Use of purpurins, chlorins and purpurin- and chlorin-containing compositions

Description

BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to the production and use of a family of purpurins, a family of chlorins and metal complexes of the purpurins and chlorins, and to purpurin- and chlorin-containing compositions. The purpurins have a reduced pyrrole ring and an unsaturated isocyclic ring fused to a pyrrole ring; the unsaturated isocyclic ring of the purpurins corresponds with a saturated ring in the chlorins. The chlorins are useful as green dyes. The chlorins and the purpurins are useful in the detection and treatment of tumors; after they have been administered systemically, e.g., intravenously, they localize preferentially in a tumor. After they have been administered, and have localized in a tumor, their presence can be detected by illumination with ultra violet light, which causes them to fluoresce. The chlorins and purpurins of the invention can also be used to treat tumors; after they have been administered and have localized, irradiation with light of a wave length at which they show an absorbance peak causes a reaction which damages or destroys the tumor where they have localized. The purpurin- and chlorin-containing compositions are solutions thereof in an organic liquid that is physiologically acceptable for intravenous or topical administration and emulsions or suspensions of such solutions and water or saline or other solutions.
2. The Prior Art
Four purpurins having an unsaturated isocyclic ring fused to a reduced pyrrole ring are known to be reported in the prior art, a communication to the editor by Woodward et al., JACS, Vol. 82, pp. 3800 et seq., 1960, where they are disclosed as intermediates in the synthesis of chlorophyll, and journal articles by Witte et al., Angew, Chem. Internat. Edit./Vol. 14, No. 5, pp. 361 et seq., 1975, and Arnold et al., Journal of the Chemical Society, Perkin Transactions I, pp. 1660 et seq., 1979. No utility for purpurins is suggested by either Witte et al. or Arnold et al. In addition, European patent application EP142,732 (C.A. 103: 123271S) discloses certain chlorins of a different family and that they accumulate preferentially in cancer cells removed from hamsters that had been infected with pancreatic cancer.
Purpurins and chlorins are similar in structure to porphyrins. One porphyrin, called protoporphyrin IX, can be separated from blood. Hematoporphyrin can be produced from protoporphyrin IX; a chemical mixture derived from hematoporphyrin, called hematoporphyrin derivative, and often abbreviated "HpD", can be administered intravenously and used in the manner described above for the detection and treatment of tumors. The exact composition of HpD, however, is not known; in fact, it is a mixture of many different porphyrins and related compounds (see, for example, Porphyrin Photosensitization, edited by David Kassel and Thomas J. Dougherty, Plenum Press, New York and London, 1983, pp.3-13). As a consequence, the chlorins and purpurins of the instant invention are preferred over HpD for this use because they are single, known compounds. In addition, the chlorins and purpurins have absorbance peaks at longer wavelengths and show greater absorbances, by comparison with HpD; the longer wavelength peaks are advantageous because light of the longer wavelengths is capable of greater penetration of tissue, while the greater absorbances are desirable because less light energy is required to cause a given degree of reaction. U.S. Pat. No. 4,878,891 ("Judy et al." , granted Nov. 7, 1989) discloses the sterilization of blood and other body fluids and tissues, using either HpD or a composition containing about 90 percent of dihematoporphyrin ether as a photosensitizer. The photosensitizer is administered, e.g., intravenously, to a blood donor or a patient, and, after a suitable time, a blood or the like sample is removed from the donor or patient, and is irradiated with light of a suitable wavelength. Alternatively, the photosensitizer is added to a sample of blood or the like, and the sample is irradiated after a suitable time.
U.S. Pat. Nos. 5,093,349 ("Pandey et al.", Mar. 3, 1992) and 5,079,262 ("Kennedy et al.", Jan. 7, 1992) disclose both the systemic and the topical administration of photosensitizers and irradiation with light of a suitable wavelength.
A published European patent application is understood to disclose certain dimers where two phlorin, porphyrin, chlorin or the like moieties are linked to one another through an ether group, and the use of those dimers in the manner described above for the detection and treatment of tumors. Finally, Porphyrin Photosensitization, edited by David Kassel, Plenum Press, New York and London, 1985, pp. 213-228, discloses dimers which are esters of certain chlorins with hematoporphyrin; the dimers are said to localize as described above, even though the chlorins from which they are produced do not.

BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a structural formula for metal complexes of a family of purpurins in accordance with the instant invention; in these complexes, an unsaturated isocyclic ring is fused to a reduced pyrrole ring.
FIG. 2 is a structural formula for metal complexes of a family of chlorins in accordance with the instant invention; in these complexes, an isocyclic ring which corresponds with the unsaturated isocyclic ring of the purpurins of FIG. 1 is saturated.
FIG. 3 is a structural formula for a family of porphyrins which can be used to produce purpurins having the formula of FIG. 7.
FIG. 4 is a structural formula for pyrroles from which porphyrins having the structure of FIG. 3 can be produced.
FIG. 5 is a structural formula for dipyrromethanes which are intermediates for the production of porphyrins from pyrroles.
FIG. 6 is a structural formula for metal complexes of porphyrins having the formula of FIG. 3.
FIG. 7 is a structural formula for the family of purpurins having the structure of the complexes of FIG. 1.
FIG. 8 is a structural formula for the family of chlorins having the structure of the complexes of FIG. 2.
FIGS. 9-13 are structural formulas for five different carboxy purpurins, each of which can be used in the synthesis of different position isomers of purpurins having the structure of FIG. 7.
FIGS. 14-18, FIGS. 19-23, and FIGS. 24-28 are structural formulas for intermediates in the synthesis of purpurin position isomers having the formulas of FIGS. 29-33 from the carboxy purpurins having the formulas of FIGS. 9-13.
FIGS. 34-38 are structural formulas for purpurins according to the invention having two isocyclic rings fused to pyrrole rings.
FIGS. 39-43 are structural formulas for chlorins, some of which are according to the invention, which can be produced from purpurins having the formulas of FIGS. 29-33.
FIGS. 44-48 are structural formulas for metal complexes of purpurins having the formulas of FIGS. 29-33.
FIGS. 49-53 are structural formulas for metal complexes of chlorins having the formulas of FIGS. 39-43.
FIGS. 54-58 are structural formulas for metal complexes of purpurins having the formulas of FIGS. 34-38.

BRIEF DESCRIPTION OF THE INVENTION
The instant invention is a method for detecting a tumor or treating a patient which comprises administering intravenously, intramuscularly, subcutaneously, intraperitoneally or topically an effective amount of a purpurin, chlorin or metal complex to a human or animal patient. A suitable time after administration of the purpurin, chlorin or metal complex, e.g., 24 hours, the relevant region of the patient can be irradiated with ultra violet or with visible light of a wavelength at which the purpurin or chlorin has an absorbance peak. The purpurin has the structure of any of FIGS. 7, 14-18 or 29-38 of the attached drawings; the purpurin metal complex has the structure of any of FIGS. 1, 44-48 or 54-58, of the attached drawings; the chlorin has the structure of any of FIGS. 8, 19-28 or 39-43 of the attached drawings; and the chlorin metal complex has the structure of any of FIGS. 2 or 49-53, of the attached drawings. In the drawings, M represents a metal cation that is complexed with two of the nitrogens of the purpurin or chlorin and is, for example, Ag, Al, Ce, Co, Cr, Dy, Er, Eu, Fe, Ga, Gd, Hf, Ho, In, La, Lu, Mn, Mo, Nd, Pb, Pd, Pt, Pt, Rh, Sb, Sc, Sm, Sn, Tb, .sup.99m Tc, Th, Ti, Tl, Tm, U, V, Y, Yb, Zn or Zr, each of R10 through R13 and R16 is hydrogen, and each of R1 through R9, R14 and R15 is:
H or CHO,
an alkyl group other than t-butyl having from 1 to 4 carbon atoms,
an alkylene group having from 2 to 4 carbon atoms,
a group having the formula R.sub.2 N(R.sub.3).sub.2 where R.sub.2 is a bivalent aliphatic hydrocarbon radical having from 1 to 4 carbon atoms, wherein any carbon to carbon bond is either a single or a double bond, and not more than one is a double bond; R.sub.3 is hydrogen or an alkyl group having from 1 to 2 carbon atoms and the two R.sub.3 groups can be the same or different,
a group having the formula R.sub.2 N(R.sub.4).sub.3 A where R.sub.2 is a bivalent aliphatic hydrocarbon radical having from 1 to 4 carbon atoms, wherein any carbon to carbon bond is either a single or a double bond, and not more than one is a double bond; A is a physiologically acceptable anion; and R.sub.4 is an alkyl group having from 1 to 2 carbon atoms and the three R.sub.4 groups can be the same or different,
a group having the formula R.sub.2 OH were R.sub.2 is a bivalent aliphatic hydrocarbon radical having from 1 to 4 carbon atoms, wherein any carbon to carbon bond is either a single or a double bond, and not more than one is a double bond,
an amino acid moiety which is a part of an amide produced by reaction between an amine function of a naturally occurring amino acid and a carboxyl function of the purpurin or chlorin,
CO.sub.2 R', CH.sub.2 CO.sub.2 R' or CH.sub.2 CH.sub.2 CO.sub.2 R', where R' is hydrogen or an alkyl group other than t-butyl having from 1 to 4 carbon atoms,
a monoclonal antibody moiety which is attached to the purpurin or chlorin moiety through a carbonyl which is a part of an amide produced by reaction between an amine function of a monoclonal antibody and a CO.sub.2 R' CH.sub.2 CO.sub.2 R' or CH.sub.2 CH.sub.2 CO.sub.2 R' group of the purpurin or chlorin, and wherein the moiety is of a monoclonal antibody which selectively binds to malignant tumors, or
in the purpurins and purpurin metal complexes of FIGS. 14-18, 29-38, 44-48 and 54-58 and in the chlorins and chlorin metal complexes of FIGS. 24-28, 39-43 and 49-53 R1 can be a bivalent aliphatic hydrocarbon radical having from 2 to 4 carbon atoms wherein both of the valences of the radical are attached to the same carbon atom thereof and to a carbon atom of the purpurin, chlorin, or metal complex, and in the purpurins and purpurin metal complexes of FIGS. 29-33 and of FIGS. 44-48, both R1 and R2 can be bivalent aliphatic hydrocarbon radicals having from 2 to 4 carbon atoms wherein both of the valences of the radical are attached to the same carbon atom thereof and to a carbon atom of the purpurin or metal complex, with the proviso that not more than one of R1 through R9, R14 and R15 is CHO, a group having the formula R.sub.2 N(R.sub.3).sub.2, a group having the formula R.sub.2 N(R.sub.4).sub.3 A, an amino acid moiety or a monoclonal antibody moiety.
