WO1994010172A1 - Wavelength-specific photosensitive porphacyanine and expanded porphyrin-like compounds and methods for preparation and use thereof - Google Patents

Abstract

A group of novel expanded porphyrin-like compounds, porphacyanine (Pc) and porphacyanine-like compounds, having absorption maxima in the range of 400-850 nanometers is useful in detecting and treating targeted tissues, cells and viruses. The use of the Pc of the invention permits the irradiation to comprise wavelengths other than those absorbed by blood. The Pc of the invention may also be conjugated to target-specific moieties such as immunoglobulins or fragments thereof to target specific tissues or cells for the radiation treatment. Use of these materials permits greater depth of treatment penetration and greater specificity in targeting tissues or cells. Coupled with an appropriate paramagnetic ion or radioisotope, the Pc of the invention is suitable for use in nuclear magnetic resonance imaging and radioimaging. Novel methods for synthesizing the Pc of the invention are disclosed.

Description

WAVELENGTH-SPECIFIC PHOTOSENSITIVE PORPHACYANINE AND

EXPANDED PORPHYRIN- IKE COMPOUNDS AND METHODS

FOR PREPARATION AND USE THEREOF

Field of the Invention The invention relates to novel expanded wavelength- specific photosensitive porphacyanine and expanded porphyrin- like compounds, novel methods for their preparation and the use of these compounds to mediate the detection or destruction of target cells or tissues by irradiation. Specifically, the invention relates to the preparation of porphacyanine and other porphacyanine-like compounds and derivatives thereof having absorption maxima in the range of 250-850 nanometers (nm) and their use to mediate the irradiation of cells or tissues to be detected or destroyed, and to the use of these compounds or conjugates thereof to focus the effects of the irradiation on particular target tissues. In addition, the invention relates to the use of porphacyanine and porphacyanine-like compounds and derivatives thereof in radioimaging and magnetic resonance imaging methods.

Background of the Invention Although considerable effort has been devoted to the synthesis and study of porphyrins and other tetrapyrrolic macrocycles, much less is known about the larger aromatic pyrrole-containing systems, the so-called "expanded porphyrins." Such systems, by virtue of containing a greater number of π electrons, additional coordinating heteroatoms and a larger central binding core, may offer advantages over the porphyrins. The pursuit of these compounds began several decades ago with the first reported synthesis of sapphyrin from tripyrrane dicarboxylio acid and bipyrroledicarboxaldehyde. (Woodward, R.B., Aromaticity Conference, Scheffield, England,

SUBSTITUTE SHEET 1966; see also, Broadhurst et al . , J. Chem . Soc . Perkins Trans . (1972) 1:2111 and Bauer et al . (1983) J. Am. Chem . Soc . 105:6429) . The synthesis of smaragdyrin from bipyrroledicarboxaldehyde and pyrroledipyrromethane dicarboxylic acid was reported in 1970 by M.M. King. (Ph.D. Dissertation, Harvard University, Cambridge, MA)

The uranyl complex of superphthalocyanine is another pentapyrrolic macrocyclic compound of historical importance. This compound was prepared by direct template condensation of dicyanobenzene with uranyl dichloride, however, the free base is unstable (Day et al . (1975) J^". Am . Chem. Soc . 97:4519). Demetalation resulted in contraction of the ring to form phthalocyanine (Marks, T.J. and D.R. Stojakovic (1978) J. Am. Chem . Soc . 100:1695) . Gossauer synthesized the first hexaphyrin by condensing a bis-α-tripyrrane with a tripyrrane dialdehyde, followed by oxidation (Bull . Soc . Chim . Belg. (1983) 92:793). Of the six methine bridges present in the hexaphyrin, two have E configuration ( Id. ) . Charriere reported that hexaphyrin forms bimetallic complexes with several transition metals (1987,

Thesis, University de Fribourg, Suisse) . Another hexapyrrolic system, rubyrin, has been recently synthesized and structurally characterized (Sessler et al . (1991a) Angew. Chem. In t . Ed . Engl . 30:977) . Vinylogous porphyrins or platyrins are another important class of pyrrole-containing macrocycles first described by R.A. Berger and E. LeGoff (Tetra. Lett . (1978) 44:4225; see also, LeGoff, E. and O.G. Weaver (1987) J. Org . Chem . 52:711; and Franck et al . (1988) Proc . SPIE Int . Soc . Opt . Eng. , Ser. 5, 997:107). These compounds are generally synthesized by reacting a dipyrromethane with a vinylaldehyde- substituted dipyrromethane (Beckman et al . (1990) Angew. Chem . In t . Ed . Engl . 29:1395). Bisvinylogous expanded porphyrins were further expanded to tetravinylogous porphyrins in which all four of the normally one atom meso bridges are enlarged.

Tetravinylogous porphyrins are made by an acid-catalyzed self- condensation of the N-protected, pyrrole-substituted allyl alcohol. Tetravinylogous porphyrins have a very intense Soret-

SUBSTITUTE SHEET like band shift of more than 150 nm from that of the normal porphyrins (Gosmann, M. and B. Franck (1986) Angew. Chem. Int .

Ed . Engl . 25:1100; Knϋbel, G. and B. Franck (1988) Angew. Chem .

Int . Ed. Engl . 27:1170) . In addition, the synthesis of bisvinylogous porphycene has recently been reported (Jux et al . (1990) Angew. Chem. Int . Ed. Engl . 29:1385; Vogel et al . (1990)

Angew. Chem. Int . Ed. Engl . 29:1387) .

Schiff-base compounds represented by texaphyrin are another class of pyrrole containing macrocyles (Sessler et al . (1987) J. Org. Chem . 52:4394; Sessler et al . (1988) J. Am. Chem.

Soc . 110:5586). Texaphyrin is synthesized by acid-catalyzed condensation of tripyrrane dialdehyde with o-phenylenediamine.

Several analogs of texaphyrin have been prepared using similar strategies (Sessler et al . (1991b) Abstract of the 201st Natl. Soc. Mtg., Inorganic Division; Sessler et al . (1992) Inorg.

Chem. 28:529) .

The use of porphyrins, combined with irradiation, for the detection and treatment of malignant cells has, by this time, some considerable history. (See, e.g., PORPHYRIN PHOTOSENSITIZATION (Kessel, D. et al . , eds. Plenum Press, 1983).

Certain porphyrins seem "naturally" capable of localizing malignant cells. When irradiated, porphyrins have two properties which make them useful. First, when irradiated with ultraviolet or visible light, they may fluorescence, and thus be useful in diagnostic methods related to detection of malignancy

(see, for example, Kessel et al . , supra; Gregory, H.B. Jr. et al . , Ann . Surg. (1968) 167:827-829).

In addition, when irradiated with ultraviolet (UV) , visible, or near-infrared light, certain porphyrins exhibit a cytotoxic effect on the cells in which they are localized (see, for example, Diamond, I. et al., Lancet (1972) 2:1175-1177;

Dougherty, T.J. et al . , Cancer Research (1978) 38:2628-2635;

Dougherty, T.J. et al . , THE SCIENCE OF PHOTO MEDICINE 625-638 (J.D.

Regan &. J.A. Parrish, eds., 1982); Dougherty, T.J. et al., CANCER : PRINCIPLES AND PRACTICE OF ONCOLOGY 1836 - 1844 (V . T . DeVita Jr . et al . , eds., 1982). Certain of the expanded porphyrins such as sapphyrin, texaphyrin and vinylogous porphyrins possess unique long-wavelength and singlet oxygen producing properties which

SUBSTITUTE SHEET make them attractive as potential photosensitizers for use in tumor phototherapy (Maiya et al . (1990) J. Phys . Chem . 94:3597; Sessler et al . (1991c) SPIE Soc . 1426:318; Franck et al . , supra) . While the conjugation of certain porphyrins, such as hematoporphyrin, to immunoglobulins specific for targeted cells may refine the ability of certain porphyrins to home to the desired cells or tissue, this still does not solve another problem ancillary to this general therapeutic approach, namely that the wavelength for irradiation required to activate certain porphyrins, which is in the range of 630 nm, is also an energy which is readily absorbed by other porphyrins and natural chromophores normally present in the blood and other tissues. Therefore, the depth of the effective treatment has been limited to a few millimeters because of blocking effects of light- absorbing natural chromophores such as hemoglobin. Accordingly, it would be desirable to administer compounds to mediate the effects of irradiation which can be excited at longer wavelengths thus avoiding the blocking effects of natural chromophores present throughout the subject organism.

