Indium photosensitizers for PDT

The invention disclosed herein involves a phototherapeutic pyrrolic core complexed with a non-radioactive Indium atom. Complexation of Indium by the pyrrolic core forms a metalopyrrolic compound which influences enables these compounds to localize at target sites a phototherapy. Such functionally aids in both detection and phototherapy of disease sites, or provides functionality that binds to site specific receptors of a target area such that the therapy is improved.

BACKGROUND OF THE INVENTION

Porphyrins and related pyrrolic macrocycles, particularly tetrapyrrolic macrocycles, as well as many other light absorbing compounds are currently receiving a great deal of attention with regard to photosensitized medicine, especially in the field of Photodynamic therapy (PDT) 1 . The therapy necessarily involves the localization of a photosensitizing agent at or near a site of disease. The sensitizer, upon illumination in the presence of oxygen, produces cytotoxic species of oxygen such as singlet oxygen or oxygen radicals, which destroy the diseased cells. As the sensitizer is innocuous at the therapeutic dose, and only becomes active on illumination with light of a specific wavelength, PDT offers the possibility of a level of control or selectivity in the treatment of diseases not found with other current methods (e.g. conventional chemotherapy). Photodynamic therapy has wide application to modern medicine, targeting diseases such as cancers, cardiovascular restenosis and plaques, psoriasis, viral infections, benign prostate hyperplasia and diabetic retinopathy. In addition, photodynamic therapy may also be useful for the sterilization of blood, an area of increasing concern, especially now with the advent of AIDS and the transmission of HIV through blood transfusions.

Most research on PDT has centered on a complex mixture consisting of ill-defined porphyrin dimers, trimers and oligomers 2 , which is marketed under the designation Photofrin II . This complex mixture has recently been recommended for approval in the treatment of obstructed endobronchial tumors by the Food and Drug Administration Advisory Panel. Although the mixture has demonstrated the potential benefits of PDT, it has by virtue of its composition a number of associated disadvantages. For example, each of its components has varied subcellular localizations, depending on structure and inherent differences in photophysical properties. This makes the interpretation of pharmacokinetic data difficult. In addition, the composition of the oligomers is often difficult to reproduce and changes, depending on the conditions under which a solution 3 thereof is stored. Hence, the true active components may vary considerably. The mixture has a less than optimal light absorption profile (630 nm, 3,000 cm 1 M 1 ). Numerous studies on light penetration through tissues show that longer wavelength light penetrates deeper into tissues 4 . While not all applications of PDT require deep penetration of light, many applications of PDT require the maximum depth of light penetration possible (for example the treatment of brain tumors). The mixture has a severe adverse normal skin response to light, which often lasts for up to 12 weeks 5 after therapy. During this time patients must avoid strong light, as otherwise severe burns and edema occur.

Several well characterized second Generation sensitizers (SnET2 6 , ZnPc 7 , BPDMA 8 , THPC 9 ) are currently in phase I/II clinical trials and the continued development of new sensitizers that show improved therapeutic efficacy is crucial to the future progress of the therapy. The development of new photosensitizers that possess all the basic requirements to be effective PDT drugs is not an easy task. While the optimal photophysical properties of a second generation drug are well defined, the factors which aid in the localization of photosensitizers to tissues are not. Several investigators have attempted to ascertain structure-activity relationships in ring systems that lend themselves to chemical modification without dramatically influencing the photophysical properties of the compounds. Woodburn and coworkers, working on hematoporphyrin based analogues, have proposed that anionic compounds tend to localize in lysosomes, while cationic photosensitizers tend to localize in the mitochondria. Pandey and coworkers have suggested that the lipophilicity of the compound is important, and have demonstrated in a chlorophyll derived series that PDT effects vary with the length of the ether carbon chain. Unfortunately, many of the correlation's found in one group of photosensitizing compounds do not transfer to different groups of photosensitizing compounds. Thus, while structure-activity relationships are valuable for a particular class of compound whose geometry and spatial arrangements vary only slightly, a different class of compound, that inherently has its own spatial and geometric parameters, must have its own structure-activity relationships investigated. This in itself is a large time consuming process with ultimately no guarantees of enhanced localization or improved PDT efficacy for the modified compound.

The instant invention is based upon the discovery of a single simple chemical modification of compounds having a pyrrolic core involving the coordination of a non-radioactive indium salt into the central cavity of the pyrrolic core to produce an indium pyrrolic complex, which markedly enhances the biological efficacy of compounds as photosensitizers for PDT.

Two isotopes of indium occur naturally: In 113 and In 115 ; the former (natural abundance 4.23%) has no radioactivity, while the latter (natural abundance 95.77%) has a half life of 6 10 14 years, and, as a result, is also considered to be non-radioactive. Other indium nuclides have half lives ranging from 50.0 days (for In 114m ) to 0.2 second (for In 109m 2).

