OF PHTHALOCYANINE DERIVATES FOR THE NON- PHOTODYNAMI C TREATMENT OF DISEASES

FIELD OF THE INVENTION

The invention relates to the field of phthalocyanine derivatives and in particular to the novel use of phthalocyanine derivatives of general formula (I), given hereinafter, for the preparation of pharmaceutical compositions useful in the non- photodynarnic diagnosis and treatment of infections of various origin and diseases characterised by cell hyperproliferation. STATE OF THE ART Molecules containing the phthalocyanine chromofluorophore macrocycle have long been known to produce, by interaction with visible light, reactive oxygen species such as radicals or singlet oxygen, which are cytotoxic towards viruses, bacteria, fungi and eukaryotic cells. Therefore, given also their abiiity to preferentially localise in pathologically affected areas, phthalocyanine compounds have been used for some time in so-called photodynamic therapy, commonly known as "PDT" therapy. PDT therapy consists of administering said compounds, allowing them to localise in the area to be treated, 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, pp. 145-147, 1985. Other photosensitising agents useful in PDT are zinc phthalocyanine complexes and their conjugates described in US patent no. 5,965,598, in the name of the Applicant. These compounds have proved to be effective photosensitising agents in PDT treatment for both tumours and various infections. However, in many cases the pathologically affected parts involve internal organs, or in any event are not easily accessible to light.

From this the need arises to identify compounds able to manifest their cytotoxic action in darkness without having to irradiate the affected area with light. SUMMARY OF THE INVENTION Contrary to the knowledge acquired to date in the state of the art and above illustrated with regard to the therapeutic applications of phthaiocyanines, the Applicant has now found that the phthalocyanine derivatives of formula (I) given hereinafter, are also active in the dark. This obviously opens up previously

inconceivable possibilities regarding 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.

Therefore the present invention provides the use of the phthalocyanine derivatives of general formula (I)

in which M is selected from 2H and a metal selected from the group consisting of Zn, Si(OR') ₂ , Ge(OR') ₂ and AIOR ¹ , wherein R ^! is selected from H and aikyl groups having from 1 to 15 carbon atoms, R is selected from H, groups comprising at least one quaternary ammonium substituent, groups comprising at least one aliphatic amino substituent, and groups suitable for conjugation to specific carriers,

R ₁ , equal or different from R, is selected from H ₁ groups comprising at least one aliphatic amino substituent and groups comprising at least one quaternary ammonium substituent,

R ₂ and R ₃ , equal or different from one another, are selected from H, alkoxy groups having from 1 to 10 carbon atoms, thioaikoxy groups having from 1 to 10 carbon atoms, groups comprising at least one aliphatic amino substituent and groups comprising at least one quaternary ammonium substituent, with the proviso that: a) at least one amongst R ₅ Ri, R ₂ and R ₃ is a group comprising at least one aliphatic amino substituent or a group comprising at least one quaternary ammonium substituent and, when R, R ₁ , R ₂ and R ₃ are groups comprising at least

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

16(17), 23(24); and their pharmaceutically acceptable salts, for the preparation of pharmaceutical compositions useful in the non- photodynamic treatment of microbial infections and diagnosis and treatment of diseases characterised by cell hyperproliferation.

The characteristics and advantages of the invention will be illustrated in detail in the following description.

DETAILED DESCRIPTION OF THE INVENTION According to the invention, the expression "group comprising at least one quaternary ammonium substituent or one aliphatic amino substituent" means preferably a

(X) _n R ₄ group, wherein X is selected from the group consisting of O, -CH2-, CO, S,

