MXPA99010169A - Ethylene glycol esters of monohydrobenzoporphyrin derivatives as photoactive agents - Google Patents

Abstract

New compounds useful in photodynamic therapy are of formula (1) or (2), (3) or (4) and the metallated and/or labeled and/or conjugated forms thereof wherein each R1 is independently alkyl(1-6C);each n is independently an integer of 0-6;and R2 is vinyl or a derivative form thereof.

Description

ESTERES DE ETILENGLICOL DE DERIVADOS DE MONOHIDROBENZOPORFIRINA AS PHOTOACTIVE AGENTS TECHNICAL FIELD The invention relates to compounds useful in photodynamic therapy (PDT) and related applications. In particular, it concerns ethylene glycol esters of monohydrobenzoporphines. BACKGROUND OF THE ART Photodynamic therapy (PDT) generally involves the administration of compounds that are capable of absorbing light, typically in the visible range, but also in the near ultraviolet, followed by irradiation of places in the subject for which a toxic or inhibitory effect. PDT was initially developed using hermatoporf-irin and related compounds in the treatment of tumors, as it appeared that these compounds would "stay" in places that contain rapidly dividing cells. The tumor can then be irradiated with light absorbed by the hermatoporphyrin and can result in the destruction of the surrounding tissue. PDT has since shown to be useful in the treatment of atherosclerotic plaques, restenosis, bloodstream infections, rheumatoid arthritis, psoriasis and in the treatment of ocular conditions not necessarily limited to tumors. The U.S. Patent No. 5,171,749 and patents issued in related applications, US Patents. Nos. 5,283,255; 5,399,583; 4,883,790; 4,920,143; and 5,095,030; all of which are incorporated herein by reference, describe and claim a class of photoactive compounds useful in PDT referred to as monohydrobenzoporphines, or "BPDs'd. This class is obtained by the Diels-Alder reaction of an alkyne monohydrate or desubst. Tissue with protoporphine-IX and the resulting compounds can also be isomerized, reduced and / or derivatized to obtain a broad class of BPDs As discussed in these / patents, a particularly useful subclass results from hydrolysis or partial hydrolysis of the ester groups of the 2-carboxyethyl side chains in the C and D rings. Esterification as protection of these groups during the Diels-Alder reaction results in initial products which contain 2-carboxyethyl groups. easy of these esters can be easily conducted, - leaving any of the carbaloxy groups associated with the Diels-Alder product obtained from a virtually completely dehydrolyzed dicarbaloxyalkine. This resulted in four compound species, BPD-MA, BPD-MB, BPD-DA and BPD-DB as depicted in Figure 1; this figure taken from the U.S. Patent. No. 5,171,749. In this embodiment, R1 and R2 are carbaloxy groups, typically carbometpxy or carboethoxy, and R is (1-6C) alkyl. It was found that BPD-MA has useful properties for PDT and is currently in clinical development. However, there remains a need for additional specific forms of photoactive agents which expand the repertoire of photoactive compounds for the variety of indications to which PDT is applied, as previously mentioned. The present invention provides compounds in which rings C and D contain ethylene glycol esters of the esterification substituents. These compounds have pharmacokinetic properties which are advantageous in some cases where PDT is used. SUMMARY OF THE INVENTION The compounds of the invention are new - useful additions to the repertoire of compounds that find application in photodynamic therapy and the related methodologies that use photoactive compounds. The presence of ethylene glycol esters in these molecules gives them characteristics that allow the expansion of the scope of the conditions under which the photoactive compounds are used to adjust the treatment. Thus, in one aspect, the invention is directed to the compounds of the formula and the metalated and / or labeled and conjugated forms thereof where R1 is (1-6C) alkyl, preferably methyl, n is an integer of 0-6, preferably 2, and R2 is vinyl or a derivative thereof, preferably vinyl. The invention is also directed to the compounds of the formula and the metalated and / or tagged and conjugated forms thereof where R1, n and R2 are defined as previously described. These analogies are derived from protoporphyrin III and protoporphyrin XIII respectively, in a manner similar to that in which the compounds of formulas 1 and 2 are derived from protoporphyrin IX. The invention also includes isomers of the various forms of the formulas 1-4 which result from the non-rearranged Diels-Alder condensation products (i.e. 1,4-dienes) as described in the U.S. Patent. No. 4,883,790 incorporated there for reference. In other aspects, the invention relates to methods of diagnosis and treatment using the compounds of formula 1, 2, 3 or 4 or their isomers 1,4-dienes or mixtures thereof.

- BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the compounds of the prior art, BPD-MA, BPD-MB, BPD-DA and BPD-DB. Figure 2 shows the kinetics of B-EA6 uptake by L1210 cells. Figure 3 shows the kinetics of B-EA6 release by L1210 cells. Figure 4 shows a graphical representation of the pharmacokinetics of B-EA6 in vi vo. Figure 5 shows a comparison of the kinetics of B-EA6 taking by normal splenocytes and L1210 cells. Figure 6 shows the time course of PDT using B-A6 in mice compared to mice treated with BPD-MA and BPD-MB. Figure 7 shows the effect of B-EA6 on the microvasculature in the mice. Figure 8 shows a comparison of the plasma spectra of BPD-MA and B-EA6. Figures 9A and 9B show the cytotoxic effect of photodynamic treatment using A-EA6 compared to BPD-MA in L1210 cells and dendritic cells. Figure 10 shows the comparative effects of A-EA6 and BPD-MA in decreasing surface expression of MHC1 receptors. Figure 11 shows the effect of photodynamic therapy using A-EA6 and BPD-MA on stress pathway and mitogenic kinases in H60 cells. Figure 12 shows the comparative effect of PDT using A-EA6 and BPD-MA in caspase activation in HL60 cells. Figure 13 shows the comparative effect of PDT using A-EA6 and BPD-MA in DNA fragmentation in HL60 cells. MODES FOR CARRYING OUT THE INVENTION The compounds of the invention are related to those set forth in the BPD patents cited above, but differ in that they contain ethylene glycol esters in the substituents in the C and D rings. These compounds can be prepared by simple hydrolysis. of the carboxyalkyl or carboxyl substituents and the re-esterification of the resulting carboxy groups in the C and D rings of the benzoporphyrins, or can be obtained directly by transesterification. It will be noted that compounds 1 and 2 are individual species of the genus, described in the US patents. previously referenced, obtained by means of a process which comprises a reaction of Diels-Alder with protoporphyrin IX. Compounds 3 and 4 are prepared in a completely analogous manner but using protoporphyrin III or protoporphyrin XIII as substrates for the Diels-Alder reaction. Because protoporphyrin IX is not symmetric with respect to rings A and B, two possible products result depending on whether or not the addition of Diels-Alder occurs in ring A or B. On the other hand, protoporphins III and XIII are symmetrical with respect to these rings, and in addition only one product results in each case regardless of the site of the addition. In the compounds of the invention, R2 is preferably vinyl, but may also be a derivative thereof. The vinyl group in ring A or B is easily derived into other R2 moieties by addition or oxidation. The products of the addition or oxidation can be substituted if they are replaced, they are added as functional groups, for example, -Br can be replaced by -OH.- OR ", -NH2, -NHR", -NR, etc. R 'is a radical - hydrocarbon. For example, one of the added substituents may be hydrogen and the other halo, hydroxy, lower alkoxy, amino, or an amide, sulfhydryl, or an organosulfide, or an additional hydrogen. The compounds of the invention include various groups such as R 2 including substituents which provide additional porphyrin ring systems or related porphyrin. In this way, R2 can be a vinyl, CHORd -CHO, -COORd -CH (OR ') CH 3, - CH (OR') CH 20 R -CH (SR ') CH 3, -CH (NR') 2 CH 3, -CH (CN) CH 3, - CH (COOR ') CH 3, -CH (OOCR ') CH 3, -CH (NR' COR ') CH 3, - CH (CONR' 2) CH 3, CH (halo) CH 3, or -CH (halo) CH 2 (halo) wherein R 1 is H , or a hydrocarbon radical (1-6C) optionally substituted with a heteroatom substitute or wherein R2 is an organic group lower than 12C that results from the direct or indirect derivation of the vinyl group, or where R2 is a group containing the 1-3 nuclei of tetrapyrrole type. As used herein, the term "alkyl" ("alkyl") refers to a straight or branched saturated hydrocarbon chain which may be, if it contains a sufficient number of carbon atoms, cyclic or containing a portion - cyclical Typical examples are methyl, ethyl, t-butyl, cyclohexyl and the like. A "hydrocarbon radical" refers to a monovalent substitute containing only carbon and hydrogen which may be a straight or branched chain, saturated or unsaturated, aromatic or non-aromatic or both, and cyclic or non-cyclic. Thus, a hydrocarbon radical of 1-10C could