NO300777B1 - Photosensitizers, processes for their preparation, pharmaceutical compositions containing them, and their use - Google Patents

Description

This invention relates to photosensitizers, more specifically it relates to new transition metal photosensitizers.

In photodynamic therapy of cancer, certain dye compounds (eg, hematoporphyrin derivative, chloroaluminum phthalocyanine sulfonate) are given to an individual with a tumor. To a certain extent, these dye compounds are taken up by the tumor tissue, and after selective irradiation with the right light source, the tumor tissue is destroyed via the dye-dependent photo-formation of toxic material such as singlet oxygen.

A large number of phthalocyanine (Pc) derivatives have been proposed as potential therapeutic photodynamic (PDT) agents. Most biological studies of Pc compounds in connection with PDT have been carried out with water-soluble sulfonated metal phthalocyanines [as reported by I. Rosenthal, Photochem. Photobiol. 53(6), 859-870 (1991)]. These compounds are generally obtained by sulfonation of the corresponding metallo-phthalocyanine or by template synthesis using the corresponding sulfonated precursors and a metal salt. Both template synthesis and direct sulfonation lead to mixtures of

PCs containing a variety of isomers and/or different degrees of sulfonation. This is a particular disadvantage with respect to pharmaceutical applications by drug control authorities

are increasingly strict in their requirements for substantially pure compounds.

Metallized Pcs have been found to have better photosensitizing action compared to metal-free Pcs when the metal is a main group element with a filled d-shell (eg Al, Zn, Sn, In). It has been reported by W.S. Chan et al., Photochem. Photobiol., 45, 757-761 (1987) that transition metal complexes of Pcs have been found to be inactive (eg

Cu, Co, Ni, V, Pd).

There remains a need for photosensitizers that can be produced in pure isomeric form and that show a good level of activity.

The present invention provides new transition metal -phthalocyanine-type derivatives of formula I, where M is a second- or third-row transition metal with a d<6->low spin electronic configuration,

X is hydrogen, alkyl, aloxy, halide or adjacent X's can together form -C4H4-,

each R is a ligand selected from phosphine, arsine, amine, isocyanide, nitrile, thiolate, hydrazine, cyanide, thiocyanate, phenolate, sulfide, and aniline groups with a water-soluble residue, and

Q is nitrogen or -CY-, where Y is hydrogen, alkyl, alkoxy or halide, in water-soluble salt or acid form.

Second- and third-row transition metals with a d<6->low-spin electronic configuration include Tc<1>, Re<1>, Ru11, Os11, Rh111, Ir111, PdIV and Pt<I>v.

Preferably, M is selected from Ru, Rh, Os or Ir. Suitable ligands R include triphenylphosphine or triethylphosphine, and solubilizing groups are suitably sulphonate or carboxylate groups. When R contains an amine, it may be a straight or branched chain amine, or an aromatic amine such as pyridine. Preferred R-ligands are triphenylphosphine, mono-, di- or tri-sulfonate, 4-pyridine ethanesulfonate, 3-pyridine sulfonate, triphenylphosphine monocarboxylate, 4-isocyanobenzoate, nicotinic acid, taurine or amino acids.

Preferably, the compound is in salt form with counterions which are preferably K<+>, Na<+> or quaternary ammonium.

The compounds of formula I are new, and can be prepared by a method consisting of reacting a metal phthalocyanine compound with formula II

where M, Q and X are as defined above, and A is an amine, preferably a pyridine group, CO (carbon monoxide) or a coordination solvent, e.g. benzonitrile, with a salt of the ligand R, and isolation of the product compound of formula

IN.

Many of the reactants of formula II and the salts of the ligand R are known from the literature. However, compounds of formula II wherein A is amine, benzonitrile, methyl cyanide and other coordinating solvent are believed to be novel and form part of the present invention. Although such compounds can be prepared by methods analogous to those previously known, the invention further provides a method for preparing the new compounds of formula II, in which A is benzonitrile by reacting M phthalocyanine-bis(amine) with benzonitrile. The amine complex can be prepared by reacting MC^-xI^O, where x is 2 or 3, with phthalonitrile, and then with ammonia.