The purpurin, chlorin or metal complex can have a structure that is enriched in an atom that can be detected by nuclear magnetic resonance or that is radioactive and can be detected by an instrument for measuring ionizing radiation. The atom that can be detected by nuclear magnetic resonance can be, for example, .sup.15 N or .sup.13 C, while the atom that can be detected by an instrument for measuring ionizing radiation can be .sup.14 C. The purpurin, chlorin or complex, in all cases, can have the formula of any of FIGS. 1, 2, 7, 8 or 14-58 where M and R1 through R16 have the meanings set forth above.
The method of the invention is for the treatment of malignant tumors, of non-malignant lesions, e.g., of the vagina or bladder, and of such cutaneous lesions as are involved in psoriasis; the method involves the administration of a chlorin, a purpurin or a complex, and illumination of the area which requires treatment. The purpurin, chlorin or complex can be injected intravenously, intramuscularly, subcutaneously, intraperitoneally, or topically, or it can be applied topically. Dosages can vary widely, but the in vivo test data reported herein indicate that the intravenous administration of up to 7 mg per kg of body weight is safe. The purpurins, chlorins and complexes can be formulated in lotions, suspensions or pastes for localized treatment, e.g., of superficial tumors or skin disorders. The topical application can also be of a solution, e.g., in DMSO or ethanol. The purpurins, chlorins and metal complexes can also be used in vitro, e.g., to sterilize blood that has been removed from a human or animal patient or donor, or to sterilize tissue that has been removed from a human or animal patient or donor.
In a further aspect, the invention is a method for treating human or animal patients which involves administering a purpurin, chlorin or complex having the formula of any of FIGS. 1, 2, 7, 8 or 14-58 where M and R1 through R16 have the meanings set forth above, and then treating the affected region with X rays. There are indications that the chlorins and the like are X ray sensitizers which increase the therapeutic ratio of X rays.
OBJECTS OF THE INVENTION
It is, therefore, an object of the invention to provide a method for detecting a tumor or treating a patient which comprises administering intravenously, intramuscularly, subcutaneously, intraperitoneally or topically an effective amount of a purpurin, chlorin or metal complex to a human or animal patient.
It is a further object to provide a method for the treatment of non-malignant lesions or of such cutaneous lesions as are involved in psoriasis by the topical application of a solution in DMSO or the like of one of the foregoing purpurins, chlorins or metal complexes, followed by illumination of the affected region.
It is another object of the invention to provide a method for detecting and treating tumors which comprises administering one of the foregoing purpurins or chlorins to a human or animal patient, followed by illumination of the region affected with ultraviolet, with visible light or with X rays, scanning of the region affected by nuclear magnetic resonance, or scanning of the region affected with an instrument that measures ionizing radiation.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Examples 1 through 3 and A through G hereof set forth the best mode presently contemplated by the inventors for producing purpurins, chlorins and metal complexes. The in vivo test procedures describe the best mode insofar as this invention is directed to solutions of the purpurins, chlorins and metal complexes in an organic liquid and to the production of such solutions, and the in vitro and in vivo test procedures describe the best mode insofar as the invention is directed to the use of purpurins, chlorins and metal complexes for the detection and treatment of tumors.
In the examples, and elsewhere herein, the term "percent v/v" means percent by volume; the term "percent w/w" means percent by weight; the term "alkyl group" is used in its ordinary sense to mean a monovalent, saturated, aliphatic hydrocarbon radical; the term "alkylene group" is used in its ordinary sense to mean a monovalent, aliphatic hydrocarbon radical which has one carbon to carbon double bond and in which any other carbon to carbon bond is a single bond; all temperatures are in .degree.C.; and the following abbreviations have the meanings indicated: mg=milligram or milligrams; g=gram or grams; kg=kilogram or kilograms; ml=milliliter or milliliters; L=liter or liters; cm=centimeter or centimeters; .epsilon.=molar absorptivity; and mw=milliwatts.
EXAMPLE 1
The production of a novel purpurin according to the invention (hereafter "Purpurin I") is described in this example. The synthesis involves the production of several pyrroles which are identified and assigned trivial names in the following table:
______________________________________Compound Structure (referring to attached drawings)______________________________________Pyffole I FIG. 4, where A is CO.sub.2 CH.sub.2 C.sub.6 H.sub.5 B is CH.sub.2 CH.sub.3 C is CO.sub.2 CH.sub.2 CH.sub.3 D is CH.sub.3Pyffole II FIG. 4, where A is CO.sub.2 CH.sub.2 C.sub.6 H.sub.5 B is CH.sub.2 CH.sub.3 C is CO.sub.2 CH.sub.2 CH.sub.3 D is CH.sub.2 OCOCH.sub.3Pyffole III FIG. 4, where A is CO.sub.2 CH.sub.2 C.sub.6 H.sub.5 B is CH.sub.2 CH.sub.3 C is COCH.sub.3 D is CH.sub.3Pyffole IV FIG. 4, where A is CO.sub.2 CH.sub.2 C.sub.6 H.sub.5 B is CH.sub.2 CH.sub.3 C is CH.sub.2 CH.sub.3 D is CH.sub.3Pyrrole V FIG. 4, where A is CO.sub.2 CH.sub.2 C.sub.6 H.sub.5 B is CH.sub.2 CH.sub.3 C is CH.sub.2 CH.sub.3 D is HPyffole VI FIG. 4, where A is CO.sub.2 CH.sub.2 C.sub.6 H.sub.5 B is CH.sub.2 CH.sub.3 C is CH.sub.2 CH.sub.3 D is CH.sub.2 0C0CH.sub.3______________________________________
The synthesis also involves the production of several dipyrromethanes which are identified and assigned trivial names in the following table:
______________________________________Compound Structure (referring to attached drawings)______________________________________Dipyrro- FIG. 5, where A is CO.sub.2 CH.sub.2 C.sub.6 H.sub.5methane I B is CH.sub.2 CH.sub.3 C is CO.sub.2 CH.sub.2 CH.sub.3 E is CH.sub.2 CH.sub.3 F is CH.sub.2 CH.sub.3 G is CO.sub.2 CH.sub.2 C.sub.6 H.sub.5Dipyrro- FIG. 5, where A is CO.sub.2 CH.sub.2 C.sub.6 H.sub.5methane II B is CH.sub.2 CH.sub.3 C is CH.sub.2 CH.sub.3 E is CH.sub.2 CH.sub.3 F is CH.sub.2 CH.sub.3 G is CO.sub.2 CH.sub.2 C.sub.6 H.sub.5Dipyrro- FIG. 5, where A is CHOmethane III B is CH.sub.2 CH.sub.3 C is CH.sub.2 CH.sub.3 E is CH.sub.2 CH.sub.3 F is CH.sub.2 CH.sub.3 G is CHO______________________________________
The synthesis also involves the production of two porphyrins and six porphyrin complexes, all of which are identified and given trivial names in the following table:
______________________________________Compound Structure (referring to attached drawings)______________________________________Porphyrin I FIG. 3, where R and R10-12 are hydrogen R1 and R3-R8 are CH.sub.2 CH.sub.3 R2 is CO.sub.2 HPorphyrin FIG. 6, where R and R10-12 are hydrogenComplex II R1 and R3-R8 are CH.sub.2 CH.sub.3 R2 is CO.sub.2 H M is NiPorphyrin FIG. 6, where R is CHOComplex III R1 and R3-R8 are CH.sub.2 CH.sub.3 R2 is CO.sub.2 H R10-R12 are H M is NiPorphyrin FIG. 6, where R, R11 and R12 are HComplex IV R1 and R3-R8 are CH.sub.2 CH.sub.3 R2 is CO.sub.2 H R10 is CHO M is NiPorphyrin FIG. 6, where R, R10 and R12 are HComplex V R1 and R3-R8 are CH.sub.2 CH.sub.3 R2 is CO.sub.2 H R11 is CHO M is NiPorphyrin FIG. 6, where R, R10 and R11 are hydrogenComplex VI R1 and R3-R8 are CH.sub.2 CH.sub.3 R2 is CO.sub.2 H R12 is CHO M is NiPorphyrin FIG. 6, where R, R10 and R12 are hydrogenComplex VII R1 and R3-R8 are CH.sub.2 CH.sub.3 R2 is CO.sub.2 H R11 is CH.dbd.CHCO.sub.2 CH.sub.2 CH.sub.3 M is NiPorphyrin FIG. 3, where R, R10 and R12 are hydrogenVIII R1 and R3-R8 are CH.sub.2 CH.sub.3 R2 is CO.sub.2 H R11 is CH.dbd.CHCO.sub.2 CH.sub.2 CH.sub.3______________________________________
Purpurin I and other purpurins that were produced in the course of the synthesis thereof are identified and the other purpurins are assigned trivial names in the following table:
______________________________________Compound Structure (referring to attached drawings)______________________________________Purpurin I FIG. 34, where R1-R5, R7 and R8 are CH.sub.2 CH.sub.3 R9 is CO.sub.2 CH.sub.2 CH.sub.3 R 13 is H R15 is CO.sub.2 CH.sub.2 CH.sub.3Purpurin II FIG. 9, where R1-R5, R7 and R8 are CH.sub.2 CH.sub.3 R9 is CO.sub.2 CH.sub.2 CH.sub.3Purpurin III FIG. 13, where R1-R6 and R8 are CH.sub.2 CH.sub.3 R9 is CO.sub.2 CH.sub.2 CH.sub.3Purpurin IV FIG. 14, where R1-R5, R7 and R8 are CH.sub.2 CH.sub.3 R9 is CO.sub.2 CH.sub.2 CH.sub.3Purpurin V FIG. 7, where R1-R5, R7 and R8 are CH.sub.2 CH.sub.3 R6 is (O.dbd.C)CH.sub.2 CO.sub.2 CH.sub.3 R9 is CO.sub.2 CH.sub.2 CH.sub.3 R10-R13 are hydrogen______________________________________
Production of Pyrrole I
Pyrrole I is produced from a saturated aqueous solution containing one gram equivalent of sodium nitrite, a 5 percent w/w solution in glacial acetic acid containing one gram equivalent of benzyl propionylacetate, a suspension in glacial acetic acid of one gram equivalent of ethyl acetoacetate and four gram equivalents of zinc dust. The sodium nitrite solution is added dropwise, with stirring, to the benzyl propionylacetate solution at a rate sufficiently slow that the temperature of the solution that is formed does not exceed 20.degree.. After the resulting solution stands at room temperature of about 22.degree. for thirty minutes, the zinc dust is added in increments to the ethyl acetoacetate suspension at such a rate that the exothermic reaction which occurs heats the slurry to about 65.degree.; the foregoing solution is then added dropwise, with stirring, to the ethyl acetoacetate/zinc slurry. The reaction mixture is maintained at 65.degree. during the addition and for an hour after completion of the addition of the sodium nitrite/benzyl propionylacetate solution; stirring is continued during the additional hour. The hot solution is then separated from the zinc by decantation. The Pyrrole I is precipitated by pouring the hot solution into ice water, and is recovered by filtration and air dried.