SO, and -NR ₅ wherein R ₅ is selected from H and C-1-C1 ₅ alky!; and R ₄ is

in which Y is selected from the group consisting of C1-C ₁₀ aikyl and phenyl, possibly substituted, or Y forms with the Z group, to which it is bound, 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, -CH ₂ N and -CONHCH ₂ CH ₂ N; R ₆ and R ₇ , equal or different from one another, are selected from the group consisting of Ci-C ₁₅ alkyl and phenyl or form with the Z group, to which they are bound, a saturated or unsaturated heterocycle, optionally substituted, which may comprise up to two heteroatoms selected from the group consisting of N, O and S; R ₈ and R ₉ , equal or different from each other, are selected from the group consisting of H, C ₁ -C _1S alkyi, and R ₁₀ COOEt or R ₁₀ COOMe groups in which R ₁₀ is C ₁ -C ₁₅ alkyl; m, n, p, w, t and u, independently from one another, are 0 or 1 ; and v is an integer between 1 and 3, with the proviso that only one amongst n, w, t and u is simultaneously 0. According to the invention, of the groups comprising at least one aliphatic amino substituent, groups selected from the following are preferred:

CONHCH ₂ CH ₂ NH ₂ CH ₂ CH ₂ CONHCH ₂ CH ₂ NH ₂

CONH(CHj) ₆ NH ₂ IH ₂ CH ₂ CONH(CH ₂ J ₅ NH ₂

CONHCH ₂ CH ₂ N(CH ₃ J ₂ CH ₂ CH ₂ CONHCH ₂ CH ₂ N(CH ₃ ) ₂

CONH(CH _E ) ₅ N(CH ₃ ) ₂ H ₂ CH ₂ CONH(CH ₂ J ₅ N(CH ₃ ) ₂

According to the invention, of the groups comprising at least one quaternary ammonium substituent, groups selected from the following are preferred:

CONHCH2CH ₂ N(CH ₃ )3 -CH ₂ CH ₂ CONHCH ₂ CH ₂ N(CH3)3

-CONH(CH ₂ ) ₅ N(CH ₃ ) ₃ CH ₂ CH ₂ CONH(CH ₂ ) ₅ N(CH ₃ ) ₃

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

As groups comprising at least one quaternary ammonium substituent, the following groups are particularly preferred:

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

The expression "saturated or unsaturated heterocycle" means preferably a heterocycle selected from the group consisting of morpholine, piperidine, pyridine, pyrimidine, piperazine, pyrrolidine, pyrroline, imidazole, aniline and julolidine. The expression "group suitable for conjugation to specific carriers" means any group suitable for covalently binding to bio-organic carriers such as amino acids, polypeptides, proteins, antibodies, polysaccharides and aptamers, able to facilitate transport of the phthalocya nines to particular targets; the aforesaid expression preferably indicates a group selected from the group consisting of -COOH, -SH, - NH ₂ , -CO-CH ₂ -Br, -SO ₂ Cl, maleimide, hydrazine, phenol, imidate, biotin, optionally bound to the phthalocyanine nucleus through a suitable spacer (X) _p -W, where X and p are defined as above and VV is selected from CrCi ₀ alkyl, aryl, and CrC ₅ arylalkyl.

When R is a group suitable for conjugation to specific carriers, as defined above, Ri is preferably equal to H and R^ and R ₃ are selected from H, groups comprising at least one aliphatic amino substituent, and groups comprising at least one quaternary ammonium substituent, provided that at least one of R ₂ and R ₃ is different from H.

The phthalocyanine derivatives of formula (I) can be prepared starting from the corresponding amino derivatives, which in their turn can be prepared from

commercially available products by known procedures, such as those described in US Patent No. 5,965,598, in European Patent No. 1 164 135 and in European Patent No. 1 381 611 , all in the name of the Applicant. Preferred are: - di- and mono-substituted compounds of formula (I), i.e. compounds in which: R is selected from groups comprising at least one quaternary ammonium substituent and groups comprising at least one aliphatic amino substituent, as defined above, R ₁ = R or Ri is H, and R ₂ = R3 = H;

- the compounds of formula (!) tetra-substituted with the same substituents, i.e. the compounds in which the R and R ₂ substituents are the same and selected from groups comprising at least one quaternary ammonium substituent and groups comprising at least one aliphatic amino substituent defined as above, and R ₁ = R ₃ = H; and

- the tetra-substituted compounds of formula (I) suitable for conjugation, in which R is selected from groups suitable for conjugation to specific carriers, as defined above, R ₂ is selected from groups comprising at least one quaternary ammonium substituent and groups comprising at least one aliphatic amino substituent, and R ₁

= R ₃ = H.