Appropriately, the starting metal phthalocyanine is mixed

or -naphthalocyanine in an organic solvent, such as mixed xylenes, with an excess, e.g. 2-10 times stoichiometric, of an organically soluble form of the water-solubilizing ligand under an inert atmosphere, such as argon. The reaction is preferably carried out by heating, e.g. at reflux for approx. 2 days. The product can be isolated by adding a co-solvent to the reaction mixture. If necessary, the solubility of the product can be increased by replacing the counterions in a generally known manner.

It is believed that the present invention, by introducing a water-solubilizing axial ligand in place of the usual substitution on the periphery of Pc to achieve water solubility, enables the synthesis of isomeric pure compounds. The new compounds are found to be active in vitro and in vivo tests for photosensitizing activity described below.

The present invention also provides a pharmaceutical composition comprising a compound of formula I in admixture or combination with a pharmaceutical carrier or diluent. The invention also encompasses the use of a compound of formula I for the preparation of a medicament for photodynamic therapy". The therapy consists in the mammal being administered an effective dose of a compound of formula I or a pharmaceutically acceptable salt thereof, and the tumor being exposed to light irradiation.

The pharmaceutical mixtures can be formulated according to well-known principles, and can, if desired, be in the form of a unit dose determined according to usual pharmacological methods. The unit dosage forms can provide a daily dose of active compound in a single dose or in a number of smaller doses. The dosage range can be determined using standard pharmacological methods and is expected to be in the range of 1 to 50 mg per day. kilograms of body weight. Other active compounds may be used in the compositions or administered separately, or adjunctive therapy may be included in a course of treatment for a patient. The pharmaceutical mixtures can suitably be available in the form of solutions or suspensions for injection, or in forms for topical application including application in the oral cavity. Suitable carriers and diluents are well known in the art, and the compositions may contain excipients and other ingredients to provide easier or more efficient administration.

After administration to the patient, photodynamic therapy can be carried out in the usual way using light sources and delivery systems known in the field. See e.g. Phys. With. Biol.

(1986), 31, 4, 327-360.

The invention will now be illustrated through the following examples which particularly describe aspects of the invention, but in no way limit its scope.

The sodium salt of triphenylphosphine monosulfate [Na(I)] and 4-pyridineethanesulfonic acid was purchased, and Ru(Pc)(pyridine)2 was prepared by a literature method. (N.P. Farrell et al. Inorg. Chim. Acta. 28 L144-L146, 1978).

EXAMPLE 1

Co[ Ru( Pc) bis( triphenylphosphine monosulphate trihydrate (compound Al

The tetrabutylammonium salt of triphenylphosphine monosulfonate [TBA(I)] was prepared by mixing 0.2 g of Na(I) and 0.23 g of tetrabutylammonium bisulphate [TBA(HS04)] in basic aqueous solution and extracting TBA( I) in methylene chloride. After washing the organic phase with water, solvent was removed to give 0.34 g of TBA(I) as a yellow oil.

TBA(I) (0.34 g, 0.58 mmol) and Ru(Pc)(Py) 2 (0.15 g, 0.20 mmol) were mixed in a mixed xylene solvent (10 mL) and the mixture heated at reflux for two days. Several times during the reaction time, a portion of the solvent was allowed to evaporate to remove pyridine, and sufficient xylenes added to keep the reaction volume at 10 mL. After cooling to room temperature, diethyl ether was added to precipitate the blue solid. This material was dissolved in a minimum volume of ethanol and filtered. Potassium acetate (0.5 g) in 5 ml of ethanol is added to this solution. The resulting crystalline blue solid was collected, washed with ethanol and diethyl ether and air dried. Yield = 0.22 g. 78% based on Ru.

