SMALL CATIONIC ORTHO-5,15-DI-HETEROARYL-PORPHYRINS DERIVATIVES AND THEIR APPLICATIONS IN PHOTOINACTIVATION OF MICROORGANISMS

Abstract

The present invention relates to small cationic ortho-5,15-di-heteroaryl porphyrin derivatives, in particular porphyrins, chlorins or bacteriochlorin of formula (I) or pharmaceutically acceptable salts thereof.

This invention also relates to the use of the above-mentioned cationic ortho-5,10-di-heteroaryl porphyrin derivatives of Formula (I) or a pharmaceutically acceptable salts thereof, in photodynamic inactivation of microorganisms, where the referred derivatives are able to treat the same in the presence of an adequate light.

The present invention also describes pharmaceutical compositions comprising one or more of the cationic ortho-5,10-di-heteroaryl porphyrin derivatives, in particular prophyrins, chlorins or bacteriochlorins of Formula (I), or pharmaceutically acceptable salts thereof, for the treatment of bacterial and/or fungi and/or yeasts and/or viral infections, in humans or animals.

Claims

1. Cationic ortho-5,15-di-heteroaryl porphyrin derivatives, namely porphyrins, chlorins or bacteriochlorin of Formula I: ##STR00012## wherein: represents a carbon-carbon single bond or a carbon-carbon double bond; M is H.sub.2 or a metal ion selected from Mg, Al, Si, Zn, Pd, Ag, In; R is chosen from unsubstituted or substituted alkyl, unsubstituted or substituted heteroalkyl, unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, provided that R has fewer than 12 atoms; For n=0, then Z.sub.1, Z.sub.2 and Z.sub.3 are each independently chosen from oxygen or from YR″, where Y are atoms in the ring each independently chosen from carbon, sulfur or nitrogen, and R″ is bonded to Y and each independently chosen from hydrogen or from R; For n=1, then Z.sub.1, Z.sub.2 and Z.sub.3 are each independently chosen from YR″, where Y are atoms in the ring each independently chosen from carbon, sulfur or nitrogen, R″ is bonded to Y, and R′ and R″ are each independently chosen from hydrogen or from R; or pharmaceutically acceptable salts thereof; for use in the photodynamic inactivation of microorganisms.

2. Cationic ortho-5,15-di-heteroaryl porphyrin derivatives for the use according to claim 1, in particular porphyrins of Formula (Ia): ##STR00013## wherein represents a carbon-carbon single bond or a carbon-carbon double bond; M is H.sub.2 or a metal ion selected from Mg, Al, Si, Zn, Pd, Ag, In; R is chosen from unsubstituted or substituted alkyl, unsubstituted or substituted heteroalkyl, unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, provided that R has fewer than 12 atoms; For n=0, then Z.sub.1, Z.sub.2 and Z.sub.3 are each independently chosen from oxygen or from YR″, where Y are atoms in the ring each independently chosen from carbon, sulfur or nitrogen, and R″ is bonded to Y and each independently chosen from hydrogen or from R; For n=1, then Z.sub.1, Z.sub.2 and Z.sub.3 are each independently chosen from YR″, where Y are atoms in the ring each independently chosen from carbon, sulfur or nitrogen, R″ is bonded to Y, and R′ and R″ are each independently chosen from hydrogen or from R; or pharmaceutically acceptable salts thereof.

3. Cationic ortho-5,15-di-heteroaryl porphyrin derivative for use according to claim 2, wherein in Formula (Ia): M is Zn Z.sub.1 is YR″ where Y is nitrogen in the ring bond to Z.sub.2 and to carbon, and R″ is methyl bound to Y; Z.sub.2 and Z.sub.3 are carbon; R is methyl; n=0, such that the derivative has Formula (IIa); ##STR00014## or pharmaceutically acceptable salts thereof.

4. Cationic ortho-5,15-di-heteroaryl porphyrin derivatives as described in claim 1, in particular bacteriochlorins of Formula (Ib): ##STR00015## wherein: represents a carbon-carbon single bond or a carbon-carbon double bond; M is H.sub.2 or a metal ion selected from Mg, Al, Si, Zn, Pd, Ag, In; R is chosen from unsubstituted or substituted alkyl, unsubstituted or substituted heteroalkyl, unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, provided that R has fewer than 12 atoms; For n=0, then Z.sub.1, Z.sub.2 and Z.sub.3 are each independently chosen from oxygen or from YR″, where Y are atoms in the ring each independently chosen from carbon, sulfur or nitrogen, and R″ is bonded to Y and each independently chosen from hydrogen or from R; For n=1, then Z.sub.1, Z.sub.2 and Z.sub.3 are each independently chosen from YR″, where Y are atoms in the ring each independently chosen from carbon, sulfur or nitrogen, R″ is bonded to Y, and R′ and R″ are each independently chosen from hydrogen or from R; or pharmaceutically acceptable salts thereof.

5. Cationic ortho-5,15-di-heteroaryl bacteriochlorins according to claim 4, wherein in Formula (Ib): M is Zn; Z.sub.1 is YR″ where Y is nitrogen in the ring bond to Z.sub.2 and to carbon, and R″ is methyl bound to Y; Z.sub.2 and Z.sub.3 are carbon; R is methyl; n=0, or pharmaceutically acceptable salts thereof.

