tests of complex IIIc encapsulated in cubic liquid crystal nanoparticles at different concentrations on a metastatic lung adenocarcinoma (Calu-3) after 30 minutes of irradiation with LED light and in the dark;

[0179] - Figure 24 Show transmission electron microscopy images of cubic liquid crystal nanoparticles loaded with complex IIIb at low temperature;

[0180] - Figure 25 Show the SAXS diffraction pattern of cubic liquid crystal nanoparticles loaded with complex IIIb, where the Bragg peaks corresponding to the bicontinuous cubic phases Im3m and Pn3m are indicated by dashed vertical lines and solid vertical lines, respectively;

[0181] - Figure 26 Show the results of stability tests of cubic liquid crystal nanoparticle formulations. Examples

[0182] Three examples of Ru(II) complexes containing two benzo[i]dipyrido[3,2-a:2’,3’-c]phenazine (dppn) units are described below. These exemplary complexes can be represented by the following general formula:

[0183]

[0184] And are designated as IIIa, IIIb, and IIIc, respectively, depending on whether R is a methyl group, a carboxylate group, or a morpholinocarbonyl group. Thus, complexes IIIa, IIIb, and IIIc have the following formulas, where the charge n+ is omitted:

[0185]

[0186] According to the method of the present invention, the synthetic route for obtaining the above complex is schematically shown below, including steps a), b) and c), respectively producing intermediates I and II and the desired Ru(II) complex (III):

[0187]

[0188] Even in this case, for simplicity, the charge of complex III is omitted.

[0189] Synthesis

[0190] Step (a): Preparation of polymer precursor "I"

[0191] In all three cases, this common intermediate is prepared by refluxing commercial ruthenium(III) chloride RuCl3·xH2O and paraformaldehyde in a 1.4:1 w / w ratio in 90% formic acid for 6 hours. The resulting polymer I, which is a light yellow powder, is isolated from hexane by trituration. The polymer I precursor is obtained in 80% yield.

[0192] (b) Preparation of trans-Cl[Ru(NN)Cl2(CO)2] intermediate "II"

[0193] The NN ligands corresponding to complexes IIIa, IIIb and IIIc are inserted into the coordination sphere of the Ru(II) center, yielding the corresponding intermediate complex II trans-Cl[Ru(NN)Cl2(CO)2]. Depending on the solubility of the specific NN ligand used, the polymer precursor I is reacted with the NN ligand in a 1:1 molar ratio in hot methanol or dimethylformamide. The intermediate complex II is obtained by simple hot filtration of the reaction mixture, with a high yield between 55% and 75%.

[0194] (c) Preparation of complexes "IIIa - IIIc" from the corresponding intermediate II

[0195] In the third step of the synthetic routes of complexes IIIa, IIIb and IIIc, in each case, two equivalents of dppn are allowed to react with the corresponding precursor II in refluxing 2-methoxyethanol as the solvent in the presence of 5 equivalents of trimethylamine N-oxide, resulting in the formation of the complex III of the formula [Ru(NN)(dppn)2] n+ (n = 0, 2). The complex is then precipitated by adding a 0.1 M solution of KPF6, filtered under vacuum and collected in high purity. If necessary, the complex is subsequently purified by flash chromatography on silica gel using a gradient mixture of dichloromethane and methanol as the eluent. Thus, the desired product, as the hexafluorophosphate [Ru(NN)(dppn)2](PF6)2, is obtained in a yield range between 50% and 78%.

[0196] The following gives a detailed description of step (c) for synthesizing each of the complexes IIIa, IIIb, and IIIc.

[0197] [Ru(4,4'-dimethyl-2,2'-bipyridine)(benzo[i]dipyrido[3,2-a:2',3'-c]phenazine)2] [PF6]2 (IIIa) 。

[0198] To a solution of trans-Cl[Ru(4,4'-dimethyl-2,2'-bipyridine)Cl2(CO)2] (80 mg, 0.19 mmol) in 8 mL of degassed 2-methoxyethanol was added benzo[i]dipyrido[3,2-a:2',3'-c]phenazine (dppn) (129 mg, 0.39 mmol) and trimethylamine N-oxide (106 mg, 0.95 mmol). The reaction mixture was stirred under reflux in a nitrogen atmosphere for 4 h. After cooling to room temperature, the addition of 3 mL of 0.1 M KPF6 solution allowed for the complete precipitation of IIIa. The precipitate was filtered and washed with water and chloroform. The crude product was purified by flash column chromatography on silica gel (eluent: starting from DCM:MeOH 50:1 with 10% acetone to DCM:MeOH 30:1 with 10% acetone) to afford IIIa as a red powder in 78% yield.