Analysis for <C>g8H5oK2N8P20gRuS2

Calculated: C, 57.13; H, 3.53; N, 7.84

Found: C, 57.13; H, 3.63; N, 8.05

EXAMPLE 2

K2 [ Ru( Pc ) bis( 4- pyridineethanesulfonatl tetrahydrate

The tetrabutyl ammonium salt of 4-pyridine ethanesulfonate [TBA-(II)] was prepared by mixing TBA[HS04] (6 g) and 3 g of the pyridine in water and adding 30% NaOH solution until the pH was greater than 11. This gave an oil which was subjected to rotary evaporation in a mixed xylene solvent. The residue was dissolved in methylene chloride, filtered and extracted three times with water. Removal of the solvent gave a pale green oil.

TBA(II) (0.72 g, 1.68 mmol) and Ru(Pc)(Py) 2 (0.14 g, 0.18 mmol) were dissolved in 25 mL of mixed xylene solvent and the mixture heated to reflux under N 2 for two days, which gave an almost colorless solution. The reaction mixture was cooled to room temperature and the resulting blue solid collected. Yield of [TBA] 2 [Ru(Pc)(4-pyridineethanesulfonate) 2 ] = 0.23 g. This material was dissolved in ethanol (12 mL) and filtered. Addition of potassium acetate (0.35 g) in ethanol (3.5 mL) precipitated a blue solid which was collected, washed with ethanol and diethyl ether and air dried. Yield = 0.174 g. 87% based on Ru.

Analysis for C4gH4oK2N-L00ioRuS2

Calculated: C, 48.62; H, 3.55; N, 12.33

Found: C, 48.46; H, 3.64; N, 12.06

EXAMPLE 3

Na2[ Ru( Pc) bis( taurine) 1hexahydrate

Taurine (0.28 g) and 0.20 mL of 10N NaOH in water were stirred in 10 mL of ethanol for 2 hours. RuPc(benzonitrile) 2 (0.30 g) and 10 mL of toluene were added and the resulting mixture was refluxed under nitrogen overnight. The solvents were removed and the residue was washed with toluene and methylene chloride. The remaining solid was dissolved in methanol and the solution filtered. The addition of toluene caused the product to precipitate. The solid was collected, washed with methylene chloride and air-dried. Yield = 0.25 g (70%).

Analysis for C3g<H>4o<N>10Na2012RuS2

Calculated: C, 42.56; H, 3.97; N, 13.79

Found: C, 42.55; H, 3.85; N, 13.59

Many other compounds within the scope of the invention

was produced by using methods analogous to those in test

vein above. The structures are indicated in abbreviated form in the table

len that follows the in vitro test section below. Most of the compounds are characterized by their light absorption maxima (Xma]^sQ) and molar extinction coefficients (e M" <1>cm_1).

Utaanas materials

Ruthenium phthalocvanine bis( amine) Å

Ruthenium trichloride hydrate [RuC^-xI^O) (2.32 g) was heated in 25 mL of pentanol until the solution had turned completely blue and all the water had distilled off. The solution was added over 3 minutes to 6.5 g of phthalonitrile and 0.54 g of hydroquinone dissolved in 20 ml of boiling pentanol. The resulting orange suspension was refluxed for 3 days under slow purging with ammonia gas. The reaction mixture was allowed to cool to room temperature and the purple solid filtered off. The solid was washed with methanol and methylene chloride repeatedly, until the washings were almost colorless. The solid was air dried. The yield of 6.58 g of RuPc(NH3)2 was used without further purification.

Ruthenium phthalocyanine bis(benzonitrile) monohydrate

The RuPc(NH3)2 was refluxed in 75 mL of PhCN for one day

under nitrogen. The mixture was diluted to 1 liter with hot chloroform, stirred at reflux for one hour. The solution was filtered hot, and 3 liters of methanol were added to the filtrate. A first crop of product, 5.39 g, was filtered off after 16 hours. A second harvest was isolated by reducing the filtrate to 2.5 liters by boiling. This gave an additional 1.48 g of product. The total yield was 6.87 g, 84%, purple crystals.