THE PRIOR ART

Tetrapyrroles containing Indium 111 (half life 2.81 days) and other metals are known in the art, being disclosed, by way of example, in Japanese Kokai 3-261786, 1991, Mauclaire et al., U.S. Pat. No. 5,268,371 and in Maier et al., U.S. Pat. No. 5,674,467, which also disclose their use for diagnostic imaging. The metal-tetrapyrrole compounds are all water soluble, and contain functional groups capable of coupling a biologically active molecule, such as an antibody, to the tetrapyrrolic nucleus. The compounds usually are prepared by reacting a porphyrin derivative with a solution of a salt of the metal to be complexed at a temperature and for a time sufficient to obtain the metal tetrpyrrole compound. For example, when a metal salt of indium 111 is heated for three hours at 110 C. in a porphyrin solution to which a mixture of acetic acid and sodium acetate has been added, an In 111 porphyrin is produced. Although the preparation of such metalloporphyrins is known and the compounds have been discussed in the context of radiocontrasting agents, surprisingly, so far as is known, there has been no report on the efficacy of indiumporphyrins as therapeutic photosensitizers. In view of the substantial prior art involving indium tetrapyrroles, it is indeed surprising to discover that complexes of non-radioactive indium with tetrapyrroles exhibit a marked improvement in their cytocidal effect in vivo when compared with other metallotetrapyrrole complexes.

R1, R2, R4 and R6 are methyl, R8, R10, R11 and R12 are H, and R7 and R9 are CH 2 CH 2 COR13 (where R13 is a residue which results when H is removed from an amino acid), R3 and R5 are ethyl, and In is not radioactive, i.e., is In 113 or In 115 .

Japanese Kokai 5-97857 discloses compounds having the following structure

Where R1 is CH(OR)Me, R is alkyl, R2 is a residue derived by removing H from an amino acid, and M is 2H, Ga, Zn, Pd, In or Sn.

The Following References Are Cited Above

1. For an overview of photodynamic therapy see Photodynamic Therapy of Neoplastic Disease , Vol. I and II, Ed. Kessel, D., CRC Press, 1990.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide pyrrolic compounds that absorb light at long wavelengths for use in photodynamic therapy and diagnosis of disease states.

It is another object to provide a reaction product of (A) P where P is a pyrrolic derivative and (B) In 113 X 3 and/or In 115 X 3 (where X is a charge balancing ion, either organic or inorganic) such as to provide the reaction product (C) PIn 113 n X n and/or PIn 115 n X n , where indium is coordinated to the pyrrolic derivative and n 1, 2, 3.

It is yet another object of the invention to provide a reaction product of (C) PIn 113 n X n and/or PIn 115 n X n with a neucleophile Y, such that a reaction product (D) PIn 113 n Y n X z and/or PIn 115 n Y n X z is obtained (where Y is a charge balancing ion, either organic or inorganic n 1,2,3 and z 0, 1, 2, 3).

It is still another object to provide compounds PIn 113 X and/or PIn 115 X that may be used to treat diseases such as atherosclerosis, restenosis, cancer, cancer pre-cursors, non-cancerous hyperproliferating diseases, psoriasis, macular degeneration, glaucoma and viruses, benign prostate hyperplasia, rheumatoid arthritis and to aid in the diagnosis of these disease states.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides derivatives of photoactivatable compounds, some of which are shown in the following Examples, in Table 1, and in FIGS. 1-5 via the introduction of non-radioactive indium into the macrocycle. The resulting products can be used to diagnose and treat disease states. Additionally, the ligand on the indium complex may then be further chemically or biochemically modified ex-vivo or in-vivo to form a different pyrrolic indium complex which is capable of binding to important blood plasma components or cell transport proteins that enhance sensitizer uptake at the diseased tissue site.

Additional functionality around the pyrrolic structure can be used to attach biomolecules, examples of which may be antibodies, growth hormones and growth factors or other disease specific molecules, or to aid such factors as water solubility, lipophilicity or hydrophobicity which are known factors that influence drug uptake in tumor disease tissue.

The following Examples are presented solely for the purpose of disclosing and illustrating the invention, and are not to be construed as limiting. In all of the procedures described in the examples, indium, where used, was the natural material, a mixture of about 4.23% In 113 and 95.77% In 115 . The following abbreviations are used in the examples: mL means milliliter or milliliters, g means gram or grams; mg means milligram or milligrams; g means microgram or micrograms.

Preparation of Indium Methyl pyropheophorbide from methyl pyropheophorbide

Methyl pyropheophorbide (0.5 g) was dissolved in acetic acid (100 mL); Indium chloride (0.5 g) and anhydrous sodium acetate (0.5 g) were added, and the solution was refluxed for 3 hrs. The solution was taken to dryness by rotary evaporation and the solid residue dissolved in dichloromethane/water (100 mL/100 mL). The organic layer was washed several times with 0.5 N HCl/water (200 mL), collected and dried over anhydrous sodium sulfate (20 g). The organic layer was filtered and evaporated to dryness. The resulting residue was crystallized from methanol/dichloromethane. Yield 0.45 g.

Preparation of Indium tert-butyl phthalocyanine

one of R2 and R3 is tert-butyl while the other and R1 and R4 are H; and M is In 113 Cl, In 115 Cl or a mixture of In 113 Cl and In 115 Cl.

4tert-butyl-1,2-dicyanobenzene (2 g) and Indium chloride (2.0 g) were mixed thoroughly in a round bottom flask with stirring. The mixture was heated at 160 C. for 2 hrs and the reaction vessel cooled to room temperature. The crude phthalocyanine was extracted from the resulting green solid using hot dichloroethane and the green solution evaporated by rotary evaporation. The resulting solid was dissolved in 7% acetone/dichloromethane and chromatographed on silica using 7% acetone/dichloromethane as eluent. The major green fraction was collected and recrystallized from methanol/dichloromethane. Yield, 1.1 g.