[0199] [Ru(4,4'-dicarboxylic acid-2,2'-bipyridine)(benzo[i]dipyrido[3,2-a:2',3'-c]phenazine)2] (IIIb)

[0200] To a solution of trans-Cl[Ru(4,4'-dicarboxylic acid-2,2'-bipyridine)Cl2(CO)2] (80 mg, 0.17 mmol) in 8 mL of degassed 2-methoxyethanol was added benzo[i]dipyrido[3,2-a:2',3'-c]phenazine (dppn) (113 mg, 0.34 mmol) and trimethylamine N-oxide (95 mg, 0.85 mmol). The reaction mixture was stirred under reflux in a nitrogen atmosphere for 6 h. After cooling to room temperature, the crude product was filtered and washed with water, and then triturated with CHCl3 to afford (IIIb) as a red powder in 50% yield.

[0201] [Ru(4,4'-(diylbis(morpholinocarbonyl))-2,2'-bipyridine)(benzo[i]dipyrido[3,2-a:2', 3'-c]phenazine)2][PF6]2 (IIIc)

[0202] To a solution of trans-Cl[Ru(4,4’-(diylbis(morpholinocarbonyl)-2,2’-bipyridine)Cl2(CO)2] (100 mg, 0.16 mmol) in 8 mL of degassed 2-methoxy-ethanol was added benzo[i]dipyrido[3,2-a:2’,3’-c]phenazine (dppn) (106 mg, 0.32 mmol) and trimethylamine N-oxide (116 mg, 1.04 mmol). The reaction mixture was stirred under N2 atmosphere at reflux for 4 h. After cooling to room temperature, 2 mL of 0.1 M aqueous KPF6 was added to allow complete precipitation of complex I He. The crude product was filtered and washed with water, methanol, and diethyl ether. Subsequently, it was purified by flash chromatography on silica gel (eluent: starting with DCM:MeOH 30:1 with 10% acetone) to afford complex IIIc as a red solid in 62% yield.

[0203] The three-step synthetic method gives the final product in a yield range of 22% to 47%. The pure product was obtained and characterized by its nuclear magnetic resonance 1 1H, 1 1H 1 1H COSY, 13 13C NMR and HR MS.

[0204] Characterization of the synthesized complexes: NMR

[0205] The NMR characterization data of the three complexes IIIa - IIIc synthesized as described above are shown below. The NMR characterization spectra of the three complexes are shown in Figures 1 - 13 . The NMR spectra were recorded using a Bruker Advance 400 MHz.

[0206] Complex IIIa

[0207] 1 H-NMR (400 MHz, (CD3)2CO): δ 9.83 (d, J c-b = 8.0 Hz, 2H, Hc), 9.73 (d, J c’-b ’ = 8.0 Hz, 2H, Hc’), 9.23 (s, 2H, Hd), 9.20 (s, 2H, Hd’), 8.81 (s, 2H, H3 / H3’), 8.67 (d, J = 4.0 Hz, 2H, Ha), 8.62 (d, J a-b = 4.0, 2H, Ha’), 8.49 - 8.44 (m, 4H, He / He’), 8.18 (dd, J 1 b-c = 8.0 Hz J 2 b-a = 4.0 Hz, 2H, Hb), 8.12 (d, JH2-H3 = 4.0 Hz, 2H, H6 / H6’), 7.95 (dd, J 1 b’-c’ = 8.0 Hz J 2 b’-a’ = 4.0 Hz, 2H, Hb’), 7.87 - 7.80 (m, 4H, Hf / Hf’), 7.38 (d, J H3-H2 = 4.0 Hz, 2H, H5 / H5’), 2.61 (s, 6H, -CH3) ppm。

[0208] 13 C-NMR (100 MHz, (CD3)2CO): δ 157.59, 155.08, 154.71, 152.34, 152.26, 152.13, 151.45, 141.51, 139.11, 135.80, 134.31, 134.23, 131.85, 131.77, 129.27, 129.19, 128.85, 128.80, 128.67, 128.45, 128.27, 125.89, 20.91 ppm。

[0209] HR-MS (ESI+) m / z: For C 56 H 36 N 10 Ru[M - 2PF6 - ] 2+ the calculated value 475.10791, found: 475.10791。