Analysis for C4gH28<N>ioORu

Calcd: C, 65.94; H, 3.37; N, 16.72

Found: C, 65.58; H, 3.33; N, 16.78

Photochemical and biological studies Singlet oxygen formation

Singlet oxygen formation was determined by photodegradation of nitrosoanaline dye followed by the method of Kraljic and Mohsni ( Photochem. PhotoBiol. , 28., 577-581 (1978) ). A stable buffer of oxygen saturated with 50 mM Na-phosphate pH = 7, 5. 16 mM imidazole (recrystallized from benzene) was mixed 1:1 with the test compound in water and 2.5 µl/ml N,N-dimethyl-p-nitroso-aniline was added in the dark.

100 years 1 of the test solution was plated in triplicate on a Corning 96-well plate and exposed to light from a Kodak dias projector with an unfiltered 300 W 82U FHS incandescent lamp producing blackbody radiation at 1700K at a distance of 3 5 cm from the plate, which is kept at 4°C in a cold box for 15 minutes of irradiation.

Cleavage of the nitroso dye is followed at 450 nm with a Dynatech MR600 microplate reader (test X 450 nm ref. X 540 nm).

Attached fig. 3 shows the relationship of singlet oxygen formation to PC50 for various ruthenium phthalocyanine compounds according to the invention and aluminum phthalocyanine sulfonate. In the figure, the abbreviations for the ligands are used to represent the entire molecule, e.g. RuPc(TPPMS)2 = TPPMS. A full list of abbreviations is provided below.

The described compounds produce singlet oxygen in an amount that varies tenfold with respect to molar sensitizer concentration. However, there is no correlation between the degree of singlet oxygen production and the efficacy of the compounds as measured by the PC50 values of the compounds. The rate of singlet oxygen production for aluminum phthalocyanine sulfonate is of the order of one to two logs better than any of the sensitizers listed and yet the efficiency of many

of these compounds higher than the aluminum compound. This

suggests improved absorption of these compounds or a new site of action inside the cells.

Phototoxicity (light)

Phototoxicity experiments follow the procedure used by Glassberg et al. (E. Glassberg, L. Lewandowski, G. Lask and J. Vitto, Invest Dermatol. 94. 604-610 (1990). Adherent cells (HeLa RCA. SKOV OVCAR) are seeded at 4 x 10<4> cells per well in a 94-well Corning microtiter plate in RPMI 164 0 and FCS for four hours.The sensitizer is added to the same media in a fixed concentration range in triplicate, and the cells are incubated for 24 hours at 37°C with 5% C02.

The media is removed, the cells are washed three times with D-PBS, and then covered with D-PBS (200 µl) and exposed to light from the same source used in the singlet oxygen measurement. The irradiation time can be varied between 1 and 15 minutes, at which time D-PBS is removed and fresh medium RPMI-1640 is added and the cells are incubated as before for 16 hours.

<3>H-thymidine (50 µl) in RPMI-1640 + FCS is added, and the cells are incubated for an additional 4 hours, at which time they are harvested on glass filter mats, and the incorporation of <3>H-thymidine is determined relative to an untreated (no sensitizer added) control.

Cvtotoxicity

This follows the procedure for phototoxicity, except that the cells are never exposed to light. The measurement is relative to an untreated control.

Results

The in vitro results obtained with compound A, K 2 [RuPc-(TPPMS) 2 ] are shown in comparison with chloroaluminum phthalocyanine sulfonate obtained from Porphyrin Products, Logan, Utah, in Fig. 1 on the attached drawings.

Compounds of the invention were determined and characterized by their light absorption maxima and their molar extinction coefficients according to standard experimental procedures. Most of the compounds were tested for their biological activity according to standard, known experimental procedures. PC5q represents phototoxic concentration 50%, which is the concentration of a compound which, when exposed to a light dose as described herein, causes a 50% reduction in cancer cell growth relative to an untreated control population. The phototoxicity index, PI, is the ratio of PC50 to the cytotoxic concentration 50%, where growth is reduced by 50% without a light dose being given.