include cyclopentane, 2-pentenyl, 3-butynyl, 2,4-dimethylhexyl, and the like. In some embodiments of the invention, the hydrocarbon radical can be substituted with a heteroatom-containing substitute. Substituents include -OR, -NR2, -SR, -COOR, CONR2, -OOCR, -NRCOR, -SOR, -S02R, -S03R, halo, -CN, and the like, wherein R is H or alkyl (1-6C) ). Cyclic amines include pyridyl, pyrimidyl, thiazolyl, quinolyl, and others. In this way, they can include single ring or combined ring systems and can contain additional heteroatoms. It will be noted that the compounds of the invention contain at least one chiral center and thus can exist in various stereoisomeric forms. If desired, the stereoisomers, which include the enantiomers, can be separated using standard techniques in the art; however, mixtures per cluster or mixtures containing more than one diastereomer can be used. The compounds as indicated in formulas 1-4, in addition, are representative of the individual optical isomers, enantiomers, or diastereomers as the case may be, as well as mixtures of these individual chiral isomers. If desired, the compounds of the invention can be prepared in metallated forms by treating the tetrapyrrole-type core with an appropriate ion such as magnesium ion, zinc ion, tin ion and the like, to obtain a metal compound. The metal ion can also be a radioactive label. Generally, the metal ion is inserted using the appropriate salts under conditions standard in the art. For example, the zinc ion can be introduced by treating the compound with zinc acetate in 1: 1 methylene chloride: methanol. The compounds may also contain a label, including radioisotopes, chromophores, and fluorescent labels. The labeling of radioisotopes is generally useful when the compounds are followed in vi or are used for specific waste labeling. The useful cationic residues that are radioisotopes include technetium, gallium and indium. In addition, radioisotopes of heteroatoms, such as 131I or 32P, can be used on the molecule itself, or the inclusion of 14C to label the molecule. As described in detail in the previously published BPD-related patents, the compounds of the invention can be coupled, if desired, towards an objective agent which will direct the molecule towards a specific tissue or agent. Target agents include antibodies, receptors, receptor-binders and the like. The linking of the target agent with the component is conducted using standard techniques. By "conjugated form" is meant a compound of formulas 1-4 coupled to a target agent, as described above. Preferred embodiments of the compounds of formulas 1-4 include those wherein both n are equal to 2, or those wherein both R 1 are ethyl or methyl, preferably methyl, and those wherein R 2 is vinyl. They are particularly the compounds of the formula ß-EAß Both A-EA6 and B-EA6 have been prepared. Both are effective photosynthesizers; It seems that A-EA6 is the easiest to formulate. The various forms of the compounds of the invention can be used in photodynamic therapy techniques generally known in the art. As stated in the Background section previously, photodynamic therapy can be conducted using a plethora of protocols and for a variety of indications. In addition, compounds of this type exhibit pharmacological activity in the absence of light in some cases. Standard pharmaceutical compositions, which include liposomal compositions as preferred, are used in the applications. The following examples are intended to illustrate but not limit the invention. While the Examples illustrate and demonstrate the surprising pharmacokinetic properties of two members of the species of the invention, A-EA6 and B-EA6, it is expected that the remaining compounds described by the formulas 1-4 will have similar variations in these properties. Accordingly, the small class of compounds contained in the present invention offer valuable additions to the repertoire of photodynamic agents useful in the treatment of the various conditions to which this therapy has been directed. Example 1 Preparation of two forms of EA6 A. To prepare B-EA6, the starting material is BPD-DB as the dimethyl ester -ie, BPD-DB as shown in Figure 1 where R1 and R2 are both COOMe one R "is vinyl, 2.0 g (2.7 mM) BPD-DB in 50 ml of ethylene glycol and 100 ml of dichloromethane was added with 1.0 ml of sulfuric acid.The