[0210] Analysis, for C 56 H 36 F 12 N 10 Calculated for P2Ru: C 54.24, N 11.30, H 2.93; found C 54.00, N 10.53, H 3.24

[0211] Complex IIIb

[0212] 1 H-NMR (400 MHz, DMSO): δ 9.62 (d, J c-b = 8.0 Hz Hz, 2H, Hc), 9.56 (d, J c’-b ’ = 8.0 Hz, 2H, Hc’), 9.27 (s, 2H, Hd), 9.22 (s, 2H, Hd’), 8.87 (s, 2H, H3 / H3’), 8.50 - 8.40 (m, 2H, Hf and Hf’), 8.35 (d, J a-b = 8.0 J a-b= 4.8 Hz, 2H, Ha), 8.31 (d, J a’-b’ = 4.8 2H, Ha’), 8.08 (dd, 2H, Hb), 7.93 - 7.84 (m, 4H, Hb’ and H6 / H6’), 7.82 - 7.75 (m, 4H, He and He’), 7.68 (d, J = 5.2 Hz, 2H, H5 / H5’) ppm.

[0213] Low solubility (0.5 mM in DMSO) prevented the 13 recording of 13C-NMR.

[0214] HR-MS (ESI+) m / z: For C 56 H 32 N 10 O4RU [M - 2PF6’] 2+ calculated value 505.08188, found: 505.08177.

[0215] Analysis, for C 56 H 30 N 10 Calculated values for C66.73, N 13.90, H 3.00; found C 62.56, N 12.62, H 3.89.

[0216] Complex IIIc

[0217] 1 H-NMR (400 MHz, (CD3)2CO): δ 9.83 (d, J c-b = 8 Hz, 2H, Hc), 9.74 (d, J c’-b’ = 8 Hz, 2H, Hc’), 9.22 (s, 2H, Hd), 9.18 (s, 2H, Hd’), 8.98 (s, 2H, H3 / H3’), 8.76 (d, J a-b = 4 Hz, 2H, Ha), 8.62 (d, J a’-b’ = 4 Hz, 2H, Ha’), 8.48 - 8.40 (m, 6H, H6 / H6’, He / He’), 8.15 (dd, J 1 b-c = 4 Hz, J 2 b-a = 8 Hz, 2H, Hb), 7.97 (dd, J 1 b’-c’ = 4 29 Hz, J 2 b’-a ’ = 8 Hz, 2H, Hb’), 7.85 - 7.79 (m, 4H, Hf / Hf’), 7.53 (d, JH3-H2 = 4 Hz, 2H, H5 / H5'), 3.71 (bs, 8H, -CH2 morpholine), 3.60 (bs, 4H, -CH2 morpholine), 3.50 (bs, 4H, -CH2 morpholine) ppm.

[0218] 13 C-NMR (100 MHz (CD3)2CO) δ 165.4; 157.9; 154.8; 154.4; 153.1; 151.4; 145.4; 140.9; 140.9; 138.6; 135.3; 134.1; 134.1; 131.4; 131.3; 128.7; 128.3; 128.2; 128.1; 128.1; 127.7; 125.4; 122.9 ppm.

[0219] HRMS (ESI+) m / z: For C 64 H 46 N 12 O4RU[M - 2PF6 - ] 2+ Calculated value 574.13987, found: 574.13897.

[0220] Analysis, for C 64 H 46 N 12 F 12 Calculated values for C53.45, N 11.69, H 3.22; found C 53.28, N 11.04, H 3.42.

[0221] Characterization of the synthesized complexes: UV-Vis absorption spectra

[0222] As shown in Table 1, the complex of formula III shows a broad 1 MLCT transition centered between 440 nm and 450 nm in acetonitrile, and a diagnostic 1 TT - TT* transition of the aromatic part of the ligand centered between 408 nm and 320 nm. Figure 14 The absorption spectra of complexes IIIa - c are shown graphically. All recorded absorption spectra shown in this invention were recorded using a PerkinElmer Lambda 6 spectrophotometer.

[0223] Determination of singlet oxygen quantum yield

[0224] Complexes IIIa - c are effective in generating singlet oxygen ( 1The ability of (O2) is a key factor in evaluating their potential for use as photosensitizers in photodynamic therapy. In this regard, the singlet oxygen quantum yields of complexes IIIa-c were generated by irradiating acetonitrile solutions of the complexes at 1270 nm 1 in the manner of the direct phosphorescence signal of O2, and determined using dichloro(1,10-phenanthroline)ruthenium(II) ([Ru(phen)3Cl2]) as a reference standard. These measurements were carried out using a Horiba FluoroMax Plus spectrofluorometer. The values are shown in Table 1.