Preparation of Indium Benzoporphyrin derivative from Benzoporphyrin derivative dimethyl ester

Where M is In 113 Cl, In 115 Cl, or a mixture of In 113 Cl and In 115 Cl in indium benzoporphyrin derivative dimethyl ester and 2H in Ring B isomer benzoporphyrin derivative dimethyl ester.

Ring B isomer, Benzoporphyrin derivative dimethyl ester (100 mg) was dissolved in acetic acid (50 mL) and Indium chloride (0.2 g) and anhydrous sodium acetate (0.2 g) were added; the solution was refluxed for 3 hrs. The solution was taken to dryness by rotary evaporation and the solid residue dissolved in dichloromethane/water (100 mL/100 mL). The organic layer was washed several times with 0.5 N HCl/water (200 mL), collected and dried over anhydrous sodium sulfate (20 g). The organic layer was filtered and evaporated to dryness. The resulting residue was chromatographed on silica using 5% MeOH/dichloromethane as eluent and the major green fraction collected. The solvent was removed by rotary evaporation and the solid crystallized from hexane/dichloromethane. Yield 50 mg.

Where each R2 is OH, each R1, R3, R4 and R5 is Hydrogen, In is a mixture of In 113 Cl and In 115 Cl, and Z is Cl.

Tetrakis (3-hydroxyphenyl)chlorin (100 mg) was dissolved in acetic acid and the solution purged with argon. Indium chloride (0.2 g) and anhydrous sodium acetate (0.2 g) was added and the solution refluxed for 3 hrs under argon. The solution was taken to dryness by rotary evaporation and the solid residue dissolved in dichloromethane/water (100 mL/100 mL). The organic layer was washed several times with 0.5 N HCl/water (200 mL), collected and dried over anhydrous sodium sulfate (20 g). The organic layer was filtered and evaporated to dryness. The resulting residue was chromatographed on silica using 5% MeOH/dichloromethane as eluent and the major green fraction collected. The solvent was removed by rotary evaporation and the solid crystallized from hexane/dichloromethane. Yield 60 mg.

Each of R1 through R8 is ethyl, In Octaethyl benzochlorin, M is 2H, and in Indum octaethyl benzochlorin

M is In 113 Z, In 115 Z or a mixture of In 113 Z, and In 115 Z;

Z is Cl.

Octaethylbenzochlorin (100 mg) was dissolved in acetic acid and Indium chloride (0.2 g) and anhydrous sodium acetate (0.2 g) were added. The solution was refluxed for 3 hrs under argon. The solution was taken to dryness by rotary evaporation and the solid residue dissolved in dichloromethane (10 mL). The resulting solution was chromatographed on silica using 5% MEOH/dichloromethane as eluent and the major green fraction collected. The solvent was removed by rotary evaporation and the solid crystallized from hexane/dichloromethane. Yield 110 mg.

Each of R1 through R8 is ethyl In Octaethyl benzochlorin, M is 2H, and in Indium Octaethyl benzochlorin M is a mixture of In 113 Cl and In 115 Cl

OEBCSO 2 NH(CH 2 ) 3 OH (100 mg) was dissolved in dimethylformamide (DMF) (25 mL) and InCl 3 (100 mg) was added. The solution was refluxed for 6 hrs and the DMF was removed by rotary evaporation. The residue was dissolved in dichloromethane (10 mL) and the resulting solution was chromatographed on silica using 5% MeOH/dichloromethane as eluent. A minor fore running green band was collected and discarded and the more polar major green fraction was collected. The solvent was removed by rotary evaporation and the solid crystallized from hexane/dichloromethane. Yield 52 mg.

Preparation of

Where each of R1 through R8 is ethyl M is In 113 Z, In 115 Z, or a mixture of In 113 Z and In 115 Z in In Octaethyl benzochlorin and 2H in Octaethyl benzochlorin R is

Z is Cl

(400 mg) was dissolved in acetic acid (150 mL) and Indium chloride (0.4 g) and anhydrous sodium acetate (0.3 g) were added. The solution was refluxed for 3 hrs under argon. The solution was taken to dryness by rotary evaporation and the solid residue dissolved in dichloromethane (10 mL). The resulting solution was chromatographed on silica using 5% acetone/dichloromethane as eluent and the major green fraction collected. The solvent was removed by rotary evaporation and the solid crystallized from hexane/dichloromethane. Yield 420 mg.

Each of R1 through R8 is ethyl In Octaethyl benzochlorin, M is 2H, and in Indium Octaethyl benzochlorin M is a mixture of In 113 Cl And In 115 Cl

OEBCSO 2 NHC(CH 2 OH) 3 (100 mg) was dissolved in bromobenzene (50 mL) and Indium chloride (0.2 g) and anhydrous sodium acetate (0.2 g) were added. The solution was refluxed for 5 hrs under argon. The solution was taken to dryness by rotary evaporation and the solid residue dissolved in dichloromethane (10 mL). The resulting solution was chromatographed on silica using 5% acetone/dichloromethane as eluent and the major green fraction collected. The solvent was removed by rotary evaporation and the solid crystallized from hexane/dichloromethane. Yield 105 mg.

