ES2367371T3 - USE OF FTALOCIANINE DETERIVED FOR NON-PHOTODYNAMIC TREATMENT OF DISEASES. - Google Patents

Abstract

Use of phthalocyanine derivatives of general formula (I) ** Formula ** in which M is Zn, R is selected from H, groups containing at least one quaternary ammonium substituent, groups comprising at least one aliphatic amino substituent and Suitable groups for covalent binding to bio-organic vehicles chosen from the group consisting of amino acids, polypeptides, proteins, antibodies, polysaccharides and aptamers, R1, same or different from R1 is selected from H, groups comprising at least one aliphatic amino substituent and groups containing at least one quaternary ammonium substituent, R2 and R3, the same or different from each other, are selected from H, alkoxy groups having 1 to 10 carbon atoms, thioalkoxy groups having 1 to 10 carbon atoms , groups comprising at least one aliphatic amino substituent and groups containing at least one quaternary ammonium substituent, with the proviso that: a) at least one between R, R1 R2 and R3 s ea a group comprising at least one aliphatic amino substituent or a group comprising at least one quaternary ammonium substituent and, when R, R1, R2 and R3 are groups comprising at least one aliphatic amino substituent or groups containing at least one quaternary ammonium substituent, or R and R2 are groups comprising at least one aliphatic amino substituent or groups containing at least one quaternary ammonium substituent and R1 and R3 are H, said groups comprising at least one aliphatic amino substituent or containing said groups at least one quaternary ammonium substituent, be the same; b) when R and R1 are both different from H, they are in positions 1,4 or 2,3, while when only one between R and R1 is different from H, it is in position 1 or 2; c) when R2 and R3 are both different from H, they are in positions 8, 11, 15, 18, 22, 25 or 9, 10, 16, 17, 23, 24 while when only one between R2 and R3 is different of H, be in positions 8 (11), 15 (18), 22 (25) or in positions 9 (10), 16 (17), 23 (24); and its pharmaceutically acceptable salts, for the preparation of pharmaceutical compositions for the non-photodynamic treatment of microbial infections and the treatment of diseases characterized by cellular hyperproliferation.

Description

Field of the Invention

[0001] The invention relates to the field of phthalocyanine derivatives and in particular to the novel use of phthalocyanine derivatives of general formula (1), hereinafter given, for the preparation of pharmaceutical compositions useful in the diagnosis and treatment Non-photodynamic infections and various origins and diseases characterized by cellular hyperproliferation.

State of the art

[0002] It has long been known to produce molecules that contain the phthalocyanine chromofluorophore macrocycle through interaction with visible light, reactive oxygen species, such as oxygen radicals or singles, that are cytotoxic to viruses, bacteria, fungi and eukaryotic cells .

[0003] Therefore, also given their agility to preferentially position themselves in pathologically affected areas, phthalocyanine compounds have been used for some time in the so-called photodynamic therapy, commonly known as "PDT" therapy. PDT therapy consists in the administration of said compounds, allowing them to be located in the area to be treated and then irradiating said area with light in the visible region of the spectrum. Examples of compounds used in PDT therapy are described by Ben-Hur E. et al. in Int. J Radiat. Biol., Vol. 47, p. 145-147, 1985. Other photosensitive agents useful in PDT are zinc phthalocyanine complexes and their conjugates described in US Patent No. 5,965,598, in the Applicant's name. These compounds have been shown to be effective photosensitive agents in PDT treatment for both tumors and various infections. However, in many cases the pathologically affected parts involve internal organs, or in no case are easily accessible with light.

[0004] EP 1 164 135 and EP 0 906 758 describe compounds that are within the scope of this general formula for use as a sensitizer in PDT and diagnosis.

[0005] EP 1 356 813 describes a photodynamic synergistic effect between phthalocyanines and chelating compounds such as EDTA.

[0006] JP 09 059279 describes that a family of positively charged phthalocyanines containing quaternary ammonium groups has antimicrobial properties that are not associated with photodynamic activation. The central metal is iron, cobalt, nickel or copper.

[0007] US 5 281 616 describes the antimicrobial activity of a neutral phthalocyanine chelated with SiCl2.

[0008] From this the need arises to identify compounds capable of manifesting their cytotoxic action in the dark without having to radiate the affected area with light.

Summary of the invention

[0009] Unlike the knowledge acquired to date in the state of the art and which has been illustrated above with respect to the therapeutic applications of phthalocyanines, now, the Applicant has discovered that the phthalocyanine derivatives of formula (I) given hereinafter, they are also active in the dark. Obviously, this opens up previously inconceivable possibilities with respect to the diagnostic and therapeutic use of said compounds, which could also be used in the internal regions of the human body that are inaccessible to light.

[0010] Therefore, the present invention provides the use of the phthalocyanine derivatives of the general formula

(one)

image 1

wherein M is selected from 2H and a metal selected from the group consisting of Zn, Si (OR ') 2, Ge (OR') 2 and AlOR ', in which R' is selected from H and alkyl groups having 1 to 15 carbon atoms, R is selected from H, groups containing at least one quaternary ammonium substituent, groups that they comprise at least one aliphatic amino substituent and groups suitable for conjugation with vehicles specific, R1, equal to or different from R, is selected from H, groups comprising at least one aliphatic amino substituent and groups containing at least one quaternary ammonium substituent, R2 and R3, the same or different from each other, are selected from H, alkoxy groups having 1 to 10 carbon atoms, thioalkoxy groups having 1 to 10 carbon atoms, groups comprising at least one amino substituent aliphatic and groups containing at least one quaternary ammonium substituent, with the condition of:

a) at least one of R, R1, R2 and R3 is a group comprising at least one aliphatic amino substituent