In addition to phototherapy, expanded porphyrins are useful in magnetic resonance imaging (MRI) . MRI is a noninvasive, nonionizing method that allows normal and abnormal tissue to be observed and recognized at the early stages of development. At this time MRI has a significant drawback, however, in that the degree of signal enhancement for diseased versus normal tissues is often insufficient to allow this method to be used in many clinical situations. To overcome this problem, considerable effort is underway to develop contrast reagents for MRI. Paramagnetic metal complexes, such as those derived from gadolinium(III) (Gd) have recently proven particularly efficient in clinical trials.

To date, the coordination of gadolinium in MRI contrast agents has been achieved using carboxylate-type ligands. ( See, for example, Lauffer, R.B. (1987) Chem. Rev. 87:901; Kornguth et al . (1987) J. Neursurg. 66:898; Koenig et al . (1986) Invest. Radiol . 21:697; Chacheris et al . (1987) Inorg. Chem . 26:958; Loncin et al. (1986) Inorg. Chem. 25:2646;

SUBSTITUTE SHEET Chang, CA. and V.C. Sekhar (1987) Inorg. Chem. 26:1981). The known systems are all of high thermodynamic stability but high intrinsic lability. Certain expanded porphyrins, on the other hand, can form stable complexes with Gd(III) which does not form stable complexes with normal porphyrins. As a result, they provide an improved approach as MRI contrast agents. Sessler et al . reported that texaphyrin forms an extremely stable Gd(III) complex, in vi tro (Sessler et al . (1989) Inorg. Chem . 28:3390). In addition to Gd(III), texaphyrin has been reported to form complexes with a variety of transition metals such as Cd and Eu (Sessler, J.L. and A.K. Burrell (1992) Top . Cur. Chem . 161:177) .

Disclosure of the Invention The invention provides novel light-absorbing compounds suitable for use in detecting and/or treating target tissues, cells and pathogens. The invention also provides for novel methods for preparing the novel expanded porphyrins (porphacyanines) , which either increase the yield of the porphacyanine or greatly simplify the synthetic process. These compounds may be administered in relatively low dosage due to their capability to absorb radiation in an energy range outside of that normally absorbed by the components present in high concentration in blood or other tissues, in particular the porphyrin residues normally associated with hemoglobin and myoglobin. Therefore, by providing these novel expanded porphyrin-like compounds with higher wavelengths of activation, the irradiation treatment can be conducted at a wavelength at which the native chromophores do not compete for photons with the active compounds. This results in greater depth of penetration of the light. These compounds are preferentially retained in target tissues and cells as compared to nontarget tissues and cells. Accordingly, when labeled with or conjugated to a radioisotope or paramagnetic ion porphacyanine and porphacyanine-like compounds are particularly useful as radioimaging and magnetic resonance image contrast agents, respectively. In addition, the increased stability of the available metal-binding core and the greater number of atoms available to bind the metals render porphacyanine and

SUBST - 6 - porphacyanine-like compounds particularly useful as magnetic resonance contrast agents. Another advantage of porphacyanine and the other novel porphacyanine-like compounds of the present invention and conjugates thereof comprising a metallic element is their ability to luminesce when exposed to UV, visible or near infrared radiation. Thus, these compounds are particularly useful for detecting lesions and tumors. This collection of fluorescent derivatives is referred to herein as "porphacyanine"

and "porphacyanine-like compounds." Porphacyanine is exemplified by the compound of

Formula I, wherein R is a noninterfering substituent including, but not limited to, the group consisting of substituted and unsubstituted alkyl, alkenyl, alkynyl; substituted and unsubstituted aryl; alkyl or aryl sulfonyl; alkyl or aryl cyano; halogen; cyano; nitro; amino; carboxy; carbalkoxy or the ester, amide or salt thereof and the like.

As used herein, carboxy is, as conventionally defined, -COOH, and carbalkoxy is -C00R, wherein R is alkyl (1-6C) . As used herein, alkyl is a saturated straight or branched chain hydrocarbon of 1-6 carbon atoms such as methyl, ethyl, 2-methylpentyl, t-butyl, n-propyl, and so forth.

Aryl (6-10C) is phenyl optionally substituted with 1-3 substituents independently selected from halo (fluoro, chloro, bromo or iodo) , lower alkyl (1-4C) or lower alkoxy (1-4C) . Alkoxy is -OR wherein R is alkyl as herein defined.

SUBSTITUTE SHEET The aryl (6-10C) or alkyl (1-6C) sulfonyl moieties have the formula S0₂R wherein R is alkyl or is aryl as above- defined.

The invention compounds also include the salts, esters and amides of -COOH. For use in vivo these salts, esters and amides must be pharmaceutically acceptable and nontoxic; this requirement in not germane to in vitro use. "Salts, esters, and amides" refers to salts derived from inorganic or organic bases, including pharmaceutically acceptable nontoxic inorganic and organic bases, and alkyl esters or amides derived from alcohols or primary or secondary amines of the formula ROH or RNH₂ or R₂NH wherein R is alkyl as herein defined, suitable inorganic bases include sodium, potassium, lithium, ammonium, calcium, and magnesium, hydroxides, and the like. Particularly preferred are the potassium and sodium salts. Pharmaceutically acceptable organic nontoxic bases include primary, secondary, tertiary and quaternary amines including cyclic amines, and basic ion- exchange resins. Examples include isopropylamine, trimethylamine, ethanolamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, glucosamine, theobromine, purines, piperazine, polyamine resins, and the like.

The salt derivatives are prepared by treating the free acids with an appropriate amount of pharmaceutically acceptable base. The reaction can be conducted under anhydrous conditions or in water, alone or in combination with an inert, water- miscible organic solvent, at a temperature of from about 0°C to about 100°C, preferably at room temperature at a suitable molar ration of invention compound to base. Typical inert, water- miscible organic solvents include methanol, ethanol, isopropanol, butanol, acetone, dioxane or tetrahydrofuran.

The salt derivatives can be reconverted to their respective free acids by acidifying with an acid, preferably an inorganic acid, e . g. , hydrochloric acid, sulfuric acid and the like, at a temperature of from about 0°c to about 50°C, preferably at room temperature.

The esters are prepared by esterifying the corresponding free acids with an alcohol reagent corresponding

SUBSTITUTE SHEE^' to the desired ester. This reaction is conducted in the presence of a strong acid, such as boron trifluoride, hydrogen chloride, sulfuric acid, p-toluenesulfonic acid, and the like. Since the alcohol reagent used in the esterification is a liquid at the reaction temperature, the alcohol reagent can be the reaction solvent. Optionally, the reaction can be carried out in an inert organic solvent in which the free acids and the alcohol reagent are soluble, such as a hydrocarbon solvent, e . g. , hexane, isooctane, decane, cyclohexane, benzene, toluene, xylene, a halogenated hydrocarbon solvent, e . g. , methylene chloride, chloroform, dichloroethane; or an ether solvent, e.g., diethyl ether, dibutyl ether, dioxane, tetrahydrofuran, and the like. The reaction is conducted at from about 0°C to the reflux temperature of the reaction mixture, preferably using hydrogen chloride at a temperature of from 15°C to about 35°C.

The product is isolated by conventional means such as diluting the reaction mixture with water, extracting the resulting aqueous mixture with a water-immiscible inert organic solvent such as diethyl ether, benzene, methylene chloride, and the like, combining the extracts, washing the extracts with water to neutrality, and then evaporating under reduced pressure.