reaction was stirred for 18 h at room temperature. The reaction was then added to a stirred mixture of 100 ml, 5% aqueous ammonium acetate and 100 ml of dichloromethane.The organic layer was isolated and then washed twice with 50 ml of water.The solvent was removed by The dark green residue was then chromatographed on 75 g of alumina (deactivated with 5% water) and extracted with solvents with a gradient of 0.5% -5.0% methanol in dichloromethane.The solvent of the fractions containing the The product was then removed by rotary evaporation, the residue was dried in vacuo at night to provide 2.02 gr. (89%) of the analytically pure green compound initially converted sold title. B. In a manner similar to that established in paragraph A, but substituting BDP-DA for BPD-DB, the isomeric form, A-EA6, was prepared. Example 2 Comparison of the Uptake and Release of B-EAß and BPD-MA by L1210 cells. BPD-MA or B-EA6 were incubated at 3 μg / ml in the presence of 10% fetal bovine serum with ld / ml of L1210 cells, a mouse leukemia cell line. The intracellular content of the photosynthesizers was measured by the fluorescence of the cell lysates on several occasions. The maximum concentration reached was 145.9 ng / 106 cells for B-EA6 and 149.5 ng / 106 for BPD-MA. The withdrawal period is shown in Figure 2 as a percentage of the cellular content in 60 min by which the time taken has reached a maximum in both cases. As shown, B-EA6 is taken more quickly and reaches 80% of its maximum concentration after only 5 min. and reached its maximum intake within 15 min. The release kinetics of these drugs from L1210 cells was measured by preloading the cells at 3μg / ml for 1 hr and then placing the cells in a drug-free medium containing 10% fetal bovine serum. The rest of the content of the intracellular drug was measured at various time points by causing lysis in the cells and measuring the fluorescence. As shown in Figure 3 (again as a percentage of intracellular start content), BPD-MA and B-EA6 showed different release kinetics. The initial release of B-EA6 was much faster, but the release was more complete in the case of BPD-MA. The pharmacokinetics in B-EA6 were not expected to be faster than those of BPD-MA. While the higher retention of B-EA6 could be attributed to its increased size compared to BPD-MA, the fastest transfer across the cell membrane was not expected.

Use 3 Comparison of pharmacokinetics in vi tro. Either BPD-MA or B-EA6 was administered by intravenous injection to the DBA / 2 mice at a dose of 4 mg / kg using 3 mice per time point. The drug content of plasma, skin, liver and kidney was determined by fluorescence in the tissue extracts. Figure 4 shows the result plotted as a percentage of the concentration in the relevant tissue 15 min after injection. As seen in Fig. 4, neither BPD-MA nor B-EA6 accumulated in plasma, liver or kidney; however, BPD-MA accumulated on the skin within the first 3 hours; B-EA6 no. The faster accumulation of B-EA6 as compared to BPD-MA, as confirmed here by a faster cleaning of all tissues, is an advantage. The light treatment can be carried out shortly after the injection of the photosynthesis and due to the rapid cleaning, prolonged photosensitivity of eyes or skin will not be exhibited. In this way, treated subjects can continue normal lives without special precautions such as avoiding light - bright and use dark glasses. The half-life of B-EA6 and BPD-MA in various tissues was then calculated within 15 min-3 hr and the results are shown in Table 1: Table 1: Average tissue lives of B-EA6 and BPD- MA T \ 4 * (15 min - 3 hr) Fabric B-EA6 BPD-MA Liver 0.6 2.4 Spleen 0.8 10.9 Kidney 0.8 5.6 Skin 1.9 0 * * Muscle 11.1 NDT Plasma 0.6 2.0 * not shown in hours ** concentration of BPD-MA in the skin increased more than 3 hr. ND = not determined The half-life of BPD-MA in this period could not be calculated on the skin due to its increased concentration during the 3 hr period. As shown in Table 1, generally, B-EA6 has a much shorter half-life than BPD-MA in most tissues. The lack of accumulation of B-EA6 in normal skin compared to BPD-MA was not expected and indicates a faster cleaning than that of BPD-MA. As previously stated, this is advantageous as long as skin photosensitivity is the only recognized lateral effect of photodynamic therapy using photosintetizers.