The results are listed in the table below. The abbreviations used to identify the compounds are indicated after the table. PC50 is determined by a relatively simple in vitro measurement at the cellular level, which is generally indicative of photosensitizing activity. However, since some photosensitizers work at a tissue/organ level, inactivity in the PC50 measurement, as shown for certain compounds below, does not determine that the compounds are not active.

Abbreviations Core structures

In vivo studies

The photodynamic threshold dose in normal rat liver was used as a model. This has been developed by Singh and Wilson at McMaster University and used to study both chloroaluminum phthalocyanine sulfonate (ASPc) and Photofrin (M.S. Patterson et al., Photochem. Photobiol. 51. 343-349, 1990, and T.J. Farrell et al., Proc. SPIE. 1426. 146-155, 1991).

Approach

1. Inject the compound at 2.5 mg/ml into the tail vein of adult Wistar rats at doses of 5, 10, 20 mg/kg. 2. 24 hours later subject the liver to laparotomy and irradiate the surface in up to four locations with a 5 mm diameter beam of light from an argon dye laser operating at 650 nm: input power = 40 m Watt; input fluence = 200 mW per cm<2>; treatment time = 5 or 10 minutes (60, 120 Joules per cm2). 3. 24 hours later, inject Evans Blue intravenously, euthanize after 15 minutes, cut liver through each irradiation spot and measure the depth of necrosis shown by color exclusion.

Results

The measured depths of necrosis are indicated in table 1 below and shown in fig. 2 together with the most comparable data for AISPc and Photofrin (which was given ip not iv). Compound A is clearly shown to be photodynamically active in vivo. The depth of necrosis at similar drug and light levels is comparable to that of previously studied agents, including the current clinical photosensitizer, Photofrin.

Claims (13)

1. Chemical compound, characterized in that it is a transition metal phthalocyanine or -naphthalocyanine derivative of formula I where M is a transition metal from the second or third row of transition metals in the periodic table with a d^-low-spin electron configuration, X is hydrogen, alkyl, alkoxy, halide, or adjacent X's can together form -C4H4-, each R is a ligand selected from phosphine, arsine, amine isocyanide, nitrile, thiolate, hydrazine, cyanide, thiocyanate, phenolate, sulfide, and aniline groups with a water solubilizing residue, and Q is nitrogen or -CY-, where Y is hydrogen, alkyl, alkoxy or halide, in water-soluble salt or acid form. 2. Connection according to claim 1, characterized by atMer Ru, Rh, Os or Ir. 3. Connection according to claim 2, characterized in that M is Ru. 4. Compound according to claim 1, 2 or 3, characterized in that R is selected from triphenylphosphine, triethylphosphine or amine with one or more sulphonate or carboxylate groups. 5. Connection according to claim 4, characterized in that R is selected from triphenylphosphine and pyridine with one or more sulfonate or carboxylate groups. 6. Compound according to each of the preceding claims, characterized in that the phthalocyanine structure is substituted with eight methyl or methoxy groups. 7. Connection according to claim 1, characterized in that it is Z2[Ru(Pc)bis(triphenylphosphine monosulphate)], where Z is a counterion. 8. Process for producing a compound of formula I according to claim 1, characterized in that a compound of formula II wherein M, Q and X are as defined in claim 1, and A is an amine, CO or a coordinating solvent, is reacted with a salt of the ligand R, and isolation of a compound of formula I. 9. Method according to claim 8, characterized in that the counterion in the product compound of formula I has been replaced in order to obtain a stronger desired salt form of the compound of formula I. 10. Method according to claim 8 or 9, characterized in that the reaction is carried out in organic solvent under an inert atmosphere. 11. Method according to claim 8, 9 or 10, characterized in that the salt of the ligand R is used in excess. 12. Pharmaceutical mixture, characterized in that it comprises a compound of formula I according to each of claims 1 to 7 in mixture or in association with a pharmaceutically acceptable carrier or diluent. 13. Use of a compound of formula I according to each of claims 1 to 7 for the production of a drug for photodynamic therapy.