Optima! results in terms of non-photodynamic activity have been observed for the aforesaid compounds of formula (I) tetra-substituted with the same substituents, and for the di- and mono-substituted compounds, but in particular for the di- and mono-substituted compounds.

For the purposes of the present invention, the expression "non-photodynamic activity" means cytotoxic activity in the absence of light radiation, i.e. in darkness; while the expression "diseases characterised by cell hyperproliferation" means for example tumours and pre-cancerous and proliferative pathologies, such as psoriasis, actinic keratosis, atheroma, endoarterial hyperplasia and prostatic hyperplasia.

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

The best results have been observed with fungal infections.

According to the invention, the present compounds of formula (I) are used for the

preparation of pharmaceutical compositions as single active principles or in combination with one or more further active principles and/or in combination with a metal chelating agent; such pharmaceutical compositions may also comprise excipients and pharmaceutically acceptable diiuents. Particularly preferred are the pharmaceutical compositions of the invention comprising, in addition to a compound of formula (I), a metal chelating agent preferably selected from metai chelating agents having specificity for Ca ²⁺ and

Mg ²⁺ ions, such as i ^-diaminocyclohexane-NXN'.N'-tetraacetic acid (CDTA), diethyienetriaminepentaacetic acid (DTPA), ethylenediamine-N,N _t N',N'-tetraacetic acid (EDTA), citric acid and its salts. Compositions comprising EDTA are particularly preferred.

The present compositions are preferably formulated as aqueous solutions, creams, gels, ointments, liposomal formulations, tablets, capsules, suspensions, etc., although formulations other than the aforestated are be considered as included within the scope of the invention. Topicai administration is the preferred route, but the present compositions can also be used for parenteral, oral, nasal administration, etc.

In said pharmaceutical compositions, the dosages of active principle can vary for example between 0.1 and 20 mg of the product of formula (!) per Kg body weight, preferably ranging between 0.2 and 5 mg per Kg body weight.

The following non-limiting examples of the present invention are given by way of illustration.

EXAMPLE 1 {2-(trimethviammonium)-1-f(trimethylammonium) methyliethoxy) dichloride in position 2 [Compound 1] a) Synthesis of 2-(2-(dimethylaminoV14(dimethylamino) methyliethoxy) zinc phthalocyaninate

Under nitrogen atmosphere 48 g of 4-{2-(dimethylamino)-1-[(dimethylamino) methyl]ethoxy} phthalonitrile (0.18 moi) and 68 g of 1 ,2-dicyanobenzene (0.53 mol) are dissolved in 420 ml of DMF.

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 ⁰ C then maintained at this temperature for 20 hours, shielded from light, under nitrogen atmosphere and under vigorous stirring. The reaction mixture is then cooled to about 5O ⁰ C and treated with 800 ml of deionised H ₂ O; the suspension thus obtained is filtered and the recovered solid washed with H ₂ O in portions (2 x 400 ml) and with 8/2 acetone/ethyl ether (2 x 500 ml).