The pharmacokinetics were also determined using a mouse tumor model in vi vo. Groups of 10 DBA / 2 mice containing rbdomyosarcoma Ml tumors were injected intravenously with a liposomal formulation of BPD-MA at various doses of 0.75-1.5mg / kg. The tumors were irradiated with a laser light of 690 nm at 50 or 150 J / cm2 several times after the injection. The results, as shown in Table 2, were determined in terms of the percentage of mice in each group that were free-from tumors on day 7 after injection. As shown in Table 2, mice treated with BPD-MA showed substantial survival rates when the post-injection times ranged from 15-180 min. On the other hand, mice treated with B-EA6 showed no response in 30 min or 180 min; however, significant responses were obtained when the irradiation was delivered after only 15 min. These data demonstrate that PDT using B-EA6 will be effective in the early treatment with light. The lack of effect of the post-injections in later occasions indicates, again, the fast cleaning of B-EA6 which is advantageous for - the previously established reasons Table 2: Results of the bioassay PDT Conditions Percentage of Tumor-free on e 1 Day 7 * Time Dose Dose of BPD-MA B-EA6 Drug ** post IV light *** (mg / kg) (min) (J / cm2) 0.75 15 50 (4/5) 50% 30 50 70% 0% 1. 0 15 50 100% 90% 30 50 90% 0% 1. 5 180 150 70% 0% * tumor model = MI tumor in DBA / 2 mice - each PDT condition was tested in 10 animals ** the drugs were formulated liposomatically and injected intravenously. *** Laser light of 690 nm As shown in Table 2, the mouse treated with BPD-MA showed a substantial survival rate when the percentage of post-injection time varied from 15-180 min. On the other hand, the mouse treated with B-EA6 showed no response at 30 min or at 180 min.; however, the significant response was obtained when radiation was given after only 15 min. These data demonstrate that PDT using B-EA6 will be effective in early treatments with light. The lack of post-injection effect at later times indicates, again, the rapid fading of B-EA6 at a disadvantage for the reasons stated above. Example 4 Removal of LD50 with and without serum Either of the two B-EA6 or BPD -MA was incubated for 1 hr with L1210 cells in a range of concentrations and exposed to broad-spectrum light 9 / cm2. This determination was made in the absence of serum and in the presence of 10% serum. The results are shown in Table 3.

Table 3: LDC values Without serum 10% serum BPD-MA 3.7 ng / ml 54.0 ng / ml B-EA6 4.7 ng / ml 19.7 ng / ml As shown, BPD-MA and B-EA6 have comparable B-EA6 values in the absence of serum; however, in the presence of serum, B-EA6 shows a substantially better retention of efficacy. In most cases, the presence of serum greatly reduces the photoactivity of the agents used in PDT, such as BPD-MA. Surprisingly, B-EA6 shows more affinity for cell membranes than for plasma compounds and is thus very slightly affected by the presence of serum in the cell environment. In this way, in vi vo, your activity - - it may be higher than that of BPD-MA and other compounds in this family. The effectiveness of B-EA6 in exerting a cytotoxic effect on L1210 cells was tested in detail by incubating the cells with B-EA6 at various concentrations for 1 hr in the absence of serum. After the excess drug was removed, the cells were exposed to broad-spectrum light 9J / cm2 (380-750 nm) and cell survival was determined by the MTT assay (Mosmann, T. et al. J Immunol Meth (1983) 65: 55-63).

The percentage of dead cells was calculated with reference to the survival of cells exposed only to light. At a concentration of approximately 7 ng / ml, 80% of the cells died; At 15 ng / ml, almost 100% of the cells did not survive. As stated above, the LDS0 for B-EA6 is approximately 4.7 ng / ml. The somewhat lower effect of B-EA6 compared to BPD-MA in vi tro makes even more unexpected the comparatively higher activity of B-EA6 compared to BPD-MA in vi in the presence of serum as demonstrated in Example Four.