Each of R1 through R8 is ethyl In Octaethyl benzochlorin, M is 2H, and in Indium Octaethyl Benzochlorin M is a mixture of In113Cl and In115Cl

OEBCSO 2 NH (CH 2 ) 4 CH(NH 2 )CO 2 H (100 mg) was dissolved in acetic acid and Indium chloride (0.2 g) and anhydrous sodium acetate (0.2 g) were added. The solution refluxed for 3 hours under argon. The solution was taken to dryness by rotary evaporation and the solid residue dissolved in dichloromethane (10 mL). The resulting solution was chromatographed on silica using 5% MeOH/dichloromethane as eluent and the major green fraction collected. The solvent was removed by rotary evaporation and the solid crystallized from hexane/dichloromethane. Yield 110 mg.

M is 2H in 2-desvinyl-2-acetyl Pyropheophorbide and a mixture of In 113 Cl and In 115 Cl in Indium-2-desvinyl-2-acetyl pyropheophorbide.

2-Desvinyl-2-acetyl pyrropheophorbide (100 mg) was dissolved in acetic acid and Indium chloride (0.2 g) and anhydrous sodium acetate (0.2 g) were added. The solution was refluxed for 3 hrs under argon. The solution was taken to dryness by rotary evaporation and the solid residue dissolved in dichloromethane (10 mL). The resulting solution was chromatographed on silica using 5% acetone/dichloromethane as eluent and the major green fraction collected. The solvent was removed by rotary evaporation and the solid crystallized from hexane/dichloromethane. Yield 100 mg.

M is 2H in 2-desvinyl-2-formyl Pyropheophorbide and a mixture of In 113 Cl and In 115 Cl in Indium-2-desvinyl-2-formyl pyropheophorbide.

2-Desvinyl-2-formyl pyropheophorbide (100 mg) was dissolved in acetic acid (20 mL) and Indium chloride (100 mg) was added. Diisopropylethylamine (0.3 ml) was added and the solution refluxed until no more starting material remained by TLC (5% acetone/dichloromethane). The acetic acid was removed by rotary evaporation and the residue dissolved in dichloromethane and washed with 10% NH 4 Cl (2 50 mL). The organic phase was separated, dried over sodium sulfate filtered and rotoevaporated to dryness. The residue was dissolved in dichloromethane and purified by chromatography on silica using 5% acetone/dichloromethane as eluent followed by 20% acetone/dichloromethane as eluent. A less polar green fraction was collected and recrystallized from dichloromethane/hexane. A second more polar green fraction was collected and discarded. Yield of (27) 62 mg.

M is 2H in Chlorin e6 Trimethyl eater and a mixture of In 113 Cl and In 115 Cl in Indium-Chlorin e6 trimethyl ester

Chlorin e6 trimethyl ester (150 mg) was dissolved in acetic acid (20 mL) and Indium chloride (150 mg) added. Diisopropylethylamine (0.3 ml) was added and the solution refluxed until no more starting material remained by TLC (5% acetone/dichloromethane). The acetic acid was removed by rotary evaporation and the residue dissolved in dichloromethane and washed with 10% NH 4 Cl (2 50 mL). The organic phase was separated, dried over sodium sulfate, filtered and rotoevaporated to dryness. The residue was dissolved in dichloromethane and purified by chromatography on silica using 5% acetone/dichloromethane as eluent followed by 20% acetone/dichloromethane as eluent. The major green fraction was collected and re-purified by chromatography on silica using 5% acetone/dichloromethane as eluent. The major green fraction was collected and recrystalized from dichloromethane/hexane. Yield, 67 mg

Various compounds comprised essentially of a non-radioactive indium atom complexed with the inner nitrogens of a pyrrolic core have been evaluated biologically as photosensitizers. The results of some of this evaluation are summarized below.

In Vitro Biological Evaluation of Functionalized Benzochlorins

The in vitro biological evaluation of certain photosensitizers was determined, using standard procedures. Several of the photosensitizers had the following structure:

where each of R1 through R8 was ethyl and R and M had various meanings.

The identities of a first group of the compounds tested, all of which had the foregoing structure, of an arbitrary Sensitizer Number assigned for reference purposes, and the Example (if any) where their preparation is described are set forth below:

Identity of Identity of R1 Sensitizer Example M in fore- in foregoing Number (if any) going formula formula 22 2H Vinyl 23 SnCl 2 Vinyl 24 InCl Vinyl 25 InCl Ethyl 26 10 InCl COCH 3 27 11 InCl CHO 28 12 Pd COCH 3 A third group of the sensitizers had the following formula:

Identity of Identity of R1 Identity of R2 Sensitizer Example M in fore- in foregoing in foregoing Number (if any) going formula formula formula 29 a mixture of Vinyl CH 2 CO 2 CH 3 In 113 Cl and In 115 Cl 30 a mixture of Ethyl CH 2 CO 2 CH 3 In 113 Cl and In 115 Cl 31 a mixture of Vinyl CO 2 CH 3 In 113 Cl and In 115 Cl 32 a mixture of Ethyl CO 2 CH 3 In 113 Cl and In 115 Cl For the Biological Evaluation, Chinese hamster lung fibroblasts (V-79) obtained from the American Tissue Culture Collection were grown in D-MEM supplemented with 10% fetal calf serum at 37 C., 5% CO 2 , and 95% humidity. These cells were used to evaluate the sensitizers for dark and light toxicity, and cellular uptake. Medium was replaced with 5% fetal calf serum supplemented medium during sensitizer incubations.