Production of Pyrrole II
Pyrrole II is produced from 6 g lead tetra-acetate and a solution of 4 g Pyrrole I in 2 ml acetic anhydride and 100 ml glacial acetic acid. The lead tetra-acetate is added to the Pyrrole I solution and dissolved by warming the resulting reaction mixture on a steam bath for about 10 minutes. The solution so formed is stirred at room temperature of about 22.degree. for 16 hours. Dropwise additions of ice are then made to precipitate the Pyrrole II, which is recovered from the reaction mixture by filtration and washed with distilled water.
Production of Pyrrole III
Pyrrole III is produced from a saturated aqueous solution containing one gram equivalent of sodium nitrite, a 5 percent w/w solution in glacial acetic acid containing one gram equivalent of benzyl propionyl-acetate, a suspension in glacial acetic acid of one gram equivalent of 2,4-pentanedione and four gram equivalents of zinc dust. The sodium nitrite solution is added dropwise, with stirring, to the benzyl propionyl-acetate solution at a rate sufficiently slow that the temperature of the solution that is formed does not exceed 20.degree.. After the resulting solution stands at room temperature of about 22.degree. for thirty minutes, the zinc dust is added in increments to the 2,4-pentanedione suspension at such a rate that the exothermic reaction which occurs heats the slurry to about 65.degree.; the foregoing solution is then added dropwise, with stirring, to the 2,4-pentanedione/zinc slurry. The reaction mixture is maintained at 65.degree. during the addition and for an hour after completion of the addition of the sodium nitrite/benzyl propionyl-acetate solution; stirring is continued during the additional hour. The hot solution is then separated from the zinc by decantation. The Pyrrole III is precipitated by pouring the hot solution into ice water, and is recovered by filtration and air dried.
Production of Pyrrole IV
Pyrrole IV is produced from 1.1 gram equivalents of boron trifluoride etherate and a 5 percent w/w solution in glacial acetic acid containing one gram equivalent of Pyrrole III; the Pyrrole III solution also contains 2 gram equivalents of sodium borohydride. The Pyrrole III solution is cooled with ice while the boron trifluoride etherate is added thereto dropwise, with stirring. After the addition is complete, the reaction mixture is allowed to warm to room temperature of about 22.degree. and then to stand two hours at room temperature; stirring is continued throughout. Excess sodium borohydride is then destroyed by cautious additions of glacial acetic acid. The reaction mixture, a solution, is then poured into ice water; Pyrrole IV which precipitates is recovered by filtration and air dried.
Production of Pyrrole V
Pyrrole V is produced from a 5 percent w/w solution in dichloromethane containing 1 gram equivalent of Pyrrole IV and a 5 percent w/w solution in dichloromethane containing 4 gm equivalents of sulfuryl chloride. The Pyrrole IV solution is diluted with about 10 percent v/v diethyl ether and the sulfuryl chloride solution is added to the diluted Pyrrole IV solution. The reaction is conducted at room temperature with stirring, which is commenced before the sulfuryl chloride solution addition is started, and continued for about one hour after that addition is completed. Solvent is then removed from the reaction mixture, leaving a pale yellow oil. The oil is dissolved in a solution of water in acetone containing 20 percent v/v water and the solution is heated under reflux. The solution becomes acidic rapidly. After 20 minutes under reflux, enough sodium acetate to neutralize the acid and a small excess is added to the solution; heating is continued until the acetone is vaporized and an oil separates from the aqueous phase which remains. Upon cooling of the reaction mixture to room temperature, the oil forms a crystallized solid ("Pyrrole VII": Pyrrole v, except that D is CO.sub.2 H) which is suspended in a 5 percent w/w solution in glacial acetic acid and 4.8 percent w/w acetic anhydride containing 3 equivalents of anhydrous sodium acetate per equivalent of the Pyrrole VII. The resulting suspension is heated gently to 80.degree. and stirred while a 5 percent w/w solution in glacial acetic acid containing 1 equivalent of iodine monochloride (based upon the Pyrrole VII) is added dropwise thereto. When the iodine monochloride addition is complete, the solution which has formed is cooled and mixed with an equal volume of water; hypophosphorous acid is added to remove excess iodine; and the solid which forms is recovered by filtration, washed with water, dried and suspended with 0.2 g platinum oxide in tetrahydrofuran. The resulting suspension is hydrogenated until the uptake of hydrogen ceases. The solution of Pyrrole V in tetrahydrofuran which results is separated from the platinum oxide by filtration; the tetrahydrofuran is replaced under reduced pressure with methanol; and the Pyrrole V is recovered by filtration.
Production of Dipyrromethane I
Dipyrromethane I is produced from a 5 percent w/w solution in dichloromethane containing one g equivalent Pyrrole II, one g equivalent Pyrrole V and about 2 g Montmorillonite clay. The clay is added to the Pyrrole II solution, and the slurry which results is stirred for about 10 minutes. The clay is then separated by filtration and washed with dichloromethane; the wash is combined with the filtrate; evaporation of the dichloromethane leaves the Dipyrromethane I.
Production of Pyrrole VI
Pyrrole VI is produced from 6 g lead tetra-acetate and a solution of 1 g equivalent Pyrrole IV in 2 ml acetic anhydride and 100 ml glacial acetic acid. The lead tetra-acetate is added to the Pyrrole II solution, and the resulting reaction mixture is warmed on a steam bath to dissolve the lead tetra-acetate. The solution which is formed is stirred for 16 hours at room temperature of about 22.degree., after which time the Pyrrole VI is precipitated by dropwise addition of ice water, separated from the liquid by filtration, washed with water and air dried.
Production of Dipyrromethane II
Dipyrromethane II is produced by dissolving Pyrrole VI in methanol containing about 0.05 percent w/w HCl to make a 5 percent w/w solution, and heating the solution under reflux for five hours. The reaction product is cooled to room temperature of about 22.degree. and poured into ice water to precipitate the Dipyrromethane II, which is then recovered by filtration and air dried.
Production of Dipyrromethane III
Dipyrromethane III is produced from 2 g Dipyrromethane II, 50 ml absolute ethanol containing 0.05 percent w/w triethyl amine, 0.1 g charcoal coated with 5 percent w/w palladium, 3 ml trifluoroacetic acid and 1.0 g p-nitobenzoyl chloride dissolved in 1.0 g dry dimethyl formamide. The Dipyrromethane II is dissolved in the absolute ethanol and the palladium on charcoal is added to the resulting solution. The Dipyrromethane II is then hydrogenated in a sloping manifold hydrogenator in which a slight positive pressure of hydrogen is maintained until there is no longer an uptake of hydrogen. The palladium on charcoal is then separated from the reaction mixture by filtration, and the solvent is evaporated, leaving a solid white residue. The white residue is powdered finely and added, under nitrogen, to the trifluoroacetic acid at a temperature of 45.degree.; the reaction mixture is maintained at 45.degree., with stirring, until the evolution of CO.sub.2 subsides and for an additional 3 minutes, and is then poured into 30 ml 56 percent w/w aqueous ammonium hydroxide to which 5 g crushed ice has been added. The aqueous mixture which results is extracted with dichloromethane, which is then evaporated; the red oil which remains after evaporation of the dichloromethane is immediately dissolved in 3 ml dry dimethylformamide, and the solution which results is cooled to and maintained at 0.degree., with stirring, while the solution of p-nitrobenzoyl chloride in dimethyl formamide is added dropwise, and for 30 minutes after completion of the addition. Am addition of 20 ml diethyl ether is made 15 minutes after completion of the addition of the dimethyl formamide solution, and, 15 minutes later, solids which have precipitated are separated from the liquid by filtration and added, with stirring, to 50 percent w/w aqueous ethanol containing about 3 g sodium carbonate, which has been heated to 70.degree.. After 15 minutes of stirring, Dipyrromethane III is separated from the ethanol/water solution by filtration and air dried.
Production of Porphyrin I
Porphyrin I is produced from two solutions, one a 5 percent w/w solution in dry tetrahydrofuran containing 0.05 percent w/w triethyl amine and 1 g equivalent Dipyrromethane I, and the second a 5 percent w/w solution in dichloromethane containing 5 percent v/v methanol, 0.05 percent w/w p-toluencsulfonic acid and 1 g equivalent of Dipyrromethane III, using 5 percent w/w, based on the weight of the Dipyrromethane I, 5 percent w/w palladium on charcoal as a hydrogenation catalyst. The palladium on charcoal is added to the Dipyrromethane I solution and hydrogenation is carried out in a sloping manifold hydrogenator in which a slight positive pressure of hydrogen is maintained until the uptake of hydrogen stops. The palladium on charcoal is then separated from the reaction mixture by filtration and washed with dilute ammonium hydroxide. The tiltrate is evaporated to dryness in vacuo, and the product which remains is dissolved in the ammonia washings; 12 percent w/w acetic acid is added to the resulting solution to adjust the pH to 4 and the temperature thereof is lowered to about 5.degree. to cause precipitation of an intermediate diacid. The diacid is then dissolved in the second solution and the resulting reaction mixture is allowed to stand in the dark at room temperature of about 22.degree. for 24 hours, after which time a methanolic solution containing about 1 g zinc acetate dihydrate is added thereto. The solution is allowed to stand in the dark at room temperature for another 72 hours, after which time the solvent is removed by evaporation, and the solid which remains is dissolved in aqueous dioxane containing 3 equivalents KOH per equivalent of Porphyrin I. The resulting solution is refluxed for four hours, cooled, and diluted with distilled water; the Porphyrin I is extracted from the solution with dichloromethane; the dichloromethane is evaporated; and the residue is recrystallized from a 50 percent v/v solution of methanol in dichloromethane.
Production of Porphyrin Complex II
A 5 percent w/w solution in a mixed dichloromethane-methanol solvent containing 20 percent v/v methanol, 1 g equivalent of Porphyrin I and 2 g equivalents of nickel acetate is refluxed for 16 hours. The solvent is then evaporated until the Porphyrin Complex II precipitates; the product is recovered by filtration and air dried.