[0225] - Table 1 -

[0226] Chemical-physical characterization of ruthenium complexes IIIa-c: by (a) direct measurement of the phosphorescence signal of 1 O2 at 1270 nm and (b) Maximum absorption, molar absorption coefficient, and singlet oxygen quantum yield obtained by indirect UV-visible measurement with DHN probe 。

[0227]

[0228] Using 1,5-dihydroxynaphthalene (DHN) as 1 an indirect chemical probe for O2 and [Ru(phen)3]Cl2 as a commercial standard, confirmation was obtained that singlet oxygen ([[]] 1 O2) was generated with such high quantum yields by three IIIa-c molecular systems. In the DHN assay, in the presence of 1 O2, the probe was selectively and quantitatively oxidized to give the corresponding photooxidation product 5-hydroxy-1,4-naphthoquinone (juglone). The generation of 1 O2 was evaluated by monitoring the decrease in the absorption band of DHN at λ = 297 nm and the corresponding increase in the juglone band at λ = 427 nm. Figure 15 Shown are UV-Vis titrations obtained for IIIa-c in acetonitrile with increasing irradiation times using an LED lamp with an emission spectrum between 400 and 500 nm. As Figure 15 shown, irradiation of IIIa-c resulted in a strong decrease in the absorption of DHN and a simultaneous increase in the absorption band of juglone. By plotting the semi-logarithmic graph of In(A t / A0) versus the irradiation time studied, the rate constant (k obs ) of the photooxidation process was obtained, as Figure 16 shown, and compared with the rate constant of the photooxidation process found for [Ru(phen)3]Cl2 under the same experimental conditions. On this basis, 1 the quantum yield of the formation of O2 can be obtained via the following equation

[0229]

[0230] where ΦΔ is from the photosensitizer 1The quantum yield of O2 production, k is the rate constant obtained from DHN measurements, I abs represents the absolute value of the integral of the absorption of the photosensitizer in the spectral emission region (400 nm to 500 nm) of the LED lamp, and the subscript ST represents the commercial standard [Ru(phen)3]Cl2.

[0231] Considering that through [Ru(phen)3]Cl2 1 The standard quantum yield of O2 production is 0.38, and the k obtained for complex IIIa obs is 1.85x 10 -3 , the k obtained for complex IIIb obs is 2.71x 10 -3 , the k obtained for complex IIIc obs is 1.83x 10 -3 and the k obtained for the reference obs is 9.41x 10 -4 ( Figure 16 ), the quantum yields of O2 production for complexes IIIa-c are 0.44, 0.47 and 0.42 respectively, which are consistent with the quantum yields obtained by directly measuring the phosphorescence signal (Table 1). 1 The ability of complexes IIIa-c to bind DNA was evaluated on calf thymus DNA (ct-DNA), monitoring the changes in the UV-Vis spectrum of the 322 nm band when titrating a fixed concentration (10 μM) of the complex in a buffer solution (10 mM Tris-HCl, 50 mM NaCl, pH 7.2) with increasing amounts of the biopolymer. At each addition, the sample was incubated at room temperature for 5 minutes, and the UV-Vis spectrum was recorded using a Tris-HCl buffer solution containing an equal amount of ct-DNA as a blank (to eliminate the inherent contribution of ct-DNA itself to the absorbance). The binding constants found for complexes IIIa-c are shown in Table 2.

[0232] Interaction with calf thymus DNA

[0233]

[0234] - Table 2 -

[0235] The binding constant (K b ) of the ruthenium complex synthesized by the present invention with ct-DNA

[0236] Kb(ct-DNA) IIIa 7,49x 105 IIIb 2,34x 106 IIIc 8,75x 105

[0237] Figure 17 As Figure 17As shown, the successive addition of ct-DNA (0 μM to 3.4 μM) results in a hypochromic effect on the entire absorption spectrum of complexes IIIa-c. The intrinsic binding constant K of complexes IIIa-c with ct-DNA was determined using the following equation b

[0238]

[0239] where [DNA] is the concentration of ct-DNA in base pairs, and the apparent absorption coefficient ε a 、ε f and ε b are the molar extinction coefficients of A obs / [Ru], the unbound complex, and the Ru complex in the fully DNA-bound form, respectively. The binding constant (K b ) was obtained as the ratio of the slope and intercept of the linear regression plotted of [DNA] / |ε a –ε f / against [DNA], as Figure 18 shown.