Alternatively, the alkyl esters can be prepared by transesterification, according to methods known in the art. It is preferred in preparing the esters via transesterification to go from a lower ester to a higher ester, e. g. , from the methyl ester, for example, to the isoamyl ester, for example. However, by using a substantial excess of a lower alcohol, a higher ester can be transesterified to a lower ester; thus, for example, by using a substantial excess of ethanol, the hexyl ester is converted by transesterification to the ethyl ester.

In still another alternative, the ester can be prepared by reacting the free acid form with the appropriate diazo alkane, such as diazomethane, diazo-n-hexane, or diazo-i- propane in an aprotic organic solvent at low temperature. The amides are obtained by activation of the carboxylic acid residue, for example by thioxylchloride, and treating with the appropriate amine.

SUBSTITUTE SHEET Examples of target cells and tissues within the present invention include, but are not limited to, tumors, including blood tumors, malignant bone marrow, virally-infected blood cells or bone marrow, dysplastic cells or tissues, sites of inflammation or infection, hyperproliferative tissue such as psoriatic plaque or papillomavirus lesions (warts) or neointimal hyperplasia lesions, hypervascularization such as portwine stains and hemangiomas, atherosclerotic plaque, hair follicles, free viruses, bacteria, protozoa or other pathogenic parasites. In still another aspect, the invention relates to a method for inactivating certain viruses, bacteria, protozoa and other pathogenic parasites using porphacyanine and/or the porphacyanine-like compounds. Targeted pathogens contemplated by the present invention include enveloped viruses such as human cytomegaloviruses, Epstein-Barr virus, Marek's disease herpes virus, human herpes simplex viruses, varicella-zoster virus, members of the family Poxviridae, members of the family Hepadnaviridae such as human hepatitis A virus (HAV) , human hepatitis B virus (HBV) and non-A, non-B hepatitis viruses, including human hepatitis C virus, members of the family Orthomyxoviridae such as influenza virus types A, B and C, members of the family Retroviridae such as human T cell leukemia viruses, human immunodeficiency viruses, and members of the family Flaviviridae such as tick-borne encephalitis virus or yellow fever virus.

Another class of pathogen contemplated by the present invention includes parasites such as Plasmodium malariae, P . falciparum, P. ovale, P. vivax and Trypanosoma cruzi .

The eradication of bacteria is also contemplated by the present invention including Bacillus subtilis, Streptococcus faecalis, Pseudomonas spp. , Mycobacterium spp . and other opportunistic organisms treatable by photodynamic activation.

In addition, porphacyanine and porphacyanine-like compounds within the present invention can be conjugated to target-specific moieties (Tsm) such as immunoglobulins including polyclonal and monoclonal antibodies and fragments thereof, H2-agonists, steroids including estrogen and testosterone, sugars such as mannose and peptides such as T-cell receptors and

SUBSTITUT - alpha-beta heterodimers. Conjugation of porphacyanine and porphacyanine-like compounds to Tsms may facilitate their concentration in a desired target tissue.

Thus, in one aspect, the invention relates to a conjugate of the formula Tsm-L-Pc where Tsm represents a target- specific moiety such as an immunoglobulin or a hormone, Pc represents a porphacyanine derivative having an absorption maximum in the range of 400-850 nm, and L represents either a covalent bond linking these components or a linking moiety covalently linked to each of the Tsm and Pc.

Preferably, the Pc is selected from a group consisting of porphacyanine and porphacyanine-like derivatives obtained using methods to cyclize mono-, di and oligo-pyrrolic precursors to give macrocyles containing ^ . In other aspects, the invention relates to methods for effecting cytotoxicity against target cells using porphacyanine and porphacyanine-like compounds in the presence of UV, visible or near infrared light either alone or as the conjugates described above. In yet additional aspects, the invention relates to methods for detecting diseased tissues using porphacyanine and porphacyanine-like compounds or conjugates thereof. Porphacyanine and the porphacyanine-like compounds of the present invention can be labeled with or conjugated to a radioisotope for radioimaging (scintigraphic imaging) or a magnetic resonance image enhancing agent, for use as a diagnostic imaging agent. Examples of radioisotopes which would be useful labels for porphacyanine and porphacyanine-like compounds include Iodine-123, Iodine-131, Technetium-99m, Indium-Ill and gallium-67. Examples of compounds which would be useful for MRI imaging enhancement when conjugated to porphacyanine and porphacyanine-like compounds include paramagnetic ions of elements such as Gd, Mn, Eu, Dy, Pr, Pa, Cr, Co, Fe, Cu, Ni, Ti, and V. In further aspects, the invention relates to pharmaceutical compositions containing these active ingredients.

SUBSTITUTESHEET Brief Description of the Drawings Figure 1 shows the structure of a porphacyanine of Formula I.

Figure 2 illustrates one method for synthesizing a porphacyanine of Formula I.

Figure 3 illustrates another method for synthesizing a porphacyanine of Formula I.

Figure 4 illustrates a method for synthesizing a porphacyanine of Formula I. Figure 5 shows the structure of a porphacyanine- like compound within the present invention.

Figure 6 shows the structure of another porphacyanine- like compound within the present invention.

Figure 7 shows the structure of yet another porphacyanine-like compound within the present invention. Figure 8 shows the structure of still another porphacyanine-like compound within the present invention.

Figure 9 illustrates the emission wavelength of the free base of a porphacyanine of Formula I when excited at 456 nm (solvent is THF) .

Figure 10 illustrates the emission wavelength of a protonated porphacyanine of Formula I when excited at 456 nm (solvent is THF and acetic acid).

Figure 11 illustrates a novel method for synthesizing a porphacyanine of Formula I.

Figure 12 illustrates a novel, simplified method for synthesizing a porphacyanine of Formula I.

Figure 13 illustrates a novel method for synthesizing porphacyanines within the present invention containing substituted or unsubstituted phenyl groups on the two bridge carbon atoms.

Figure 14 illustrates a novel method for synthesizing asymmetrical porphacyanines within the present invention containing a substituted or unsubstituted phenyl groups on one of the two bridge carbon atoms.

SUBSTITUTE SHEET Modes of Carrying Out the Invention Porphacyanine and the Porphacyanine-like Compounds

All of the compositions of the invention employ as the light-absorbing compounds novel expanded porphyrin-like macrocycle derivatives, namely porphacyanine and porphacyanine- like compounds, which have a light absorption maximum in the range of 400-850 nm. Porphacyanine and porphacyanine-like compounds are macrocyles wherein at least one of the bridges of the known polypyrrolic macrocycles is replaced by ^^ ^^x _ Typical examples of the nuclei of such compounds are shown in Figures 1 and 5-8.

Specific preparation of a porphacyanine useful in the invention is achieved by adding lead tetraacetate to 2-benzyloxycarbonyl-3,4-diethyl-5-methyl pyrrole in glacial acetic acid. Ethylene glycol is added to reduce any remaining Pb(IV) . Water is added and the 5-acetoxymethyl- 2-benzyloxycarbonyl-3,4-diethylpyrrole (Compound A in Figure 3) is collected by filtration and washed with additional water. The 5-acetoxymethyl-2-benzyloxycarbonyl-3,4-diethylpyrrole is added to acetic acid in water and heated. The solid product, is precipitated as large chunks when the foregoing solution cools to room temperature. Water is added and the product is collected by filtration and then washed with additional water. The filtrate is extracted with CH₂Cl₂ and then evaporated to - produce a solid product. The solid products are combined, and then recrystallized from a solution of CH₂C1₂ and hexanes.

5,5' -bis (benzyloxycarbonyl) -3,3' -4,4' -tetraethyl-2,2' - dipyrromethane (Compound B of Figure 3) in tetrahydrofuran (THF) is stirred under hydrogen in the presence of Pd/C and triethylamine. After the catalyst is filtered through celite, the filtrate is evaporated to dryness resulting in dicarboxylic acid. The dicarboxylic acid is dissolved in

N,N-dimethylformamide and heated to boiling under argon. The solution is chilled and an excess of chilled benzoyl chloride is added to the bis-α-free dipyrromethane (Compound B) . The reaction mixture is s.tirred and the solid product collected by filtration. The solid product is added to water and basified using NaHC0₃ and heated to 60°C. The pale yellow product

SUBSTΓΓUTE SHEET crystallizes from the solution and is filtered and washed with water.