Production of Porphyrin Complexes III-VI
A mixture of the porphyrin complexes identified above is prepared from 1 g Porphyrin Complex II, 28 ml freshly distilled phosphorus oxychloride, 20 ml dry dimethyl formamide and 750 ml dry 1,2-dichloroethane. The dimethyl formamide is cooled on an ice bath, and the phosphorus oxychloride is added thereto dropwise. The solution which results is allowed to stand at room temperature of about 22.degree. for 30 minutes, and is then warmed to 50.degree.. The Porphyrin Complex II is dissolved in the 1,2-dichloroethane, and the resulting solution is added dropwise, with stirring, to the phosphorus oxychloride; the addition is made over a period of about 30 minutes. The reaction mixture is maintained at about 50.degree., with stirring, for an additional 2 hours. The organic and the aqueous phases are then separated, and the aqueous phase is extracted with dichloromethane. The organic phase and the dichloromethane extract are then combined, and evaporated to dryness. The solid which remains is recrystallized from a solvent composed of equal parts by volume of dichloromethane and methanol, yielding a mixture of Porphyrin Complex III, Porphyrin Complex IV, Porphyrin Complex V and Porphyrin Complex VI. The mixture of complexes is separated by silica gel chromatography, using dichloromethane containing 1 percent v/v methanol as the eluant.
Production of Porphyrin Complex VII
A solution of 506 mg Porphyrin Complex V and 1.024 g. (carbethoxymethylene)triphenylphosphorane in 50 ml xylene is heated under reflux for 18 hours. The solution is cooled; the xylene is removed in vacuo; and the solid which remains is dissolved in the minimum amount of dichloromethane and chromatographed on silica gel. A minor fraction of Porphyrin Complex V and a major red fraction are recovered. The solvent is removed from the red fraction; the solid which remains is recrystallized from a solvent composed of equal parts by volume of dichloromethane and methanol, yielding Porphyrin Complex VII.
Production of Porphyrin VIII
A solution is prepared by dissolving 621 mg Porphyrin Complex VII in 10 ml concentrated (96.7 percent w/w) sulfuric acid; after the solution stands for 2 hours at room temperature of about 22.degree., an addition of 100 ml dichloromethane is made thereto, followed by saturated aqueous sodium bicarbonate to neutralize the sulfuric acid. The organic layer is collected, washed and dried; the solvent is then vaporized. The crude product which remains is recrystallized from a solvent composed of equal parts by volume of dichloromethane and methanol, yielding Porphyrin VIII.
Production of Purpurin II and Purpurin IIl
A solution of 100 mg Porphyrin VIII in 20 ml glacial acetic acid is heated under reflux in a nitrogen atmosphere for 24 hours. The solution is then cooled; the acetic acid is removed in vacuo; and the remaining product is dissolved in the minimum amount of dichloromethane and chromatographed on silica gel, yielding a major green fraction from which the solvent is removed. The solid which remains is recrystallized from 50 percent v/v dichloromethane and methanol, yielding a mixture of Purpurin II and Purpurin III which are separated by silica gel chromatography using dichloromethane containing 1 percent v/v methanol as the elutant.
Production of Purpurin V
Purpurin V is produced from 59 mg Purpurin II, 25 mg N,N'-carbonyldiimidazole, 100 mg zinc acetate, 25 mg sodium hydride and 87 mg methyl t-butyl malonate. The Purpurin II is dissolved in 5 ml dichloromethane and refluxed for one hour with the N,N'-carbonyldiimidazole; the zinc acetate dissolved in 5 ml methanol is then added and the resulting reaction mixture is warmed gently for 5 minutes. After an addition of 25 ml dichloromethane, the solution which forms is washed three times with 50 ml portions of water, dried over MgSO.sub.4 and evaporated under vacuum; the solid residue which remains after evaporation of the solvents is maintained at an absolute pressure of 0.1 mm Hg for 30 minutes, and is then dissolved in dichloromethane. The resulting solution is then added to a malonate anion solution prepared by adding the sodium hydride and the methyl t-butyl malonate to 10 ml tetrahydrofuran, and the reaction mixture is stirred for 40 minutes at room temperature of about 22.degree. and added to 50 ml chloroform and 20 ml 1 normal hydrochloric acid. The organic phase is separated from the aqueous phase, washed twice with 50 ml portions of water, dried over MgSO.sub.4 and evaporated; the residue is stirred at room temperature of about 22.degree. with 5 ml trifluoroacetic acid for 40 minutes and the resulting product is mixed with 100 ml water and 50 ml chloroform. The organic layer is separated from the aqueous layer, washed twice with 50 ml portions of water, dried over MgSO.sub.4, and evaporated to dryness. The residue is dissolved in the minimum amount of dichloromethane containing 5 percent v/v acetone and purified by elutriation on alumina. Purpurin V is recovered by evaporating the solvents from the elutriate.
Production of Purpurin IV
Purpurin IV is prepared from 68 mg Purpurin V and 110 mg thallium trifluoroacetate. A solution of the Purpurin V in 20 ml dry dichloromethane and 20 ml dry tetrahydrofuran is treated with a solution of the thallium trifluoroacetate in 10 ml dry tetrahydrofuran. After 2 minutes, the solution which results is placed in sunlight for about 10 minutes until a sample examined spectrophotometrically shows the expected shift of the Soret absorption band. The solution is then treated briefly with SO.sub.2 gas, stirred for about 1 minute with about 1/2 ml 37 percent w/w hydrochloric acid, diluted with 50 ml dichloromethane, and washed three times with 100 ml portions of water. The solvents are then removed by evaporation; the residue is dissolved in dichloromethane containing 5 percent v/v methanol; the solution is chromatographed on alumina; and the Purpurin V is crystallized from the solvent and recovered by filtration.
Production of Purpurin I
A solution of 60 mg sodium borohydride in 10 ml methanol is added dropwise to a solution of 200 mg Purpurin IV in 5 ml dichloromethane; the resulting solution is stirred at room temperature of about 22.degree. for 2 hours, and is poured into 100 ml water. The organic phase is separated from the aqueous phase; the solvent is removed from the organic phase; and the solvent is evaporated. The residue is dissolved in 50 ml chloroform containing 25 percent v/v methanol; a 10 mg addition of p-toluene sulfonic acid is made; and the reaction mixture is refluxed for 6 hours. Water is then added to the reaction mixture; the organic layer is collected; and the solvent is removed by evaporation. The residue is dissolved in 5 ml dichloromethane containing 2 percent v/v methanol; the resulting solution is chromatographed on silica gel; and Purpurin I is recovered by evaporating the solvent from the chromatographed solution.
Purpurin II and Purpurin III are both produced from Porphyrin VIII in one step of the procedure described above as Example 1; Purpurin V, Purpurin IV and Purpurin I are then produced from Purpurin II. It will be appreciated that Purpurin III can be substituted for Purpurin II to produce the following isomers of Purpurin I, Purpurin IV and Purpurin V:
______________________________________Compound Structure (referring to attached drawings)______________________________________Purpurin VI FIG. 38, where R1-R6 and R8 are CH.sub.2 CH.sub.3 R9 is CO.sub.2 CH.sub.2 CH.sub.3 R 13 is H R15 is CO.sub.2 CH.sub.2 CH.sub.3Purpurin VII FIG. 18, where R1-R6 and R8 are CH.sub.2 CH.sub.3 R9 is CO.sub.2 CH.sub.2 CH.sub.3Purpurin VIII FIG. 7, where R1-R6 and R8 are CH.sub.2 CH.sub.3 R7 is (O.dbd.C)CH.sub.2 CO.sub.2 CH.sub.3 R9 is CO.sub.2 CH.sub.2 CH.sub.3 R10-R13 are hydrogen______________________________________
Similarly, Porphyrin Complex III, Porphyrin Complex IV, Porphyrin Complex V and Porphyrin Complex VI are all produced in one step of the procedure; Porphyrin Complex VII, Porphyrin VIII, Purpurin II and Purpurin III are then produced from Porphyrin Complex V. It will be appreciated that Porphyrin Complex IV can be substituted for Porphyrin Complex V to produce the following isomers of Porphyrin Complex VII, Porphyrin VIII, Purpurin I, Purpurin II, Purpurin IV and Purpurin V:
______________________________________Compound Structure (referring to attached drawings)______________________________________Porphyrin FIG. 6, where R, R11 and R12 are hydrogenComplex IX R1 and R3-R8 are CH.sub.2 CH.sub.3 R2 is CO.sub.2 H R10 is CH.dbd.CHCO.sub.2 CH.sub.2 CH.sub.3 M is NiPorphyrin X FIG. 3, where R, R11 and R12 are hydrogen R1 and R3-R8 are CH.sub.2 CH.sub.3 R2 is CO.sub.2 H R10 is CH.dbd.CHCO.sub.2 CH.sub.2 CH.sub.3Purpurin IX FIG. 35, where R1-R3 and R5-R8 are CH.sub.2 CH.sub.3 R9 is CO.sub.2 CH.sub.2 CH.sub.3 R 13 is H R15 is CO.sub.2 CH.sub.2 CH.sub.3Purpurin X FIG. 10, where R1-R3 and R5-R8 are CH.sub.2 CH.sub.3 R9 is CO.sub.2 CH.sub.2 CH.sub.3Purpurin XI FIG. 15, where R1-R3 and R5-R8 are CH.sub.2 CH.sub.3 R9 is CO.sub.2 CH.sub.2 CH.sub.3Purpurin FIG. 7, where R1-R3 and R5-R8 are CH.sub.2 CH.sub.3 R4 is (O.dbd.C)CH.sub.2 CO.sub.2 CH.sub.3 R9 is CO.sub.2 CH.sub.2 CH.sub.3 R10-R13 are hydrogen______________________________________
Similarly, it will be appreciated that Porphyrin Complex VI can be substituted for Porphyrin Complex V to produce the following isomers of Porphyrin Complex VII, Porphyrin VIII and Purpurin II:
______________________________________Compound Structure (referring to attached drawings)______________________________________Porphyrin FIG. 6, where R, R10 and R11 are hydrogenComplex XI R1 and R3-R8 are CH.sub.2 CH.sub.3 R2 is CO.sub.2 H R12 is CH.dbd.CHCO.sub.2 CH.sub.2 CH.sub.3 M is NiPorphyrin FIG. 3, where R, R10 and R11 are hydrogenXII R1 and R3-R8 are CH.sub.2 CH.sub.3 R2 is CO.sub.2 H R12 is CH.dbd.CHCO.sub.2 CH.sub.2 CH.sub.3Purpurin FIG. 11, where R1-R7 are CH.sub.2 CH.sub.3XIII R9 is CO.sub.2 CH.sub.2 CH.sub.3Purpurin FIG. 12, where R1-R4 and R6-R8 are CH.sub.2 CH.sub.3XIV R9 is CO.sub.2 CH.sub.2 CH.sub.3______________________________________
In a like manner, Purpurins XIII and XIV can be substituted for Purpurin II to produce other isomers of Purpurin I:
______________________________________Compound Structure (referring to attached drawings)______________________________________Purpurin FIG. 36, where R1-R7 are CH.sub.2 CH.sub.3XV R9 is CO.sub.2 CH.sub.2 CH.sub.3 R 13 is H R15 is CO.sub.2 CH.sub.2 CH.sub.3Purpurin FIG. 16, where R1-R7 are CH.sub.2 CH.sub.3XVI R9 is CO.sub.2 CH.sub.2 CH.sub.3Purpurin FIG. 7, where R1-R7 are CH.sub.2 CH.sub.3XVII R8 is (O.dbd.C)CH.sub.2 CO.sub.2 CH.sub.3 R9 is CO.sub.2 CH.sub.2 CH.sub.3 R10-R13 are hydrogenPurpurin FIG. 37, where R1-R4 and R6-R8 are CH.sub.2 CH.sub.3XVIII R9 is CO.sub.2 CH.sub.2 CH.sub.3 R 13 is H R15 is CO.sub.2 CH.sub.2 CH.sub.3Purpurin FIG. 17, where R1-R4 and R6-R8 are CH.sub.2 CH.sub.3XIX R9 is CO.sub.2 CH.sub.2 CH.sub.3Purpurin FIG. 7, where R1-R4 and R6-R8 are CH.sub.2 CH.sub.3XX R5 is (O.dbd.C)CH.sub.2 CO.sub.2 CH.sub.3 R9 is CO.sub.2 CH.sub.2 CH.sub.3 R10-13 are hydrogen______________________________________
It will be appreciated that other purpurins having the structures of FIGS. 7, 9-18, 29-33 and 34-38 can be produced by the method of Example 1 from porphyrins having an appropriate structure, if available, or synthesized from dipyrromethanes having an appropriate structure, if available; further, the requisite dipyrromethanes can be synthesized by the method set forth from available pyrroles or from pyrroles synthesized as described. Purpurins so produced have the structure of one of the indicated figures of the drawings where each of R1 through R8 is H,
an alkyl group other than t-butyl having from 1 to 4 carbon atoms,
an alkylene group having from 2 to 4 carbon atoms,
a group having the formula R.sub.2 N(R.sub.3).sub.2 where R.sub.2 is a bivalent aliphatic hydrocarbon radical having from 1 to 4 carbon atoms, wherein any carbon to carbon bond is either a single or a double bond, and not more than one is a double bond; R.sub.3 is hydrogen or an alkyl group having from 1 to 2 carbon atoms and the two R.sub.3 groups can be the same or different,
a group having the formula R.sub.2 N(R.sub.4).sub.3.sup.+ A.sup.- where R.sub.2 is a bivalent aliphatic hydrocarbon radical having from 1 to 4 carbon atoms, wherein any carbon to carbon bond is either a single or a double bond, and not more than one is a double bond; and R.sub.4 is an alkyl group having from 1 to 2 carbon atoms and the three R.sub.4 groups can be the same or different,
a group having the formula R.sub.2 OH were R.sub.2 is a bivalent aliphatic hydrocarbon radical having from 1 to 4 carbon atoms, wherein any carbon to carbon bond is either a single or a double bond, and not more than one is a double bond, or
CO.sub.2 R', CH.sub.2 CO.sub.2 R' or CH.sub.2 CH.sub.2 CO.sub.2 R' where R' is H, or an alkyl group other than t-butyl having from one to four carbon atoms.
In the purpurins so produced, R9 and R15 are usually CO.sub.2 CH.sub.3 or CO.sub.2 CH.sub.2 CH.sub.3, their identity being determined by that of the precursor porphyrin (see steps of producing Porphyrin Complex VII, Porphyrin VIII, and Purpurins II and III in Example 1; the identity of the CO.sub.2 CH.sub.2 CH.sub.3 groups in Purpurin II and in Purpurin III was determined by that of the -ethoxycarbonylvinyl moiety in Porphyrin VIII), and R10 through R14 are hydrogen.
Where any of R1 through R16 of any of the foregoing purpurins is CO.sub.2 H, that moiety can be reacted with an amino acid moiety, which can be a monoclonal antibody, to form an amide. Example 2 is illustrative of such reactions:
EXAMPLE 2
A purpurin coupled to a monoclonal antibody is produced from 20 mg Purpurin II dissolved in 1.25 ml water and 0.8 ml N,N-dimethyl formamide, 20 mg 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. HCl dissolved in 0.6 ml water and 15 mg monoclonal antibody dissolved in 5 ml distilled water. The Purpurin II solution is added to the carbodiimide hydrochloride solution, and the combined solution is mixed with the monoclonal antibody solution. After 30 minutes, the reaction is quenched by adding 0.05 ml monoethanol amine, and the conjugated material, i.e., the amide of the monoclonal antibody and Purpurin II, is dialyzed exhaustively at 4.degree. against 0.001N phosphate buffered saline, pH 7.4.
The procedure of Example 2 can be used to couple other proteins, other amino acids, to Purpurin II and to other purpurins having a CO.sub.2 H group. Examples of preferred amino acids include lysine, histidine, arginine, cystine, serine, aspartic acid, aspartic acid esters, glutamic acid and glutamic acid esters. Five of these amino acids have the formula ##STR1## where R has the meaning indicated below: ##STR2## The formula for aspartic acid is given below: ##STR3## The preferred aspartic acid and glutamic acid esters are esters of lower alkyl alcohols, most desirably those other than t-butyl having from 1 to 4 carbon atoms.
It is known that some monoclonal antibodies, after they have been administered to a patient, for example intravenously, localize in tumor cells, specifically in malignant tumor cells. It is also known that some purpurins and some chlorins, after administration, localize in a similar manner and can then be detected with ultra violet light, which causes them to fluoresce, or illuminated with visible light of a wavelength at which they show an absorbance peak, which causes them to undergo a reaction which destroys the tumor in which they have localized; as is subsequently demonstrated in Examples 3-9 hereof, this is also true of several purpurins having the formula of FIG. 7 of the attached drawings and of several chlorins having the formula of FIG. 8. It will be appreciated, therefore, that a purpurin or chlorin with a monoclonal antibody which localizes in this way chemically attached thereto has an enhanced capability to localize and the same capability for detection and destruction of a tumor where it has localized, by comparison with the parent purpurin or chlorin.
EXAMPLE 3
The production of a purpurin (hereafter "Purpurin NT2") from nickel meso-formyl octaethyl porphyrin is described in this Example. The production of nickel meso-formyl octaethyl porphyrin is described in a journal article by R. Grigg et al., J. Chem. Soc. Perkin Trans I, 1972, pp. 1789, 1798; it has the formula of FIG. 6 of the attached drawings where R1 through R8 are ethyl, R is CHO, R10 through R12 are hydrogen, and M is Ni. Two intermediates were produced in the Example 3 procedure, nickel meso-(-ethoxycarbonylvinyl)octaethyl porphyrin, which has the formula of FIG. 6 of the attached drawings where R1 through R8 are ethyl, R is CH.dbd.CHCO.sub.2 CH.sub.2 CH.sub.3, R10 through R12 are hydrogen, and M is Ni, and meso(-ethoxycarbonylvinyl)octaethyl porphyrin, which has the formula of FIG. 3 of the attached drawings where R1 through R8 are ethyl, R is CH.dbd.CHCO.sub.2 CH.sub.2 CH.sub.3, and R10 through R12 are hydrogen. Purpurin NT2 has the formula of FIG. 7 of the attached drawings where R1 through R8 are ethyl, R9 is CO.sub.2 CH.sub.2 CH.sub.3, and R10 through R13 are hydrogen.
Production of nickel meso-(-ethoxycarbonylvinyl)octaethyl porphyrin
A solution of 506 mg nickel meso-formyl octaethyl porphyrin and 1.024 g (carbethoxymethylene)triphenylphosphorane in 50 ml xylene was heated under reflux for 18 hours. The solution was cooled; the xylene was removed in vacuo; and the solid which remained was dissolved in the minimum amount of dichloromethane and chromatographed on silica. A minor fraction of nickel octaethyl porphyrin and a major red fraction were recovered. The solvent was removed from the red fraction; the solid which remained was recrystallized from a solvent composed of equal parts by volume of dichloromethane and methanol, yielding 455 mg small brown needles. The product was identified by nuclear magnetic resonance as nickel meso-(-ethoxycarbonylvinyl)octaethyl porphyrin; it showed visible spectrum absorbance peaks at 405, 530 and 565 nanometers (94 180, 18 604, 27 790).
Production of Meso-(-ethoxycarbonylvinyl)octaetheyl porphyrin
A solution was prepared by dissolving 621 mg nickel meso-(-ethoxycarbonylvinyl)octaethyl porphyrin in 10 ml concentrated (96.7 percent w/w) sulfuric acid; after the solution stood for 2 hours at room temperature of about 22.degree., an addition of 100 ml dichloromcthane was made thereto, followed by saturated aqueous sodium bicarbonate to neutralize the sulfuric acid. The organic layer was collected, washed and dried; the solvent was then vaporized. The crude product which remained was recrystallized from a solvent composed of equal parts by volume of dichloromethane and methanol, yielding 552 mg small reddish-brown crystals which were identified by nuclear magnetic resonance as meso-(-ethoxycarbonylvinyl)-octaethyl porphyrin. The production of this porphyrin is disclosed in a Journal article by Fuhrhop et al., Ann. Chern., 1976, pp. 1539-1559.
Production of Purpurin NT2
A solution of 100 mg meso-(-ethoxycarbonylvinyl)octaethyl porphyrin in 20 ml glacial acetic acid was heated under reflux in a nitrogen atmosphere for 24 hours. The solution was then cooled; the acetic acid was removed in vacuo; and the remaining product was dissolved in the minimum amount of dichloromethane and chromatographed on silica, yielding a major green fraction from which the solvent was removed. The solid which remained was recrystallized from 50 percent v/v dichloromethane and methanol yielding 68 mg purple microcrystals which were identified by nuclear magnetic resonance as Purpurin NT2, and found to have visible spectrum absorbance peaks at 433, 453, 503, 530, 568, 648 and 695 nanometers (89 509, 89 509, 14 571, 12 143, 18 908, 10 582, 42 673).
EXAMPLE 4
Production of Zn Purpurin NT2
A solution was prepared by dissolving 20 mg Purpurin NT2 in a mixed solvent composed of 15 ml dichloromethane and 5 ml methanol and 100 mg zinc acetate was added to the solution; the mixture which resulted was refluxed for about 4 minutes until the electronic spectrum of the reaction mixture indicated that chelation was complete. The reaction mixture was then concentrated to 7 ml and allowed to cool to room temperature of about 22.degree.. Product which precipitated was recovered by filtration, dissolved in a mixed solvent composed of 5 ml dichloromethane and 2 ml methanol, and recrystallized, yielding 18 mg Zn Purpurin NT2 in the form of microcrystals. The Zn Purpurin NT2, a metal complex, has the formula of FIG. 1 of the attached drawings where R1 through R8 are ethyl, R9 is CO.sub.2 CH.sub.2 CH.sub.3, R10 through R13 are hydrogen and M is Zn; the compound has visible spectrum absorbance peaks at 413, 435, 535, 578, 618 and 663 nanometers (195 270, 219 498, 14 052, 18 886, 28 588, 86 733).