The product is subjected to chromatographic purification on silica gel (eiuent: 9/1 THF/DMF), foliowed by re-precipitation from DMF {400 ml)/Et ₂ O (1 ,6 !)/n-hexane (12 I) to obtain 35.6 g of product (yield = 27%). ¹ H-NMR (300 MHz, DMSO-d ₆ ): δ = 9.28-9.25 (m, 6H) ₁ 9.14 (d, 1 H ₁ J = 8.4 Hz) ₁ 8.87(s, 1 H), 8.21-8.19 (m, 6H), 7.79 (d, 1 H ₁ J = 8.4Hz) ₁ 5.17 (t, 1 H ₁ J = 5 Hz) 2.86 (d, 4H, J = 5 Hz), 2.5 (s, 12H) ppm. ESl-MS: m/z 721 [(M+H)*]. b^ Synthesis of 2-{2-ftrimethylammoniumV1-f(trimethylammonium) methvilethoxy) zinc phthalocvaninate diiodide

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

¹ H-NMR (300 MHz ₁ DMSO-d ₆ ): δ = 9.46-9.43 (m, 7 H), 9.19 (bs = 1H), 8.29-8.27 (m, 6H) ₁ 8.17 (d, 1 H, J = 10 Hz), 6.20-6.10 (m, 1 H), 4.22-4.04 (m = 4H), 3.41 (s, 18H) ppm. c) Synthesis of 2-f2-(trimethylammonium)-1-r(trimethylammonium) methvi]ethoxy) zinc phthalocvaninate dichloride

10.1 g of this product are dissolved in 250 ml of a 4/1 MeOH/DMF mixture. The solution is passed through a chromatography column having 500 g of Amberlite ^®

IRA 400 (Cl) resin as the stationary phase, previously washed with an aqueous solution made acid with 0.5 M HCI and conditioned with 4/1 MeOH/DMF, Ethy! ether (2 I) is slowly added to the eiuate (about 500 ml) while maintaining under stirring, and the obtained suspension is allowed to rest for 1 hour at 4°C 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.

¹ H-NMR (300 MHz, DMSO-d ₆ ): δ = 9.43-9.40 (m, 7 H), 9.19 (bs = 1 H), 8.26-8.22

(m, 6H), 8.17 (d, 1 H, J = 10 Hz), 6.20-6.18 (m, 1 H), 4.19-4.17 (m = 4H) ₅ 3.42 (s,

18H) ppm. ¹³ C-NMR (75 MHz, DMSO-d ₆ ): δ = 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.

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

ESI-MS: m/z 375 [(M-2CI) ^2*3 By using the procedure previously described in example 1 , the following phthalocyanine derivatives of formula (I) were also prepared:

EXAMPLE 2

Compound of general formula (I) in which M is Zn, R ₁ =R ₂ =Rs=H and R= {2-

(dimethylamino)-1-[(dimethylamino) methyl]ethoxy} in position 2 [Compound 2] EXAMPLE 3

Compound of general formula (I) in which M is Zn, and R= {2-

(trimethylammonium)-1-[(trimethy!ammonium) methyi]ethoxy} diiodide in position 1

[Compound 3]

EXAMPLE 4 Compound of general formula (I) in which M is Zn, and R= [2-(/V- methyi-1-piperidinium) ethoxy] iodide in position 1 [Compound 4]

EXAMPLE 5

Compound of general formula (I) in which M is Zn, [3-

(λ/,/v ^" ,λMrimethylamrnonium) phenoxy] iodide in position 2 [Compound 5] EXAMPLE 6

Compound of general formula (I) in which M is Zn, [4-

(A/,λ/,λ/-trimethylammonium) phenoxy] iodide in position 1 [Compound 6]

EXAMPLE 7

Compound of general formula (I) in which M is Zn, and R= [N- methyipyridinium-3-oxy) iodide in position 1 [Compound 7]

EXAMPLE 8 Compound of general formula (I) in which M is Zn, and R= {2,4,6- tris-[(W,λ/-dimethylamino) methyl]phenoxy} in position 2 [Compound 8]

EXAMPLE 9

Compound of general formula (I) in which M is Zn, R ₁ =R ₂ =Ra=H and R= {2,4,6-tris-

[(N,N,λ/-trimethylamrnonium) methyljphenoxy} trichloride in position 2 [Compound 9] EXAMPLE 10