or a group comprising at least one quaternary ammonium substituent and, when R, R1, R2 and R3 are groups comprising at least one aliphatic amino substituent or groups containing at least one quaternary ammonium substituent, or R and R2 are groups comprising at least one aliphatic amino substituent or groups containing at least one quaternary ammonium substituent and R1 and R3 are H, said groups comprising at least one aliphatic amino substituent or said groups comprising at least one quaternary ammonium substituent, being the same ; b) when R and R1 are both different from H, they are in positions 1,4 or 2,3, while when only one between R and R1 is different from H, it is in position 1 or 2; c) when R2 and R3 are different from H, they are in positions 8, 11, .15, 18, 22, 25 or 9, 10, 16, 17, 23, 24 while when only one between R2 and R3 is different of H, be in positions 8 (11), 15 (18), 22 (25) or in positions 9 (10), 16 (17), 23 (24);

and its pharmaceutically acceptable salts, for the preparation of pharmaceutical compositions useful in the non-photodynamic treatment of infections microbial and the diagnosis and treatment of diseases characterized by cellular hyperproliferation.

[0011] The features and advantages of the invention will be illustrated in more detail in the following description.

Detailed description of the invention

[0012] According to the invention, the term "group comprising at least one quaternary ammonium substituent or an aliphatic amino substituent" preferably refers to a group (X) pR4, in which X is selected from the group consisting in O, -CH2-, CO, S, SO and -NR5, wherein R5 is selected from H and C1-C15 alkyl; and R4 is where Y is selected from the group consisting of C1-C10 alkyl and phenyl, possibly substituted, or Y forms with the

image 1

5 group Z, to which it is attached, a saturated or unsaturated heterocycle, optionally substituted, which may comprise up to 2 heteroatoms selected from the group consisting of N, O and S; Z is selected from the group consisting of -N, -CH2N and -CONHCH2CH2N; R6 and R7, the same or different from each other, are selected from the group consisting of C1-C15 alkyl and phenyl or form with the group Z, to which they are attached, a saturated or unsaturated heterocycle, optionally substituted, which may

10 comprising up to two heteroatoms selected from the group consisting of N, O and S; R8 and R9, the same or different from each other, are selected from the group consisting of H, C1-C15 alkyl and R10COOEt or R10COOMe group in which R10 is C1-C15 alkyl; m, n, p, w, t and u, independently of each other, are 0 or 1; and v is an integer between 1 and 3, with the proviso that only one between n, w, t and u is simultaneously 0.

[0013] According to the invention, of the groups comprising at least one aliphatic amino substituent, groups selected from those indicated below are preferred: [0014] According to the invention, of the groups containing at least a quaternary ammonium substituent, groups selected from those indicated below are preferred:

image 1

image 1

image 1

image 1

[0015] As the groups comprising at least one aliphatic amino substituent, the following groups are particularly preferred:

image 1

[0016] As the groups containing at least one quaternary ammonium substituent, the following groups are particularly preferred:

image 1

[0017] According to a preferred embodiment of the invention, M is Zn.

[0018] The term "saturated or unsaturated heterocycle" preferably refers to a heterocycle selected from the group consisting of morpholine, piperidine, pyridine, pyrimidine, piperazine, pyrrolidine, pyrroline, imidazole, aniline and julolidine.

[0019] The expression "suitable group for conjugation with specific vehicles" refers to any group

Suitable for covalently binding to bio-organic vehicles chosen from the group comprising amino acids, polypeptides, proteins, antibodies, polysaccharides and aptamers; capable of facilitating the transport of phthalocyanines to particular targets; the expression mentioned above preferably indicates a group selected from the group consisting of -COOH, -SH, -NH2, -CO-CH2-Br, -SO2Cl, maleimide, hydrazine, phenol, imidate, biotin, optionally linked to the phthalocyanine nucleus through a suitable separator (X) pW, in which X and p are

20 defined as above and W is selected from C1-C10 alkyl, aryl and C1-C5 arylalkyl. [0020] When R is a group suitable for conjugation with specific vehicles, as defined above, R1 is preferably equal to H and R2 and R3 are selected from H, groups comprising at least one aliphatic amino substituent and groups containing at least one quaternary ammonium substituent, provided that at least one of R2 and R3 is different from H.

[0021] Phthalocyanine derivatives of formula (I) can be prepared starting from the corresponding amino derivatives, which in turn can be prepared from commercially available products by known methods, such as those described in US Pat. No. 5,965,598, in European Patent No. 1164 135 and in European Patent No. 1381 611, all in the name of the Applicant.

[0022] Preferred:

- di- and mono-substituted compounds of formula (I), ie compounds in which: R is selected from groups containing at least one quaternary ammonium substituent and groups comprising at least one aliphatic amino substituent, as defined previously, R1 = R or R1 is H and R2 = R3 = H;

-

the compounds of formula (I) tetra-substituted with the same substituents, that is the compounds in which the R and R2 substituents are the same and are selected from the groups containing at least one quaternary ammonium substituent and groups comprising at least an aliphatic amino substituent defined as above, and R1 = R3 = H; Y

-

tetra-substituted compounds of formula (I) suitable for conjugation, in which R is selected from the groups suitable for conjugation with specific vehicles, as defined above, R2 is selected from groups containing at least one substituent of quaternary ammonium and groups comprising at least one aliphatic amino substituent, and R1 = R3 = H.

[0023] Optimum results have been observed for the non-photodynamic activity for the compounds of the tetra-substituted formula (I) mentioned above with the same substituents, and for the di- and mono-substituted compounds, but in particular for di- and mono-substituted compounds.

[0024] For the purposes of the present invention, the term "non-photodynamic activity" refers to cytotoxic activity in the absence of light radiation, ie in the dark; while the expression "diseases characterized by cellular hyperproliferation" refers, for example, to tumors and precancerous and proliferative pathologies, such as psoriasis, actinic keratosis, atheroma, endoarterial hyperplasia and prostatic hyperplasia.

