Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
  • C07D487/22—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains four or more hetero rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
  • A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
  • A61K41/0071—PDT with porphyrins having exactly 20 ring atoms, i.e. based on the non-expanded tetrapyrrolic ring system, e.g. bacteriochlorin, chlorin-e6, or phthalocyanines
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
  • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
  • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/001—Preparation for luminescence or biological staining
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents

Definitions

• the invention relates to the use of light absorbing compounds to mediate the destruction of unwanted cells or tissues or other undesirable materials by irradiation. Specifically, the invention relates to the use of Diels-Alder derivatives' of porphyrin having absorption maxima in the range 700-820 nanometers to mediate the irradiation of materials to be destroyed, and to the use of these compounds conjugated to target-specific ligands, such as receptor-specific ligands, or immunoglobulins or their immunospecific fragments, to focus the effects of the irradiation on particular targets.
• target-specific ligands such as receptor-specific ligands, or immunoglobulins or their immunospecific fragments
• HPD hematoporphyrin and its acetylated derivative mixture hematoporphyrin derivative (HPD) sys- temically, combined with irradiation, for the detection and treatment of malignant cells has, by this time, some considerable history.
• HPD is a mixture of porphyrins including hematoporphyrin itself, hydroxyethyl vinyl deuteroporphyrin, protoporphyrin, and dihe atoporphyrin ethers. (See, e.g., "Porphyrin Photosensitization", Kessel, D. , et al, eds. (1983) Plenum Press.)
• HPD seems "naturally" capable of localizing in malignant cells.
• it has two properties which make it useful.
• More pertinent to the present invention is the capacity of HPD, when irradiated with visible light, to exhibit a cytotoxic effect on the cells in which it is localized (see, for example, Diamond, I., et al, Lancet (1972) 2:1175-1177; Dougherty, T.J., et al, Cancer Research (1978) 3_8:2628-2635; Dougherty, T.J., et al, "The Science of Photo Medicine” (1982) J.D. Regan & J.A. Parrish, eds., pp. 625-638; Dougherty, T.J., et al, "Cancer: Principles and Practice of Oncology” (1982) V.T.
• a purified form of the active component(s) of HPD is obtained by adjustment of pH to cause aggregation and recovery of the aggregate, as disclosed in U.S. Pat ⁇ ent 4,649,151.
• the purified form, called DHE in the patent, is marketed under the trademark Photofrin® II and has been used in a manner completely analogous to HPD.
• the porphyrins can be used in other in vivo and in vitro applications.
• photosens .-izers are useful in the detection and treat ⁇ ment of atherosclerotic plaques as described in U.S. Pat. Nos. 4,512,762 and 4,577,636.
• U.S. Pat. Nos. 4,500,507 and 4,485,806 describe the use of radiolabeled porphyrin compounds, including HPD, for tumor imaging.
• hematoporphyrin was conjugated to a monoclonal antibody specific to an antigen associated with a human leukemia (CAMAL) and the conjugates were shown to mediate the irradiation-induced killing of leukemic cells specifically, in vitro (Mew, D., et al. Cancer Research (1985) 45:4380-4386) .
• BPD desig ⁇ nated hydro-monobenzoporphyrins and their derivatives
• the compounds of the present invention which are di-Diels-Alder adducts of dienophiles to diagonal rings in the porphyrin system add to the repertoire of useful photosensitizers which have absorption maxima in the non-interfering range of 700-820 nm, which permit reduced dosage and enhanced light penetration.
• the invention provides light absorbing com ⁇ pounds capable of exhibiting light-mediated cytotoxic -6- and diagnostic effects.
• these compounds may be administered in in vivo relatively low dosage due to their capability to absorb radiation whose energy range is outside of that normally absorbed by the components present in high concentration in the blood or other tissues, in particular, the porphyrin residues normally associated with hemoglobin and myoglobin. Therefore, by providing these modified porphyrins for in vivo treatment at lower concentration, hypersensitivity of nontarget tissues is reduced, and the irradiation treatment can be conducted at a wave ⁇ length at which the native chromophores do not compete for photons with the active compounds, resulting in greater depth of penetration of the light. Similar advantages accrue in in vitro treatment of colored mate ⁇ rials, such as blood samples.