6. Cationic ortho-5,15-di-heteroaryl porphyrin derivatives as described in claim 1, in particular chlorins of Formula (Ic): ##STR00016## wherein represents a carbon-carbon single bond or a carbon-carbon double bond; M is H.sub.2 or a metal ion selected from Mg, Al, Si, Zn, Pd, Ag, In; R is chosen from unsubstituted or substituted alkyl, unsubstituted or substituted heteroalkyl, unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, provided that R has fewer than 12 atoms; For n=0, then Z.sub.1, Z.sub.2 and Z.sub.3 are each independently chosen from oxygen or from YR″, where Y are atoms in the ring each independently chosen from carbon, sulfur or nitrogen, and R″ is bonded to Y and each independently chosen from hydrogen or from R; For n=1, then Z.sub.1, Z.sub.2 and Z.sub.3 are each independently chosen from YR″, where Y are atoms in the ring each independently chosen from carbon, sulfur or nitrogen, R″ is bonded to Y, and R′ and R″ are each independently chosen from hydrogen or from R; or pharmaceutically acceptable salts thereof.

7. Cationic ortho-5,10-di-heteroaryl chlorins according to claim 6, wherein in Formula (Ic): M is Zn; Z.sub.1 is YR″ where Y is nitrogen in the ring bond to Z.sub.2 and to carbon, and R″ is methyl bound to Y; Z.sub.2 and Z.sub.3 are carbon; R is methyl; n=0, or pharmaceutically acceptable salts thereof.

8. Cationic ortho-5,15-di-heteroaryl porphyrin derivatives for use according to claim 1, wherein the microorganisms are bacteria.

9. Cationic ortho-5,15-di-heteroaryl porphyrin derivatives for use according to claim 1 wherein the microorganisms are in biofilms.

10. Cationic ortho-5,15-di-heteroaryl porphyrin derivatives described in claims 1-7 for use in the treatment of infectious diseases caused by microorganisms including bacteria, fungi, yeasts, viruses or protozoa.

11. Cationic ortho-5,15-di-heteroaryl porphyrin derivatives described in claims 1-7 for use as disinfectants and/or antiseptics and/or in prevention of infectious diseases caused by microorganisms.

12. A pharmaceutically composition comprising at least one of the derivatives described in claims 1-7 and a pharmaceutically acceptable carrier.

13. A pharmaceutically composition according to claim 12, additionally including a small molecule inhibitor of pathogen efflux systems and/or a small polycationic molecular species that disrupts the outer membrane of the microorganism and/or an antimicrobial peptide and/or a species that undergoes electron transfer to the photosensitizer triplet state to generate reactive radicals and potentiate the photodynamic inactivation of the microorganism.

14. The pharmaceutically composition described in claims 12-13 for use in the treatment of bacterial and/or viral and/or fungi infections.

15. The pharmaceutically composition described in claims 12-13 for use in topical therapies.

16. A method of eliminating microorganisms in a real patient environment comprising the steps of: selecting at least one cationic ortho-5,15-di-heteroaryl porphyrin derivative described in claims 1-7, preparing a pharmaceutical composition according to claims 12-13, applying said pharmaceutical composition to the target intended for the elimination of the microorganisms, allowing time for said porphyrin derivative to accumulate in said targeted microorganisms; and, irradiating said targeted microorganisms with light of an appropriate wavelength to activate said porphyrin derivative to destroy said microorganisms.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Without intent to limit the disclosure herein, this application presents attached drawings of illustrated embodiments for an easier understanding.

[0069] FIG. 1: Chem 3D minimized structure of Formula (IIa) where the atoms shown in black have an excess of positive charge

[0070] FIG. 2: Structures of the molecular species of Formula (I), (Ia), (Ib) and (Ic).

[0071] FIG. 3: Structures of the molecular species of Formula (II), (IIa), (IIb) and (IIc).

[0072] FIG. 4: Structures of the molecular species of Formula (III) and (IV).

[0073] FIG. 5. General synthetic approach for the synthesis of precursors and compounds of Formula (Ia)

[0074] FIG. 6. General synthetic approach for the synthesis of precursors and compounds of Formula (IIa)

[0075] FIG. 7. Normalized absorption spectrum of compound with Formula (IIa) using water as solvent.

[0076] FIG. 8: PDI of planktonic bacteria for various photosensitizer concentrations 0 μM (blank); 0.1 μM (oblique lines) and 1 μM (crosses or horizontal lines) with 60 min incubation and a light dose of 1.36 J/cm.sup.2 at 415 nm.

[0077] FIG. 9. PDI of planktonic bacteria for various photosensitizer concentrations 0 μM (blank); 0.1 μM (oblique lines) and 1 μM (crosses), with 60 min incubation and a light dose of 1.36 J/cm.sup.2 at 415 nm.

[0078] FIG. 10. Confocal microscopy of biofilms incubated with the molecular species of formula (IIa) where the fluorescence of the molecular species of Formula (IIa) in the biofilm is shown in red and the endogenous fluorescence of planktonic bacteria is in green.

[0079] FIG. 11. PDI of S. aureus biofilms under white light (40 mW/cm.sup.2) using 5.2 nM of molecular species with structure (IIa).

[0080] FIG. 12. Dark cytotoxicity (oblique lines) and phototoxicity (white bars) under a light dose of 5 J/cm.sup.2 of molecular species with Formula (IIa) towards on fibroblasts (A) and keratinocytes (B).

DESCRIPTION OF THE EMBODIMENTS

[0081] Referring to the drawings, herein are described optional embodiments in more detail, which however are not intended to limit the scope of the present application.