[0025] The compounds of formula (I) have been shown to be particularly useful in the treatment of microbial infections caused by Gram- and Gram + bacteria, and fungi, and preferably Gram + bacteria and fungi.

[0026] The best results have been observed with fungal infections.

[0027] In accordance with the invention, the present compounds of formula (I) are used for the preparation of pharmaceutical compositions as the only active ingredients or together with one or more additional active ingredients and / or together with a metal chelating agent; said pharmaceutical compositions may also comprise pharmaceutically acceptable excipients and diluents.

[0028] Particularly preferred are the pharmaceutical compositions of the invention which comprise, in addition to a compound of formula (I), a metal chelating agent preferably selected from metal chelating agents having specificity for Ca2 + and Mg2 + ions, such as acid 1, 2-diaminocyclohexane-N, N, N ', N'tetraacetic acid (CDTA), diethylenetriaminepentaacetic acid (DTPA), ethylenediamine-N, N, N', N'-tetraacetic acid (EDTA), citric acid and its salts. Particularly preferred are compositions comprising EDTA.

[0029] The present compositions are preferably formulated in the form of aqueous solutions, creams, gels, ointments, liposomal formulations, tablets, capsules, suspensions, etc., although formulations other than those which have been considered to be included within the scope of the invention indicated above. Topical administration is the preferred route, but the present compositions can also be used for parenteral, oral, nasal, etc. administration.

[0030] In said pharmaceutical composition, the doses of the active ingredients may vary, for example, between 0.1 and 20 mg of the product of formula (I) per Kg of body weight, preferably vary between 0.2 and 5 mg per Kg of body weight.

[0031] The following examples of the present invention are given by way of illustration.

Example 1

Preparation of the compound of formula (I) in which M is Zn R1 = R2 = R3 = H and R = {2- (trimethylammonium) 1 - [(trimethylammonium) methyl] ethoxy} dichloride in position 2 [Compound 1]

a) Synthesis of 2- {2-dimethylamino-1-methyl] ethoxy} zinc phthalocyaninate

[0032] 48 g of 4- {2- (dimethylamino) -1 - [(dimethylamino) methyl] ethoxy} phthalonitrile (0.18 mol) and 68 g of 1,2-dicyanobenzene (0) are dissolved in a nitrogen atmosphere , 53 mol) in 420 ml of DMF.

[0033] 32.4 g of Zn (AcO) 2 (0.18 mol) and 136 ml of DBU (0.90 mol) are added and the reaction mixture is brought to 130 ° C and then maintained at this temperature for 20 hours, protecting it from light, in an atmosphere of nitrogen and with vigorous agitation.

[0034] Then, the reaction mixture is cooled to about 50 ° C and treated with 800 ml of deionized H2O; The suspension obtained in this way is filtered and the recovered solid is washed in portions with H2O (2 x 400 ml) and with 8/2 acetone / ethyl ether (2 x 500 ml).

The product is subjected to purification by chromatography on silica gel (eluent: 9/1 of THF / DMF) followed by precipitation again in DMF (400 ml) / Et2O (1.6 L) / n-hexane (12 I) to obtain 35.6 g of the product (yield = 27%).

[0036] 1H NMR (300 MHz, DMSO-d6):  = 9.28-9.25 (m, 6H), 9.14 (d, 1H, J = 8.4 Hz), 8.87 (s , 1H), 8.21-8.19 (m, 6H), 7.79 (d, 1H, J = 8.4 Hz), 5.17 (t, 1H, J = 5 Hz) 2.86 ( d, 4H, J = 5 Hz), 2.5 (s, 12H) ppm.

[0037] ESI-MS: m / z 721 [(M + H) +].

b) Synthesis of 2- {2- (trimethylammonium) -1 - [(trimethylammonium) methyl] ethoxy} zinc phthalocyaninate diiodide

[0038] 30.2 g (0.042 mol) of 2- {2- (dimethyl amino) -1 - [(dimethylamino) methyl] ethoxy} zinc phthalocyanate, obtained as described above in point a), are dissolved in 900 ml of NMP. 60 ml of MeI (1 mol) are added and the solution is kept at room temperature for 72 hours, protected from light, in a nitrogen atmosphere and under vigorous stirring. The reaction mixture is diluted with 1.4 I of MeOH and then treated with 5.5 I of ethyl ether. The suspension obtained in this way is left under stirring for 30 minutes, allowed to stand for 1 hour and then filtered. The recovered solid is washed with ethyl ether (4 x 0.5 I) to finally obtain 47 g of the wet product, which is discovered to be 2- {2- (trimethylammonium) [(trimethylammonium) methyl] ethoxy} phthalocyaninate diiodide zinc.

[0039] 1H NMR (300 MHz, DMSO-d6):  = 9.46-9.43 (m, 7 H), 9.19 (sa = 1H), 8.29-8.27 (m, 6H ), 8.17 (d, 1H, J = 10 Hz), 6.20-6.10 (m, 1H), 4.22-4.04 (m = 4H), 3.41 (s, 18H) ppm.

c) Synthesis of 2- {2- (trimethylammonium) -1 - [(trimethylammonium) methyl] ethoxy} zinc phthalocyaninate dichloride

[0040] 10.1 g of this product are dissolved in 250 ml of a 4/1 mixture of MeOH / DMF. The solution is passed through a chromatography column that has 500 g of Amberlite® IRA 400 (CI) resin as the stationary phase, previously washed with an aqueous solution made acidic with 0.5 M HCl and conditioned with 4/1 of MeOH / DMF. Ethyl ether (2 L) is added to the eluate (approximately 500 ml) while stirring, the suspension obtained is allowed to stand for 1 hour at 4 ° C and then filtered; The recovered solid is washed with ethyl ether (4 x 500 ml) to obtain, after drying, 6.9 g of the title product.