EXAMPLE 5
Production of "Chlorin NT2H2"
A solution was prepared by dissolving 100 mg Purpurin NT2 in 20 mg tetrahydrofuran and adding 2 drops of triethylamine; with stirring, an addition of 20 mg palladium on charcoal was made and the mixture which resulted was hydrogenated at room temperature of about 22.degree. for 5 hours in a sloping manifold hydrogenator in which a slight positive pressure of hydrogen was maintained. The palladium on charcoal that was used was composed of 10 percent w/w of palladium and 90 percent w/w of charcoal. The palladium on charcoal was filtered from the colorless reaction mixture, and the filtrate was stirred vigorously while exposed to air until the solution turned brown, about 21/2 hours. The solvent was then removed in vacuo, and the residue was dissolved in the minimum dichloromethane containing 1 percent v/v of methanol and chromatographed on silica. A major blue band was collected; the solvent was removed; and the crude product was dissolved in 5 ml dichloromethane containing 1 percent v/v of methanol and recrystallized, yielding 72 mg brown microprisms which were identified by nuclear magnetic resonance as Chlorin NT2H2, a compound having the formula of FIG. 8 of the drawings where R1 through R8 are ethyl, R9 is CO.sub.2 CH.sub.2 CH.sub.3, and R10 through R13 are hydrogen. Chlorin NT2H2 was found to have absorbance peaks in the visible spectrum at 403, 500, 535, 558, 610 and 660 nanometers (114 650, 23 532, 5 662, 4 246, 8 493, 39 455). The Chlorin NT2H2 zinc complex was prepared by the method described in Example 4; it was found to have absorbance peaks in the visible spectrum at 408, 515, 545, 590 and 633 nanometers (145 474, 9 858, 5 377, 15 832, 59 444).
The nickel complex of Chlorin NT2H2 was also prepared by the method described in Example 4, except that nickel acetate was substituted for the zinc acetate. The nickel complex of Chlorin NT2H2 was found to have absorbance peaks in the visible spectrum at 405, 498, 533, 588 and 630 nanometers (145 779, 11 034, 8 693, 19 392, 64 146).
The zinc and nickel complexes have the formula of FIG. 2 of the drawings where R1 through R8 are ethyl, R9 is CO.sub.2 CH.sub.2 CH.sub.3 and R10 through R13 are hydrogen. M is Zn for the zinc complex and Ni for the nickel complex.
EXAMPLE 6
Production of Purpurin NT2 and Purpurin NT1
A solution of 100 mg meso-(-ethoxycarbonylvinyl)octaethyl porphyrin in 20 ml glacial acetic acid was heated under reflux in air for 24 hours. The solution was allowed to stand at room temperature of about 22.degree. until it cooled; the solvent was removed in vacuo; and the residue was dissolved in the minimum dichloromethane containing 1 percent v/v of methanol and chromatographed on silica. First and second major green bands were collected; the solvent was removed from the first band; and the crude product was dissolved in 4 ml dichloromethane containing 1 percent v/v of methanol and recrystallized, yielding 40 mg "Purpurin NT1 ", a compound having the formula of FIG. 7 of the drawings where R1 is .dbd.CHCH.sub.3, R2 through R8 are ethyl, R9 is CO.sub.2 CH.sub.2 CH.sub.3 and R10 through R13 are hydrogen. Purpurin NT1 was identified by nuclear magnetic resonance; it has absorbance peaks in the visible spectrum at wavelengths of 438, 510, 540, 583, 653, and 715 nanometers (104 158, 9 450, 11 130, 15 540, 9 020, 42 629).
The solvent was also removed from the second green band, and the crude product was dissolved in 4 ml dichloromethane containing 1 percent v/v of methanol and recrystallized, yielding 39 mg Purpurin NT2, which was identified by nuclear magnetic resonance.
Purpurin NT1 was hydrogenated by a procedure similar to that described above in Example 5, yielding, after work-up and chromatographic purification as there described, 65 mg Chlorin NT2H2.
The procedure described in Example 3 has been used to produce other purpurins. Typical ones of the starting materials used and the intermediates and purpurins produced are set forth tabularly in Examples 7, 8 and 9.
EXAMPLE 7
______________________________________Compound Name Formula of______________________________________Starting Nickel meso-formyletio porphyrin I FIG. 6*MaterialFirst Nickel meso-(-ethoxycarbonylvinyl)- FIG. 6*intermediate etio porphyrin ISecond Meso-(-ethoxycarbonylvinyl)-etio FIG. 3*intermediate porphyrin I"Purpurin FIG. 7*ET2"______________________________________ *Where: R1, R3, R5, and R7 are CH.sub.3, R2, R4, R6, and R8 are CH.sub.2 CH.sub.3. In the starting material, R is CHO and M is Ni. In the first intermediate, R is CH.dbd.CHCO.sub.2 CH.sub.2 CH.sub.3 and M is Ni. In the second intermediate, R is CH.dbd.CHCO.sub.2 CH.sub.2 CH.sub.3. In Purpurin ET2, R9 is CO.sub.2 CH.sub.2 CH.sub.3 and R10 through R13 are hydrogen.
The production of nickel meso-formyletio porphyrin I is disclosed in a Journal article by Johnson et al., J. Chem. Soc. (c) 1966, p.794.
EXAMPLE 8
______________________________________Compound Name Formula of______________________________________Starting Nickel meso-formyl coproporphyrin I FIG. 6*Material tetramethyl ester*First Nickel meso-(-ethoxycarbonylvinyl)- FIG. 6*intermediate coproporphyrin I tetramethyl esterSecond Meso-(-ethoxycarbonylvinyl)- FIG. 3*intermediate coproporphyrin I tetramethyl ester"Purpurin FIG. 7*JP1"______________________________________ *Produced as subsequently described herein. *Where: R1, R3, R5, and R7 are CH.sub.3 and R2, R4, R6, and R8 are CH.sub.2 CH.sub.2 CO.sub.2 CH.sub.3. In the starting material, R is CHO and M is Ni. In the first intermediate, R is CH.dbd.CHCO.sub.2 CH.sub.2 CH.sub.3 and M is Ni. In the second intermediate, R is CH.dbd.CHCO.sub.2 CH.sub.2 CH.sub.3. In Purpurin JP1, R9 is CO.sub.2 CH.sub.2 CH.sub.3 and R10 through R13 are hydrogen.
The nickel meso-formyl coproporphyrin I tetramethyl ester starting material used in the procedure of Example 8 was produced from a commercially available material, coproporphyrin I tetramethyl ester (formula of FIG. 3 of the attached drawings); nickel coproporphyrin I tetramethyl ester (formula of FIG. 6 where M is Zn) was produced therefrom. In both cases, R1, R3, R5 and R7 are CH.sub.3 and R2, R4, R6 and R8 are CH.sub.2 CH.sub.2 CO.sub.2 CH.sub.3.
The Ni Coproporphyrin I Tetramethyl ester was prepared from a solution of 100 mg coproporphyrin I tetramethyl ester in a mixed solvent composed of 50 ml dichloromethane and 5 ml methanol and 100 mg nickel acetate. A mixture which was prepared by adding the nickel acetate to the solution was refluxed for about 12 hours until the electronic spectrum of the reaction mixture indicated that chelation was complete. The reaction mixture was then concentrated to 7 ml and allowed to cool to room temperature of about 22.degree.. Product which precipitated was recovered by filtration, dissolved in a mixed solvent composed of 5 ml dichloromethane and 2 ml methanol, and recrystallized, yielding 98 mg Ni coproporphyrin I tetramethyl ester. The compound showed absorbance peaks in the visible spectrum at 392, 515 and 552 nanometers; the relative intensities at these peaks were 20.19, I and 2.56, respectively.
The Nickel-meso-formyl coproporphyrin I tetramethyl ester was prepared from: 2.8 ml freshly distilled phosphorus oxychloride, 2 ml dry dimethyl formamide, a solution of 100 mg nickel-coproporphyrin I tetra-methyl ester in 75 ml dry 1,2-dichloroethane and 75 ml saturated aqueous sodium acetate. The dimethyl formamide was cooled on an ice bath, and the phosphorus oxychloride was added thereto dropwise. The solution which resulted was allowed to stand at room temperature for 30 minutes, and was then warmed to 50.degree.. The nickel-coproporphyrin I tetramethyl ester solution was then added dropwise, with stirring, over 30 minutes to the phosphorus oxychloride solution. The reaction mixture was maintained at about 50.degree. , with stirring, for an additional 2 hours, during which time a change in color from red to green was observed. The sodium acetate solution was then added to the reaction mixture, and stirring was continued for an additional 2 hours. The organic and the aqueous phases were then separated, and the aqueous phase was extracted with dichloromethane. The organic phase and the dichloromethane extract were then combined, and evaporated to dryness. The solid which remained was recrystallized from a solvent composed of equal parts by volume of dichloromethane and methanol, yielding 86 mg red microcrystals which were identified by nuclear magnetic resonance as nickel-mesoformylcoproporphyrin I tetramethyl ester. Absorbance peaks were found in the visible spectrum at 400, 420, 558 and 645 nanometers, with relative intensities of 10.10, 8.69, 1.02 and 1, respectively.
EXAMPLE 9
______________________________________ FormulaCompound Name of______________________________________Starting Nickel meso-formyloctaethylporphyrin* FIG. 6*MaterialFirst Nickel meso-(-methoxycarbonylvinyl)- FIG. 6*intermediate octaethylporphyrinSecond Meso-(-methoxycarbonylvinyl)- FIG. 3*intermediate octaethylporphyrin"Purpurin FIG. 7*GG2"______________________________________ *Where: R1 through R8 are CH.sub.2 CH.sub.3. In the starting material, R is CHO and M is Ni. In the first intermediate, R is CH.dbd.CHCO.sub.2 CH.sub.3 and M is Ni. In the second intermediate, R is CH.dbd.CHCO.sub.2 CH.sub.3. In Purpurin GG2, R9 is CO.sub.2 CH.sub.3 and R10 through R13 are hydrogen
The procedure of Example 5 has been used to hydrogenate Purpurin ET2 and Purpurin JP1, producing Chlorin ET2H2 and Chlorin JP1H2, respectively, where the isocyclic ring (to which the R9 substituent is attached) is saturated. The chlorins had the same substituents as the starting purpurins, but the structure of FIG. 8 instead of that of FIG. 7.