Preparation of the compound of general formuia (i) in which M is Zn. R^=R ₃ =H,

R=R ₁ = ■ff3-(dimethylaminotohenyr)thio). with R and Ri in positions 2 and 3

[Compound 10] a) Synthesis of 2,3-bis{[3-(dimethylamino)phenvπthio> zinc phthalocyaninate 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 thus obtained mixture was heated to 120 ⁰ C and maintained under stirring for 3 hours under inert atmosphere and shielded from light, returned to room temperature then poured into 50 ml of deionised water. The blue precipitate thus obtained was separated by centrifugation then subjected to two chromatographic passages on silica gel (flash (THF) and MPLC (3/2 n-hexane/THF)) to give 33 mg of the desired product (yield = 19%). ¹ H-NMR (300 MHz, Cf ₆ -DMSO): δ 8.98-8.96 and 8.81-8.73 (8H, two m), 8.12-8.07 (6H ₁ m), 7,46 (1 H, dd, Hz), 2.99 (12H, s).

UV-vis (DMF): nm (%) 685 (100), 345 (28.1 ), 613 (17.3). FAB ⁺ -MS: m/z 880 [(C ₄₈ H ₃₄ N ₁₀ S ₂ Zn)+H] ⁺ b) Synthesis of 2,3~bisff3-(trimethylammonium)phenvπthio) zinc phthalocvaninate diiodide

1 mi of iodomethane was added to a solution of Zinc 2,3-bis{[3- (dimethylamino)phenyl]thio}-phthalocyaninate (50 mg, 0.06 mmol) in 3 ml of

anhydrous NMP. The solution was maintained at room temperature for 8 hours, under stirring and shielded from light, then treated with ethyl ether. The blue precipitate thus obtained was separated from the mother liquors, redissolved in methanol and reprecipitated from ethyl ether. 44 mg of the desired product were obtained (yield = 85%).

¹ H-NMR (300MHz, Cf ₆ -DMSO): δ ppm 9.45-9.41 (4H, m), 9.34 (2H, s), 9.25-9.22 (2H ₁ m), 8.46 (2H, bs), 8.35-8.27 (6H, m), 8.13-8.09 <1 H, m), 7.88-7.74 (6H, m), 3.35 (18H, s). ¹³ C-NMR (75 MHz ₅ DMSO-d ₆ ) £ 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), λ ₆₉₀ = 126000 M ^"1 crτϊ1b. ESi-MS: m/z 454 [(C ₅₀ H ₄₀ N ₁₀ S ₂ Zn)-2I+2H] ²⁺ By using the procedure described above in Example 10, the following compound was also prepared: EXAMPLE 11

Compound of general formula (I) in which M is Zn, R ₂ =Rs=H, R=R ₁ = {3-(N,N,N- trimethylarnmonium)phenoxy] iodide, with R and Ri in positions 2 and 3 [Compound 11] EXAMPLE 12

Preparation of compound of general formula (I) in which M is Zn, R ₁ =Ry=H, R=R?= [3-(Af,A/,λ/-trimethylammonium)phenoxy1 tetraiodide, with R and R? in positions 2.9(10)λ 6(17),23(24) [Compound 12] a) Synthesis of 2.9(10116(17),23(24Hetra[3-(λ/.λ/-dimethylamino)phenoxy1 Zinc phthalocyaninate

DBU (29 ml - 194 mmol) and anhydrous Zn(OAc) ₂ (3.48 g - 19 mmoi) were added to 3-(W,λkJimethylarnino)phenoxy] phthalonitrile (10 g - 38 mmol); the mixture thus obtained was brought to 16O ⁰ C and maintained at this temperature for 4 hours, under stirring, under inert atmosphere and shielded from light. After having returned the mixture to room temperature, it was treated with 200 ml of deionised water and the solid obtained separated and washed with water and methanol. The crude product was then subjected to chromatographic purification (silica gel,