[0041] 1H NMR (300 MHz, DMSO-d6):  = 9.43-9.40 (m, 7 H), 9.19 (sa = 1H), 8.26-8.22 (m, 6H ), 8.17 (d, 1H, J = 10 Hz), 6.20-6.18 (m, 1H), 4.19-4.17 (m = 4H), 3.42 (s, 18H) ppm.

[0042] 13C NMR (75 MHz, DMSO-d6): 156 = 156.20, 154.40, 154.17, 153.95, 153.63, 153.08, 141.07, 139.09, 138, 93, 134.42, 130.18, 124.72, 123.15, 120.31, 111.04, 69.36, 67.42, 54.57 ppm.

[0043]

UV-vis (DMF) max (%): 672 (100), 606 (9), 341 (11).

[0044]

ESI-MS: m / z 375 [(M-2Cl) 2 + 1

[0045]

Using the procedure described previously in Example 1, the preparations were also prepared.

following phthalocyanine derivatives of formula (I):

Example 2

[0046] Compound of general formula (I) in which M is Zn, R1 = R2 = R3 = H and R = {2- (dimethylamino) -1 [(dimethylamino) methyl] ethoxy} in position 2 [Compound 2 ]

Example 3

[0047] Compound of general formula (I) in which M is Zn, R1 = R2 = R3 = H and R = {2- (trimethylammonium) 1 - [(trimethylammonium) methyl] ethoxy} diiodide in position 1 [ Compound 3]

Example 4

[0048] Compound of general formula (I) in which M is Zn, R1 = R2 = R3 = H and R = iodide of [2- (N-methyl-1piperidinium) ethoxy] in position 1 [Compound 4]

Example 5

[0049] Compound of general formula (I) in which M is Zn, R1 = R2 = R3 = H and R = iodide of [3- (N, N, N-trimethylammonium) phenoxy] at position 2 [Compound 5]

Example 6

[0050] Compound of general formula (I) in which M is Zn, R1 = R2 = R3 = H and R = iodide of [4- (N, N, N-trimethylammonium) phenoxy] at position 1 [Compound 6]

Example 7

[0051] Compound of general formula (I) in which M is Zn, R1 = R2 = R3 = H and R = iodide of (N-methylpyridinium-3oxy) in position 1 [Compound 7]

Example 8

[0052] Compound of general formula (I) in which M is Zn, R1 = R2 = R3 = H and R = {2,4,6-tris - [(N, N-dimethylamino) methyl] phenoxy} at position 2 [Compound 8]

Example 9

[0053] Compound of general formula (I) in which M is Zn, R1 = R2 = RJ = H and R = (2,4,6-tris - [(N, N, N-trimethylammonium) methyl] phenoxy) trichloride in position 2 [Compound 9]

Example 10

Preparation of the compound of general formula (I) in which M is Zn, R2 = R3 - = H, R = R1 = {[3 (dimethylamino) phenyl] thio}, with R and R1 in positions 2 and 3 [Compound 10]

a) Synthesis of 2,3-bis {[3- (dimethylamino) phenyl] thio} zinc phthalocyaninate

[0054] DBU (1.22 g. 7 mmol) was added to a solution of 4,5-bis {[3- (dimethylamino) phenyl] thio} phthalonitrile (107 mg, 0.2 mmol), 1,2- dicyanobenzene (77 mg, 0.6 mmol) and Zn (OAc) 2 (183 mg, 0.8 mmol) in anhydrous DMF (6 ml). The mixture obtained in this way was heated to 120 ° C and kept under stirring for 3 hours in an inert atmosphere and protected from light, returned to room temperature and then poured into 50 ml of deionized water. The blue precipitate obtained from this report was separated by centrifugation and then subjected to two chromatographic steps on silica gel (ultrafast (THF) and MPLC (3/2 n-hexane / THF)) to give 33 mg of the product desired (yield = 19%).

[0055] 1H NMR (300 MHz, d6-DMSO): ,9 8.98-8.96 and 8.81-8.73 (8H, two m), 8.12-8.07 (6H, m), 7.46 (1H, dd, J1 = J2 = 8.4 Hz), 7.06-7.04 (2H, m), 6.93 (1H, d, J = 9.6 Hz), 2.99 (12H, s).

[0056] UV-vis (DMF): nm (%) 685 (100), 345 (28.1), 613 (17.3).

[0057] FAB + -MS: m / z 880 [(C48H34N10S2Zn) + H] + b) Synthesis of 2,3-bis {[3- (trimethylammonium) phenyl] thio} zinc phthalocyaninate diiodide

[0058] 1 ml of iodomethane was added to a solution of zinc 2,3-bis {[3- (dimethylamino) phenyl] thio) -phthalocyanate (50 mg, 0.06 mmol) in 3 ml of anhydrous NMP. The solution was kept at room temperature for 8 hours, under stirring, protected from light and then treated with ethyl ether. The blue precipitate obtained in this way separated from the mother liquor, was dissolved again in methanol and re-precipitated in ethyl ether. 44 mg of the desired product were obtained (yield = 85%).

[0059] 1 H NMR (300 MHz, d6-DMSO):  ppm. 9.45-9.41 (4H, m); 9.34 (2H, s), 9.25-9.22 (2H, m), 8.46 (2H, sa), 8.35-8.27 (6H, m), 8.13-8, 09 (1H, m), 7.88-7.74 (6H, m), 3.35 (18H, s).

[0060] 13C NMR (75 MHz, DMSO-d6)  154.40, 154.04, 152.78, 150.22, 149.10, 138.58, 138.38, 138.27, 138.81, 136.32, 132.23, 131.95, 130.53, 130.29, 130.20, 126.85, 123.27, 123.12, 122.82, 120.38, 57.26. UV-vis (DMF): nm (%) 690 (100), 334 (34.6), 613 (17.0), 690 = 126000 M-1cm-1b.