• photoactive compounds are modified porphyrins which, by virtue of their derivatization, undergo a shift in absorption maxima so that they appear green rather than red, indicating their absorption of wavelengths in the red-orange range. They confer sensi ⁇ tivity on target cells at concentrations greater than 10-fold lower than those required for hematoporphyrin (Hp) or HPD.
• the compounds of the invention are derived from protoporphyrin II and other A/C divinyl analogs—i.e., analogs corresponding to modifications of protoporphyrin IX which contain vinyl substituents in the A and C rather than the A and B rings, or the analo ⁇ gous B/D divinyl which have vinyl substituents on the B and D rings.
• protoporphyrin II and other A/C divinyl analogs i.e., analogs corresponding to modifications of protoporphyrin IX which contain vinyl substituents in the A and C rather than the A and B rings, or the analo ⁇ gous B/D divinyl which have vinyl substituents on the B and D rings.
• the compounds of the invention are obtained by Diels-Alder reactions with both of the vinyl groups, resulting in fused six-membered rings attached both to the A and C (or B and D) rings.
• the resulting deriva ⁇ tives are of the same oxidation state as bacteriochlorin (or bacteriochlorophyll) . Because of their structural derivation, they have been designated herein A/C (or B/D) hydro-dibenzoporphyrin compounds as is further explained below.
• A/C or B/D hydro-dibenzoporphyrin com ⁇ pounds of the invention are selected from a group of derivatives obtained using Diels-Alder reactions of eth- ylene or acetylene dienophiles with protoporphyrin II, or other A/C or B/D divinyl porphyrins under conditions which effect a reaction at both available conjugated, nonaromatic diene structures present in the relevant porphyrin ring system (rings A and C or B and D).
• the formulas shown in Figure 1 represent the A/C and B/D hydro-dibenzo-porphyrins of the invention.
• the modified A/C or B/D hydro-dibenzo- porphyrins of the invention can be used per se or can be conjugated to specific ligands reactive with a target, such as receptor-specific ligands or immunoglobulins or immunospecific portions of immunoglobulins, permitting them to be more concentrated in a desired target tissue or substances.
• a target such as receptor-specific ligands or immunoglobulins or immunospecific portions of immunoglobulins
• the invention relates to a com ⁇ pound of the formulas 1-1 through 1-6 in Figure 1, which compound is fluorescent and photosensitizing, wherein at least one of R 1 and R 2 is selected from the group consisting of carbalkoxy (2-6C); aryl (6-10C); alkyl (1-6C) or aryl (6-10C) sulfonyl; cyano; and -CONR 5 CO-, wherein R 5 is aryl (6-10C) or alkyl (1-6C); and the other R 1 and R is selected from the group consisting of the aforesaid substituents and H; and wherein each R 3 and R 4 is independently selected from the group consisting of substituted or unsubstituted alkyl (1-6C); and substituted or unsubstituted omega-carboxyalkyl (2-6C) and the esters, amides, and salts thereof.
• R 1 and R 2 is selected from the group consisting of carbalkoxy (2-6C); aryl (6-10C); al
• the invention also relates to labeled forms of these compounds.
• the invention is directed to methods of locating or effecting cytotoxicity, i.e. photosensitizing, with respect to target materials using the A/C or B/D hydro-dibenzoporphyrins of the invention either alone or as conjugates.
• cytotoxicity i.e. photosensitizing
• A/C or B/D hydro-dibenzoporphyrins are localized specifically in vivo to certain target tissues, where their presence can be detected by fluorescence, or by other means when the invention compounds are provided with additional or alternate labeling.
• the specificity of the compounds can be further enhanced by conjugation to ligands specific for the target.
• the compounds when the compounds are irradiated in situ using light in the range of 700-820 nm, photoactivation results in cytotoxicity to the surrounding tissue.
• Cells to which the A/C or B/D hydro-dibenzoporphyrin is normally attracted include tumor cells, and neoplastic cells in general, as well as bacteria and other diseased tissues.
• the method can be applied either in vitro or in vivo, and, when applied in vivo, can be topical or systemic.