3,3' ,4,4' -tetraethyl-5,5' -diformyl-2,2' -dipyrromethane (Compound C of Figure 3) in ethanol is bubbled with argon and then hydroxylamine hydrogen chloride and sodium acetate are added. This mixture is heated under argon and then the solvent is removed and the product dried overnight in vacuo . The bis- oxime is dissolved in acetic anhydride and saturate with argon. The crude bisnitrile product (Compound D of Figure 3) is obtained as a black solid after removal of acetic anhydride and dried under vacuum. The product is purified by silica gel column with 0.5% methanol in CH₂C1₂, followed by an alumina column with 10-20% EtOAc. Evaporation of the solvent yields 3,3' -4,4' -tetraethyl-5,5' -cyanodipyrromethane as pale pink crystals.

In one embodiment (Figure 3), the 3,3'- 4,4' -tetraethyl-5,5' -cyanodipyrromethane is then dissolved in THF and added to a THF suspension of LiAlH₄. The resulting mixture is stirred and water is added. A solid product forms which is filtered off. The bis-amine product is obtained after evaporation of the solvent by drying under vacuum. The bis- amine is dissolved in anhydrous methanol and bis-aldehyde is added. The solution is bubbled and brought to reflux with nitrogen. Lead thiocyanate (Pb(SCN₂)) is added and the solution is refluxed. Oxygen gas is bubbled through the solution at room temperature. After evaporation of the solvent, the crude porphacyanine product is dried under vacuum. The product is purified by Al₂0₃ column with ethyl acetate in CH₂C1₂. The green eluent is collected and concentrated. Crystals of the porphacyanine macrocycle are obtained after evaporation of the solvent.

In an alternative embodiment, the 3,3'- 4,4' -tetraethyl-5,5' -cyanodipyrromethane in anhydrous THF is added to a THF suspension of LiAlH₄ under nitrogen at 0° C. The mixture is stirred and water is added to quench the reaction and the precipitate is filtered off. The golden colored solution is transferred to a two-neck flask containing equimolar portions of Pb(SCN)₂ and anhydrous sodium sulphate. Anhydrous methanol is

SUBS added and the mixture is brought to reflux. The color gradually changes from purple to dark green. The reaction is stopped and air is bubbled slowly through the solution. The crude product was dissolved in methylene chloride and the solid was filtered off. The volume of the green solution is r _*educed to approximately 5 ml and then charged on an alumina column and eluted with ethylacetate in CH₂Cl₂. The bright green eluent containing the porphacyanine is collected and evaporated to dryness. In yet another alternative (Figure 4) , the unique linking group "^{/ N} ^^N" is formed by preforming and dehydrating an amide linkage as shown in Figure 4.

Thus, there are obtained compounds shown as Formula I of Figure 1 wherein each R can independently be substituted or unsubstituted alkyl, alkenyl, alkynyl; substituted or unsubstituted aryl; alkyl or aryl sulfonyl; alkyl or aryl cyano; halogen; cyano; nitro; amino; carboxy; carbalkoxy or the ester, amide or salt thereof and the like.

In a novel method for preparing a porphacyanine of Formula I (Figure 11), the 3,3' -4,4' -tetraethyl-

5,5' -cyanodipyrromethane in anhydrous THF is added to a THF suspension of LiAlH₄ under nitrogen at 0° C. The mixture is stirred and water is added to quench the reaction and the precipitate is filtered off. A ten-fold excess of 2,3-dichloro- 5,6-dicyano-l,4-benzoquinone (DDQ) in a THF/CH₂Cl₂ suspension under nitrogen at 0° C is added to the 3,3' -4,4' -tetraethyl- 5,5' -bisaminomethyl-2,2' -dipyrromethane in anhydrous THF/CH₂C1₂. The solution turns dark green immediately. The crude product is dissolved in methylene chloride and the solid filtered off. The volume of the green solution is reduced to approximately 5 ml and then charged on an alumina column and eluted with ethylacetate in CH₂Cl₂. The bright green eluent containing the porphacyanine is collected and evaporated to dryness. A significant increase in yield (48%) is obtained from this synthetic process as compared to the yield obtained via air oxidation.

In another novel method for preparing a porphacyanine of Formula I (Figure 12,) 3,3' -4,4' -tetraethyl-5,5' -diformyl- 2,2' -dipyrromethane is suspended in dry EtOH. The resulting ethanol solution is chilled at 0°C and ammonia gas is bubbled through it. The flask is then placed in an oil bath at 60°C for 72 hours. The reaction is stopped and cooled at 0°C. Ethanol is removed on the roto-vap, the residue is chromatographed on neutral Alumina with dichloromethane. This greatly simplified synthetic process yields 20% or 4.6 mg of octaethylporphacyanine. Another 1.5 mg of product is obtained from the oxidation of the more polar component by DDQ. In yet another embodiment, porphacyanines containing a phenyl group on each of the two bridge carbon atoms are prepared by the reaction scheme shown in Figure 13. Thus, there are obtained compounds shown in Figure 13 wherein each X can independently be substituted or unsubstituted alkyl, alkenyl, alkynyl; substituted or unsubstituted aryl; alkyl or aryl sulfonyl; alkyl or aryl cyano; halogen; cyano; nitro; amino; carboxy; carbalkoxy or the ester, amide or salt thereof and the like.

In still another embodiment, porphacyanines containing a phenyl group on one of the two bridge carbon atoms are prepared by the reaction scheme shown in Figure 14.

Target-Specific Moieties

Porphacyanine and the porphacyanine-like compounds alone are particularly useful as targeting agents in that they specifically localize to certain diseased tissues and cells such as tumors. This targeting ability may be enhanced by coupling a porphacyanine to certain moieties that specifically bind to epitopes or receptors located on the surface of such tissues or cells. Thus, target-specific moieties within the present invention include steroids such as estrogen and testosterone and derivatives thereof, peptides comprising T-cell receptors or alpha-beta heterodimers, saccharides such as mannose for which monocytes and macrophages have receptors and H2 agonists.

Target-specific moieties which comprise a component of conjugates within the present invention may also consist of immunospecific components. The Tsm may be derived from polyclonal or monoclonal antibody preparations and may contain

SUBSTITUTE SHEET whole antibodies or immunologically reactive fragments of these antibodies such as (F(ab¹)₂, Fab, or Fab' fragments. Use of such immunologically reactive fragments as substitutes for whole antibodies is well known in the art. See, for example, Spiegelberg, H.L., Immunoassays in the Clinical Laboratory (1978) 3:1-23.

Polyclonal antisera are prepared in conventional ways by injecting a suitable mammal with antigen to which antibody is desired, assaying the antibody level in serum against the antigen, and preparing antisera when the titers are high. Monoclonal antibody preparations may also be prepared conventionally such as by the method of Koehler and Milstein using peripheral blood lymphocytes or spleen cells from immunized animals and immortalizing these cells either by viral infection, by fusion with myelomas, or by other conventional procedures, and screening for production of the desired antibodies by isolated colonies. Formation of the fragments from either monoclonal or polyclonal preparations is effected by conventional means as described by Spiegelberg, H.L., supra . Particularly useful antibodies exemplified herein include the monoclonal antibody preparation CAMAL-1 which can be prepared as described by Malcolm et al . (Ex. Hema tol . (1984) 12:539-547); polyclonal or monoclonal preparations of anti-Mi antibody as described by Mew et al., (J^". Immunol . (1983) 130:1473-1477) and B16G antibody which is prepared as described by Maier et al . (J. Immunol . (1983) 131:1843) and Steele et al . ( Cell Immunol . (1984) 90:303) .

The foregoing list is exemplary and certainly not limiting; once the target tissue is known, antibody specific for this tissue may be prepared by conventional means. Therefore, the invention is applicable to effecting toxicity against any desired target.