The procedure of Example 6 has been used to produce other zinc and nickel complexes. The purpurin and chlorin starting materials, the zinc or nickel compound used, and the complexes produced are set forth below:
______________________________________Starting Purpurin Zincor Chlorin or nickel Compound Complex Produced______________________________________Purpurin ET2 Zinc acetate ZnPurpurin ET2 Nickel acetate NiPurpurin GG2 Zinc acetate ZnPurpurin GG2 Nickel acetate NiChlorin ET2H2 Zinc acetate ZnChlorin ET2H2 Nickel acetate Ni______________________________________
Additional peak absorbance data (visible spectrum, wavelengths in nanometers) are given below.
______________________________________Compound Wavelengths (relative intensities)______________________________________Purpurin ET2 406(16.69), 424(15.26), 502(1.36), 531(1)Chlorin ET2H2 400(70.16), 498(5.53), 530(1.29), 555(1)Zn Chlorin ET2H2 401(20.36), 530(1), 568(1.18), 630(3.20)Purpurin ET2 434(16.44), 530(1), 576(1.31)612(1.77)Ni Purpurin ET2 434(5.14), 657(1)Ni Chlorin ET2H2 404(11.70), 497(1), 622(4.41)Purpurin JP1 409(22.41), 504(1.67), 541(1.21) 567(1.08), 647(1), 691(3.79)Chlorin JP1H2 401(14.53), 650(1)Purpurin GG2 406(12.94), 427(19.18), 500(1), 526(1), 565(1.89), 637(5.25)Zn Purpurin GG2 436(8.33), 616(1), 661(3.43)Ni Purpurin GG@ 427(4.20), 648(1)______________________________________
In vitro and in vivo testing of purpurins and chlorins produced as described in Examples 3 through 9 was also carried out. For the in vitro testing, the compounds were dissolved in dimethyl sulfoxide or in a solvent that is commercially available under the trade designation PROTOSOLV, and diluted with phosphate buffer saline to a concentration of 0.010 mg per ml. The tests were conducted on FANFT (N-[4-(5-nitro-2furyl)2-thiazolyl] formamide) induced rat bladder tumor cells. Two tests were conducted, uptake and toxicity.
The uptake test involved incubating the FANFT induced rat bladder tumor cells with a solution of a purpurin or with a solution of a chlorin at a concentration of 0.010 mg per ml for one hour, temperature 37.degree., followed by removing of the incubation media, three washings of the cells with phosphate buffered saline, and extracting and quantitating of the purpurin or chlorin retained by the cells. The procedure as used in investigating the use of HpD in rat tumor cells is described in detail in a journal article by Garbo et al., Analytical Biochemistry, Vol. 151 (No. 1), pp. 70-81, 1985.
The toxicity test involved the incubation and washing steps of the uptake test, followed by illumination of the cells with red light of a wavelength greater than 590 nanometers. Cell survival was then determined by Trypan Blue exclusion, a technique described in a journal article by Schneck, R., Arch. Path. (Lab. Med.) 35, p. 857, 1943.
The uptake test was positive for Purpurin NT2 and for Chlorin NT2H2. The results of the toxicity test are given in the following table, together with the results of toxicity testing of HpD, of phosphate buffer saline and of the solvent system in which the purpurin or chlorin was dissolved.
______________________________________Test Solution Average Viability______________________________________Purpurin NT2 46Chlorin NT2H2 51HpD 42Phosphate Buffer saline 93Mixed solvent 96______________________________________
The in vivo testing was conducted on male Fisher 344 rats weighing 135 to 150 g in whom the transplantable FANFT (N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide tumor system had been implanted. (Use of this system is reported by Selman, S. H., et al., Cancer Research, pp. 1924-1927, May, 1984.) Two tumors were implanted into the subcutaneous tissue of the abdominal wall of each test animal; when the testing was carried out, each tumor was about 1 cm in diameter.
The purpurins and chlorins tested were dissolved in a commercially available non-ionic solubilizer and emulsifier obtained by reacting ethylene oxide with castor oil in a ratio of 35 moles of ethylene oxide per mole of castor oil, diluting the resulting solution with 1,2-propanediol, and producing an emulsion with the resulting solution and 0.9 percent w/w aqueous sodium chloride solution. The specific non-ionic solubilizer used is available from BASF under the designation CREMOPHOR EL; it is composed of fatty acid esters of polyglycols, glycerol polyglycols, polyethylene glycols and ethoxylated glycerol. The test solutions were prepared from 50 mg purpurin or chlorin, 1 or 2 ml warm solubilizer (enough to dissolve the test compound), enough 1,2-propanediol to make a solution of the purpurin or chlorin in a mixed diol/solubilizer solvent containing 32.9 percent w/w solubilizer; finally, enough 0.9 percent w/w aqueous sodium chloride was added to make 10 ml test solution so that the final concentration of the purpurin or chlorin in the test solution was 5 mg per ml. Each test solution was made, with mechanical shaking and stirring, by dissolving the purpurin or chlorin in the solubilizer, diluting the resulting solution with the indicated amount of 1,2-propanediol, and adding the sodium chloride solution to the diluted solution. A control solution was also prepared for use with each test solution. The control was identical with the test solution except that it contained no purpurin or chlorin. The test solutions were prepared in air, but it is believed that a nitrogen atmosphere would be advantageous because it would minimize the chance of a reaction with oxygen.
The testing involved injecting each rat with a solution of the purpurin or chlorin under test, dosage 4 mg purpurin or chlorin per kg of body weight or 10 mg purpurin or chlorin per kg of body weight or with the same volume of the appropriate control, irradiating one of the two tumors with light for 30 minutes, sacrificing the animals, and examining the tumors. The injections were made via the dorsal tail vein. The irradiation of one of the tumors occurred either four or twenty four hours after each rat was injected while the other of the two tumors was shielded by an opaque box.
Tumor temperature and body core temperature were monitored, using thermistors, one placed into the tumor and one placed intrarectally. Tumor temperature was kept within 2.degree. of body core temperature by directing a jet of cool air over the tumor.
The light source was a slide projector that had a 500 watt bulb fitted with a red filter which is available from Corning Glass Works under the designation 2418. The light was reflected 90 by a silvered mirror, and was focused onto the tumor with a secondary condensing lens. The light intensity on the tumor was monitored, using a photometer/radiometer that is available from United Detector Technology under the designation "UDT #351", and was maintained at 200 mw per cm.sup.2.
Six rats were injected with the purpurin or chlorin test solution and two were injected with the appropriate control solution.
Four hours after the irradiation, three of the rats that had been injected with the test solution and one of the rats that had been injected with the control were sacrificed by an intracardiac injection of saturated aqueous potassium chloride solution. Twenty four hours after the irradiation, another three of the rats that had been injected with the test solution and the other rat that had been injected with the control were sacrificed in the same way. During the testing, the rats were under barbiturate anesthesia (65 mg per kg body weight).
The tumors were then excised, placed in 10 percent w/w phosphate-buffered formalin and cut into three sections perpendicular to their long axis. The tumors were then embedded in paraffin and cut into sections five microns in width. The sections were stained with hematoxylin and eosin.
Histologic examination of the stained sections revealed approximately comparable areas of hemorrhage and tumor cell necrosis in specimens removed four hours after irradiation from animals that had been injected with Purpurin NT2, with Purpurin GG2, and with Purpurin ET2. However, tumor cells which appeared to be viable were observed. Only minor hemorrhage and tumor cell necrosis were observed in specimens removed four hours after irradiation from animals that had been injected with Purpurin JP1 but much greater hemorrhage and necrosis were observed in specimens that had been injected with Purpurin ZnET2 and even more in specimens that had been injected with Chlorin SnET2H2. Tumor necrosis was extensive in specimens removed twenty four hours after irradiation from animals that had been injected with Purpurin NT2, with Purpurin GG2, with Purpurin ET2, with Purpurin JP1, with Purpurin ZnET2 and with Chlorin SnET2H2; no viable tumor cell was observed in specimens from animals that had been injected with Purpurin ZnET2 and Chlorin SnET2H2, while a few were observed in specimens from animals that had been injected with Purpurin NT2 and with Purpurin ET2, and more were observed in specimens from animals that had been injected with Purpurin GG2 and with Purpurin JP2. No change in the tumors was observed in the specimens that were removed from animals that had been injected with the control solution. Tumor necrosis was complete in specimens removed from animals that had been injected with purpurin NT1 both four hours after irradiation and twenty four hours after irradiation. However, the irradiation was found to have caused extensive liver damage to some of the animals. The liver damage is believed to have occurred because there was residual Purpurin NT1 in the liver which was unintentionally irradiated. The in vivo testing, however, indicated that Purpurin NT1 is highly effective when properly used.
The in vivo test procedure described above has also been used to evaluate solutions in which the Purpurin NT2, Purpurin GG2, Purpurin NT1, Purpurin JP1, Purpurin ET2, Purpurin ZnET2 and Chlorin SnET2H2 were replaced by Chlorin NT2H2 and by Chlorin ET2H2. Histologic examination of the stained sections from rats into which the Chlorin NT2H2 and ET2H2 solutions had been injected indicated that these chlorins were substantially equivalent in this test and were similar to Purpurin NT2, to Purpurin GG2 and to Purpurin ET2, the only difference observed being that hemorrhage within the tumors was less pronounced with the chlorins.