• the invention relates to conjugates of the formulas Re*-L-A/C, Re*-L-B/D, Ig-L-A/C and Ig-L-B/D wherein Re* represents a ligand which is specific to, and capable of, binding a receptor at a cell surface, Ig represents an immunoglobulin or an immunologically reactive portion thereof, A/C and B/D represents a compound of the invention as defined above having an absorption maximum in the range of 700-820 nanometers, and L represents either a covalent bond linking these components or a linking moiety covalently linked to each of the Re* or Ig and invention compound.
• Re* represents a ligand which is specific to, and capable of, binding a receptor at a cell surface
• Ig represents an immunoglobulin or an immunologically reactive portion thereof
• A/C and B/D represents a compound of the invention as defined above having an absorption maximum in the range of 700-820 nanometers
• L represents either a covalent bond linking these components or
• the invention is also directed to tripartite complexes which include Re*-L-A/C; Re*-L-B/D; Ig-L-A/C; or Ig-L-B/D further conjugated to or associated with a label.
• the label may be bound either to the targeting component or to the A/C or B/D or both.
• the invention relates to pharmaceutical compositions containing these active ingredients.
• Figure 1 shows the structures of A/C and B/D compounds of the invention.
• Figures 2A-2D show absorption spectra of sev ⁇ eral invention compounds.
• compositions of the invention employ, as the light absorbing moiety, one or more derivatives of the protoporphyrin ring system which has a light absorption maximum in the range of 700-820 nanometers.
• Figures 2A-2D show the absorption spectra of some of the compounds of the invention shown in Fig ⁇ ure 1; all have absorptions close to 800 nm.
• this shift is achieved by effec ⁇ tively saturating one of the two ir-bonds in two of the four pyrrole rings which constitute the typical porphyrin system.
• two of the diagonally positioned pyrroles (A and C or B and D which are, in this case, equivalent,) contain vinyl substitu ⁇ tions such that the exocyclic ⁇ -bond is conjugated to one of the two ⁇ r-bonds in the ring.
• a Diels-Alder reaction involving these conju ⁇ gated systems with an ethylene or acetylene dienophile results in six-membered rings fused to the A and C or B and D rings.
• the resultant of this addition is shown in Figure 1 as formulas 1-1 and 1-2 (for addition of acety ⁇ lene dienophiles) and as formulas 1-5 and 1-6 (for addi ⁇ tion of ethylene dienophiles).
• Rearrangement of the ir system in the hexadiene ring obtained in formulas 1-1 and 1-2 results, as shown, in the compounds of formulas 1-3 and 1-4; reduction provides an alternative route to the compounds of formulas 1-5 and 1-6.
• compounds of the formulas 1-5 and 1-6 can be provided directly by reaction of the protoporphyrin system with an ethylene dienophiles. All of these compounds provide the desired shift in absorption maximum.
• Reaction of protoporphyrin II or other A/C or B/D divinyl analogs with, for example, diethyl acetylene dicarboxylate (DEAD)—results in the compounds shown as formulas 1-1 and 1-2 of Figure 1, wherein R and R 2 rep ⁇ resent the substituents on the original acetylene- derived Diels-Alder reagent, R C CR , in this case, carboethoxy.
• DEAD diethyl acetylene dicarboxylate
• A/C hydro-dibenzoporphyrin refers generically to compounds of formulas 1-1, 1-3 and 1-5;
• B/D hydro-dibenzoporphyrin refers generically to compounds of formulas 1-2, 1-4 and 1-6.
• R x 1 and R9 are each, i.ndependently, electron-withdrawing substituents, and are, most com ⁇ monly, carbalkoxy (2-6C); aryl (6-10C); alkyl (1-6C) or aryl (6-10C) sulfonyl; -CONR 5 CO- wherein R 5 is aryl (6-10C) or alkyl (1-6C); cyano; or any other activating substituents.
• R and R" may optionally be H while the other is an electron withdrawing substituent as set forth above of sufficient strength to facilitate the Diels-Alder reaction.
• R 1 and R 2 are preferably carbalkoxy groups such as carboethoxy.
• carboxy is, as conventionally defined, -COOH, and carbalkoxy is -COOR, wherein R is alkyl (1-6C).
• alkyl (1-6C) is a satu ⁇ rated straight or branched chain hydrocarbon of 1-6 car ⁇ bon atoms such as methyl, ethyl n-hexyl, 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.)