Linkage

Conjugation of the target-specific moiety to a porphacyanine within the present invention can be effected by any convenient means and requires that at least one of x-R_g contain a carboxylic group. A direct covalent bond between

SUBSTITUTESHEET these moieties may be effected, for example, using a dehydrating agent such as a carbodiimide, in which case L represents a covalent bond. A particularly preferred method of covalently binding a porphacyanine to the target-specific moiety is treatment with l-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDCI) in the presence of a reaction medium consisting essentially of dimethylsulfoxide (DMSO) . A preparation using this preferred procedure is illustrated in Example 5 below. Of course, other dehydrating agents such as dicyclohexylcarbodiimide or diethylcarbodiimide could also be used as well as conventional aqueous and partially aqueous media.

The active moieties of the conjugate may also be conjugated through linker compounds which are bifunctional, and are capable of covalently binding each of the two active components. A large variety of these linkers is commercially available, and a typical list would include those found, for example, in the catalog of the Pierce Chemical Co. These linkers are either homo- or heterobifunctional moieties and include functionalities capable of forming disulfides, amides, hydrazones, and a wide variety of other linkages.

Other linkers include polymers such as polyamines, polyethers, polyamine alcohols, derivatized to the components by means of ketones, acids, aldehydes, isocyanates, or a variety of other groups.

The techniques employed in conjugating the active moieties of the conjugate include any standard means and the method for conjugation does not form part of the invention. Therefore, any effective technique known in the art to produce such conjugates falls within the scope of the invention, and the linker moiety is accordingly broadly defined only as being either a covalent bond or any linker moiety available in the art or derivable therefrom using standard techniques.

Administration and Use The porphacyanine or conjugates thereof are formulated into pharmaceutical compositions for administration to the subject using techniques known in the art generally. A summary of such pharmaceutical compositions may be found, for example, in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Publishing Co., Easton, Pennsylvania, latest edition) .

Porphacyanine, porphacyanine-like compounds and conjugates thereof within the present invention would normally be administered systemically, in particular by injection. Injection may be intravenous, subcutaneous, intramuscular, or even intraperitoneal. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid form suitable for solution or suspension in liquid prior to injection, or as emulsions. Suitable excipients are, for example, water, saline, dextrose, glycerol and the like. Of course, these compositions may also contain minor amounts of nontoxic, auxiliary substances such as wetting or emulsifying agents, pH buffering agents and so forth.

Systemic administration can also be implemented through implantation of a slow release or sustained release system, by suppository, or, if properly formulated, orally. Formulations for these modes of administration are well known in the art, and a summary of such methods may be found, for example, in REMINGTON'S PHARMACEUTICAL SCIENCES ( supra) .

If the treatment is to be localized, such as for the treatment of superficial tumors or skin disorders, porphacyanine, porphacyanine-like compounds or the active conjugates thereof may be topically administered using standard topical compositions involving lotions, suspensions, pastes, or creams.

The quantity of conjugate or porphacyanine derivative to be administered depends on the choice of active ingredient, the condition to be treated, the mode of administration, the individual subject, and the judgment of the practitioner.

Depending on the specificity of the preparation, smaller or larger doses may be needed. Dosages in the range of about 0.05-

10 mg/kg are suggested for systemic administration. Dosages in the range of about 0.01-20% concentration of active ingredient, preferably 1-5%, are suggested for topical administration. A total daily dosage in the range of about 10-300 mg are suggested for oral administration. The foregoing ranges are merely

SUBSTITUTE SHEET suggestive, as the number of variables in regard to an individual treatment regime is large and considerable excursions from these recommended values are expected.

The radioimaging (scintigraphic imaging) method of the present invention is practiced by injecting an individual parenterally with an effective amount of the porphacyanine radioimaging agent. By parenterally is meant, e . g. , intravenously, intraarterially, intrathecally, interstitially or intracavitarily. It is contemplated that an individual will receive a dosage of from about 1 mCi to 50 mCi of the radioimaging agent, the amount being a function of the particular radioisotope and mode of administration. For intravenous injection, the amount are normally: about 10-40 mCi, preferably about 20 mCi of Tc-99m; about 2-5 mCi, preferably about 4 mCi of In-Ill or Ga-67.

The radioimaging agent is conveniently provided as an injectable preparation, preferably a sterile injectable preparation for human use, for targeting the agent to diseased tissue or cells, preferably comprising: a sterile injectable solution containing an effective amount of the radiolabeled agent in a pharmaceutically acceptable sterile injection vehicle, preferably phosphate buffered saline (PBS) at physiological pH and concentration. Other pharmaceutically acceptable vehicles may be utilized as required for the site of parenteral administration.

A representative preparation to be parenterally administered in accordance with this invention will normally contain about 0.1 to 20 mg, preferably about 2 mg, of radiolabeled agent in a sterile solution. Once enough isotope has been deposited at the target site, scanning is effected with either a conventional planar and/or SPECT gamma camera, or by use of a hand held gamma probe used externally or internally to localize the inflammation or the lesion. The scintigram is normally taken by a gamma imaging camera having one or more windows for detection of energies in the 50-500 KeV range.

Magnetic resonance imaging (MRI) is effected in an analogous method to radioimaging except that the imaging agents

SUBSTITUTE ς u i !ET will contain MRI enhancing species rather than radioisotopes. It will be appreciated that the magnetic resonance phenomenon operates on a different principle from radioimaging. Normally the signal generated is correlated with the relaxation times of the magnetic moments of protons in the nuclei of the hydrogen atoms of water molecules in the region to be imaged. The magnetic resonance image enhancing agent acts by increasing the rate of relaxation, thereby increasing the contrast between water molecules in the region where the imaging agent accretes and water molecules elsewhere in the body. However, the effect of the agent is to increase both T_χ and T₂, the former resulting in greater contrast, while the latter results in lesser contrast. Accordingly, the phenomenon is concentration dependent, and there is normally an optimum concentration of a paramagnetic species for maximum efficiency. The optimum concentration will vary with the particular agent used, the locus of imaging, the mode of imaging, i.e., spin-echo, saturation-recovery, inversion-recovery and for various other strongly T_x dependent or T₂ dependent imaging techniques, and the composition of the medium in which the agent is dissolved or suspended. These factors, and their relative importance are known in the art. See, e . g. , Pykett, Scientific American (1982) 246:78, and Runge et al. , Am . J. Radiol . (1987) 141:1209. The MRI method of the invention is practiced by injecting an individual parenterally with an effective amount of an MRI contrast agent comprising porphacyanine or a porphacyanine-like compound within the present invention coupled to a metallic element such as gadolinium. It is contemplated that an individual will receive a dosage of contrast agent sufficient to enhance the MRI signal at the targeted site by at least about 20%, preferably 50-500%, the amount being a function of the particular paramagnetic species and the mode of administration.

Again, a contrast agent within the present invention is conveniently provided as an injectable preparation for use, preferably a sterile injectable preparation for human use, for targeting an MRI agent to diseased tissues or cells, preferably comprising: a sterile injectable solution containing an

SUBSTITUTE SHEET effective amount of the contrast agent in a pharmaceutically acceptable sterile injection vehicle, preferably phosphate buffered saline. Other conventional pharmaceutically acceptable vehicles for parenteral administration may be utilized as required for the site of parenteral admini _*stration.

A representative preparation to be parenterally administered in accordance with this invention will normally contain about 0.1 to 20 mg, preferably about 2 mg of contrast agent, in a sterile solution.

Examples

The following examples are intended to illustrate the invention but not to limit it scope.