[0061] ESI-MS: m / z 454 [(C50H40N10S2Zn) -2I + 2H] 2+

[0062] Using the procedure described above in Example 10, the following compound was also prepared:

Example 11

[0063] Compound of general formula (I) in which M is Zn, R2 = R3 = H, R = R1 = iodide of {3- (N, N, N-trimethylammonium) phenoxy], with R and R1 in positions 2 and 3 [Compound 11]

Example 12

Preparation of the compound of general formula (I) in which M is Zn, R1 = R3 = H, R = R2 = (3 (N, N, N-trimethylammonium) phenoxy tetraiodide, with R and R2 in positions 2, 9 (10), 16 (17), 23 (24) [Compound 12]

a) Synthesis of 2.9 (10), 16 (17), 23 (24) -tetra [3- (N, N-dimethylamino) phenoxyl zinc phthalocyanate

[0064] DBU (29 ml -194 mmol) and anhydrous Zn (OAc) 2 (3.48 g-19 mmol) were added to 3- (N, N-dimethylamino) phenoxy] phthalonitrile (10 g-38 mmol); The mixture obtained in this way was brought to 160 ° C and maintained at this temperature for 4 hours, under stirring, in an inert atmosphere and protected from light. After returning the mixture to room temperature, it was treated with 200 ml of deionized water, the solid obtained was separated and washed with water and methanol. Then, the crude product was subjected to purification by chromatography (silica gel, 98/2 CH2Cl2 / MeOH v / v). The eluate containing the desired compound as a mixture of positional isomers was concentrated dissolved in CH2Cl2 and re-precipitated in n-hexane to give 7.62 g of a pure isomeric mixture (yield = 72%). UV-Vis (DMF) max (nm) 681 ( = 70300 M-1 cm-1) 612, 356

[0065] 1 H NMR (200 MHz, DMSO-d6)  ppm 9.01-8.90 (m, 4H), 8.51-8.45 (m, 4H), 7.82-7.73 (m , 4H), 7.49-7.36 (m, 4H), 6.85-6.73 (m, 12H), 3.05-3.02 (m, 24H).

[0066] 13C NMR (300 MHz, DMSO-d6)  ppm 159.71, 159.47, 158.33, 158.21, 153.06, 152.53, 152.23, 152.03, 151.77 , 151.36, 139.91, 132.89, 131.16, 131.02, 124.23, 120.32, 110.76, 109.17, 107.97, 107.83, 104.59

[0067] FAB-MS m / z 1117 [M + H] +.

b) Synthesis of 2.9 (10), 16 (17), 23 (24) -tetra [3- (N, N, N-methylammonium) phenoxy] zinc phthalocyanate tetraiodide

[0068] An excess of iodomethane (16 ml) was added to a solution of 2.9 (10), 16 (17), 23 (24) -tetra [3- (N, N, N-dimethylamino) phenoxy phthalocyanate ] zinc (6.32 g-5.65 mmol) in NMP (158 ml) and the mixture was kept under stirring for 120 hours, at room temperature and protected from light, then diluted with methanol (320 ml) and treated with ethyl ether (1.3 1) to give a green precipitate corresponding to the desired product in the form of an isomeric mixture (9 g, 95% yield).

[0069] UV-Vis (DMF) max (nm) 677 ( = 161000 M-1 cm -1), 609, 353;

[0070] 1 H NMR (200 MHz, DMSO-d6)  ppm 9.55-9.43 (m, 4H), 9.09-9.02 (m, 4H), 8.22-8.15 (m , 4H), 8.07-7.76 (m, 12H), 7.62-7.52 (m, 4H) 3.77 and 3.75 (2s, 36H)

[0071] 13C NMR (200 MHz, DMSO-d6)  ppm 157.84, 157.67, 152.50 (m), 148.85, 140.00 (m), 134.00, 131.77, 124 , 70, 121.30 (m), 120.18, 119.89, 115.99, 115.80, 112.70, 112.42, 56.60

[0072] ESI-MS m / z 388 [M -41-CH3] 3+, 573 [M -41-2CH3] 2+ 1132 [M -41-3CH3] +.

[0073] Using the procedure described previously in Example 12 and Example 1c), the following phthalocyanine derivatives of formula (I) were also prepared:

Example 13

[0074] Compound of general formula (I) in which M is Zn, R1 = R3 = H, R = R2 = [3- (N, N, N-trimethylammonium) phenoxy] chloride, with R and R2 in positions 1 , 8 (11), 15 (18), 22 (25) [Compound 13]

Example 14

[0075] Compound of general formula (I) in which M is Zn, R1 = R3 = H, R = R2 = [4- (N, N, N-trimethylammonium) phenoxy chloride], with R and R2 in positions 1 , 8 (11), 15 (18), 22 (25) [Compound 14]

Example 15

[0076] Compound of general formula (I) in which M is Zn, R = R1 = R2 = R3 = [3- (N, N, N-trimethylammonium) phenoxy] chloride, with R, R1, R2, R3 in which positions 2,3,9,10,16,17,23,24 [Compound 15]

Example 16

Evaluation of antimicrobial activity in the dark

a) In vitro experiments

[0077] An in vitro non-photodynamic antimicrobial activity evaluation was performed for Compounds 1-12 prepared as described above using the following procedure.

[0078] Candida albicans cells (ATCC 10231) were cultured in liquid Sabouraud medium (Difco Laboratories, Detroit, Ml) at 37 ° C under aerobic conditions. Cells were collected in the stationary growth phase (3,000 g, 15 min), washed with phosphate buffer saline (PBS) and diluted to give a final concentration of 1 x 10 6 cells / ml, corresponding to an absorbance of 0 , 12 to 630 nm.