• aryl (6-10C) or alkyl (1-6C) sulfonyl moi ⁇ eties have the formula SO2R wherein R is alkyl or is aryl as above-defined.
• R 3 and R 4 represent substituents present on the porphyrin used in the reaction or substituents derived therefrom.
• all R 4 are methyl and both R 3 are 2-carboxyethyl (-CH2CH2COOH) .
• R 3 and R are ordinarily not relevant to the progress of the Diels-Alder reaction (although it should be noted that while they do not ordinarily influence the course of the Diels-Alder reac ⁇ tion by altering the nature of the diene substrate, their influence on other factors, such as suitable solu ⁇ bility characteristics, lack of interference with the progress of the reaction, and effectiveness and absorp ⁇ tion spectrum of the resulting product, are relevant).
• R and R 4 are substituted or unsubstituted alkyl (1-6C) , or substituted or unsubstituted ⁇ -carboxyalkyl (2-6C) or the esters, amides or salts thereof.
• the substitutents may include, for example, halogen as above-defined, and/or other non- reactive substituents.
• Alkyl is as above defined.
• Omega carboxyalkyl (2-6C) refers to substituents of the formula -(CH2)nC00H wherein n is 1-5.
• the invention compounds also include the salts, esters and amides of -COOH.
• esters and amides must be pharmaceutically acceptable and non-toxic; this requirement is not ger ⁇ mane to in vitro use.
• Salts, esters, and amides refers to salts derived from inorganic or organic bases, including phar ⁇ maceutically 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, sec ⁇ ondary, tertiary and quaternary amines including cyclic amines, and basic ion-exchange resins.
• Examples include isopropyla ine, trimethylamine, ethanolamine, dicyclohexylamine, lysine, arginine, histidine, caf ⁇ feine, procaine, choline, betaine, glucosamine, theobromine, purines, piperazine, piperidine, polyamine resins, and the like.
• the salt derivatives are prepared by treating the free acids with an appropriate amount of pharmceutically acceptable base.
• the reaction is con ⁇ ducted 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, preferaby at room temperture at a suitable molar ratio 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.
• 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 corre ⁇ sponding to the desired ester. This reaction is con ⁇ ducted 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 esterificaion is a liquid at the reaction temperature, the alcohol reagent can be the reaction solvent.
• a strong acid such as boron trifluoride, hydrogen chloride, sulfuric acid, p-toluenesulfonic acid, and the like. Since the alcohol reagent used in the esterificaion is a liquid at the reaction temperature, the alcohol reagent can be the reaction solvent.
• the reaction can be car ⁇ ried 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, chloro ⁇ form, dichlorethane; 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, prefera ⁇ bly 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-i iscible inert organic colvent, such as diethyl ether, benzene, methylene chloride, and the like, com ⁇ bining the extracts, washing the extracts with water to neutrality, and then evaporating under reduced pressure.
• a water-i iscible inert organic colvent such as diethyl ether, benzene, methylene chloride, and the like
• the alkyl esters can be pre ⁇ pared 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 sub ⁇ stantial 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.
• the ester can be prepared by reacting the free acid form with the appro ⁇ priate diazo alkane, such as diazomethane, diazo-n-hexane, or diazo-i-propane in an aprotic organic solvent at low temperature.
• 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 and treating with the appropri ⁇ ate amine.
• the starting porphyrin A/C or B/D divinyl com ⁇ pounds are then converted to the invention compounds by di Diels-Alder reactions.
• T and D rings These compounds have absorption maxima in the 720-750 nm range.
• hydro-dibenzoporphyrins which directly result from the Diels-Alder reaction can also be isomerized in a manner as described for the correspond ⁇ ing hydro-monobenzoporphyrins by Morgan et al. J Chem Soc Chem Commun (1984) pp. 1047-1048; and Pangka et al. J Orq Chem (1986) 5_1:1094, both incorporated herein by reference, to compounds of formulas shown as 1-3 and 1-4 of Figure 1. Rearrangement is by treatment with suit ⁇ able reagents such as triethylamine (TEA) in methylene chloride or 1,5-diaza bicyclo[5.4.0] undec-5-ene (DBU). The stereochemistry of the product is determined by the choice of reagent.