In the first phase, the combination of intracellular and extracellular concentration of photosensitizer required for lethal damage to 50% of the cells in culture (DC(50)) on light exposure was determined by plating cells at a density of 100 cells/cm 2 and incubating for 1-3 hours to allow attachment of the cells. Cells were incubated with various concentrations of sensitizer ( C 0.01-1.0 M) delivered in non-toxic concentrations of DMSO/5% dextrose solution or in some cases in egg yolk phosphatidase (EYP) for 16-24 hours. The cells were then irradiated with laser light using a tunable Lexel argon pumped dye laser at the band I absorption of the photosensitizer. Total power density was adjusted to 12.5 mW/cm 2 and a total light dose of 1.25 J/cm 2 was applied. After treatment, cells were washed with HBSS, refed with 10% FBS supplemented DMEM with phenol red and incubated for 3-5 days to allow colony formation. Cells were fixed in methanol, stained with Giemsa, and colonies were counted. Plating efficiency was determined using a control colony and the mean percent survival fraction plotted against the sensitizer concentration and the DC50 established (Table 5).

Several photosensitizers were screened by a standard cellular uptake protocol. A 10 M stock solution of photosensitizer in EYP was prepared by dilution of the photosensitizer stock (1 mM) using 5% dextrose solution. Monolayer V-79 cells in the log phase of growth in 12-well tissue culture plates were incubated with 0.2 mL of the 10 M solution of photosensitizer for time periods of 0, 12, 36, 48 hrs. Following incubation the cells were washed with PBS (1 mL) three times, then washed with HBSS (1 mL) and detached using 100 L of trypsin. Individual wells containing cells were lysed by three freeze thaws.

Spiking experiments were performed to confirm that the sensitizer could be adequately recovered using standard procedures. The sensitizer was then extracted from the cell debris with DMSO, centrifuged and the supernatant examined for fluorescence. Sensitizer concentration was determined by comparison to a standard curve. Results are expressed in ug/ug protein. Untreated monolayers are used to determine cell count. Treated cells of each time point were used to determine the protein concentration using the BCA assay. The results for the photosensitizers studied are presented in Table 2. Results shown are the mean of the three experiments for each timepoint.

As can be clearly seen for the compounds tested, indium compounds had clearly a much lower DC(50)(light) values, regardless of the functionality attached to the chlorin.

In Vivo Assay of Photosensitizers on Tumors

Female C3H/HeJ mice (8-9 weeks old) were subjected to trochar implantation of BA mammary carcinoma on the hind leg of the animals. Animals having tumors ca 5 mm in diameter were entered into the in vivo screen. For each new photosensitizer, a group of at least three mice were used to ascertain a drug dose that caused a response as described in table 3 when treated with the appropriate wavelength of light. Generally in this series, animals were injected with 1 mole of sensitizer/Kg of body weight, formulated in egg yolk phosphatidate (EYP), via the tail vein. At a defined time period later the animals were irradiated with light from a laser source tuned to the wavelength of activation of the sensitizer. The power density of the laser was set at 75 mW/cm 2 with a total light dose of 200 J/cm 2 . The spot size was 1 cm diameter. Results for each photosensitizer are shown in Table 3.

Selected Kaplan-Meier curves for two different benzochlorin metal classes are shown in FIGS. 1 and 2 of the drawings. Survival curves for SnOEBC are shown in FIG. 1 , while the structure of SnOBEC is shown in FIG. 3 . Survival curves for InOECB are shown in FIG. 2 , while the structure of InOEBC is shown in FIG. 4 .

Examples of compounds having a pyrrolic core composed of at least two pyrroles, and which can be substituted as described in the preceding paragraph to produce additional compounds according to the instant invention are named in Table 1, and defined by reference to FIGS. 1-58 , which follow the Table.

Naturally occurring or synthetic porphyrins and derivatives thereof ( FIG. 2 )

Examples and illustrations from the literature outlined in Table 1 and FIGS. 1 to 57 of types of photosensitizers that may be used in photodynamic therapy or imaging and are applicable to the insertion of indium into the central core are as follows:

Dipyrromethenes have been used widely as intermediates in the synthesis of porphyrins (for example The Porphyrins Ed. D. Dolphin, Academic Press, 1978, Volume II 215-223; Volume I Chapter IV, 101-234. References within these volumes provide actual experimental details. These compounds can be coordinated with metal salts to produce metallo complexes (for example A. W. Johnson, I. T. Kay, R. Price, K. B. Shaw, J. Chem. Soc, Perkin Trans I, 3416-3424, 1959; S. M. Bloom, P. P. Garcia; J.H. Boyer, L. R. Morgan U.S. Pat. No. 5,189,029; date of patent Feb. 23, 1993; L. R. Morgan, J. H. Boyer, U.S Pat. No. 5,446,157). As shown in FIG. 1 , these molecules can be synthesized such that a wide variety of functionality can be directly attached to the basic diyrromethene ring structure. Such functionality can be used to increase water solubility, lippophilicity, conjugation to biomolecules such as antibodies or proteins, to increase the wavelength of absorption of the molecules (by increasing the conjugation of the macrocycle). As such, these molecules can be used for light activated photochemistry or diagnosis.