[0079] Samples (1 ml) were prepared by adding to the PBS adequate volumes of the phthalocyanine stock solutions in DMSO (the final concentrations of the phthalocyanines tested varied from 1 to 50 M) so that the final amount of DMSO in Sample out of 5% v / v. Then, the samples were incubated at 37 ° C for 1 h. Cell survival was determined by serial 10-fold dilution in PBS of the sample aliquots (10 µl) and then plated in triplicate on Sabouraud dextrose agar (Difco). After incubation of the plates at 37 ° C for 24 h, the colony forming units (CFU / ml) were counted and the minimum bactericidal concentration (MBC) was determined, corresponding to the concentration of the active substance causing a log4 log10 reduction of CFU / ml).

[0080] Control experiments were performed in the absence of the phthalocyanine derivative. All experiments were repeated three times.

[0081] The MBC values of the compounds tested, as well as those discovered with the same protocol for two known antibiotics, nystatin and amphotericin, are indicated in the following Table 1.

Table 1

Compound

MBC (m)

one

5

2

5

3

one

4

10

5

10

6

10

7

10

8

10

9

10

10

 5

eleven

 10

12

 10

Nystatin

10

Amphotericin

10

[0082] From the results of Table 1 above, it is evident that the present phthalocyanine derivatives

5 have a non-photodynamic antimicrobial activity equivalent to that of the two antibiotics taken as a reference, which are the most commonly used antifungal antibiotics due to their outstanding fungal activity. Compounds 1-3 and 10 have shown MBC values even higher than those of nystatin and amphotericin.

b) In vivo experiments

[0083] An animal model of infection (S. aureus wound infection in guinea pig) was used to evaluate the in vivo activity of Compounds 1, 9, 13, 14 and 15 prepared as described above, compared to a Classic antibiotic regimen for the treatment of infections caused by Gram + bacteria, which consists of the administration of Imipenem, the most used and effective antimicrobial agent for this type of infections.

[0084] Next, the experimental protocol is indicated.

[0085] Animals: Dunkin-Hartley guinea pigs (weight: 450 to 500 g) were used throughout the study.

[0086] Bacteria: Staphylococcus aureus (ATCC 6538) was used. Bacterial colonies were collected after growing overnight in TSA (Soy Tripticasa Agar) and suspended in phosphate buffered saline (PBS, pH = 7.4) to achieve conventional turbidity (A600 reading of 0.4). Serial dilutions were made 10 times and 2 times to prepare a series of inoculums. The inoculum of 2 x 107 cells / ml was mixed in a 1: 1 ratio (vol / vol) with dextran microbeads (Cytodex, Sigma Chemicals Co., St. Louis, Mo) and used in

25 these experiments. The triplicate and average counts were determined to determine the precise size of the bacterial inoculum.

[0087] Guinea pig model: The animals' dorsal hair was removed and a mesh was drawn designating 8 areas for intermuscular inoculation. Each site of 2 guinea pigs for the treatment groups was inoculated with 0.2 ml of the

30 suspensions with bacterial bed. Control guinea pigs received PBS (vehicle). After inoculation, the guinea pigs were returned to their cages. After one day, 0.1 ml of PBS solutions of said Compounds of the invention that have been selected above, or of Imipenem in each abscess were administered.

[0088] A day later, the guinea pigs were sacrificed; using a sterile technique the abscesses were removed,

35 weighed and homogenized in 3 ml of saline solution for 2 min (Ultraturrax). The homogenates were diluted and plated on blood agar. Plates were incubated at 37 ° C for 24 h and the number of colony forming units (CFU / ml) was recorded. The bacterial count means of the groups treated with the compound were compared with the vehicle treated group to give the percentage of inhibition of abscess development. The means by weight of the abscesses of the groups treated with the compound were compared with the

40 group treated with vehicle to give the percentage of abscess reduction. All experiments were repeated twice on different days.

[0089] The results obtained are summarized in the following Tables 2-7, in relation to Compounds 1, 9, 13, 14, 15 and imipenem, respectively.

Four. Five

Table 2 - Compound 1

Concentration (g / kg)

% CFU inhibition % abscess reduction

0.08

 0 0

0.16

48.2  2.4 11.6  0.5

0.8

54.7  8.1 11.5  1.9

1.6

69.0  6.7 23.0  3.9

16

71.5  7.7 20.1 5.2

160

89.4  3.8 25.6 ,3 4.3

Table 3 - Compound 9

Concentration (g / kg)

% CFU inhibition % abscess reduction

0.002

 0 0

0.1

4.8  3.0 0

0.2

45.3  1.2 1 0.6  0.8

one

55.1  6.6 9.9  2.4

2

55.9  7.2 11.7  1.3

twenty

80.0  8.1 30.6  12.0 -

200

94.5  5.2 30.9  5.7

Table 4 - Compound 13

Concentration (g / kg)

% CFU inhibition % abscess reduction

0.044

 0.0 0.0

0.22

56.4  7.3 16.6  3.8

0.44

57.8 ,6 9.6 16.8  6.3

4.4

73.2  9.8 20.3  5.5

44

64.6  11 20.1  6.2

440

80.4  9.4 25.3  8.7

Table 5 - Compound 14

Concentration (g / kg)

% CFU inhibition % abscess reduction

0.44

39.7  8.1 9.5  4.3

4.4

10.0  5.5 1.5  1.0

44

57.9  8.1 9.8  4.1

440

80.0  9.5 22.8  4.2

Table 6 - Compound 15

Concentration (g / kg)

% CFU inhibition % abscess reduction

0.41

5.7  1.5 0

2.05

5.5  3.1 0

4.1

38.8  1.9 1.9  0.5

41

59.4  9.6 20.6  7.2

410

62.2  9.0 21.8  6.2

Table 7 - Imipenem

Concentration (g / kg)

% CFU inhibition % abscess reduction

83200

 77.0113.5 3.6  1.0

Total dose of 1152 mg / kg in 3 days * (384 mg / kg / day)

64.4  7.4 n.d.