Example 1 Synthesis of Porphacyanine, Figure 2 Lead tetraacetate (18.2 g, 0.041 mole) was added to a stirred solution of 2-benzyloxycarbonyl-3,4-diethyl-5-methyl pyrrole (10.1 g, 0.037 mole) in 60 ml glacial acetic acid. The mixture was warmed briefly to 60°C. Ten milliliters of ethylene glycol was added to reduce any remaining Pb(IV) . Twenty milliliters of water was added and the 5-acetoxymethyl-

2-benzyloxycarbonyl-3,4-diethylpyrrole (Compound A in Figure 2) was collected by filtration and washed with additional water. The 5-acetoxymethyl-2-benzyloxycarbonyl-3,4-diethylpyrrole was added to 80% acetic acid in 100 ml water and heated at 100°C for one hour. The solid product precipitated as large chunks when the foregoing solution cooled to room temperature. One hundred milliliters of water was added and the product was collected by filtration and then washed with additional water. The filtrate was extracted with CH₂C1₂ and then evaporated to produce a solid product. The solid products were combined, and then recrystallized from a solution of CH₂Cl₂ and hexanes. Yield: 6.6 g, 67.4%

Mol. Wt. Calcd. for C₃₃H₃₈θ₄N₂: 526.2831 High resolution MS: 526.2835

SUBSTITUTE SHE! IT ^XH NMR in CDC1₃: 1.05 (t, 6H) , 1.12 (t, 6H) , 2.43 (q, 4H) , 2.75 (q, 4H) , 3.85 (s, 2H) , 5.25 (s, 4H) , 7.28-7.40 (m, 10H) , 8.70 (br s, 2H) .

5,5' -bis(benzyloxycarbonyl) -3,3' -4,4' -tetraethyl- 2,2' -dipyrromethane (Compound B of Figure 2) (8.1 g, 0.015 mole) in 200 ml tetrahydrofuran (THF) was stirred under hydrogen at atmospheric pressure and room temperature overnight in the presence of 0.4 g 10% Pd/C and 5 drops of triethylamine. After the catalyst was filtered through celite, the filtrate was evaporated to dryness on a roto-vap resulting in dicarboxylic acid. The dicarboxylic acid was dissolved in 100 ml N,N- dimethylformamide and heated to boiling under argon for one and one-half hours. The solution was then chilled on ice and an excess of chilled benzoyl chloride (7.2 ml) was added drop-wise to the bis-α-free dipyrromethane (Compound B) . The reaction mixture was stirred for 2 hours at 5°C and the solid product collected by filtration. The solid product was added to 50 ml water and basified using NaHC0₃. The solution was heated and held at 60°C for one hour. The pale yellow product which crystallized from the solution was filtered and then washed with water.

Yield: 3.4 g, 70.0%

Mol . Wt . Calcd . for C₁₉H₂₆N₂0₂ : 314 . 1994

High resolution MS : 314 . 1994 ^XH NMR in CDC1₃ : 1 . 10 ( t , 6H) , 1 . 25 (t , 6H) , 2 . 50 ^'

(q , 4H) , 2 . 70 (q , 4H) , 4 . 00 ( s , 2H) , 9 . 55 ( s , 2H) , 10 . 90 ( s , 2H) .

3,3' ,4,4' -tetraethyl-5,5' -diformyl-2,2' -dipyrromethane (Compound C of Figure 2) (1.03 g, 0.0033 mole) in 300 ml ethanol was bubbled with argon for 20 minutes. hydroxylamine hydrogen chloride (0.51 g, 0.0073 mole) and sodium acetate (1.20 g, 0.015 mole) were added. This mixture was heated at 60°C under argon for two and one-half hours. The solvent was removed on a roto- vap and the product dried overnight in vacuo. The bis-oxime was dissolved in 5 ml acetic anhydride and saturated with argon for 30 minutes. The crude bisnitrile product (Compound D of Figure 2) was obtained as a black solid after removal of acetic anhydride and dried under vacuum overnight. The product was purified by silica gel column (40 g) with 0.5% methanol in

SUBSTITUTE SHEET CH₂C1₂, followed by an alumina column (40 g) with 10-20% EtOAc. Evaporation of the solvent yielded pale pink crystals.

Yield: 0.43 g, 42%

Mol . Wt . Calcd . for C₁₉H₂₄N₄ : 308 . 2001 High resolution MS : 308 . 2002 . K NMR in CDC1₃ : 1 . 10 ( t , 6H) , l ^'. 25 ( t , 6H) , 2 . 45 (q , 4H) , 2 . 60 (q, 4H) , 3 . 90 ( s , 2H) , 8 . 45 ( s , 2H) .

3,3' -4,4' -tetraethyl-5,5' -cyanodipyrromethane (0.053 g, 1.7 X 10^"4 mole) was dissolved in 10 ml anhydrous THF and added dropwise to a THF suspension of LiAlH₄ (0.050 g, 1.5 X 10"³ mole) under N₂ at 0°C. The resulting mixture was stirred for 30 minutes and two drops of water was added. A solid product formed which was filtered off. The bis-amine product was obtained after evaporation of the solvent by drying under vacuum overnight. The bis-amine was dissolved in 50 ml anhydrous methanol and bis-aldehyde (0.050 g, 1.6 X 10^"4 was added. The solution was bubbled with N₂ for 20 minutes and brought to reflux under N₂. Lead thiocyanate (Pb(SCN₂)) (0.055 g, 1.7 x 10^"4 mole) was added and the solution refluxed for 4 hours. Oxygen gas was bubbled through the solution at room temperature overnight. After evaporation of the solvent, the crude porphacyanine product was dried under vacuum overnight. The product was purified by Al₂0₃ column (4% water added) with 10% ethyl acetate in CH₂C1₂. The green eluent was collected and concentrated on the roto-vap. Crystals of the porphacyanine macrocycle were obtained after evaporation of the solvent.

Yield: 19.3 mg, 19.1% macrocycle

Mol. Wt. Calcd. C₃₈H₄₈N₆: 588.3941

High resolution MS: 588.3933 ^XH NMR (300 MHz, CDC1₃) , -4.50 (bs, 2H) , 2.05 (t, 12H) ,

2.13 (t, 12H) , 4.28 (q, 8H) , 4.40 (q, 8H) , 10.50 (s, 2H) , 13.0 (s, 4H) .

UV/VIS in CH₂C1₂ 455, 592, 800 nm.

Example 2 Synthesis of Porphacyanine. Figure 3

3,3' -4,4' -tetraethyl-5, 5' -cyanodipyrromethane was prepared according to the methodology of Example 1. 0.102 g,

SUBSTITUTE SHEET 3.3 X 10"⁴ mole of 3,3' -4,4' -tetraethyl-5,5' -cyanodipyrromethane in 10 ml anhydrous THF was slowly added to a 20 ml THF suspension of LiAlH₄ under N₂ at

0° C. The mixture was stirred for 30 minutes and two drops of water were added to quench the reaction. The precipitate was filtered off. The golden colored solution was transferred to a two-neck flask containing equimolar portions of Pb(SCN)₂ and anhydrous sodium sulphate. Fifteen milliliters of anhydrous methanol were added and the mixture was brought to reflux. The color of the solution gradually changed from purple to dark green. The reaction was stopped after four and one-half hours and air was bubbled slowly through the solution overnight. The crude product was dissolved in methylene chloride and the solid was filtered off. The volume of the green solution was reduced to approximately 5 ml and then charged on an alumina column (120 g, 4% water added) and eluted with 10% ethylacetate in CH₂C1₂. Two liters of the bright green eluent containing the porphacyanine was collected and evaporated to dryness. Yield: 23.4 mg, 24.1% The spectroscopic data of this compound are identical to the compound prepared in Example 1 above.

Example 3 In Vi tro Toxicitv of Porphacyanine Cells are washed three times in serum-free medium (DME) , counted and made up to a concentration of 10⁷ cells per ml.

For the "affinity" assay, in the dark, 100 μl of the cell suspension and 100 μl of the test or control compound are mixed. "Labeling" is allowed to continue for one hour at 4°C, and labeled cells are washed in the dark three times with 3 ml medium each time and resuspended in fresh medium. The resuspended cells are then subjected to light exposure at 300- 850 nm for 30 minutes.