[0082] A. Materials and Methods

[0083] All solvents were dried according to standard procedures. All commercial reagents were purchased from Sigma-Aldrich and Fluorochem and used without further purification. The .sup.1H and .sup.13C nuclear magnetic resonance (NMR) spectra were recorded on a 400 Bruker Avance spectrometer (400 and 101 MHz, respectively), using tetramethylsilane (δ=0.00 ppm) as internal standard for .sup.1H and .sup.13C. The electrospray ionization (ESI) mass spectra were obtained at the Mass Spectrometry Unit (UniMS), ITQB/iBET, Oeiras, Portugal.

[0084] Optical Absorption: The ultra-violet visible optical absorption (UV-vis) spectra used in the control of the synthesis were recorded on Hitachi U-2001 or Shimadzu 2100 spectrophotometers using spectroscopic grade solvents. The UV-Vis-NIR optical absorption was recorded with an Agilent Cary5000 UV-Vis-NIR Spectrophotometer in the determination of the molar absorption coefficient and with Shimadzu UV-2100 spectrometer in routine measurements. The absorption spectra were recorded in the wavelengths from 300 nm up to 800 nm. Molar absorption coefficients were determined by using Beer-Lambert's law. For each compound, a minimum of 6 solutions were prepared in concentrations ranging from 10.sup.−7 to 10.sup.−6 M, giving absorbance values between 0.1 and 1.

[0085] Fluorescence Emission: The fluorescence emission spectra were recorded in a Horiba Scientific Spectrofluorometer Fluoromax-4. The spectra were collected from 550 nm up to 800 nm using standard cuvettes of 1 cm of optical path. Fluorescence quantum yields (Φ.sub.F) were obtained comparing the area of integrated fluorescence of the samples with that of a reference fluorimetric compound with known Φ.sub.F, corrected by the absorption of sample and reference at the excitation wavelength and by the refractive indices of the solvents used for the standard and reference solution. Tetraphenylporphyrin (TPP) in toluene (Φ.sub.F=0.11) was used as standard. The absorbance of the solutions at the excitation wavelength was 0.01.

[0086] Singlet Oxygen Quantum Yield: The experiments were run at room temperature. The solutions were excited at 355 nm using a Nd-YAG laser (Spectra-Physics Quanta-Ray GRC-130) and the phosphorescence of singlet oxygen collected at 1270 nm in a Hamamatsu R5509-42 photomultiplier, cooled to 193 K in a liquid nitrogen chamber, after selection of the wavelength with a monochromator with 600 lines grading. A Newport filter model 10LWF-1000-B was used in the emission to avoid scattering and fluorescence. Phenalenone was used as a reference of singlet oxygen generator, Φ.sub.Δ.sup.Ref=0.98. Extrapolating to time-zero the decays of the singlet molecular oxygen emissions measured for sample and the reference solutions at a given laser intensity, we obtained a relation between emission intensities as a function of laser intensity, that is identical to the relation between the singlet molecular oxygen quantum yields. The singlet oxygen quantum yields were obtained by comparing the linear dependence between and the energy of the laser pulse for both the sample and the reference for the same absorption of sample and reference at the excitation wavelength, taking into account the singlet oxygen quantum yield of phenalenone.

[0087] n-Octanol:PBS partition ratio: a modification of the shake-flask method was employed to determine the equilibrium concentrations of the photosensitizer in n-octanol and in phosphate-buffered saline (PBS) mixed in equal volumes, using the typical fluorescence band of the same photosensitizer and the ratio of the fluorescences after dilution by the same factor with dimethylsulfoxide.

[0088] Photobleaching experiments: Photodecomposition experiments were made dissolving the photosensitizers in water with 9% dimethylsulfoxide. A volume of 3 mL was placed in a cuvette and irradiated ensuring that all the light hits the solution. The porphyrins were irradiated using a LED light with emission at 415 nm and an effective output power of 0.27 mW. Photodecomposition quantum yield (Φ.sub.pd) is defined as the ratio between the rate of disappearance of photosensitizer molecules v.sub.d and the rate of absorption of photons v.sub.p.

[0089] Phototoxicity towards bacteria was evaluated in vitro. Assays were performed with the following bacteria: Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 29213. Further assay were performed with clinical antibiotic-resistant strains from the Centro Hospitalar da Universidade de Coimbra, namely Staphylococcus aureus methicillin-resistant (MRSA) strain Sa1CHUC recovered from the skin of a burnt patient (resistant to all beta-lactamic antibiotics), and Acinetobacter baumannii 141HUC isolated from an exudate of a burn wound and highly resistant to all beta-lactamics (penicillins, cephalosporins, monobactams and carbapenems), quinolones and the aminoglycosides gentamicin and netilmicin. The planktonic bacteria cells were cultured in Mueller Hinton (MH) agar (Sigma Aldrich) at 37° C. overnight. Cell density was adjusted to the 0.5 McFarland standard in sterile water, which corresponds to approximately 1.5×10.sup.8 CFU/mL. For PDI experiments cell suspensions of bacteria were incubated with various concentrations of the photosensitizers for 1 h, in the dark, at room temperature, using a 96 well plate. Then, the plate was illuminated with a blue light LED (420 nm, 4 mJ/s). Cells incubated with photosensitizers in the dark were covered with aluminum foil for the same time as the PDI groups (1 h). After illumination (or dark incubation) samples were shaken, diluted in PBS and mixed. Aliquots were taken from each well and streaked in MH agar in duplicate for CFU determination and incubated for 37° C./18-24 h in the dark. After 24 h, the colonies were counted and CFU determined. The experiments were performed in triplicate. Statistical analysis was performed with GraphPad Prism 6.