[0240] Activity of compound IIIa

[0241] The activity of compound IIIa as a photosensitizer in photodynamic therapy was evaluated on two in vitro tumor models: squamous cell carcinoma of the epidermis (A431) and glioblastoma multiforme (U87MG). Cells cultured in 96-well plates were treated with increasing concentrations of IIIa dispersed in the medium for one hour. At the end of the incubation, the medium was changed, and the cells were exposed to LED light (λ max 462 nm) for 30 minutes, or kept in the dark for the same amount of time. At the end of 30 minutes, the cells were returned and kept in the incubator for 24 hours, and then the metabolic activity was measured by MTT assay. Six replicates were used for each condition, and 100% cell viability was calculated from the response of untreated cells not exposed to LED light. On both models, compound IIIa showed high potency and specificity. In particular, even at nanomolar concentrations, compound IIIa induced extensive cell death after LED irradiation, while showing almost no toxicity when not photoactivated, as Figure 19 shown. The high specificity of IIIa (defined as toxicity only when exposed to light) was evident from the difference in IC 50 values, which indicated potent cytotoxic activity in the presence of light irradiation and significant inertness in the dark at the tested concentrations, as shown in Table 3. This indicates that compound IIIa has great potential as a photosensitizer in photodynamic therapy for treating various types of tumors.

[0242] - Table 3 –

[0243] From compound IIIa Average IC obtained from in vitro cytotoxicity experiments on tumor models 50 value

[0244]

[0245] Activity of compound IIIb

[0246] According to the same protocol as for compound IIIa, the activity of compound IIIb as a photosensitizer in photodynamic therapy was evaluated on an in vitro tumor model, namely squamous cell carcinoma of the epidermis (A431). Compound IIIb showed high potency and specificity. In particular, even at nanomolar concentrations, IIIb induced extensive cell death after LED irradiation, while showing almost no toxicity when not photoactivated, as Figure 20 shown. The high specificity of IIIb (defined as toxicity only when exposed to light) was evident from the difference in IC 50 values, which indicated potent cytotoxic activity in the presence of light irradiation and significant inertness in the dark at the tested concentrations, see Table 4. This indicates that compound IIIb has great potential as a photosensitizer in photodynamic therapy for treating various types of tumors.

[0247] - Table 4 -

[0248] From compound IIIb Average IC obtained from in vitro cytotoxicity experiments on tumor models 50 value

[0249]

[0250] Formulation of cubic liquid crystal nanoparticles loaded with complex IIIb: Chemical-physical characterization

[0251] A formulation of cubic liquid crystal nanoparticles loaded with complex IIIb was prepared using glyceryl monooleate, i.e., glycerol monooctanoate (monoolein, 1-monooleoyl glycerol, RYLO MG 19PHARMA, 98.1 wt.%, supplied by Danisco A / S) and Pluronic F108 (PEO132-PPO50-PEO132, purchased from Sigma Aldrich) as stabilizers. Fresh distilled water purified with a Milli-Q system (Millipore) was used to prepare each sample, and it was filtered through a hydrophilic filter with a pore size of 0.22 μm before using it to prepare each sample.

[0252] Cubosomes were prepared by melting glyceryl monooleate at 40 °C and dispersing the melted glyceryl monooleate by means of an ultrasonic bath. Then, an appropriate amount of aqueous solution of Pluronic F108 stabilizer was added to the lipid phase, and the mixture was subjected to sonication (90% amplitude; 1 s on, 1 s off) with a UP100H ultrasonic processor developed by Hiescher for cycles of 5 min, 4 min, 3 min, 2 min and 1 min.

[0253] The average hydrodynamic diameter and polydispersity index (as a measure of the particle size distribution width) were determined by dynamic light scattering using a Nano Zetasizer (Malvern Instrument). Backscattering of the sample was performed at an angle of 173° using a helium-neon laser (633 nm) at a constant temperature of 25 °C. The ζ-potential was estimated using the M3-PALS (phase analysis light scattering) technique of the Nano Zetasizer.