Example 6 Selectivity of B-EA6 for tumor cells The ability of L1210 cells to accumulate B-EA6 was compared with the ability of splenocytes to do so. B-EA6 was incubated 3μg / ml with each cell type and the cell content of B-EA6 was determined by fluorescence in the cell lysates. Figure 5 shows a comparison of the intake for the two cell types in ng / 10s cells. As shown, L1210 cells were able to take approximately 140 ng / 106 cells reaching this value after approximately 20 min. On the other hand, the splenocytes accumulated less than 20 ng / 106 cells after one hour of incubation. DBA / 2 mice suffering from Ml tumor (rhabdomyosarcoma), grown subcutaneously on their sides, were used as a model to show that B-EA6 demonstrated selectivity for tumors. The mice were administered 0.75 ng / kg of B-EA6 in a liposomal formulation intravenously. After 15 min, an area of 1 cm which included a tumor of 5 mm in diameter was exposed to a light with wavelength of 690 mm at 50 J / cm2 at 70 mW from a pump nozzle laser of 4 Argon 7 Immunomodulation using B-EA6 Balb / C mice (5-8 mice per group) were tested using the postponed skin photosensitivity test also called the contact hypersensitivity assay (CHS). The mice were painted on the side with the sensitizing agent dinitrofluorobenzene (DNFB) and 5 days later, one ear is stimulated with DNFB, while the other serves as a control. The swelling is an indicator of immune response. Mice were injected intravenously with 1 mg / kg B-EA6 liposomal and also irradiated with 15 J / cm2 of light over the whole body or exposed to ambient light. The ability of this treatment to avoid the immune response was demonstrated as demonstrated by the inhibition of ear swelling. The results showed that administering B-EA6 combined with either of the two after irradiation with 15 J / cm2 of light over the whole body or with ambient light decreased the swelling in the test ear compared to untreated mice. The swelling in both cases was only about 60% of that shown in mice - without the treatment. In a further assay to determine immunomodulation, the peripheral macrophages of mice were isolated, purified and activated by interferon-? recombinant (100 U / ml). The Activated cells were incubated for 1 hr at 37 ° C with B-EA6 in a range of combinations and then exposed to LED light of 690 nm at 5 J / cm2. The expression levels of MHC I, MHC II, CD54, CD80 and CD86 were determined 24 hr later using FITC conjugated antibodies and a cell sorter. The results are shown in Table 4 for B-EA6 at 0.5 ng / ml compared to similar experiments using BPD-MA at 2.5 ng / ml. Table 4: Effect of low dose PDT with B-EA6 on expression levels of cell surface antigens by peripheral macrofags of mice Compound MHC MHC CD54 CD80 CD86 Class I Class (ICAM- (B7-1) (B7-2) II 1) BPD-MA 99.1 79.3 105.4 93.5% 99.2% (2.5 ± 4.3% ± 10.1% ± 3.0% ng / ml) BPD-B-EA6 100.4% 71.8% 106.9% 102.3% 92.2% (0.5 ng / ml) The results in the table are given as percentage of expression compared to - cells treated only with light. As shown, both BPD-MA and B-EA6 were able to reduce the expression of MHC II, but not the rest of the ethereal surface tags. Thus, although B-EA6 has advantageous pharmacokinetics, it retains the immunomodulatory activity of BPD-MA and other compounds of this group. Effect 8 of B-EA6 in an arthritis model MRL-I mice spontaneously developed arthritis; this was accentuated by the intradermal injection of Freund's adjuvant. Various numbers of MRL-Ipr mice were treated with PDT on days 0, 10 and 20 after injection of the adjuvant. PDT consisted of 0.5 mg / kg of liposomal B-EA6 injected intravenously followed by exposure of the ventral part of the mice to red light (560-900 nm) at 80 J / cm2 in a post injection of B-EA6 from 1 hr. The mice were observed and the symptoms were noted every 5 days of 30 days. The results are shown in Figure 6 compared to mice treated in a similar manner with BPD-MA and BPD-MB. As shown in Figure 6, either measured by the incidence of clinical symptoms (ie, the percentage of mice exhibiting those symptoms) or by the change in bimalleolar ankle width in millimeters, B-EA6 (shown as circles) solids) was effective in preventing the sequelae of adjuvant injection. Again, the retention of the immunomodulatory activity of B-EA6 was demonstrated. The effect of B-EA6 on the microvasculature. The model of the testis muscle was used. B-EA6 was administered intravenously at 2 mg / kg and starting at a post-injection at 5 and 15 min, the small arteries and veins surgically exposed were irradiated with light at an intensity of 25 J / cm2 for 5 min starting in 5 min and 15 min after the injection of B-EA6. The vessels were measured in both the diameter of the red blood column and a percentage