[0090] Phototoxicity towards biofilms was evaluated after biofilm growth overnight. Assays were performed with biofilms from Staphylococcus aureus ATCC 25925. Initially, bacteria cultures were diluted 1:9 in Brain Heart Infusion (BHI) (Kasvi®, Brazil). The microorganisms were centrifuged (1500 rpm, 10 min) and washed twice with phosphate-buffered saline (PBS). Aliquots of the diluted bacterial suspensions were inoculated into 24-well flat-bottom sterile polystyrene microplates and incubated for 24 h at 37° C. For PDI experiments, the plates with biofilms were incubated with 5.2 nM of the photosensitizer for 30 min in the dark at room temperature. Wells used as controls were incubated with PBS only. After that, the plates were illuminated with a specially designed light source named Biotable®. The Biotable® was composed by 24 LED lamps that deliver a uniform light fluence rate of 30 mW/cm.sup.2 in the wavelength range between 400-650 rm. Wells used as controls were incubated with PBS only. Cells incubated with photosensitizers in the dark were covered with aluminum foil for the same time as the PDI cells (1 h). Following irradiation (or dark control), photosensitizer was carefully removed from the wells and the biofilms were washed once with PBS. The biofilms were scraped carefully, sonicated and then vortexed to homogenize the samples. Treated and untreated samples were serially diluted, plated on the MH petri dishes, and incubated for 24 h at 37° C. in the dark to allow colony formation. After this time, the colonies were counted and the colony-forming units (CFU) determined.

[0091] Toxicity towards human cell lines was evaluated in vitro using the 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT, from Sigma Aldrich) assay to estimate the viability of cells after appropriate treatment. After cell attachment, photosensitizer solutions in PBS at concentrations between 0 to 10 μM were added to the cell cultures and incubated for 1 h at 37° C. in the dark. After illumination with the Biotable® or after the equivalent time in control experiments, MTT dissolved at 5 mg/ml in PBS was added to each well (final concentration 0.5 mg/ml), and the microplates were further incubated for 3-4 hours. Medium were then discarded and 100 μl of methanol were added to the cultures and mixed thoroughly to dissolve the dark blue crystals of formazan. Formazan quantification was performed using an automatic microplate reader (Multiskan Go Thermo) by absorbance measurements at 570 nm. Each experiment was repeated three times. Data were expressed as mean absorbance value of six samples and standard error of the mean.

[0092] Confocal microscopy images were acquired using a Zeiss fluorescence confocal microscope (LSM 780 inverted model) with laser excitation (LASER Diode 405 nm). The microscope is equipped with high sensitivity GaAsP detectors for spectral imaging (400-700 nm).

[0093] B. Description of Methods of Preparation of the Compounds Precursors

[0094] The general scheme for the preparation of porphyrin precursors is depicted below (FIG. 5):

##STR00010##

[0095] Non-symmetric 5,15-disubstituted porphyrins precursors were prepared by mixture of equimolar amounts of commercially available dipyrromethane (Harvechem) with the desired heteroaromatic aldehyde dissolved in an appropriate solvent. The solvent was degassed with an inert gas and selected from dichloromethane, chloroform, carbon tetrachloride, toluene, dioxane, N-methyl-2-pyrrolidone, tetrahydrofuran or dimethoxyethane. Then, an acid was added as catalyst which was selected from organic, namely trifluoroacetic acid, p-toluenosulfonic acid, tricloroacetic acid, or inorganic namely, aluminosilicate (Al—NaY) or sulfonic clay. The reaction vessel was shielded from ambient light and stirred under inert gas, for 15 min to 6 hours, at temperature between 0° C. and 50° C. The cyclization to porphyrinogen was monitored by thin-layer chromatography (TLC). Then, the porphyrinogen was oxidized to the correspondent porphyrin. The appropriate amount of oxidant was chosen from O.sub.2, O.sub.2/light, nitrobenzene/organic acid (acids from 2 to 13 carbon atoms) or preferentially from high potential quinones, namely 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) or 2,3,5,6-tetracyano-1,4-benzoquinone, for 5 min to 10 hours, at temperature between 25° C. to 100° C. After solvent removing, the crude was dissolved in the appropriate solvent (dichloromethane, chloroform, carbon tetrachloride, toluene, dioxane, N-methyl-2-pyrrolidone, tetrahydrofuran, dimethoxyethane) and washed with a saturated solution of an appropriate base. The base can be inorganic and selected from carbonates, phosphates or organic, or can be selected from amines, preferentially trimethylamine. After that, a silica gel column chromatography was performed, using the appropriate solvent mixture (solvents can be selected from: dichloromethane, chloroform, carbon tetrachloride, toluene, dioxane, tetrahydrofuran, dimethoxyethane, ethyl ether, ethyl acetate, methanol, ethanol).

[0096] Non-symmetric metal complexes precursors of 5,15-dissubstituted porphyrin were prepared by reaction of 5,15-dissubstituted porphyrin with the selected metal salt. The 5,15-dissubstituted porphyrin was dissolved in the appropriate solvent, selected from dichloromethane, chloroform, carbon tetrachloride, toluene, dioxane, N-methyl-2-pyrrolidone, tetrahydrofuran, dimethoxyethane; dimethylformamide, and an excess (1-50 equivalents) of the selected metal salt is added. The metal salt was selected from magnesium dichloride, magnesium acetate, aluminium trichloride; zinc acetate, zinc chloride; palladium acetate, palladium dichloride, silver acetate, silver chloride; indium trichloride, indium acetate or trichlorosilane. Then, the reaction was heated up at temperatures between −25° C. to 200° C. The complexation was monitored by UV-vis and thin-layer chromatography (TLC). Once complete, the solvent was removed and the solid dissolved in the appropriate solvent, selected from dichloromethane, chloroform, carbon tetrachloride, toluene, tetrahydrofuran, ethyl ether or ethyl acetate, and the impurities are extracted with water (3-7 times). The organic layer was dried using an inorganic drying agent selected from anhydrous sodium sulfate, sodium sulfate, anhydrous calcium sulfate; anhydrous calcium sulfate; anhydrous calcium oxide. After decantation, the organic solvent was evaporated, and pure metal complex was isolated.