[0254] Due to the presence of the ruthenium complex, the macroscopic appearance of the resulting sample was that of a fluid, opaque, pale orange aqueous dispersion. The study of cubosomes loaded with complex IIIb was initiated by evaluating the encapsulation efficiency of the ruthenium complex. For this purpose, the formulation was dialyzed against 2 L of water for 2 h through a 14 kDa molecular weight cut-off tubular cellulose membrane (Sigma Aldrich), changing the water after one hour at room temperature to separate the cubosomes loaded with complex IIIb from the free complex IIIb. Then, after the formulation was disintegrated in methanol (in which all components are soluble), the encapsulation / entrapment efficiency was evaluated at 325 nm by UV-visible spectroscopy using a Synergy 4 multi-well plate reader (BioTek, Winooski, USA). Specifically, this efficiency was calculated by the following expression:

[0255]

[0256] The final concentration of IIIb was 0.02% (w / w) and the encapsulation efficiency value was 60 ± 5%. Thus, the final composition of the cubosomes loaded with complex IIIb was

[0257] Glyceryl monooleate / Pluronic F108 / Complex IIIb / Water = 3.30 / 0.03 / 0.02 / 96.65% (w / w).

[0258] Prior to any measurements by dynamic light scattering, the samples were visually inspected to check for the absence of aggregates or phase separation. Additionally, the colloidal system was characterized by an average diameter of the nanoparticles of 142 ± 1 nm, a polydispersity index of 0.13 ± 0.01, and a ζ-potential of -30 ± 2 mV.

[0259] The morphology of cubic liquid crystal nanoparticles loaded with IIIb was revealed by cryogenic transmission electron microscopy (Cryo-TEM). As Figure 24 shown, the cubic liquid crystal nanoparticles appeared as spherical nanoparticles of different sizes, and their internal structure was characterized by a dark matrix and alternating bright spots, representing lipid bilayers and water channels, respectively.

[0260] The internal nanostructure of cubic liquid crystal nanoparticles loaded with complex IIIb was evaluated by small-angle X-ray scattering (SAXS) experiments. In particular, Figure 25 the recorded SAXS diffraction pattern of cubic liquid crystal nanoparticles loaded with complex IIIb, as shown in and strongly indicated the simultaneous presence of two bicontinuous cubic phases, Pn3m and Im3m, characterized by lattice parameters of and

[0261] Information on the stability of the formulation was obtained by monitoring the mean diameter, polydispersity index PDI, and ζ potential over a 30-day period, as Figure 26 shown in A. In addition, the chemical stability of complex IIIb encapsulated in cubic liquid crystal nanoparticles was evaluated by UV-visible spectroscopy of the formulation, as Figure 26 shown in B. The study of the particle size distribution revealed the optimal stability of the formulation, as Figure 26 shown.

[0262] Indeed, during storage at 25 °C for 30 days, the mean diameter did not change significantly, showing a value of approximately 140 nm throughout the study. The polydispersity index remained almost constant and was below 0.15, which confirmed the retention of a fairly narrow size distribution during storage. In addition, the value of the ζ potential did not change, with a recorded value of approximately -30 mV. Furthermore, UV-visible analysis revealed a decrease in the concentration of complex IIIb between day 0 and day 7. However, from day 7 until the end of the study, the concentration of complex IIIb remained constant.

[0263] Activity of compound IIIb encapsulated in cubic liquid crystal nanoparticles

[0264] Therefore, according to the same protocol as for compounds IIIa and IIIb, the activity of compound IIIb encapsulated in cubic liquid crystal nanoparticles as a photosensitizer in photodynamic therapy was evaluated on an in vitro tumor model (squamous cell carcinoma of the epidermis (A431)). Even at nanomolar concentrations, compound IIIb encapsulated in cubic liquid crystal nanoparticles induced extensive cell death after LED irradiation, while showing almost no toxicity when not photoactivated, as Figure 21As shown. Encapsulation in cubic liquid crystal nanoparticles results in a decrease in the IC 50 value compared to IIIb as a free compound (Tables 4 and 5). However, incorporation into a delivery system can confer many benefits, including increased distribution in tumors after in vivo administration, increased stability and solubility, and improved crossing of biological barriers.

[0265] - Table 5 -

[0266] From in vitro cytotoxicity experiments of compound IIIb encapsulated in cubic liquid crystal nanoparticles on tumor models Obtained average IC 50 Value

[0267]

[0268] In addition, it should be noted that the above-mentioned drug represents the first example of a Ru(II) polypyridyl complex encapsulated in cubic liquid crystal nanoparticles reported in the literature to date.

[0269] The foregoing description of the embodiments of the present invention and its examples can conceptually illustrate the present invention. Others using known techniques will be able to modify and / or adjust these embodiments in this way in various applications without further research and without departing from the inventive concept, and thus, it should be understood that such adjustments and modifications will be considered equivalent to the described embodiments. Devices and materials for practicing various functions can be of various types without departing from the scope of the present invention. It should be understood that the expressions or terms used are purely descriptive and thus not restrictive.