• TAA triethylamine
• DBU 1,5-diaza bicyclo[5.4.0] undec-5-ene
• the Diels-Alder products can be selectively reduced by treating with hydrogen in the presence of palladium on charcoal to give the saturated ring analogs, shown as formulas 1-5 and 1-6 in Figure 1, corresponding to the respective Diels-Alder products of rings A and C and B and D.
• These reduced products also absorb light at 720-750 nm and are less preferred in the method of the invention than the compounds of formulas 1-3 and 1-4.
• the compounds of formulas 1-5 and 1-6 may also be prepared directly by reaction of the appropriate A/C or B/D divinyl porphyrin starting materials with an eth ⁇ ylene dienophile. Thus, they are formed by reaction of the appropriate porphyrin with a compound of the formula
• R-C CR ⁇ wherein R and R are as above defined.
• the conjugates and meth ⁇ ods of the invention include compounds having both configurations of the chiral carbons, whether the com ⁇ pounds are supplied as isolates of a single stereoisomer or are mixtures of enantiomers and/or diasteriomers. Separation of mixtures of diasteriomers m ⁇ y be effected by any conventional means; mixtures of enantiomers may be separated by usual techniques of reacting them with optically active preparations and separating the result ⁇ ing diasteriomers.
• the target-specific component can be, for example, an immunoglobulin or portion thereof or a ligand specific for receptor.
• the immunoglobulin component can be any of a variety of materials. It may be derived from polyclonal or monoclonal antibody preparations and may contain whole antibodies or immunologically reactive fragments of these antibodies such as F(ab')2, Fab, or Fab' frag ⁇ ments. Use of such immunologically reactive fragments as substitutes for whole antibodies is well known in the art. See, for example, Spiegelberg, H.L., in "Immunoassays in the Clinical Laboratory” (1978) 1:1-23.
• Polyclonal anti-sera are prepared in conven ⁇ tional ways by injecting a suitable mammal with antigen to which antibody is desired, assaying the antibody level in serum against the antigen, and preparing anti-sera 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 proce ⁇ dures, and screening for production of the desired anti ⁇ bodies 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.
• antibodies exemplified herein include the monoclonal antibody preparation CAMAL1 which can be prepared as described by Malcolm, A., et al. Ex Hematol (1984) 12:539-547; polyclonal or monoclonal preparations of anti-Ml antibody as described by Mew, D. , et al, J Immunol (1983) 130:1473-1477 (supra) and B16G antibody which is prepared as described by Maier, T. , et al, J Immunol (1983) 131:1843; Steele, J.K., et al. Cell Immunol (1984) :303, all incorpo ⁇ rated herein by reference.
• the ligand specific for receptor refers to a moiety which binds a receptor at cell surfaces, and thus contains contours and charge patterns which are complementary to those of the receptor.
• the ligand spe ⁇ cific for receptor is symbolized in the formulas of the compounds of the invention as Re*, wherein the asterisk indicates that the moiety bound in the compound of the invention is not the receptor itself, but a substance complementary to it. It is well understood that a wide variety of cell types have specific receptors designed to bind hormones, growth factors, or neurotransmitters. However, while these embodiments of ligands specific for receptor are known and understood, the phrase "ligand specific for receptor”, as used herein, refers to any substance, natural or synthetic, which binds specifi ⁇ cally to a receptor.
• ligands examples include the steroid hormones, such as progesterone, estrogens, androgens, and the adrenal cortical hormones; growth factors, such as epidermal growth factor, nerve growth factor, fibroblast growth factor, and so forth; other protein hormones, such as human growth hormone, parathyroid hormone, and so forth; and neurotransmitters, such as acetylcholine, serotonin, and dopamine. Any analog of these substances which succeeds in binding to the recep ⁇ tor is also included.
• steroid hormones such as progesterone, estrogens, androgens
• adrenal cortical hormones growth factors, such as epidermal growth factor, nerve growth factor, fibroblast growth factor, and so forth
• growth factors such as epidermal growth factor, nerve growth factor, fibroblast growth factor, and so forth
• other protein hormones such as human growth hormone, parathyroid hormone, and so forth
• neurotransmitters such as acetylcholine, serotonin, and dopamine. Any
• the conjugation of the target-cell-specific component to the A/C and B/D hydro-dibenzoporphyrins can be effected by any convenient means.