CH ₂ CI ₂ /Me0H 98/2 v/v ). The eluate containing the desired compound as a mixture of positional isomers was concentrated, dissolved in CH ₂ Ci ₂ and reprecipitated from n-hexane to give 7.62 g of pure isomeric mixture (yield = 72%). UV-Vis (DMF) λ _max (nm) 681 (ε=70300 M ^"1 cm ^'1 ) 612, 356 ¹ H-NMR (200 MHz, DMSO-d ₆ ) £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) ₁ 3.05-3.02 (m, 24H). ¹³ C-NMR (300 MHz ₁ DMSO-Cf ₆ ) δ 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 FAB-MS m/z 1117 [M+H] ⁺ . b) Synthesis of 2,9(10),16(17),23(24Hetrar3-fA/,A/./V-trimethylammonium)phen oxyl zinc phthalocvaninate tetraiodide

An excess of iodomethane (16 ml) was added to a solution of 2,9(10),16(17),23(24)-tetra[3-(/V,λ/-dimethy[amino)phenoxy] Zinc phthalocyaninate (6.32 g - 5.65 mmo!) in NMP (158 mi) and the mixture maintained under stirring for 120 hours, at room temperature and shielded from light, then diluted with methanol (320 ml) and treated with ethyl ether (1.3 I) to give a green precipitate corresponding to the desired product in the form of an isomeric mixture (9 g, 95% yield). UV-Vis (DMF) λ _max (nm) 677 (ε = 161000 M ^"1 cm ^"1 ), 609, 353;

¹ H-NMR (200 MHz, DMSO-Cf ₆ ) β 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) ¹³ C-NMR (200 MHz, DMSO-Cf ₆ ) <?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

ESI-MS m/z 388 [M -4I -CH ₃ f ⁺ , 573 [M -4I -2CH ₃ J ²⁺ , 1132 [M -4I -3CH ₃ J ⁺ . By using the procedure previously described in Example 12 and in Example 1c), the following phthalocyanine derivatives of formula (I) were also prepared: EXAMPLE 13 Compound of general formula (I) in which M is Zn, Ri=R ₃ =H, R=R ₂ = [3-(N,N,N- trimethylammonium)phenoxy] chloride, with R and R ₂ in positions 1 , 8(11), 15(18), 22(25) [Compound 13]

EXAMPLE 14

Compound of general formula (I) in which M is Zn, R ₁ =R ₃ =H, R=R ₂ = [4-{N,N,N- trimethylammonium)phenoxy] chloride, with R and R ₂ in positions 1 , 8(11), 15(18), 22(25) [Compound 14] EXAMPLE 15

Compound of general formula (I) in which M is Zn, R=R ₁ =R ₂ = R _$ = [3-(N, N, N- trirnethylammonium)phenoxy] chloride, with R, R ₁ , R ₂ , R ₃ in positions 2,3,9,10,16,17,23,24 [Compound 15] EXAMPLE 16 Evaluation of antimicrobial activity in darkness a) in vitro experiments

An evaluation of in vitro non-photodynamic antimicrobial activity was carried out for Compounds 1-12 prepared as described above, by using the following procedure. Cells of Candida albicans (ATCC 10231) were grown in Fluid Sabouraud Medium (Difco Laboratories, Detroit, Ml) at 37°C under aerobic conditions. Cells in the stationary growth phase were harvested (3,000 g, 15 min), washed with phosphate buffer saline (PBS), and diluted to a final concentration of 1x10 ⁶ cells/m! corresponding to an absorbance of 0.12 at 630 nm. Samples (1 ml) were prepared by adding to PBS suitable volumes of the phthalocyanine stock solutions in DMSO (the final concentrations of the tested phthalocyanines range from 1 to 50 μM) so that the final amount of DMSO in the sample is 5% v/v. The samples were then incubate 37 ⁰ C for 1 h. CeI! survival was determined by serial 10-fold dilution in PBS of sample aliquots (10 μl), then plated in triplicate onto Sabouraud dextrose agar (Difco). After incubation of the plates at 37°C for 24 h, colony forming units (CFU/ml) were counted and the minimal bactericidal concentration (MBC), corresponding to the concentration of active principle that cause a > 4 !og ₁₀ of reduction of CFU/ml) was determined. Control experiments were carried out in the absence of the phthalocyanine derivative. All experiments were repeated in triplicate.