Synthesis of cationic ortho-5,15-di-heteroaryl porphyrins

[0097] The cationic ortho-5,15-di-heteroaryl porphyrin (Formula Ia; M=2H) was achieved via alkylation of ortho-nitrogen atom (Formula Ia, R) using the selected halogenated substituted or unsubstituted alkyl, heteroalkyl, aryl, heteroaryl (R≤12 atoms). The selected precursor 5,15-di-heteroaryl porphyrin was dissolved in a solvent, selected from dichloromethane, chloroform, carbon tetrachloride, toluene, dioxane, N-methyl-2-pyrrolidone, tetrahydrofuran, dimethoxyethane; dimethylformamide and an excess (2 to 100 equivalents) of the selected halogenated substituted or unsubstituted alkyl, heteroalkyl, aryl, heteroaryl was added. The reaction was maintained at temperature between 20° C. and 100° C. for 1 to 96 hours. The progress of the reaction was followed by TLC. 5,15-di-heteroaryl porphyrin (Formula Ia; M=2H) was precipitated with a selected solvent. The solvent was selected from an apolar solvent namely, diethyl ether, dichloromethane, hexane, pentane, chloroform. The solid was filtrated and recrystallized from the selected solvent (methanol, ethanol, propanone, ethyl acetate) and pure cationic ortho-5,15-di-heteroaryl porphyrins (Formula I; M=2H) were isolated.

Synthesis of metal complexes of cationic ortho-5,15-di-heteroaryl porphyrins

[0098] The metal complexes of cationic ortho-5,15-di-heteroaryl porphyrin derivatives (Formula I; M=Mg, Al, Zn, Pd, Ag, In) were synthetized via alkylation of ortho-nitrogen atom (Formula Ia, R) using the selected halogenated alkyl, heteroalkyl, aryl, heteroaryl (R≤12 atoms). The selected metal complex of 5,15-di-heteroaryl porphyrin precursor was dissolved in a solvent, selected from dichloromethane, chloroform, carbon tetrachloride, toluene, dioxane, N-methyl-2-pyrrolidone, tetrahydrofuran, dimethoxyethane; dimethylformamide and an excess (2 to 100 equivalents) of the selected halogenated alkyl, heteroalkyl, aryl, heteroaryl was added. The reaction was maintained at temperature between 20° C. and 100° C. for 1 to 96 hours. The evolution of the reaction was followed by TLC. The product 5,15-bis(1,3-dimethylimidazol-2-yl)porphyrinate (Formula I; M=Mg, Al, Zn, Pd, Ag, In) was precipitated with a selected solvent. The solvent was selected from an apolar solvent namely, diethyl ether, dichloromethane, hexane, pentane, chloroform. The solid was filtrated and recrystallized from the selected solvent (methanol, ethanol, propanone, ethyl acetate) and pure cationic ortho-5,10-di-heteroaryl porphyrins (Formula I; M=Mg, Al, Zn, Pd, Ag, In) were isolated.

Synthesis of cationic ortho-5,15-di-heteroaryl bacteriochlorins (Formula Ib)

[0099] The cationic ortho-5,15-di-heteroaryl porphyrin derivatives were used as precursors to obtain the corresponding reduced bacteriochlorins. The reduction was based on the diimide reduction method using hydrazide as the hydrogen source, preferably using p-toluenesulfonyl hydrazide (p-TSH), inorganic or hindered organic bases, in solvents selected from dioxane, N-methyl-2-pyrrolidone, tetrahydrofuran, dimethoxyethane, dimethylformamide, pyridine and picoline, using a modification of the method disclosed in PCT/EP2005/012212 (16). The reduction can also take place in the absence of solvents and in the absence of bases, using a modification of the method disclosed in PCT/PT2009/000057 (17). After cooling (room temperature) the solid was dissolved in water and the excess of hydrazide was removed using Amicon device with membranes with the appropriated molecular weight cut-offs. The water was lyophilized, the solid obtained recrystallized from the selected solvent (methanol, ethanol, propanone, ethyl acetate) and pure cationic ortho-5,15-di-heteroaryl bacteriochlorins were isolated.

Synthesis of cationic ortho-5,15-di-heteroaryl chlorins (Formula Ic)

[0100] The cationic ortho-5,15-di-heteroaryl porphyrins were used to obtain the corresponding reduced chlorins. The reduction was based on the diimide reduction method using hydrazide as the hydrogen source, preferably using p-toluenesulfonyl hydrazide (p-TSH), inorganic or hindered organic bases, in solvents selected from dioxane, N-methyl-2-pyrrolidone, tetrahydrofuran, dimethoxyethane, dimethylformamide, pyridine and picoline, using a modification of the method disclosed in PCT/EP2005/012212 (16). The reduction can also take place in the absence of solvents and in the absence of bases, using a modification of the method disclosed in PCT/PT2009/000057 (17). After cooling (room temperature) the solid was dissolved in water and the excess of hydrazide was removed using Amicon device with membranes with the appropriated molecular weight cut-offs. A mixture of chlorin and small amounts of bacteriochlorin was obtained. The mixture of chlorin and small amounts of bacteriochlorin was dissolved in an appropriated solvent selected from dioxane, N-methyl-2-pyrrolidone, tetrahydrofuran, dimethoxyethane, dimethylformamide and oxidized to the corresponding chlorin. The oxidation was performed by heating the mixture at 20° C. to 100° C. in the presence of air or by adding FeCl.sub.3.6H.sub.2O (0.5-10 equivalents) followed by hydrogen peroxide (3% in water, 0.1-10 mL). The final solution was kept under stirring, at room temperature for 30 minutes to 6 hours. Once complete, the solvent was removed and the solid dissolved in in water and the excess of hydrazide was removed using Amicon device with membranes with the appropriated molecular weight cut-offs. The water was lyophilized, the solid obtained recrystallized from the selected solvent (methanol, ethanol, propanone, ethyl acetate) and cationic ortho-5,15-di-heteroaryl chlorins (Formula Ic) were isolated.