• a direct covalent bond between 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 hydro-dibenzoporphyrins to the immunoglobulin moiety is treatment with l-ethyl-3- (3-dimethylamino propyl) carbodiimide (EDCI) in the presence of a reaction medium consisting essentially of dimethyl sulfoxide (DMSO) .
• DMSO dimethyl sulfoxide
• dehydrating agents such as dicyclohexylcarbodiimide or diethylcarbodiimide could also be used as well as conventional aqueous and par ⁇ tially aqueous media.
• Nonprotein receptor ligands can be conjugated to the invention compounds according to their relevant functional groups by means known in the art.
• the active moieties of the conjugate may also be conjugated through linker compounds which are bifunc ⁇ tional, and are capable of covalently binding each of the two active components.
• linker compounds which are bifunc ⁇ tional, 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 Company, Rockford, IL.
• These linkers are either homo or heterobifunctional moieties and include functionalities capable of forming disulfides, amides, hydrazones, and a wide variety of other linkages. The most popular of these is N-succidimidyl-3-(2-pyridyldithio) propionate (SPDP).
• This reagent creates a disulfide linkage between itself and a cysteine residue in one protein and an amide link ⁇ age through the ⁇ amino on a lysine or other free amino group in the other.
• disulfide/amide-forming agents are known. See, for example, Immun Rev (1982) 2:185.
• Other bifunctional coupling agents form a thioether rather than a disulfide linkage.
• Many of these thioether-forming agents are commercially available and include reactive esters of 6-maleimidocaproic acid, 2-bromoacetic acid, 2-iodoacetic acid, 4-(N-maleimido-methyl) cyclohexane-1-carboxylic acid, and the like.
• the car- boxyl groups can be activated by combining them with succinimide or l-hydroxy-2-nitro-4-sulfonic acid sodium salt.
• a particularly preferred coupling agent is succinimidyl 4-(N-maleimido-methyl) cyclohexane-1-carboxylate (SMCC) .
• 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 invention compounds per se or the conjugates may be further derivatized to a compound or ion which labels the drug.
• labeling moieties can be used, including radioisotopes, chromophores , and fluo ⁇ rescent labels. Radioisotope labeling is preferred, as it can be readily detected in vivo.
• the compounds which are the invention A/C or B/D hydro-dibenzoporphyrins alone or which are conju ⁇ gates of these with a specific binding substance can be labeled with radioisotopes by coordination of a suitable radioactive cation in the porphyrin system.
• Useful cat ⁇ ions include technetium, gallium, and indium.
• the conjugates either or both the specific binding sub ⁇ stances tan be linked to or associated with label, or the label can be conjugated or coordinated with the A/C or B/D hydro-dibenzoporphyrin moiety itself.
• the compounds of the invention can be adminis ⁇ tered or used in in vitro methods as shown above or when complexed to appropriate metal ions.
• the A/C or B/D hydro-dibenzoporphyrin nucleus can be treated with an appropriate ion such as magnesium ion, zinc ion, stannous ion, and the like to obtain the metal complex.
• the metal ion may also be a radiolabel.
• the nature and desirability of the inclusion of a metal ion in the A/C or B/D hydro-dibenzoporphyrin nucleus depends on the specific application for which the com ⁇ pound is intended.
• the desired metal ion can be inserted using the appropriate metal salts under known conditions.
• zinc ion can be introduced by treating the com ⁇ pound with zinc acetate in 1:1 methylene chloride:methanol.
• the improved photosensitizing compounds of the invention are thus useful in general, in the manner known in the art for hematoporphyrin derivative and for DHE. These materials are useful in sensitizing neoplas- tic cells or other abnormal tissue to destruction by irradiation using visible light — upon photoactivation, the compounds have no direct effect, nor are they entered into any biological event; however the energy of photoactivation is believed to be transferred to endogenous oxygen to convert it to singlet oxygen. This singlet oxygen is thought to be responsible for the cytotoxic effect.
• the photoactivated forms of porphyrin fluorescence which fluoresce can aid in localizing the tumor.