Porphyrins that possess at least one meso-nitrogen linking atom are called azoporphyrins. The number of meso-nitrogen linking atoms may be extended from one to four. Phthalocyanines and Naphthalocyanine may be regarded as tetraazoporphyrins with extended conjugation due to annelated benzene and napthalene rings. The synthesis of mono, di, tri and tetraazoporphyrin analogues is discussed in The Porphyrins Ed. D. Dolphin, Academic Press, 1978, Volume I, Chapter 9, p 365-388; Phthalocyanines Properties and Applications, Eds. C. C. Leznoff, A. B. P. Lever. VCH Publishers Inc., 1989; The Phthalocyanines . Eds, F. H. Moser, A. L. Thomas, CRC Press, Volumes I and II, 1983. References within these volumes provide actual experimental details. The synthesis of a series of tetrabenzotriazoporphyrins and tetranapthotriazoporphyrins has recently been published (Y-H, Tse, A. Goel, M. Hue, A. B. P. Lever, C. C. Leznoff, Can. J. Chem. 71, 742, 1993.) and clearly it can be envisaged that chemistry typical of phthalocyanine chemistry and porphyrin chemistry may be applied to these compounds, such that hetero atoms may be introduced into the annelated benzene or napthalene rings.

Corroles contain an aromatic 18 electron chromophore. The synthesis and structural modifications of these compounds (such as N-alkylation) are discussed in detail in The Porphyrins Ed. D. Dolphin, Academic Press, 1978, Volume I, Chapter 9, p 357-363 and in Porphyrins and Metalloporphyrins Ed. K. M. Smith, Elsevier Publishing Company, New York, 1975, Chapter 18, p730 -749.

It will be appreciated that various changes and modifications can be made from the specific details of the invention as disclosed above, and as described in the foregoing examples and that, in its essential details, the invention is a composition which is comprised essentially of a non-radioactive indium atom complexed with the inner nitrogens of a pyrrolic core composed of at least two pyrroles, with the proviso that fully unsaturated porphyrins having the structures:

where R1, R2, R4 and R6 are methyl, R8, R10, R11 and R12 are hydrogen, and R7 and R8 are CH 2 CH 2 COR13 (where R13 is a moiety which results from the removal of H from an amino acid), and

where R1 is CH(OH)Me, R is alkyl, and R 2 is a residue derived by removing H from an amino acid are excluded.

Preferably, the invention is a composition which is comprised essentially of a non-radioactive indium atom complexed with the inner nitrogens of a tetrapyrrolic core, with the proviso that the fully unsaturated porphyrins identified above are excluded. A non-radioactive indium atom complexed with the inner nitrogens of a photosensitive compound having a dihydro or tetrahydro tetrapyrrolic core is another preferred composition of the invention.

Z is a halide, acetate, OH, alkyl, aryl, substituted aryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, or a functional group less than or equal to 100000 daltons, protein or biomolecule, with the exclusions noted above when the pyrrolic core is fully unsaturated.

Still another preferred family has the following structure:

M in the foregoing formula can also be Pd, Sn, Pt, Al, Ru, Ga.

Another preferred family has the following structure:

M is In 113 , In 115 or a mixture of In 113 and In115

Z is a halide, acetate, OH.

M in the foregoing formula can also be Pd, Sn, Pt, Al, Ru, Ga.

Another preferred family has the following structure:

Where each of A, B, C, D and E is C, N, N R (where R is alkyl charged or uncharged) or combinations thereof;

M is In 113 , In 115 or a mixture of In 113 and In 115 and

Z is a halide, acetate, OH.

M is the foregoing formula can also be Pd, Sn, Pt, Al, Zn, Ru, Ga.

Still another preferred family has the structure:

Yet another family has the structure:

M is In 113 , In 115 or a mixture of In 113 and In 115

Z is a halide, acetate, OH.

Still another family has the structure:

Where each of A-E is C, N, or N R (where R is a charged or uncharged alkyl group);

M is In 113 , In 115 or a mixture of In 113 and In 115

Z is halide, acetate, or OH.

Another family has the structure:

Another family of compounds has the structure immediately above, where each of R1, R2, R6, R7 is CO 2 H (or a salt thereof), or CO 2 R11 (where R11 is alkyl or aryl).

Each of R3 and R8 is methyl or ethyl.

M is In 113 , In 115 or a mixture of In 113 and In 115 and Z is a halide, acetate, OH.

Still another family of compounds has the structure:

Another family of compounds has the structure:

Another family has the structure:

each of R3 and R8 is methyl or ethyl.

M is In 113 , In 115 or a mixture of In 113 and In 115 ; and Z is a halide, acetate, OH.

Yet another family of compounds has the following structure;

Still another family of compounds has the structure:

Each of R5 and R12 is methyl or ethyl.

M is In 113 , In 115 or a mixture of In 113 and In 115 .

Z is a halide, acetate, or OH.

Another family of compounds has the structure:

Another family of compounds has the structure:

where each of R1 and R5 is alkyl or aryl.

Each of R2 and R6 is methyl or ethyl.

Each of R7, R8, R3, and R4 is methyl, ethyl alkyl, CH 2 CH 2 CO 2 H (or a salt thereof), or

M is In 113 , In 115 or a mixture of In 113 and In 115 .

Z is a halide, acetate, OH.

Yet another family of compounds has the structure:

Another family of compounds has the structure:

where each of R1-R8 is ethyl or methyl

M is In 113 , In 115 or a mixture of In 113 and In 115 is a halide, acetate, OH.

Another family of compounds has the structure:

where each of R1-R8 is ethyl or methyl.

M is In 113 , In 115 or a mixture of In 113 and In 115 .

Z is a halide, acetate, OH.

Another family has the structure:

Another family of compounds has the structure:

M can also be Pd, or Ga.