[0270] Activity of compound IIIc

[0271] According to the same protocol as for compounds IIIa and IIIb, the activity of compound IIIc as a photosensitizer in photodynamic therapy was evaluated on an in vitro tumor model of metastatic lung adenocarcinoma (Calu-3). Compound IIIc showed high potency and specificity. In particular, even at nanomolar concentrations, IIIc induced extensive cell death after LED irradiation, while showing almost no toxicity when not photoactivated, as Figure 22 shown. The high specificity of IIIc (defined as toxicity only when exposed to light) is evident from the difference in IC50 values, which indicates potent cytotoxic activity in the presence of light irradiation and significant inertness in the dark at the tested concentrations, see Table 6. This indicates that compound IIIc has great potential as a photosensitizer in photodynamic therapy for treating various types of tumors.

[0272] - Table 6 -

[0273] Average IC50 value obtained from in vitro cytotoxicity experiments of compound IIIc on tumor models

[0274]

[0275] Activity of compound IIIc encapsulated in cubic liquid crystal nanoparticles

[0276] According to the same protocol as for compounds IIIa and IIIb, the activity of compound IIIc encapsulated in cubic liquid crystal nanoparticles as a photosensitizer in photodynamic therapy was evaluated on an in vitro tumor model (metastatic lung adenocarcinoma (Calu-3)). Even at nanomolar concentrations, compound IIIc encapsulated in cubic liquid crystal nanoparticles induced extensive cell death after LED irradiation, while showing little toxicity when not photoactivated, as Figure 23 shown. Encapsulation of IIIc in cubic liquid crystal nanoparticles led to a decrease in the IC50 value compared to IIIc as a free compound (Tables 6 and 7). However, incorporation into a delivery system can confer many benefits, including increased distribution in tumors after in vivo administration, increased stability and solubility, and improved crossing of biological barriers.

[0277] - Table 7 -

[0278] From in vitro cytotoxicity experiments of compound IIIc encapsulated in cubic liquid crystal nanoparticles on tumor models Obtained average IC50 value

[0279]

[0280] The foregoing description of the exemplary embodiments and specific examples of the present invention so fully discloses the present invention from a conceptual perspective that others can, by applying current knowledge, modify and / or adapt such embodiments for different applications without further research and without departing from the present invention, and thus it should be understood that such adaptations and modifications should be considered equivalent to the specific embodiments and examples. Therefore, devices and materials performing different functions described herein may have different properties without departing from the scope of the present invention. It should be understood that the wording or terminology used herein is for the purpose of description and not of limitation.

[0281] References

[0282] 1) Wang, L. et al. “TT-Expansive Heteroleptic Ruthenium(II) Complexes as Reverse Saturable Absorbers and Photosensitizers for Photodynamic Therapy” Inorg. Chem. 56, 3245 - 3259 (2017).

[0283] 2) Turro et al., “Excited state dynamics of two new Ru(II)

[0284] cyclometallated dyes: Relation to cells for solar energy conversion and comparison to conventional systems” J. Phys. Chem. C (2012).

Claims

1. A ruthenium(II) coordination compound having the following formula: wherein n is selected from between 0 and 2, wherein R is a substituent group selected from the group consisting of: - a methyl group; - a phenyl group; --CH2NH2 group; --CH2OH group; - a carboxyl group -COOH; - an ester group -COOR1, wherein R1 is selected from the group consisting of: - a straight-chain or branched alkyl group having a carbon atom number in the range of 1 to 4; - an unsubstituted benzyl group; - Polyethylene glycol methoxy group H(OCH2CH2) 1-4 OH; - an amide group -CONR2R3, wherein - R2 is hydrogen and R3 is a group selected from the group consisting of: - a straight-chain or branched alkyl group having a carbon atom number in the range of 1 to 4; --(CH2)2NH2 group; - a phenyl group, or - R2 and R3 are straight-chain or branched alkyl groups having a carbon atom number in the range of 1 to 4, or - NR2R3 is a group selected from the group consisting of: - a cycloalkyl secondary amine having a carbon atom number in the range of 4 to 5; - a heterocyclic alkyl secondary amine containing a heteroatom selected between nitrogen and oxygen, and the coordination compound is used as a photosensitizer in photodynamic therapy for treating tumors.