[0101] C. Properties of the Compounds

[0102] The absorptivities of the compounds and other photophysical and photobiological properties were measured as described in the Materials and Methods. The wavelength of maximum absorption in the infrared did not vary in the concentration range studied. This is indicative of negligible aggregation between the molecules, which exist mostly as monomers at the studied concentrations in the selected solvents. Table 1 presents the molar absorption coefficient (ε.sub.max) of the most intense absorption band of a typical cationic ortho-5,15-di-heteroaryl porphyrin derivative of Formula (I), more specifically, of the porphyrin derivative of Formula (IIa) in water with 9% dimethylsulfoxide. The same table also presents the fluorescence quantum yield (Φ.sub.F), singlet oxygen generation (Φ.sub.Δ) and the logarithm of the n-octanol:water partition coefficient (log P.sub.OW). The phototoxicity of the same derivative are also presented in Table 1 for human fibroblast and human keratinocyte cell lines. Table 2 presents the phototoxicity towards bacteria, including multidrug-resistant strains and bacteria in biofilms, described in the Materials and Methods.

TABLE-US-00001 TABLE 1 Photophysical and photochemical properties of the cationic ortho-5,15-di-heteroaryl porphyrin derivative of Formula (IIa), together with phototoxicity towards human fibroblast and human keratinocyte cell lines for a light dose of 5 J/cm.sup.2. λ ε(λ)/10.sup.3 log Survival fraction at 10 μM nm M.sup.−1 cm.sup.−1 Φ.sub.F Φ.sub.Δ P.sub.ow Fibroblast Keratinocytes 407 >20 0.10 0.75 −1.16 100% 80%

TABLE-US-00002 TABLE 2 Photobiological properties the cationic ortho-5,15- di-heteroaryl porphyrin derivative of Formula (IIa) in planktonic bacteria under 1.36 J/cm.sup.2 at 415 nm and in bacterial biofilms under 5 J/cm.sup.2 in the wavelength range between 400-650 nm. log CFU log CFU at 1 μM at 5.2 nM S. E. P. S. Aureus Acinetobacter S. Aureus Aureus Coli Aerogirosa MRSA 141Hu biofilm 7 7 3 7 7 7

[0103] The intensity of its longest wavelength absorption band at 622 nm decreased by less than ΔA=0.001 after 15,000 second of illumination corresponding to an effective absorption of ca. 4 J. This places an upper limit of 0.001 to the photodecomposition quantum yield.

[0104] The typical photophysical, photochemical and photobiological properties of cationic ortho-5,15-di-heteroaryl porphyrin derivative of Formula (I) remedy the shortcoming aforementioned of current photosensitizers employed in PDI of microorganisms. In particular, the molecular species of Formula (I) can have small size, proper distribution of positive charge, solubility in biocompatible vehicles, intense light absorption, moderate fluorescence for convenient monitoring and very sigh singlet oxygen quantum yields.

[0105] The conjugation of photostability, strong absorption in the phototherapeutic window, high yield of ROS and proper distribution of positive charge density offers another advantageous technical characteristic to the porphyrin derivatives of formula (I): very low phototoxicity towards human cells but very high phototoxicity towards bacteria. Table 1 shows an example of a photosensitizer according to Formula (I) that incubated in a 10 μM concentration with human fibroblast and human keratinocyte cell lines under a light dose of 5 J/cm.sup.2 has very low phototoxicity: more 80% of the human cells survive these conditions. However, when incubated at 1 μM concentration with Gram-negative S. Aureus or Gram-negative E. Coli and exposed to 1.36 J/cm.sup.2 or light at 415 nm, the number of bacterial CFU is reduced by 7 orders of magnitude. More importantly, the same happens when said photosensitizer is used in PDI of a methicillin-resistant Staphylococcus aureus strain or an Acinetobacter baumannii strain highly resistant all beta-lactamics, including carbapenems, quinolones, gentamicin and netilmicin. The phototoxicity against a Staphylococcus aureus biofilm is even more impressive: incubation with a ca. 5 nM concentration of said photosensitizer followed by exposure to 5 J/cm.sup.2 of white light to leads to a 7 orders of magnitude reduction of bacteria in the biofilm.

[0106] The ability of porphyrin derivatives of Formula (I) to diffuse rapidly to they target, combined with their low phototoxicity towards human cells and high phototoxicity towards microorganisms, make these porphyrin derivatives especially suitable for antimicrobial and/or antiviral and/or anti-fungi and/or anti-yeasts and/or anti-protozoa medications for human or animal usage exhibiting as a main active agent one or several porphyrin derivatives described in the present invention. This type of medication, used in particular in PDI, may also contain one or several pharmaceutically acceptable excipients. Additionally, the formulation may contain small molecule inhibitors of pathogen efflux systems and/or a small polycationic molecular species that disrupts the outer membrane of the microorganism and/or an antimicrobial peptide and/or a species that undergoes electron transfer to the photosensitizer triplet state to generate reactive radicals and potentiate the photodynamic inactivation o the microorganism.