of the controls. The results are shown in Figure 7. While closure of the transient vessel could be obtained when irradiation has begun at 5 min, permanent closure was obtained when the irradiation started after 15 min. The improved ability of B-EA6 to tighten or occlude the vasculature, as demonstrated in this Example, in combination with faster pharmacokinetics, makes B-EA6 particularly advantageous in treating neovascular diseases in the eye. Example 10 Absorption Spectrum of B-EA6 BPD-MA and B-EA6 have similar absorption spectra in plasma before and after a 4 hr exposure to fluorescent light (380-750 nm). A comparison of these spectra is shown in Figure 8. The similarity of the B-EA6 spectra to the BPD-MA spectrum is advantageous because the use of BPD-MA as a useful therapeutic agent in PDT is well developed . The similarity in their spectra indicates that the same light sources can be used for B-EA6 as long as they are successful in BPD-MA treatment. Example 11 In vitro cytotoxicity of A-EA6. In a manner similar to that established in Example 5, the cytotoxicity of A-EA6 in vi tro was tested and compared with BPD-MA in two different cell lines. Either of the two cells L1210 or dendritic cell line D2SC / 1 was incubated for one hour at 37 ° C with either A-EA6 or BPD-MA. After removing the excess of - - drug, the cells were exposed to light of 690 nm at 5 J / cm2 for the dendritic cells and in light of 9 J / cm2 for the L1210 cells. Cell survival was determined 18-24 hr later using the MTT colorimetric assay described in Example 5. The percentage of dead cells was calculated by reference to cells exposed only to light. As shown in Figure 9A, A-EA6 showed a cytotoxicity comparable to that of BPD-MA with respect to L1210 cells in the absence of serum but it was ethereally more toxic in the presence of serum than of BPD-MA. The open circles represent A-EA6 plus serum; the closed circles represent BPD-MA plus serum; the open squares represent A-EA6 in the absence of serum; and closed squares represent BPD-MA in the absence of serum. As shown in Figure 9B, in dendritic cells where BPD-MA has an LDS0 of 6 ng / ml and A-EA6 has an LDS0 of 2.7 ng / ml, A-EA6 was toxic at concentrations lower than BPD-MA in presence of 5% fetal calf serum. In Figure 9B, closed circles represent BPD-MA and open squares represent A-EA6. In a similar determination, but measuring the MHC I receptors before the cytotoxicity, A- - - EA6 was effective in decreasing the expression of these receptors at lower concentrations. In this determination, the dendritic cells were incubated for 1 hour at a drug concentration lower than their LD50; 2.5 ng / ml and 5 ng / ml for BPD-MA and 1 ng / ml and 2.5 ng / ml for A-EA6. The cells were treated with light from 690 nm to 5 J / cm2 and then labeled with the appropriate antibody during a 3-hour after-treatment and evaluated by flow cytometry. The results were measured as the percentage of the average fluorescence intensity of the channel for control cells treated with light. These results are shown in Figure 10; BPD-MA gave a reduction of 18% and 29%, respectively, at 2.5 ng / ml and 5 ng / ml; A-EA6 reduced the intensity of the channel fluorescence by approximately 25% at both concentrations of 1 ng / ml and 2.5 ng / ml. The effect of A-EA6 on intracellular signaling. The study conditions set forth in Example 11 were repeated using HL-60 cells as the target and comparing the effects of A-EA6 and BPD-MA on cytotoxicity, on the kinase of the cyto pathway p70 S6K, and on the kinases of trajectory of stress c-jun and HSP27. The results are shown in Figure 11. At sublethal concentrations, A-EA6 showed stronger activation of stress path kinases and stronger inhibition of mitogenic pathway kinases. The effect on caspase activation was measured in HL60 cells as well. A-EA6 showed a stronger activation in the caspases than what BPD-MA showed. The effect is desirable as it was associated with apoptosis. Using apoptosis to remove unwanted cells causes minimal effect on surrounding normal cells and tissues. The comparison of A-EA6 with BPD-MA is shown in Figure 12. Figure 13 shows a similar comparison when fragmentation of DNA percentage was measured in HL60 cells. Again, A-EA6 was effective at lower concentrations than BPD-MA. Example 13 Photodynamic therapy in vivo using A-EA6. In a protocol similar to that established in Example 3, either of the two A-EA6 or BPD-MA was injected intravenously into mice harboring Ml tumors at a dose of 1 mg / kg. This was followed by a whole-body irradiation with 50 J / cm2 of laser light of 690 nm on several occasions after administration of the drug. The number of tumor-free animals was determined on day 7 and the results are shown in Table 5.