Still another family has the structure:

M can also be Pd or Ga.

Another family of compounds has the structure:

Still another family of compounds has the structure:

R3 and R4 are H

X is O

M is In 113 , In 115 or a mixture of In 113 and ln 115

Z is a halide, acetate or OH.

Another family of compounds has the structure:

Another family of compounds has the following structure:

Still another family of compounds has the following structure:

R3 is H

R6 is CO 2 H, CO 2 Me or an amide

M is In 113 , In 115 or a mixture of In 113 and In 115

Z is a halide, acetate, or OH.

Another family of compounds has the structure:

Still another family of compounds has the following structure:

where each of R1 and R2 is CO 2 R14 (where R14 is alkyl or aryl), CO 2 H (or a salt therof), SO 2 Ph, or CN;

R9, R11 are CH 2 CH 2 CO 2 R15 (where R15 is alkyl or H or a salt of the carboxylic acid)

M is In 113 , In 115 or a mixture of In 113 and In 115 ; Z is a halide, acetate, OH.

Still another family of compounds has the following structure:

Still another family of compounds has the following structure:

R7 H or OH

R6 Me or OH

R4 H or CO 2 CH 3

M is In 113 , In 115 or a mixture of In 113 and In 115

Z is a halide, acetate, or OH.

Another family of compounds has the structure:

Still another family of compounds has the following structure:

Another family of compounds has the following structure:

Another family of compounds has the following structure:

Where each of R1 and R2 is CO 2 R14 (where R14 is alkyl or aryl), CO 2 H (or a salt thereof), SO 2 Ph, CN or a combination thereof;

Each of R4, R7, R13, R10 is H

R9 and R11 are CH 2 CH 2 CO 2 R15 (where R15 is alkyl or H or a salt of the carboxylic acid).

M is In 113 , In 115 or a mixture of In 113 and In 115 .

Z is a halide, acetate, or OH.

Another family of compounds has the following structure:

Another family of compounds has the following structure:

where each of R1, R2, R3 and R4 is CO 2 R16 (where R16 is alkyl or aryl), CO 2 H (or a salt thereof) or SO 2 Ph, CN;

Each of R5, R8, R10 and R14 is Me.

Each of R6, R9, R12 and R15 is H

Each of R11 and R13 is CH 2 CH 2 CO 2 R16 (where R16 is alkyl or H or a salt of the carboxylic acid), or an amide

M is In 113 , In 115 or a mixture of In 113 and In 115

Z is a halide, acetate, or OH.

Still another family of compounds has the following structure:

Another family of compounds has the structure:

Another family of compounds has the following structure:

Another family of compounds has the following structure:

Another family of compounds has the following structure:

Another family of compounds has the following structure:

R3 is H

R4is Me

Each of R5 and R6 is OH

Each of A and B is O or NR7 (R7 is alkyl).

X is O or NR7 (R7 is alkyl, an amino acid, an alcohol containing group, or an ether containing group)

M is In 113 , In 115 or a mixture of In 113 and In 115

Z is a halide, acetate, or OH.

Another family of compounds has the following structure:

Another family of compounds has the following structure:

R2 is CO 2 CH 3 , CO 2 H, CO 2 R7 (R7 is an alkyl or phenyl group), or an amide

R3 is H

R4 is H or CO 2 CH 3

R6 is Me

X is O

M is In 113 , In 115 or a mixture of In 113 and In 115

Z is a halide, acetate or OH.

Another family of compounds has the following structure:

Still another family of compounds has the following structure:

R7 is Me

R3 is H

R4 and R8 are H, CO 2 CH 3 , CO 2 H (or a salt thereof), CO 2 R7 (R7 is an alkyl or phenyl group), an amide, CH 2 CO 2 CH 3 , CH 2 CO 2 H (or a salt thereof), CO 2 R9 (R9 is an alkyl or a phenyl group), or an amide

R2 is CO 2 CH 3 , CO 2 H, CO 2 R9 (R9 is an alkyl or phenyl group), or an amide

M is In 113 , In 115 or a mixture of In 113 and In 115

Z is a halide, acetate, or OH.

Another family of compounds has the following structure:

Another family of compounds has the following structure:

Each of A and B is O or NR6 (where R6 is an alkyl or phenyl group)

R5 is Me

R3 is H

R2 is CO 2 CH 3 , CO 2 H, CO 2 R6 (R6 is an alkyl or phenyl group), or an amide

X is O, NR6 (R6 is alkyl, an amino acid, an alcohol containing group, or an ether containing group)

M is In 113 , In 115 or a mixture of In 113 and In 115

Z is a halide, acetate, OH.

Another family of compounds has the following structure:

Another family of compounds has the structure:

R6 is O or NR7 (where R7 is an alkyl group)

R5 is Me

R3 is H

R4 is H or CO 2 CH 3

X is O

M is In 113 , In 115 or a mixture of In 113 and In 115

Z is a halide, acetate, or OH.

Another family of compounds has the structure:

Another family of compounds has the structure:

R5 is Me

R3 is H

M is In 113 , In 115 or a mixture of In 113 and In 115

Z is a halide, acetate, OH.

Another family of compounds has the following structure:

R4 is Me

R3 is H

Each of A and B is O or NR5 (R5 is alkyl)

X is O, NR5 (R5 is alkyl, an amino acid, an alcohol containing group, or an ether containing group)

M is In 113 , In 115 or a mixture of In 113 and In 115 .