2. The coordination compound for use according to claim 1, wherein each substituent group R is bonded to a carbon atom at a position between the 5,5'-position and the 4,4'-position of each pyridine ring selected from 2,2'-bipyridine groups:

3. The coordination compound for use according to claim 1, wherein the substituent group R is bonded to a carbon atom at the symmetric 5,5'- or 4,4'-position of the corresponding pyridine ring of the 2,2'-bipyridine group 4. A photosensitizer drug for photodynamic therapy for treating tumors, the photosensitizer drug comprising a ruthenium(II) coordination compound as a photosensitizer, wherein the ruthenium(II) coordination compound has the following formula: wherein n is selected from between 0 and 2, wherein R is a substituent group selected from the group consisting of: - hydrogen; - a methyl group; - a phenyl group; --CH2NH2 group; --CH2OH group; - a carboxyl group -COOH; - an ester group -COOR1, wherein R1 is selected from the group consisting of: - a straight-chain or branched alkyl group having a carbon atom number in the range of 1 to 4; - an unsubstituted benzyl group; - Methoxypolyethylene glycol group H(OCH2CH2) 1-4 OH; - an amide group -CONR2R3, wherein - R2 is hydrogen and R3 is a group selected from the group consisting of: - a straight-chain or branched alkyl group having a carbon atom number in the range of 1 to 4; --(CH2)2NH2 group; - a phenyl group, or - R2 and R3 are straight-chain or branched alkyl groups having a carbon atom number in the range of 1 to 4, or - NR2R3 is a group selected from the group consisting of: - a cycloalkyl secondary amine having a carbon atom number in the range of 4 to 5; - a heterocyclic alkyl secondary amine containing a heteroatom selected between nitrogen and oxygen, wherein the coordination compound is incorporated into liquid crystal lamellar lipid nanoparticles and / or non-lamellar lipid nanoparticles.

5. The medicament for photodynamic therapy according to claim 4, wherein the nanoparticles are cubic liquid crystal nanoparticles.

6. The medicament for photodynamic therapy according to claim 4, wherein the nanoparticles are selected from the group consisting of: - hexagonal liquid crystal nanoparticles; - ethosomes; - transfersomes; - solid lipid nanoparticles; - nanoemulsions; - combinations of the above.

7. The medicament for photodynamic therapy according to claim 4, wherein the nanoparticles are liposomes.

8. A method for obtaining a ruthenium (II) coordination compound having the following formula: where n is selected between 0 and 2, where R is a substituent group selected from the group consisting of: - hydrogen; - methyl group; - phenyl group; --CH2NH2 group; --CH2OH group; - carboxyl group -COOH; - ester group -COOR1, where R1 is selected from the group consisting of: - straight-chain or branched-chain alkyl groups with a carbon atom number in the range of 1 to 4; - unsubstituted benzyl group; - having a polyethylene glycol methoxy group of the formula H(OCH2CH2) 1-4 OH; - amide group -CONR2R3, where - R2 is hydrogen and R3 is a group selected from the group consisting of: - straight-chain or branched-chain alkyl groups with a carbon atom number in the range of 1 to 4; --(CH2)2NH2; - phenyl group, or - R2 and R3 are straight-chain or branched-chain alkyl groups with a carbon atom number in the range of 1 to 4, or - NR2R3 is a group selected from the group consisting of: - cycloalkyl secondary amines with a carbon atom number in the range of 4 to 5; - heterocyclic alkyl secondary amines containing heteroatoms selected between nitrogen and oxygen, The method comprises the following step sequence: - A reactive polymer precursor [Ru(CO)2Cl2] is obtained by reacting ruthenium(III) chloride with paraformaldehyde m ; - obtaining an intermediate complex trans-Cl[Ru(NN)Cl2(CO)2] by: where NN is a 2,2'-bipyridine ligand symmetrically functionalized with two identical functional R groups, React the reactive polymer precursor [Ru(CO)2Cl2] m with a predetermined symmetrically functionalized 2,2'-bipyridyl group bearing the two identical R functional groups, - obtaining the ruthenium (II) coordination compound by reacting the intermediate complex with benzo[i]dipyrido[3,2-a:2',3'-c]phenazine.

9. The method according to claim 8, wherein each substituent group R is bonded to a carbon atom at a position between the 5,5' position and the 4,4' position of each pyridine ring selected from the 2,2'-bipyridine group 10. The method according to claim 8, wherein the substituent group R is bonded to a carbon atom at the symmetric 5,5' or 4,4' position of the corresponding pyridine ring of the 2,2'-bipyridine group

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