Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
  • C07D487/22—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains four or more hetero rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/28—Compounds containing heavy metals
  • A61K31/315—Zinc compounds
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/555—Heterocyclic compounds containing heavy metals, e.g. hemin, hematin, melarsoprol
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents

Definitions

• the invention relates to the field of photosensitising compounds for therapeutic use, and in particular to novel phthalocyanine derivatives of formula (I) given hereinafter, having photosensitising characteristics and high solubility in water, useful for the photodynamic treatment of bacterial infections, in particular infections generated by Gram-negative bacteria.
• STATE OF THE ART Molecules containing the phthalocyanine chromofluorophore macrocycle are known to produce reactive oxygen species, such as radicals or singlet oxygen, by interacting with visible light.
• phthalocyanine compounds have long been used in photodynamic therapy (hereinafter abbreviated to "PDT”) for both therapeutic treatment and diagnostic purposes.
• phthalocyanine derivatives are useful for the photodynamic treatment of bacterial infections caused by Gram-negative bacteria.
• the derivatives of formula (I) in accordance with the invention although included in the general formula given in US 5,965,598, were not specifically identified therein.
• the present derivatives have also demonstrated a surprising effectiveness in photodynamic therapy PDT against bacterial infections due to Gram-negative bacteria, unexpectedly higher than similar products described in the aforesaid US patent.
• the particular antibacterial activity towards Gram- negative bacteria makes them useful for treating bacterial infections caused by this type of micro-organisms alone, and for treating mixed infections due in part to Gram-negative bacteria and in part to other bacterial species.
• Their solubility in water has also proven to be surprisingly high, with a consequent higher bio-availability and faster metabolism within the organism compared to similar products described in US 5,965,598.
• the present products of formula (I) have the ability to localise onto target cells where, following irradiation with light of determined wavelength, they produce reactive species which damage the cell itself. Because of their short duration these reactive species hit the target cell and damage it without any possibility of their spreading to nearby cells, while those that do not hit the biological target rapidly decay. By virtue of using said products, PDT therapy is therefore selective and does not lead to the phenomena of systemic or local skin phototoxicity.
• the phthalocyanine derivatives of the invention possess a higher molar extinction coefficient than the photosensitising agents currently used in therapy, this guaranteeing effective therapeutic response.
• the present products are moreover activated by tissue penetrating radiation which has a wavelength longer than 650 nm and are therefore suitable for applications in PDT therapy against localised infections, whether dermatological or of mucosal surfaces, as well as deep seated infections due to the penetration characteristics of these radiations; when irradiated, they induce the production of reactive oxygen species even in conditions of low oxygenation, this being an important requisite for products that have to enable specific treatment against anaerobic micro-organisms, well known to proliferate in oxygen poor environments.
• the products of the invention have a limited toxicity against host tissues and/or cells, but they re-activate if irradiated once more in a continuous production process of reactive species.
• Sources of light suitable for carrying out the PDT treatment are known in the art and comprise white light, suitably filtered non-coherent sources at a wavelength of preferably between 650 and 750 nm or lasers specific for the wavelength of the present compounds.
• the total quantity of light radiation used varies according to the treatment and the tissues to be treated, and generally it is comprised between 50 and 1000 J/cm 2 , preferably between 100 and 350 J/cm 2 .
• the products of the invention have showed a particularly good effectiveness against Gram-negative bacteria in contrast to other products of very similar structure which, though effective for example against Gram-positive bacteria however demonstrate a lower photodynamic activity against Gram- negative bacteria compared with the present products.
• the present phthalocyanine derivatives can be prepared starting from commercial products by the following process, also subject of the invention.
• the present process comprises the following steps: i) nucleophilic substitution of the amino alcohol of formula (III) onto the phthalonitrile of formula (II) to obtain the compound of formula (IV)
• Step ii) of reductive methylation can be carried out for example by treating the primary amine of formula (IV), dissolved in a suitable solvent, with a carbonylating agent in the presence of a reducing agent; preferred conditions are those in which the amine (IV), dissolved in acetonitrile, is treated with 30% aqueous formaldehyde and sodium cyanoborohydride.
• Step iii) of the process of the invention can be carried out in an organic solvent, preferably water miscible such as dimethylformamide (hereinafter abbreviated to DMF), using as the base a base chosen from 1 ,5-diazabicyclo[5.4.0]non-5-ene (hereinafter abbreviated to DBN), 1 ,8-diazabicyclo[5.4.0]undec-7-ene (hereinafter abbreviated to DBU) and 2-dimethylamino-ethanol (hereinafter abbreviated to DMAE), or, preferably, step iii) is carried out in the absence of solvent with a base chosen from the aforementioned.