• Typical indications include destruction of tumor tissue in solid tumors, dissolution of plaques in blood vessels (see, e.g., U.S. Patent 4,512,762); treatment of topical conditions such as acne, athletes foot, warts, papilloma, and psoriasis and treatment of biological products (such as blood for transfusion) for infectious agents, since the presence of a membrane in such agents promotes the accumulation of the drug.
• conjugates of the invention or the hydro- dibenzoporphyrins when employed alone are formulated into pharmaceutical compositions for administration to the subject or applied to an in vitro target using techniques known in the art generally.
• a summary of such pharmaceutical compositions may be found, for exam ⁇ ple, in Remington's Pharmaceutical Sciences, Mack Pub ⁇ lishing Co., Easton, Pennsylvania, latest edition.
• conjugates or compounds of the invention taken alone can be used in the systemic treatment of tumors and neoplasties made as bronchial, cervical, esophageal or colon cancer and for the diagnosis of same.
• the conjugates and A/C and B/D hydro-dibenzoporphyrins of the present invention, labeled or unlabeled, can be administered systemically, in particular by injection, or can be used topically.
• the A/C and B/D hydro-dibenzoporphyrins or conjugates can be used singly or as components of mixtures.
• 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 solu ⁇ tion or suspension in liquid prior to injection, or as emulsions.
• Suitable excipients are, for example, water, saline, dextrose, glycerol and the like.
• these compositions may also contain minor amounts of nontoxic, auxiliary substances such as wetting or emul ⁇ sifying agents, pH buffering agents and so forth.
• Systemic administration can also be imple ⁇ mented through implantation of a slow release or sus ⁇ tained 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) .
• the compounds may be used alone or may be labeled with a radioisotope or other detecting means.
• the active conjugates or A/C and B/D hydro-dibenzoporphyrins may be topically administered using standard topical compositions involving lotions, suspensions, or pastes.
• the quantity of conjugate or A/C and B/D hydro-dibenzoporphyrins derivative to be administered depends on the choice of active ingredient, the condi ⁇ tion to be treated, the mode of administration, the individual subject, and the judgment of the practitioner. Depending on the specificity of the prep ⁇ aration, smaller or larger doses may be needed.
• dosages in the range of 0.05-1 mg/kg are suggested.
• compositions which are less specific to the target tissue larger doses, up to 1-10 mg/kg may be needed.
• doses up to 1-10 mg/kg may be needed.
• the compounds of the invention can be used in the treatment of materials in vitro to destroy harmful viruses or infectious agents.
• blood plasma or blood which is to be used for transfusion or banked for future transfusion can be treated with the compounds of the invention and irradiated to effect sterilization.
• bio ⁇ logical products such as Factor VIII which are prepared from biological fluids can be irradiated in the presence of the compounds of the invention to destroy contam ⁇ inants.
• Protoporphyrin II 7,17-Bis(methoxycarbonylethyl)-2,12-divinyl- 3,8,13,18-tetramethyl-porphyrin was synthesized from acyclic precursors via dipyrromethene and a,c-biladiene intermediates, as described in "The Porphyrins" (supra) and in Scheme 1. The product was verified by UV, NMR and MS.
• Preparation B Preparation of A/C Dialkyl Analog 7,17-Diethyl-2,12-divinyl-3,8,13,18-tetra- methylporphyrin was synthesized from acyclic precursors via dipyrromethene and a,c-biladiene intermediates as in Preparation A as shown in Scheme 2, and the product was verified by UV, NMR and MS.
• the protoporphyrin-II dialkyl analog (10) of preparation B 50 mg; 0.105 mmol
• (E)- ⁇ - phenylsulphonyl-acrylonitrile (1.01 g, 5.25 mmol) were d ⁇ solved/suspended in dry toluene (20 mL), degassed by three freeze-pu p-thaw cycles and heated at 110°C in a sealed tube for three days.
• the reaction mixture was evaporated to dryness in vacuo and the residue chromato- graphed on silica gel (activity I, 70-230 mesh, 100 g) using 2% methanol-dichloromethane as eluent.
• the frac ⁇ tions absorbing at 734 nm were combined, evaporated in vacuo and further purified using a chromatotron with a 1 mm silica gel plate.
• the title compound (11) was obtained as the major product (45% yield).