* Classic regime for parenteral administration

5

10

fifteen

twenty

25

30

35

REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is only for the convenience of the reader. It is not part of

European patent document. Although special care has been taken in the compilation of references, it is not

they can exclude errors or omissions and the EPO rejects any responsibility in this regard.

Patent documents cited in the description

image 1

Patent documents not cited in the description

image 1

Claims (20)

1. Use of phthalocyanine derivatives of general formula (I) image 1 wherein M is Zn, R is selected from H, groups containing at least one quaternary ammonium substituent, groups comprising at least one aliphatic amino substituent and groups suitable for covalent binding to bio-organic vehicles chosen from the group consisting of in amino acids, polypeptides, proteins, antibodies, polysaccharides and aptamers, R1, the same or different from R1 is selected from H, groups comprising at least one aliphatic amino substituent and groups containing at least one quaternary ammonium substituent, R2 and R3, same or different from each other, are selected from H, alkoxy groups having 1 to 10 carbon atoms, thioalkoxy groups having 1 to 10 carbon atoms, groups comprising at least one aliphatic amino substituent and groups containing at least a quaternary ammonium substituent, with the proviso that: a) at least one of R, R1 R2 and R3 is a group comprising at least one aliphatic amino substituent or a group comprising at least one quaternary ammonium substituent and, when R, R1, R2 and R3 are groups comprising at least one aliphatic amino substituent or groups containing at least one quaternary ammonium substituent, or R and R2 are groups comprising at least one aliphatic amino substituent or groups containing at least one quaternary ammonium substituent and R1 and R3 are H, comprising said groups at least one aliphatic amino substituent or said groups containing at least one quaternary ammonium substituent, are the same; b) when R and R1 are both different from H, they are in positions 1,4 or 2,3, while when only one between R and R1 is different from H, it is in position 1 or 2; c) when R2 and R3 are both different from H, they are in positions 8, 11, 15, 18, 22, 25 or 9, 10, 16, 17, 23, 24 while when only one between R2 and R3 is different of H, be in positions 8 (11), 15 (18), 22 (25) or in positions 9 (10), 16 (17), 23 (24); and its pharmaceutically acceptable salts, for the preparation of pharmaceutical compositions for the non-photodynamic treatment of microbial infections and the treatment of diseases characterized by cellular hyperproliferation. 2. Use according to claim 1, wherein said group comprising at least one quaternary ammonium substituent or an aliphatic amino substituent is a group (X) pR4 in which X is selected from the group consisting of O, -CH2-, CO, S, SO and -NR5, wherein R5 is chosen from H and C1-C15 alkyl; and R4 is where image 1 Y is selected from the group consisting of C1-C10 alkyl and phenyl, possibly substituted, or Y form 5 with the group Z, to which it is attached, a saturated or unsaturated heterocycle, optionally substituted, which may comprise up to 2 heteroatoms selected from the group consisting of N, O and S; Z is selected from the group consisting of -N, -CH2N and -CONHCH2CH2N; R6 and R7, the same or different from each other, are selected from the group consisting of C1-C15 alkyl alkyl and phenyl, or form with the group Z, to which they are attached, a saturated or unsaturated heterocycle, optionally 10 substituted, which may comprise up to two heteroatoms selected from the group consisting of N, O and S; R8 and R9, the same or different from each other, are selected from the group consisting of H, C1-C15 alkyl and R10COOEt or R10COOMe groups, wherein R10 is C1-C15 alkyl; m, n, p, w, t and u, independently of each other, are 0 or 1; Y 15 v is an integer between 1 and 3, with the proviso that only one between n, w, t and u is simultaneously 0.

3.

 Use according to claim 1, wherein said group comprising at least one quaternary ammonium substituent is selected from the following groups:

Four.

 Use according to claim 1, wherein said group comprising at least one quaternary ammonium substituent is selected from the following groups:

5.

 Use according to claim 1, wherein said group comprising at least one aliphatic amino substituent is selected from the following groups:

6.

 Use according to claim 1, wherein said group comprising at least one aliphatic amino substituent is selected from the following groups:

image 1 image 1 image 1 image 1 image 1 image 1 Use according to claim 1, wherein in said compounds of formula (I) M is Zn. 8. Use according to claim 1, wherein said saturated or unsaturated heterocycle is selected from the group consisting of morpholine, piperidine, pyridine, pyrimidine, piperazine, pyrrolidine, pyrroline, imidazole, aniline and julolidine. 10 9. Use according to claim 1, wherein said group suitable for conjugation with specific vehicles is a group suitable for covalent binding to amino acids, polypeptides, proteins, antibodies, polysaccharides and aptamers. Use according to claim 9, wherein said group suitable for conjugation with specific vehicles is a group selected from the group consisting of -COOH, -SH, - NH2, -CO-CH2-Br, - SO2Cl, maleimide, hydrazine, phenol, imidate, biotin, possibly linked to the phthalocyanine core through a suitable separator (X) pW, in which X and p are as defined in claim 1 and W is chosen from alkyl C1-C10, aryl and arylalkyl C1-C5. twenty 11. Use according to claim 1, wherein R is a group suitable for conjugation with specific vehicles, R1 is equal to H and R2 and R3 are selected from H, groups comprising at least one aliphatic amino substituent and groups containing at least one quaternary ammonium substituent, provided that at least one of R2 and R3 is different from H. 25 12. Use according to claim 1, wherein said compounds of formula (I) are selected from: tetra-substituted compounds with the same substituents, ie compounds in which the R and R2 substituents are the same and are selected from the groups containing at least one Quaternary ammonium substituent and groups comprising at least one aliphatic amino substituent, as defined in the preceding claims, and R1 = R3 = H; tetra-substituted compounds of formula (I) suitable for conjugation, in which R is selected from groups suitable for conjugation with specific vehicles, as defined in the preceding claims, R2 is selected from groups containing at least one substituent of Quaternary ammonium and groups comprising at least one aliphatic amino substituent, as defined in the preceding claims, and R1 = R3 = H; and di- and mono-substituted compounds, that is, compounds in which: R is selected from groups containing at least one quaternary ammonium substituent and groups comprising at least one aliphatic amino substituent, as defined in the preceding claims. , R1 = R or R1 is H, and R2 = 40 R3 = H.