• DMF dimethylformamide
• DBN 1 ,5-diazabicyclo[5.4.0]non-5-ene
• DBU 1 ,8-diazabicyclo[5.4.0]undec-7-ene
• DMAE 2-dimethylamino-ethanol
• reaction times vary according to the scale of synthesis, while the temperatures can vary from 100 to 250°C, preferably between 130 and 180°C.
• Optimal results are obtained when step iii) is carried out in the absence of solvents, with DBU as the base, and at a temperature equal to 140°C.
• the crude product of formula (Vl) is preferably precipitated by treating the reaction mixture of step iii) with water, filtering or centrifuging the suspension then washing the recovered solid several times with water and methanol.
• the intermediate (Vl) coming from step iii) is preferably purified by column chromatography followed by re-precipitation from solvent prior to undergoing the subsequent step.
• the stationary phase for the chromatography is for example silica gel, while the mobile phase is a mixture of dichloromethane and methanol; for the re-precipitation, dichloromethane for example can be used as the solvent and n-hexane as the precipitant.
• the methyl iodide is used in a quantity between 1 and 20 equivalents per amino group to be methylated, preferably in a quantity between 5 and 11 equivalents.
• the methylation reaction is furthermore typically carried out in a solvent, preferably selected from the group consisting of DMF, dimethylsulfoxide (hereinafter abbreviated to DMSO) and N-methylpyrrolidone (hereinafter abbreviated to NMP).
• DMSO dimethylsulfoxide
• NMP N-methylpyrrolidone
• suitable Zinc(ll) salt in step iii) means for example Zinc(ll) chloride or
• Zinc(ll) acetate preferably Zinc(l I) acetate.
• the product of formula (VII) is precipitated from the solution in NMP with ethyl ether or isopropyl ether in a quantity of 8 volumes with respect to the volume of NMP, after having diluted the solution in NMP with methanol in a quantity of 2 volumes with respect to the volume of NMP.
• the exchange of iodide for chloride at step v) of the present process is preferably undertaken by a chromatography process by using a solution of the iodide and an ion exchange resin and recovering the product from the solution by evaporation, lyophilisation or precipitation.
• Ion exchange resins suitable for undertaking the present process are strong basic resins, with quaternary ammonium functional groups, for example a polystyrene based resin with a degree of cross-linking between 4 and 10%, such as Amberlite ® IRA-400 (Cl) resin.
• step v) is carried out by a chromatography process by using a solution in methanol of the iodide of formula (VII) and a suitable resin, then treating the eluate with ethyl ether to precipitate the desired chloride of formula (I).
• step v) is carried out by a chromatography process by using a solution of the iodide of formula (VII) in a 8/2 mixture of methanol/DMSO and a suitable resin, then re-precipitating from the eluate the obtained chloride of formula (I) by treating it with ethyl ether and purifying it from the residual DMSO by dissolving in methanol and re-precipitating by adding ethyl ether.
• a preferred aspect of the process is the use of an eluent based on methanol rather than water for the ion exchange chromatography in step v).
• the final chloride is found to be further purified from related compounds present, achieving a HPLC purity greater than 98%.
• the present products can be used as active principles in combination with pharmaceutically acceptable excipients and diluents for preparing pharmaceutical compositions, such as for parenteral administration and topical application.
• the compositions can be formulated for example as aqueous solutions, lotions, creams, ointments or gels.
• dosages of the active principle can vary for example between 0.1 and 20 mg of product of formula (I) per Kg body weight, preferably varying between 0.2 and 5 mg per Kg body weight.
• compositions of the invention comprising, in addition to a product of formula (I), a metal chelating agent, chosen preferably among metal chelating agents having specificity for Ca 2+ and Mg 2+ ions, such as citric acid, 1 ,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA), diethylenetriaminepentaacetic acid (DTPA) or ethylenediamine-N,N,N',N'- tetraacetic acid (EDTA).
• a metal chelating agent chosen preferably among metal chelating agents having specificity for Ca 2+ and Mg 2+ ions, such as citric acid, 1 ,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA), diethylenetriaminepentaacetic acid (DTPA) or ethylenediamine-N,N,N',N'- tetraacetic acid (EDTA).
• reaction mixture thus obtained is maintained for 22 hours under vigorous stirring at room temperature, then treated with 11.2 I of deionised H 2 O and maintained for 30 minutes under stirring.
• the mixture thus obtained is heated to 50°C and maintained under hot conditions and under stirring for 22 hours. After this period, the mixture is brought again to room temperature, treated with 12 I of deionised H 2 O and maintained under stirring for 30 minutes then the suspension obtained is filtered and the solid washed with deionized H 2 O (2 x 2 I).
• the 2 sub-batches of Zinc(ll) [1 ,8(11),15(18),22(25)-tetrakis-(4-N,N,N- trimethylammonium phenoxy)] phthalocyaninate tetraiodide obtained as aforedescribed in paragraph d), each equal to 47.2 g, are processed as follows. 47.2 g of the aforesaid product are dissolved in 2 I of 4/1 MeOH/DMSO. The solution is subjected to column chromatography whose stationary phase is prepared with 470 g of Amberlite ® IRA 400 (Cl) resin, previously washed with an aqueous solution made acid by 0.5 M HCI and conditioned with 4/1 MeOH/DMSO.

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• 2005
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