[0107] The reactive oxygen species generated by the illuminated photosensitizer molecules trigger a cascade of chemical and biological processes that culminate in the death of the bacteria and/or viruses and/or fungi and/or yeasts and/or protozoa.

[0108] The compounds of the present invention may also fluoresce with reasonable quantum yields and in the phototherapeutic window. Table 1 presents an example of a photosensitizer with Φ.sub.F=0.10. This typical fluorescence can be used to detect the presence of the compound in the target tissue and offers the possibility of using the compounds of the present invention for the visualization of infections originated by microorganisms.

EXAMPLES

[0109] This invention will now be described in more detail in the following non-limiting EXAMPLES.

Example 1. Procedure for the Preparation of 5,15-bis(1,3-dimethylimidazol-2-yl)porphyrinate zinc (II) diiodide

[0110] ##STR00011##

[0111] A solution of commercial dipyrromethane (438 mg, 3 mmol) and 1-methylimidazole-2-carboxaldehyde (330 mg, 3 mmol) in CH.sub.2Cl.sub.2 (300 ml) was degassed with a continuous stream of argon, for 10 min, before addition of catalytic amounts of TFA (153 μL, 2 mmol). The reaction vessel was shielded from ambient light and stirred under argon, for 3 hours, at T=25° C. Then, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) (2.04 g, 6 mmol) was added, at once, to the reaction mixture, and stirring at temperature of 30-50° C. was pursued for 1 hour. After removal of the solvent, the crude was dissolved in CH.sub.2Cl.sub.2 and washed with a saturated solution of sodium bicarbonate. After that, a silica gel column chromatography was performed, using dichloromethane/methanol (10:1) as eluent. After solvent evaporation 5,15-bis(1-methylimidazol-2-yl)porphyrin was isolated and after drying, under vacuum, with 19% yield (0.134 g). .sup.1H NMR (400 MHz, CDCl.sub.3): δ mixture of atropoisomers 10.35 (s, 2H), 9.43 (d, J=4.3 Hz, 4H), 9.03 (d, J=4.3 Hz, 4H), 7.73 (d, J=10.7 Hz, 2H), 7.54 (s, 2H), 3.52 (s, 6H), −3.31 (s, 2H). UV-vis (CH.sub.2Cl.sub.2): λ.sub.max/nm (log ε): 406 (4.83), 500 (3.75), 535 (3.54), 573 (3.37), 627 (3.12). ESI-MS [M+H]+ (CH.sub.2Cl.sub.2), m/z: 471.20405; calculated for [C.sub.28H.sub.23N.sub.8].sup.+: 471.20402.

[0112] Next, the precursor 5,15-bis(1-methylimidazol-2-yl)porphyrin (86 mg; 0.183 mmol) was dissolved in 10 mL of chloroform. Separately, zinc acetate (401 mg; 1.83 mmol) was dissolved in 3 mL of methanol and added to the previously solution, at temperature of 25° C., under stirring. The complexation was monitored by UV-vis and thin-layer chromatography (TLC). Once the reaction completed, the solvent was removed and the solid dissolved in dichloromethane and the excess of metal salts extracted with water. The organic layer was dried using anhydrous sodium sulfate and the solvent was removed. The solid was dried under vacuum, yielding 70 mg (81% yield) of 5,15-bis(1-methylimidazol-2-yl)porphyrinate zinc (II). UV-vis (DMSO): λ.sub.max/nm (log ε): 415 (4.29), 545 (3.14), 581 (2.94); .sup.1H NMR (400 MHz, DMSO): δ mixture of atropoisomers 10.38 (d, J=3.8 Hz, 2H), 9.66 (d, J=4.1 Hz, 4H), 9.04 (d, J=4.3 Hz, 2H), 8.94 (d, J=4.6 Hz, 2H), 8.02 (s, 2H), 7.62 (s, 2H). Finally, the quaternization of imidazoyl groups of 5,15-bis(1-methylimidazol-2-yl)porphyrinate zinc (II) (20 mg, 0.0375 mmol) was achieved via methylation of nitrogen imidazoyl atoms with a large excess of iodomethane (100 eq) using preferentially DMF as solvent (0.15 mL) at 30° C., for 12-24 hours. The progress of the reaction was followed by TLC. The product 5,15-bis(1,3-dimethylimidazol-2-yl)porphyrinate zinc (II) diiodide (Formula IIa) was precipitated with diethyl ether and, after filtration and recrystallization, using preferentially methanol or ethanol as solvent, the 5,15-bis(1,3-dimethylimidazol-2-yl)porphyrinate zinc (II) diiodide (Formula IIa) was obtained in almost quantitative yields. .sup.1H NMR (400 MHz, DMSO-D.sub.6): δ 10.73 (s, 2H), 9.81 (d, J=4.5 Hz, 4H), 9.07 (d, J=4.5 Hz, 4H), 8.50 (s, 4H), 3.70 (s, 12H). UV-vis (H.sub.2O): λ.sub.max/nm (log ε): 406 (4.37), 540 (3.09), 573 (3.34). ESI-MS [M−I]+ (MeOH), m/z: 689.06134; calculated for [C.sub.30H.sub.26IN.sub.8Zn].sup.+: 689.06111.

[0113] FIG. 7 presents the absorption spectrum of compound with Formula (IIa).