• UV ⁇ max (CH2CI2) 384, 412 (Split Soret), 488, 520, 668, 698, 734 nm, shown in Figure 2A.
• EET H NMR (CDCI3, 400 MHz): 6 -2.54 (s, 1H) , -2.52 (s, 1H), 1.70 (t, 6H), 1.99, 2.01 (s, s, 6H) , 2.01 (s, 3H) , 3.26 (s, 6H), 3.25-3.60 (m, 4H) , 3.75 (d, 2H) , 3.78-3.86 (q, 4H), 4.31 (m, 2H) , 7.00 (m, 2H) , 7.50-8.05 (m, 10H) , 9.05 (s, 2H), 9.33 (s, 2H) .
• protoporphyrin-II dialkyl analog (10) of preparation B 50 mg; 0.105 mmol
• N-phenylmaleimide 0.91 g 5.25 mmol
• Purification by column chromatography (silica gel, 2% methanol- dichloro ethane) followed by further purification using the chromatotron (silica gel; 2% methanol- dichloromethane) afforded the A/C hydro-dibenzoporphyri (12) as the major product (45%).
• Example 3 The A/C adduct of Example 3 (13) (20 mg, 0.025 mmol) was dissolved in freshly distilled dichloromethane (8 mL) and stirred in the dark with 1,8-diazabicyclo- [5.4.0]undec-7-ene (DBU). The reaction, monitored by visible"spectroscopy, was complete in 3 h. The mixture was poured into 1M hydrochloric acid, extracted with dichloromethane, the organic layer washed with brine (twice) and water (once) and dried (MgS ⁇ 4). The product was purified by chromatography on silica gel using the -35- chromatotr ⁇ n wit ' 2% methanol-dichloromethane as the eluent; yield >90% of compound 14.
• DBU 1,8-diazabicyclo- [5.4.0]undec-7-ene
• UV ⁇ max (CH2CI2) 448 ( sh), 468 (Soret), 588 (sh), 622, 702, 742, 784 nm.
• UV ⁇ max (CH2CI2) 380, 406 (split Soret), 484, 516, 668, 5 700, 738 nm.
• UV ⁇ max (CH2CI2) 446 (sh), 466 (Soret), 584 (sh), 616, 702, 744, 786 nm.
• reaction mixture was dried in a vacuum desic ⁇ cator over KOH (overnight) and dissolved in dilute aque ⁇ ous sodium hydroxide, to obtain the hydrolyzed dicarboxylic acid salt.
• UV ⁇ m a ⁇ H2O; pH-10) 406 (sh), 464 (Soret), 618, 708, 740 (wk) 790 nm.
• UV ⁇ m a ⁇ H2O; pH-3) 442 (Soret), 466 (Sh) , 624, 708, 796 nm, as shown in Figure 2D.
• the product was extracted into ethyl acetate.
• UV ⁇ max (ethyl acetate) 444 (sh), 464 (Soret), 614, 698, 740, 782 nm.
• This example describes methods of preparation for immunoconjugates of four different antibody prepara ⁇ tions with either hematoporphyrin (Hp) or the invention hydro-dibenzoporphyrins.
• Hp hematoporphyrin
• the antibodies employed are CAMAL-1, anti-Ml antibody, and B16G anti ⁇ body, all prepared as described hereinabove, and affinity-purified rabbit/anti-mouse Ig (R ⁇ MIg).
• the conjugation is con ⁇ ducted in an entirely nonaqueous solvent.
• Various invention compounds were assayed in vitro using either cell line P815 or MI-S as model sys ⁇ tems to test their photosensitizing activity.
• various concentrations of test com ⁇ pounds were added to washed suspensions of cells from cultures of the target cells and the mixtures were then irradiated using 700-820 nm light for 30 minutes.
• the results were assayed by determining cytotoxicity using direct counting using eosin-Y exclusion, a standard pro ⁇ cedure for differentiating living from dead cells.
• the cells recovered from light exposure were assayed for viability by incubating them for 18 hours in 10 ⁇ Ci/ml tritium-labeled thymidine according to the standard pro ⁇ cedure whereby thymidine incorporation is equated with viability.
• the cells were harvested and radioactivity uptake was measured by a scintillation counter. The results obtained are shown in Table 1 below; all R 4 are methyl.

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