These results show that A-EA6 is at least as effective as BPD-MA in this assay. Example 14 Immunomodulatory activity The control sides and the test mice were painted with the DMFB antigen and their ears were stimulated 5 days later by gluing the same compound. The test animals were treated with whole body PDT using BPD-MA or A-EA6, by injecting the synthesizer intravenously and then exposing the animals to the red LED light of 15 J / cm2. The percentage of suppression of ear swelling was calculated in comparison with the controls. The results are shown in Table 6 and indicate that A-EA6 had an immunomodulatory effect - - stronger in this trial than BPD-MA

Claims (11)

1. - -
2. NOVELTY OF THE INVENTION Having described the present invention, it is considered as a novelty and therefore the property described in the following claims is claimed as property. 1 . A compound of the formula
3. -NH N-v .y- ° * C '° B, C- ~ d N HN c - Kj (CHA ÍCH? • 0000 ^ 0 ^ 0-1 CQGCHjC ^ GH 1 Z and the metalated and / or tagged and / or conjugated forms thereof characterized in that each R1 is independently alkyl (1-6C); each n is independently an integer of 0-6; and R2 is vinyl or a derivative form thereof. 2. The compound according to claim 1 characterized in that R2 is vinyl, -CHOR ', -CHO, COOR', -CH (OR ') CH3, -CH (OR') CH2OR ', -CH (SR') CH3, - CH (NR ') 2CH 3, -CH (CN) CH 3, - CH (COOR') CH 3, - CH (OOCR ') CH 3, -CH (NR' COR ') CH 3, - CH (CONR' 2) CH 3, - CH (halo) CH3, or CH (halo) CH2 (halo) wherein R 'is H, or a hydrocarbon radical (1-6C) optionally substituted with a heteroatom substitute or wherein R2 is an organic group of less than of 12 carbon atoms resulting from the direct or indirect derivation of a vinyl substitute, or wherein R2 is a group containing 1-3 nuclei of the tetrapyrrole type. 3. The compound according to claim 1 or 2 characterized in that it is in a metalated form, and / or which is in the conjugated form, and / or is labeled.
4. 4. The compound according to claim 1 or 2 characterized in that it does not contain a metal ion.
5. 5. The compound of any of claims 1-4 characterized in that R2 is vinyl, and / or wherein each R1 is methyl, and / or wherein both n are 2.
6. 6. The compound according to claim 5 - - characterized in that R2 is vinyl and both R1 are methyl.
7. 7. The compound according to claim 6, characterized in that it is of the formula Ü-CAÍ and the metalated and / or tagged and / or conjugated forms thereof.
8. 8. The compound according to claim 7 which is in a metalated form, and / or which is in conjugated form, and / or which is labeled.
9. 9. The compound according to claim 7, characterized in that it does not contain a metal ion.
10. 10. A pharmaceutical composition characterized in that it comprises the compound of any of claims 1-9 in admixture with at least one pharmaceutically acceptable excipient.
11. 11. An improved method for conducting photodynamic therapy or diagnosis characterized in that the method comprises administering a photoactive compound to a subject in need of therapy or diagnosis wherein the improvement comprises the use of the compound of any of claims 1-9 as the agent photoactive