Another family of compounds has the structure:

Each of R6 and R7 is H or OH

R5 is Me

R3 is H

each of R2 and R8 is CO 2 CH 3 , CO 2 H, CO 2 R9 (R9 is an alkyl or a phenyl group), or an amide

M is In 113 , In 115 or a mixture of In 113 and In 115

Z is a halide, acetate, or OH.

Still another family of compounds has the structure:

Still another family of compounds has the structure:

R4 is Me

R3 is H

A and B are O or NR6 (R6 is an alkyl or a phenyl group)

X is O, NR6 (R6 is alkyl), an amino acid, an alcohol containing group, or an ether or amine containing group

M is In 113 , In 115 or a mixture of In 113 and In 115

Z is a halide, acetate, or OH.

Still another family of compounds has the structure:

M can also be Pd or Ga.

Yet another family of compounds has the structure:

A, B, C, D are; C, N, O , O, S, Te, P, N (R9)X (where R9 is a functional group less than or equal to 100000 daltons and X is a charge balancing ion), or combinations thereof. M is In 113 , In 115 or a mixture of In 113 and In 115 ; Z is a halide, acetate, OH, alkyl, aryl, substituted aryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, protein or biomolecule or functional group less than or equal to 100000 daltons; X is O, NR9 (where

R9 is H, alkyl (1-10 carbons), aryl, substituted aryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, an amino acid, an amino acid ester or a functional group less than or equal to 100000 daltons).

M can also be Pd or Ga.

Yet another family of compounds has the structure:

M can also be Pd or Ga.

Another family of compounds has the structure:

M can also be Pd or Ga.

Another family of compounds has the structure:

M can also be Pd or Ga.

Still another family of compounds has the following structure:

M can also be Pd or Ga

Still another family of compounds has the structure:

M can also be Pd or Ga.

Still another family of compounds has the structure:

M can also be Pd or Ga

Another family of compounds has the structure:

M can also be Ga.

Still another family of compounds has the structure:

M can also be Pd or Ga.

Yet another family of compounds has the structure:

Yet another family of compounds has the following structure:

Still another family of compounds has the following stucture:

Yet other families of compounds have the following structure:

M can also be Pd, Pt, Ga or Al.

Another family of compounds has the following structure:

M can also be Pd, Pt, Ga or Al.

Yet another family of compounds has the following structure:

M can also be Pd, Pt, Ga or Al.

Still another family of compounds has the following structure:

Another family of compounds has the following structure:

Still another family of compounds has the following structure:

Still another family of compounds has the formula:

Two other families of compounds have the structures of Compound I and II, below:

wherein M is In 113 , In 115 or a mixture of In 113 and In 115

R1, R2 and R3 can be the same or different, and each is CO 2 H, CO 2 R4, CONR4, CH 3 Y , CONR4R4, NH 2 , N(R4) 2 , or N(R4) 3 Z , where Y is halogen, OH, OR4, or a functional group having a molecular weight equal to or less than 100,000 daltons, R4 is a functional group having a molecular weight equal to or less than 100,000 daltons, and Z is a physiologically acceptable charge balancing ion with the proviso that R4 is not a mono-or di-carboxylic acid of an amino acid,

R5 is a methylene group or an ethylene group,

X is H, vinyl, ethyl, acetyl or formyl, and

Y is methyl or formyl.

In still another aspect, the invention is a free base or a metal complex having the structure of one of Compounds III and IV, below:

wherein M is 2H or a metal cation,

R1, R2 and R3 can be the same or different, and each is CO 2 H, CO 2 R4, CONR4, CH 3 Y , CONR4R4, NH 2 , N(R4) 2 , N(R4) 3 Z , or CONHR6OR7 where R6 is a bivalent moiety composed of a number, n, of alkylene groups and (n minus 1) oxygens, each oxygen linking two alkylene groups through an ether linkage,

CONHR6OR7 where R6 is a bivalent moiety composed of a number, n, of alkylene groups and (n minus 1) oxygens, each oxygen linking two alkylene groups through an ether linkage, and R7 is alkyl,

and Z is a physiologically acceptable charge balancing ion, with the proviso that there is at least one R6 group in the structure,

X is H, vinyl, ethyl, acetyl or formyl, and

Y is methyl or formyl.

In the foregoing definition, and elsewhere herein, the term alkylene means a bivalent radical derived from an alkane with the free valences on different carbon carbon atoms. i.e., C n H 2n .

In still another aspect, the invention is a metal complex having the structure of one of Compound V and VI below:

wherein M is 2H, In 113 , In 115 or a mixture of In 113 and In 115 .

R1, R2 and R3 can be the same or different, and each is

X is H, vinyl, ethyl, acetyl or formyl, and

Y is methyl or formyl.

In the foregoing discussion of the invention and in the following claims, the term functional group having a molecular weight equal to or less than 100,000 daltons has been used. This term refers to such groups as monoclonal antibodies, proteins, saccharides, oligomeric nucleosides, peptides and the like. The terms alkyl, alkenyl, alkynyl, substituted alkenyl, substituted alkynyl and aryl, when used herein and in the appended claims to refer to substituents, preferably mean groups having 1 to 10, 2 to 10, 2 to 10, 2 to 10, 2 to 10 and 6 to 10 carbons, respectively.

It will be apparent that various changes and modifications can be made from the specific disclosure of the invention set forth herein without departing from the spirit and scope thereof as defined in the following claims.