Example 2. Phototoxicity Towards Gram-Positive Staphylococcus aureus ATCC 29213, Gram-Negative Escherichia coli ATCC 25922 and Gram-Negative Pseudomonas aeruginosa ATCC 27853

[0114] This example describes the evaluation of in vitro phototoxicity against Gram-positive and Gram-negative bacteria of the cationic ortho-5,15-di-heteroaryl porphyrin with Formula (IIa). The phototoxicity was measured according to the description in the Materials and Methods section. The porphyrin with Formula (IIa) is soluble in water and its use did not require a specific formulation. There is a dose-phototoxicity response of the test compound relative to the non-treated control. FIG. 8 illustrates the CFU reduction as a function of photosensitizer concentration for the light dose of 1.36 J/cm.sup.2 at 415 nm. Nanomolar concentrations of the test compound after 60 min incubation and under low light doses produce a dramatic decrease in CFU.

Example 3. Phototoxicity Towards Multidrug Resistant Gram-Positive Bacterial Strains of Staphylococcus aureus and Multidrug Resistant Gram-Negative Bacterial Strains of Acinetobacter

[0115] This example describes the evaluation of in vitro phototoxicity against multidrug-resistant Gram-positive and Gram-negative bacteria of the cationic ortho-5,15-di-heteroaryl porphyrin with Formula (IIa). Assays were performed with clinical resistant strains from the Centro Hospitalar da Universidade de Coimbra, namely Staphylococcus aureus methicillin-resistant (MRSA) strain Sa1CHUC collected from the skin of a burnt patient (resistant to all beta-lactamic antibiotics), and Acinetobacter baumannii 141HUC highly resistant all beta-lactamics, including carbapenems, quinolones, gentamicin and netilmicin, isolated from an exudate of a burn wound. The phototoxicity was measured according to the description in the Materials and Methods section. The porphyrin with Formula (IIa) is soluble in water and its use did not require a specific formulation. There is a dose-phototoxicity response of the test compound relative to the non-treated control. FIG. 9 illustrates the CFU reduction as a function of photosensitizer concentration for the light dose of 1.36 J/cm.sup.2 at 415 nm. At 1 μM concentration of the test compound, followed by 60 min incubation and then exposure to low light doses, a dramatic decrease in CFU is observed.

Example 4. Phototoxicity Towards Biofilms of Staphylococcus Aureus

[0116] Staphylococcus aureus ATCC 25925 biofilms were grown on 24-well flat-bottom sterile polystyrene microplates. After 24 hours, the plates were observed under confocal microscopy and biofilm formation with a mean thickness of 20 μm was corroborated. Then, a 1 μM solution of the cationic ortho-5,15-di-heteroaryl porphyrin with Formula (IIa) was added and the fluorescence intensity from the biofilm was followed over the incubation time of 1 h for the said photosensitizer. FIG. 10 shows the fluorescence of the molecular species of Formula (IIa) after 30 min of incubation with biofilms.

[0117] For the phototoxicity study, Staphylococcus aureus ATCC 25925 biofilms grown on 24-well flat-bottom sterile polystyrene microplates were incubated with 5.2 nM of the cationic ortho-5,15-di-heteroaryl porphyrin with Formula (IIa) for 30 min in the dark at room temperature. Wells used as controls were incubated with PBS only. After the incubation period, the plate was illuminated with a Biotable®. Cells incubated with photosensitizer in the dark were covered with aluminum foil for the same time as the PDI cells. Following irradiation (or dark control), the biofilm was carefully removed from the wells and washed once with PBS. The biofilms were scraped carefully, sonicated and then vortexed to homogenize the samples. Treated and untreated samples were serially diluted, plated on the MH petri dishes, and incubated for 24 h at 37° C. in the dark to allow colony formation. After this time, the colonies were counted and CFU determined. The experience was made nine times. FIG. 11 shows the inactivation of the biofilms using a 5.2 nM concentration of the photosensitizer with Formula (IIa).

Example 7. Toxicity Towards Fibroblasts (HDFn-Gibco) and Keratinocytes (HaCaT) Cell Lines

[0118] This example describes the evaluation of in vitro toxicity against HDFn neonatal human dermal fibroblast and HaCaT immortalized human keratinocyte cell lines of a cationic ortho-5,15-di-heteroaryl porphyrin with Formula (IIa). Both types of cells were grown in DMEM (BioTech) with addition of 10% fetal bovine serum (Cultilab—Campinas, SP, Brazil). Before the experiments, the cells were removed by trypsinization, washed with PBS and maintained in a humidified atmosphere at 37° C. and 5% CO.sub.2. The cells were incubated in the dark with the photosensitizer of Formula (IIa) in PBS for 30 min at concentrations up to 10 μM, and then illuminated with the Biotable® to deliver 5 J/cm.sup.2 in the wavelength range between 400-700 nm. After exposure to light, the cells were washed with fresh medium and plates were returned to the incubator for 24 h. Cell viability was determined by a MTT assay performed 24 h after irradiation. The cells in the control experiment remained in the dark for the time of the illumination. FIG. 12 shows that fibroblasts and keratinocytes incubated with a 10 μM concentration of the test drug in the dark did not show a statistically significant reduction of cell viability. Exposure to a light dose of 5 J/cm.sup.2 led only to a 20% decrease in the viability of fibroblasts at the highest drug dose tested: 10 μM. These examples show that for drug and light doses where the photosensitizer of Formula (IIa) is very phototoxic towards bacteria and biofilms, it is not toxic or phototoxic towards human cells.

BIBLIOGRAPHY

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