The MBC values of the tested compounds, as well as that ones found with the same protocol for two known antibiotics, nystatin and amphotericin, are reported in

the following Table 1. Table 1

From the results in the Table 1 above, it is evident that the present phthalocyanine derivatives have a non-photodynamic antimicrobial activity equivalent to that of the two antibiotics taken as a reference, that are the most used antifungal antibiotics due to their remarkable fungicidal activity. Compounds 1-3 and 10 have shown MBC values even higher than those of nystatin and amphotericin. b) In vivo experiments An animal model of infection (S. aureus wound infection in guinea-pig) was used to assess the in vivo activity of Compounds 1 , 9, 13, 14, and 15 prepared as described above, in comparison to classic antibiotic regimen for the treatment of infections caused by Gram+ bacteria, consisting in administration of Imipenem, the most used and effective antimicrobial agent for this kind of infections. The experimental protocol is reported below.

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

Bacteria: Staphylococcus aureus (ATCC 6538) was used. Bacterial colonies were harvested after overnight growth on TSA (Tryptic Soy Agar) and suspended in

phosphate-buffered saline (PBS, pH=7.4) to achieve a standard turbidity (A ₆ oo reading of 0.4). Serial 10-fold and 2-fold diiutions were performed to prepare a range of inocula. The inoculum of 2 x 10 ⁷ cells/ml was mixed 1 :1 (vol/vol) ratio with dextran microbeads (Cytodex, Sigma Chemicals Co., St.Louis, Mo) and used in our experiments. Backcounts were determined in triplicate and averaged to determine precise size of the bacterial inoculum.

Guinea pig model: The dorsal hair of animals was removed and a grid was drawn designating 8 areas for intermuscular inoculation. Each site of 2 guinea pigs for treatment groups was inoculated with 0.2 ml of the bacterium-bead suspensions. Control guinea pigs received PBS (vehicle). After the inoculation the guinea pigs were returned to their cages. After one day, 0.1 ml of solutions in PBS of the above said selected Compounds of the invention, or of imipenem was administered in each abscess. One day later, the guinea pigs were sacrificed; by using a sterile technique abscess were removed, weighed and homogenised in 3 ml of saline solution for 2 min (Ultraturrax). Homogenates were diluted and plated onto blood agar. The 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 compound-treated groups were compared to the vehicle-treated group to give percentage inhibition of abscess development. The abscess weight means of the compound-treated groups were compared to the vehicle-treated group to give percentage abscess decreasing. All experiments were repeated two times in different days. The obtained results are summarised in the following Tables 2-7, related to Compounds 1 , 9, 13, 14, 15 and to Imipenem respectively. Table 2 - Compound 1

Table 3 - Compound 9

Concentration (μg/kg) CFU inhibition % Abscess decreasing %

0.002 0 0

0.1 4.8 ± 3.0 0

0.2 45.3 ±1.2 10.6 ±0.8

1 55.1 ±6.6 9.9 ± 2.4

2 55.9 ± 7.2 11.7±1.3

20 80.0 ±8.1 30.6 ±12.0

200 94.5 ±5.2 30.9 ± 5.7

Table 4 - Compound 13

Concentration (μg/kg) CFU inhibition % Abscess decreasing ' Vo

0.044 0.0 0.0

0.22 56.4 ±7.3 16.6 ±3.8

0.44 57.8 ± 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 (μg/kg) CFU inhibition % Abscess decreasing %

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 {μg/kg) CFU inhibition % Abscess decreasing %

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 - lmipenem

*Classic regimen for parenteral administration