13.

 Use according to claim 12, wherein said compounds of formula (I) are selected from tetra-substituted compounds with the same substituents, and di- and mono-substituted compounds as defined in claim 12.

14.

 Use according to claim 12, wherein said compounds of formula (I) are selected from di- and mono-substituted compounds as defined in claim 12.

fifteen.

 Use according to claim 1, wherein said compounds of formula (I) are selected from the following compounds:

- Compound of formula (I) in which M is Zn, R1 = R2 = R3 = H and R = {2- (trimethylammonium) -1 [(trimethylammonium) methyl] ethoxy} dichloride in position 2 [Compound 1] ,

-

compound of general formula (I) in which M is Zn, R1 = R2 = R3 = H and R = {2- (dimethylamino) -1 [(dimethylamino) methyl] ethoxy} in position 2 [Compound 2],

-

compound of general formula (I) in which M is Zn, R1 = R2 = R3 = H and R = {2- (trimethylammonium) iodide -1 [(trimethylammonium) methyl] ethoxy} at position 1 [Compound 3] ,

-

compound of general formula (I) in which M is Zn, R1 = R2 = R3 = H and R = [2- (N-methyl-1piperidinium) ethoxy] iodide in position 1 [Compound 4],

-

compound of general formula (I) in which M is Zn, R1 = R2 = R3 = H and R = iodide of [3- (N, N, N-trimethylammonium) phenoxy] at position 2 [Compound 5],

-

compound of general formula (I) in which M is Zn, R1 = R2 = R3 = H and R = iodide of [4- (N, N, N-trimethylammonium) phenoxy] at position 1 [Compound 6],

-

compound of general formula (I) in which M is Zn, R1 = R2 = R3 = H and R = iodide of (N-methylpyridinium-3-oxy) in position 1 [Compound 7],

-

compound of general formula (I) in which M is Zn, R1 = R2 = R3 = H and R = {2,4,6-tris - [(N, N-dimethylamino) methyl] phenoxy} at position 2 [Compound 8 ],

-

compound of general formula (I) in which M is Zn, R1 = R2 = R3 = H and R = {2,4,6-tris - [(N, N, Ntrimethylamm / oneium) methyl] phenoxy} trichloride in position 2 [Compound 9],

-

compound of general formula (I) in which M is Zn, R2 = R3 = H, R = R1 = {[3- (dimethylamino) phenyl] thio}, with R and R1 in positions 2 and 3 [Compound 10] ,

-

compound of general formula (I) in which M is Zn, R2 = R3 = H, R = R1 = iodide of {3- (N, N, N-trimethylammonium) phenoxy], with R and R1 in positions 2 and 3 [ Compound 11],

-

compound of general formula (I) in which M is Zn, R1 = R3 = H, R = R2 = [3- (N, N, N-trimethylammonium) phenoxy] chloride, with R and R2 at positions 2.9 ( 10), 16 (17), 23 (24) [Compound 12],

-

compound of general formula (I) in which M is Zn, R1 = R3 = H, R = R2 = [3- (N, N, N-trimethylammonium) phenoxy] chloride, with R and R2 at positions 1, 8 ( 11), 15 (18), 22 (25) [Compound 13],

-

compound of general formula (I) in which M is Zn, R1 = R3 = H, R = R2 = [4- (N, N, N-trimethylammonium) phenoxy] chloride, with R and R2 at positions 1, 8 ( 11), 15 (18), 22 (25) [Compound 14], and

-

compound of general formula (I) in which M is Zn, R = R1 = R2 = R3 = [3- (N, N, N-trimethylammonium) phenoxy] chloride, with R, R1, R2, R3 in positions 2, 3, 9, 10, 16, 17, 23, 24 [Compound 15]

16.

 Use according to claim 1, wherein said diseases characterized by cellular hyperproliferation are selected from tumors, psoriasis, actinic keratosis, atheroma, endoarterial hyperplasia and prostatic hyperplasia.

17.

 Use according to claim 1, wherein said pharmaceutical compositions are useful in the treatment of microbial infections caused by Gram- bacteria, Gram + bacteria and fungi.

18.

 Use according to claim 1, wherein said pharmaceutical compositions are useful in the treatment of microbial infections caused by Gram + bacteria and fungi.

19.

 Use according to claim 1, wherein said pharmaceutical compositions are useful in the treatment of fungal microbial infections.

twenty.

 Use according to claim 1, wherein said pharmaceutical compositions comprise, in addition to the compounds of formula (I), one or more different active ingredients and / or a metal chelating agent, optionally together with one or more pharmaceutically excipients and diluents acceptable.

twenty-one.

 Use according to claim 20, wherein said metal chelating agent is selected from the group consisting of 1,2-diaminocyclohexane-N, N, N ', N'-tetraacetic acid (CDTA), diethylenetriaminepentaacetic acid (DTPA) , ethylenediamine-N, N, N ', N'-tetraacetic acid (EDTA), citric acid and its salts.

22

 Use according to claim 21, wherein said metal chelating agent is ethylenediamine N, N, N ', N'-tetraacetic acid (EDTA).