In a "direct" assay the cells are irradiated immediately upon addition of the test or control compound. The effect of irradiation is estimated using methods appropriate to the target cells. SUBSTITUTE SHEET When human erythrocytes (RBCs) are used as test cells, the hemolysis caused by irradiation of control (hematoporphyrin, Hp) -labeled and porphacyanine (Formula I) -labeled cells is estimated visually. When the murine mastocytoma cell line P815 is used, the results are determined as follows:

The cells are labeled as above using concentrations of 10-50 ng/ml of Hp as control and the porphacyanine of Formula I as the test substance. The resuspended cells are treated with 300-850 nm light for 30 minutes and the viability resulting is estimated by direct counting using eosin-Y exclusion, a standard procedure for differentiating living from dead cells.

In other determinations conducted as above, the cells recovered from light exposure are assayed for viability by incubating them for 18 hours in 10 μCi/ml tritium-labeled thymidine according to the standard procedure whereby thymidine incorporation is equated with viability. The cells are harvested and radioactivity uptake is measured by a scintillation counter.

Example 4

Selective Binding of Porphacyanine P815 cells are incubated as described in Example 3 using 1-200 ng/ml Hp or the porphacyanine of Formula I. The cells are labeled in the dark for 30 minutes, washed free of unabsorbed porphyrins, resuspended, and then exposed to 300-850 nm light for another 30 minutes. Viability of the cells is established by tritiated thymidine incorporation after labeling with 30 μCi/ml tritiated thymidine and incubating at 37°C for 18 hours.

Example 5

Preparation of Immunoconiugates This example describes methods of preparation for immunoconjugates of four different antibody preparations with either hematoporphyrin (Hp) or a porphacyanine (Pc) of Formula I. The antibodies employed are CAMAL-1, anti-Mi antibody, and B16G antibody, all prepared as described hereinabove, and

SUBSTITUTE SHEET af f inity purified rabbit anti-mouse Ig (RαMIg) . In addition, a purif ied irrelevant monoclonal preparation (C-MAb) is used where a control is desired .

One preparation of the conj ugates is performed basically as described by Mew et al . (J. Im _*munol . ( 1983 )

130:1473). Briefly, to 220 mg Hp.0.2 HC1 (Sigma Chemical Co., St. Louis, MO) in 25 ml water and 0.8 ml N,N-dimethylformamide is added 20 mg l-ethyl-3- (3-dimethylaminopropyl) -carbodiimide HCl (EDCI) in 0.6 ml water. After 30 minutes, this solution is mixed with 15 mg of the antibody protein dissolved in 5 ml distilled water and incubated for 5 hours. During this period, the pH of the solution is monitored and adjusted to between 6 and 7. Then 50 μl of monoethanolamine is added, and the solution is allowed to stand overnight at room temperature. The solution is dialyzed against 0.001 M phosphate buffer pH 7.4 for four days with three changes per day and overnight against PBS. The conjugate of porphacyanine is analogously prepared.

In a preferred protocol, 2 ml of a dispersion in DMSO containing 5 mg each of the Hp or Pc and the dehydrating agent is prepared and stirred for 30 minutes at room temperature under nitrogen. To this is added a dispersion containing 2 mg of the appropriate immunoglobulin in 2 ml of DMSO, and the resulting mixture stirred for another 10 minutes. This mixture is then worked up by dilution in phosphate-buffered saline, pH 7.4 (PBS) by adding 5 times the volume of PBS containing 50 μl monoethanolamine, and is then dialyzed against PBS using three changed of wash.

Alternatively, 2 ml of a dispersion containing 5 mg each of Hp or Pc, a linking agent, and a dehydrating agent is prepared and stirred for approximately 15 minutes at room temperature under nitrogen. To this is then added a dispersion containing about 2 mg of the immunospecific protein in 2 ml of tetrahydrofuran and the resulting mixture stirred for another 10 minutes. The mixture is then worked up as described above. The foregoing procedures are appropriate for CAMAL-l and for the remaining antibody preparations listed above.

In addition, the following preparations are made specifically with B16G and RcκMIg:

SUBSTITUTE SHEET B16G

Eleven mg of hematoporphyrin plus 11 mg of EDCI in 4 ml spectral grade DMSO are stirred for 30 minutes under nitrogen at room temperature before the addition of 20 mg lyophilized B16G antibodies, prepared as described by Maier et al . J.

Immunol . (1983) 131:1843, in 2 ml DMSO. The resulting mixture is stirred for 40 second at room temperature and working up as described above. The resulting product contains approximately 375 μg Hp/mg B16G. A similar procedure is used substituting Pc for Hp.

RoMI

Four hundred μg of EDCI and 400 μg hematoporphyrin in 1 ml DMSO are stirred for 30 minutes under nitrogen at room temperature as above before the addition of 800 μg lyophilized RorMIg antibodies, prepared as described by Mew et al . (J.

Immunol . (1983) 130:1473) in 1 ml DMSO. The resulting mixture is stirred for 30 seconds and worked up as described above to obtain a product containing 200 g Hp/mg RαMIg. A similar procedure is used substituting Pc for Hp.

Example 6

Specificity of Immunoconiugates In Vi tro The Tsm-Hp and Tsm-Pc conjugates wherein the Tsm is comprised of an immunoglobulin are tested against cells in vi tro by mixing the conjugates with the appropriate cell types, along with suitable controls, and then exposing the labeled cells to irradiation. Procedures for carrying out this assay are described in detail in Mew et al . , Cancer Research (1985) for CAMAL-1, and by Mew et al . , J. Immunol . (1983) for Anti-Mi, both references cited hereinabove are incorporated herein by reference.

Briefly, for CAMAL-1, three cell lines, WC4, WC6 and WC2 (WC4 and WC6 produce the CAMAL antigen, but WC2 does not) , are labeled with the appropriate Tsm-Hp or Tsm-Pc preparation as described above in Example 4. The labeled cell preparations containing 10⁶ cells each are introduced to Rose chambers and

SUBSTITUTE SHEET exposed to light activation with a laser at 630 nm. The results for various preparations are then compiled.

For the anti-Mi conjugate, Ml tumor cells are used as target cells and treated with the Tsm-Hp, Tsm-Pc conjugates or drug or antibody alone or the combination of antibody and drug, but uncoupled, by incubating them in 6% Co₂ humidified incubator at 37°C for two hours. The cells are washed three times in PBS and then plated and exposed to fluorescent light overnight. The cells are assessed for viability by tritiated thymidine uptake as above.

For the B16G conjugates, A10, P815, and L1210 cells are used as target cells. (A10 cells are a T-cell hybridoma which secretes a B16G-reactive T-suppressor factor; P815 cells are also reactive with B16G.) The in vi tro study is done using a direct method employing the B16G-Hp or B16G-Pc conjugate or indirectly using unlabeled B16G antibodies and labeled RαMIg-Hp or Rα.MIg-Pc.

In a direct method, 5 x 10^s cells are suspended in 1 ml DME/Hepes containing the appropriate Tsm-drug conjugate as test or control at Hp or Pc concentrations of 320, 160, 80, 40 and 20 ng drug/ml. The cells are incubated in the dark at 37°C for 1 hour, then washed 3 times in 5 ml DME/Hepes, and then resuspended in 1 ml of the same buffer. Three 100 μl test portions of the labeled preparations are dispensed into flat bottom microtiter wells and the remainder of the cell suspensions (700 μl) are exposed to incandescent light (22.5 mW/cm²) at a distance of 20 cm for 1 hour. Then three additional 100 μl aliquots are removed to microtiter wells. Tritium-labeled thymidine diluted in DME/Hepes containing 20% FCS is then added to all microtiter wells in 100 μl aliquots so that 2 μCi of labeled thymidine is added to each well. Cultures are incubated for 18 hours at 37°C and humidified 10% C0₂ and then harvested on a MASH harvester. Thymidine incorporation is measured with an Hp scintillation counter (Tri-Carb Model 4550) . In an indirect assay, the A10 suspended cells, prepared as described above, are exposed to 50 μg/ml of either B16G or a control antibody C-MAb at 4°C for 30 minutes, washed in DME/Hepes, and then exposed for an additional 30 minutes at 4°C in the dark to varying concentrations of RαMIg-Hp or RαMIg-Pc between 2 μg/ml and 15 ng/ml of Hp or Pc. The cells are assessed for viability using labeled thymidine uptake as described above.

Example 7

In Vivo Cytotoxicity of the Porphacyanine and Conjugates Thereof The in vivo efficacy of porphacyanine (Pc) and conjugates thereof is also assessed. For the CAMAL-1 and anti-Ml conjugates, the procedures are as described in the two Mew et al . papers referenced above in Example 6. For the B16G-Hp and B16G-Pc conjugates and for the Pc (Formula I) alone, the in vivo studies are conducted as follows:

The in vivo test relies on the indirect effect of a population of T-suppressor cells on tumors, which then serve as means to assess the effectiveness of the irradiation treatment. P815 mastocytoma cells grown in syngeneic DBA/2 mice stimulate T-suppressor cells specific for the tumor. These T-suppressor cells impede the development of specific T-killer cells which would otherwise aid in the regression of the tumor. The T-cell hybridoma designated A10 above secretes a T-suppressor factor which is associated with these T-suppressor cells. Thus, selective killing of these T-suppressor cell populations through reaction with conjugates in which the Tsm is an antibody specific for the T-suppressor factor on the surface of the cells (namely B16G) should result in tumor regression in mice bearing the P815 tumors.

Therefore, in this assay, DBA/2 mice are injected in the right flank subcutaneously with 10⁴ P815 cells to incorporate the tumor. On day eight, when the tumors are palpable (approx. 25-42 sq. mm) , the mice are randomly sorted into groups of eight and injected IV with 150 μl PBS containing nothing, Hp or Pc, B16G-Hp or B15G-Pc, B16G plus either drug, B16G alone or C-MAb-Hp or C-MAb-Pc. The levels of Hp are 50 μg per animal in all cases and B16G 310 μg in all cases (where appropriate) .

;STITUTE Sr ET The animals are maintained in the dark for two hours and then exposed to strong light at 300-850 nm and 22.5 mW/cm². The animals are then treated normally and monitored on a daily basis.

Example 8

Diagnostic Imaging A 32-year old female patient develops fever and abdominal pain. The patient is maintained on antibiotic therapy for a period of one week without effect. A CAT scan fails to demonstrate any abnormal mass. Radioimaging studies are performed using Tc-99m-labeled porphacyanine. An injection of 20 mCi of the radiolabeled porphacyanine is used and the patient is scanned with a gamma camera in SPECT mode. The scan of the patient's abdomen demonstrates a focus of accumulation of Tc-99m. Surgery is performed and an abscess is found at the site of the Tc-99m activity.

Example 9 Synthesis of Porphacyanine. Figure 11 3,3' -4,4' -tetraethyl-5,5' -cyanodipyrromethane was prepared according to the methodology of Example 1. A ten-fold excess of 2,3-dichloro-5,6-dicyano-l,4-benzoquinone (DDQ) was added to 69 mg of 3,3' -4,4' -tetraethyl-5,5' -cyanodipyrromethane in 10 ml anhydrous THF. The resulting solution turned dark green immediately. The residue was chromatographed according to the methodology of Example 2. A significant increase in yield was obtained when compared to air oxidation. Yield: 32 mg, 48%

The spectroscopic data of this compound are identical to the compound prepared in Example 1 above.

Example 10

Synthesis of Porphacyanine. Simplified Method In an alternative method for synthesizing porphacyanine, the dialdehyde was prepared according to the methodology of Example 1. 25 mg of 3,3' -4,4' -tetraethyl-5, 5' - diformy1-2,2' -dipyrromethane was suspended in 30 ml dry EtOH.

SUBSTITUTE SH ET The resulting ethanol solution was chilled at 0°C and ammonia gas was bubbled through it for 30 minutes. The gas inlet was then removed and the flask placed in an oil bath at 60°C for 72 hours. The reaction was stopped and cooled at 0°C. Ethanol was removed on the roto-vap and the residue was chromatographed on neutral Alumina (4% H₂0 added) with dichloromethane.

Yield: 4.6 mg, 20%

The spectroscopic data of this compound are identical to the compound prepared in Example 1 above. Another 1.5 mg of product was obtained from the oxidation of the more polar component by DDQ.

SUBSTITUTE SHEET

Claims

Claims

1. A porphacyanine of the formula:

wherein each of R_!- ₈ is independently a noninterfering substituent and each of R₉ and R₁₀ is independently -H or

wherein each X is independently a noninterfering substituent.

2. The compound of claim 1 wherein each of Rx-R_g and each X is independently selected from the group consisting of -H, substituted or unsubstituted alkyl, alkenyl, alkynyl; substituted or unsubstituted aryl; alkyl or aryl sulfonyl; alkyl or aryl cyano; halogen; cyano; nitro; amino; carboxy; carbalkoxy and the esters, amides and salts thereof.

3. The compound of claim 1 or 2 wherein R₉ and R 1C are H.

SUBSTITUTESHEET The compound of claim 1 or 2 wherein one of R9 and R₁₀ is H and the other is

or wherein both R₉ and R₁₀ are

5. The compound of any of claims 1-4 wherein each of R^R_g is independently ethyl or H.

6. A conjugate of the formula Tsm-L-Pc wherein Tsm represents a target-specific moiety, and wherein Pc represents a porphacyanine having a light absorption maximum in the range of 400-850 nm, and wherein L represents a covalent bond or a linker moiety bound to the Tsm and Pc through covalent bonds.

7. The conjugate of claim 6 wherein the Tsm is an immunoglobulin or an immunoglobulin fragment; or is a steroid; or is a saccharide; or comprises a T-cell receptor; and/or wherein L represents a covalent bond.

8. A pharmaceutical composition which is cytotoxic to specific cells or tissues which comprises an effective amount of the compound of any of claims 1-5 or the conjugate of claim 6 or 7 in admixture with at least one pharmaceutically acceptable excipient.

SUBSTITUTE SHEET 9. The use of the compound of any of claims 1-5 or the conjugate of claim 6 or 7 in a method to impair the metabolism of or to effect the destruction of targeted cells, tissues or viruses which comprises contacting said cells, tissues or viruses with an effective amount of said compound or conjugate and irradiating the contacted cells, tissues or viruses with light in the wavelength range of 400-850 nm.

10. A magnetic resonance imaging (MRI) contrast agent comprising the compound of any of claims 1-5 and a paramagnetic ion.

11. The diagnostic contrast agent of claim 10, wherein the paramagnetic ion element is gadolinium(III) or manganese(II) .

12. The use of the agent of claim 10 or 11 in a method to detect diseased tissue which comprises administering to an animal in need of such treatment said contrast agent and then observing the distribution of the contrast agent in the animal.

13. A radioimaging agent comprising the compound of any of claims 1-5 and a radioisotope selected from the group consisting of technetium, indium and gallium.

14. The use of the agent of claim 13 in a method to detect diseased tissue which comprises administering to an animal in need of such treatment said radioimaging agent and then observing the distribution of the radioimaging agent in the animal.

15. A method to prepare a porphacyanine of claim 1 wherein each R is a noninterfering substituent, comprising the steps of: (a) adding a ten-fold excess of 2,3-dichloro-5,6- dicyano-1,4-benzoquinone to an appropriately substituted 5,5'- bisaminomethyl-2,2' -dipyrromethane to produce a crude product;

SUBSTITUTESHEET (b) purifying the crude product by chromatography on an alumina column; and then

(c) evaporating the purified porphacyanine to dryness.

16. A method to prepare a porphacyanine of claim 1 comprising the steps of:

(a) suspending appropriately substituted diformyl- 2,2' -dipyrromethane in dry ethanol;

(b) bubbling ammonia gas through the solution of step (a) ;

(c) heating the product of step (b) at least about 60°C for at least about 72 hours;

(d) cooling the product of step (c) to 0°C;

(e) purifying the crude product by chromatography on an alumina column; and then

(f) evaporating the purified porphacyanine to dryness.

SUBSTITUTE SHEET