It will be appreciated from the results reported above in Example 6 that the cyclization step of Example 1 by which Purpurin II and Purpurin III are produced could be carried out in air, rather than in nitrogen, and that the reaction product would then be a mixture of Purpurin II, Purpurin III, and the following purpurins:
______________________________________Com-pound Structure of Substituents______________________________________Purpurin FIG. 9 R1 is .dbd.CHCH.sub.3, R2-R5, R7 and R8 areXXI CH.sub.2 CH.sub.3, and R9 is CO.sub.2 CH.sub.2 CH.sub.3Purpurin FIG. 13 R1 is .dbd.CHCH.sub.3, R2-R6 and R8 areXXII CH.sub.2 CH.sub.3, and R9 is CO.sub.2 CH.sub.2 CH.sub.3______________________________________
In fact, cyclization in air can be used to produce purpurins, and metal complexes can be produced from those purpurins by the method of Example 4, with or without the modifications thereof subsequently described herein, where the purpurins and complexes have the structures of FIGS. 10-12, of FIGS. 14-18, of FIGS. 29-33, of FIGS. 34-38, of FIGS. 44-48 and of FIGS. 54-58. In all cases, a mixture of products will be produced, some in which R1 is saturated and some in which R1 is a bivalent aliphatic hydrocarbon radical having from 2 to 4 carbon atoms wherein both of the valences of the radical are attached to the same carbon atom thereof and to a carbon atom of the purpurin or metal complex. Furthermore, after cleavage of the exocyclic ring of FIGS. 9-18, oxidation in air can be used, in the purpurins and complexes of FIGS. 29-33 and of FIGS. 44-48 to convert R2 to a bivalent aliphatic hydrocarbon radical having from 2 to 4 carbon atoms wherein both of the valences of the radical are attached to the same carbon atom thereof and to a carbon atom of the purpurin or metal complex. In general, purpurins can be converted to the corresponding chlorins by the hydrogenation method described in Example 5; chlorins can be converted to the corresponding purpurins by oxidation; and chlorin metal complexes can be produced from chlorins by the method of Example 4, with or without the modifications thereof subsequently described herein. However, hydrogenation of Purpurin NT1 (where there was a double bond between the R1 substituent and a carbon of the purpurin), as described in Example 6, produced Chlorin NT2H2 (where both the double bond of the exocyclic ring and that of R1 were saturated). It will be appreciated that the R1 double bond forms in the Example 6 procedure because a hydroxyl group is introduced into the molecule and, at the temperature of reflux, the elements of water are eliminated to form the double bond. Elimination of the elements of water can be prevented in the Example 6 procedure, or, more generally, whenever R1 is to be bivalent, by cyclizing at a lower temperature; the resulting purpurin can then be hydrogenated to the corresponding chlorin and the double bond with R1 can be formed by heating.
The method of Example 4, supra, can be used to produce metal complexes of other purpurins and of various chlorins. Specifically, an equivalent amount of another purpurin or of a chlorin can be substituted for the Purpurin NT2, or copper acetate, nickel acetate, cobalt acetate, silver acetate, palladium acetate, or platinum acetate can be substituted for the zinc acetate, or both substitutions can be made. In this manner, purpurin metal complexes having the formulas of FIGS. 54-58 where M is one of the metals named above in this paragraph can be produced from purpurins having the formulas of FIGS. 34-38; chlorin metal complexes having the formulas of FIGS. 49-53 where M has the same meaning can be produced from chlorins having the formulas of FIGS. 39-43; purpurin metal complexes having the formulas of FIGS. 44-48 where M has the indicated meaning can be produced from purpurins having the formulas of FIGS. 29-33; metal complexes of purpurins having the formulas of FIGS. 9-18 can be produced; metal complexes having the structure of FIG. 1 can be produced from purpurins having the structure of FIG. 7; and metal complexes having the structure of FIG. 2 can be produced from chlorins having the structure of FIG. 8. Other complexes can be produced by the method of Example 4 from salts containing cations other than acetate, and producing complexes which have the structures of the FIGS. to which reference is made above in this paragraph, but where M does not represent merely a metal anion. Examples of salts that can be substituted for zinc acetate in the Example 4 procedure are given below, together with the identity of M in the foregoing FIGS.:
______________________________________Salt Identity of M______________________________________FeCl.sub.3 Fe(Cl)MnCl.sub.4 Mn(Cl)InCl.sub.3 IN(Cl)VCl.sub.4 * V(O)Tl(CF.sub.3 CO.sub.2).sub.3 Tl(OAc)(H.sub.2 O)SnCl.sub.2 Sn(OH).sub.2[Rh(CO)Cl].sub.2 Rh(Cl)(H.sub.2 O)______________________________________ *Using phenol as the solvent instead of glacial acetic acid.
The procedure of Example 4 can also be modified by substituting phenol for glacial acetic acid and metal chelates of pentane, 2,4-dione for zinc acetate to produce complexes of any of the foregoing purpurins and chlorins. Metals that can be So reacted (as pentane, 2,4-dione chelates) and the identity of M in the complex that is produced are set forth in the following table:
______________________________________Metal Identity of M Metal Identity of M______________________________________Al Al(acac)* Th Th(acac).sub.2Sc Sc(acac) U U(acac).sub.2Ga Ga(acac) La La(acac).sub.2In In(acac) Ce Ce(acac)Mo Mo(acac) Nd Nd(acac)Ti Ti(acac).sub.2 Sm Sm(acac)Zr Zr(acac).sub.2 Gd Gd(acac)Hf Hf(acac).sub.2 Tb Tb(acac)Eu Eu(acac) Dy Dt(acac)Pr Pr(acac) Ho Ho(acac)Yb Yb(acac) Er Er(acac)Y Y(acac) Tm Tm(acac)Lu Lu(acac)______________________________________ *The pentane, 2,4dione portion of a chelate thereof with a metal.
Complexes of any of the foregoing purpurins and chlorins can also be produced by the procedure of Example 4, substituting dimethylformamide for glacial acetic acid and CrCl.sub.2 for zinc acetate. Metal complex formation occurs at higher temperatures when dimethylformamide is used, because of its higher boiling temperature. M in the complexes is Cr(OH).
Similarly, complexes of the foregoing purpurins and chlorins can be produced by the procedure of Example 4, substituting pyridine for glacial acetic acid and PbCl.sub.2 for zinc acetate. M in the complexes is Pb.
The method of Example 4 and the modifications thereof described above can be used to produce purpurin complexes having the structures of FIGS. 44-48 from purpurins having the structures of FIGS. 29-33; to produce chlorin complexes having the structures of FIGS. 49-53 from chlorins having the structures of FIGS. 39-43; to produce purpurin complexes having the structures of FIGS. 54-58 from purpurins having the structures of FIGS. 34-38; to produce purpurin complexes of FIG. 1 from purpurins having the structure of FIG. 7; and to produce chlorin complexes of FIG. 2 from chlorins having the structure of FIG. 8.
The following example illustrates the production of Chlorin I, a compound having the structure of FIG. 19 where R1-R5, R7 and R8 are CH.sub.2 CH.sub.3, Chlorin II, a compound having the structure of FIG. 24 where R1-R5, R7 and R8 are CH.sub.2 CH.sub.3 and R14 is CH.sub.3, and Purpurin XXIII, a compound having the structure of FIG. 29 where R1-R5, R7 and R8 are CH.sub.2 CH.sub.3, R10 and R12 are H, R14 is CH.sub.3 and R15 is CO.sub.2 CH.sub.2 CH.sub.3.
EXAMPLE 10
Production of Chlorin I and Chlorin II
A 5 percent w/w solution of 100 mg Purpurin IV in dichloromethane containing 25 percent v/v methanol is caused to react by vigorous stirring in air while illuminated with visible light. The reaction is continued, with periodic monitoring by visible light spectroscopy, until the spectrum indicates that no Purpurin IV remains in the solvent. The dichloromethane/methanol solvent is then evaporated; a 4 ml portion of a 5 percent w/w solution of sodium methoxide in methanol is mixed with the residue and refluxed for 2 hours; and the solution is cooled. A 5 ml portion of water is then mixed with the reaction product; an organic layer which forms is separated from the aqueous layer and treated in vacuo to remove solvent; and the crude product which remains is dissolved in about 3 ml dichloromethane containing 1 percent v/v methanol and chromatographed on silica gel. The CHO group of Chlorin I is then reduced to CH.sub.3, for example by the method described above as Example 5, producing Chlorin II.
Production of Purpurin XXIII
A solution of 60 mg sodium borohydride in 10 ml methanol is added dropwise to a solution of 200 mg Chlorin II in 5 ml dichloromethane; the resulting solution is stirred at room temperature of about 22.degree. for 2 hours, and is poured into 100 ml water. The organic phase is separated from the aqueous phase; the solvent is removed from the organic phase; and the solvent is evaporated. The residue is dissolved in 50 ml chloroform containing 25 percent v/v methanol; a 10 mg addition of p-toluene sulfonic acid is made; and the reaction mixture is refluxed for 6 hours. Water is then added to the reaction mixture; the organic layer is collected; and the solvent is removed by evaporation. The residue is dissolved in 5 ml dichloromethane containing 2 percent v/v methanol; the resulting solution is chromatographed on silica gel; and Purpurin XXIII is recovered by evaporating the solvent from the chromatographed solution.
The procedure of Example 10 can also be used to produce other chlorins and other purpurins. For example, Purpurin XI can be substituted for Purpurin IV and Chlorin III, Chlorin W and Purpurin XXIV can then be produced by the method of Example 10; these compounds are identified in the following table:
______________________________________Com-pound Structure (referring to attached drawings)______________________________________Chlorin FIG. 20, where R1-R3 and R5-R8 are CH.sub.2 CH.sub.3IIIChlorin FIG. 25, where R1-R3 and R5-R8 are CH.sub.2 CH.sub.3, andIV R14 is CH.sub.3Purpurin FIG. 30, where R1-R3 and R5-R8 are CH.sub.2 CH.sub.3,XXIV R10 and R11 are H R14 is CH.sub.3, and R15 is CO.sub.2 CH.sub.2 CH.sub.3______________________________________
Similarly, Purpurin XVI can be substituted for Purpurin IV and Chlorin V, Chlorin VI and Purpurin XXV can then be produced by the method of Example 10; these compounds are identified in the following table:
______________________________________Compound Structure (referring to attached drawings)______________________________________Chlorin V FIG. 21, where R1-R7 are CH.sub.2 CH.sub.3Chlorin VI FIG. 26, where R1-R7 are CH.sub.2 CH.sub.3, and R14 is CH.sub.3Purpurin XXV FIG. 31, where R1-R7 are CH.sub.2 CH.sub.3, R11 and R12 are H, R14 is CH.sub.3, and R15 is CO.sub.2 CH.sub.2 CH.sub.3______________________________________
In a like manner, Purpurin XIX can be substituted for Purpurin IV and Chlorin VII, Chlorin VIII and Purpurin XXVI can then be produced by the method of Example 10; these compounds are identified in the following table:
______________________________________Com-pound Structure (referring to attached drawings)______________________________________Chlorin FIG. 22, where R1-R4 and R6-R8 are CH.sub.2 CH.sub.3VIIChlorin FIG. 27, where R1-R4 and R6-R8 are CH.sub.2 CH.sub.3, andVIII R14 is CH.sub.3Purpurin FIG. 32, where R1-R4 and R6-R8 are CH.sub.2 CH.sub.3,XXVI R10 and R11 are H, R14 is CH.sub.3, and R15 is CO.sub.2 CH.sub.2 CH.sub.3______________________________________
Finally, Purpurin VII can be substituted for Purpurin IV and Chlorin IX, Chlorin X and Purpurin XXVII can then be produced by the method of Example 10; these compounds are identified in the following table:
______________________________________Compound Structure (referring to attached drawings)______________________________________Chlorin FIG. 23, where R1-R6 and R8 are CH.sub.2 CH.sub.3IXChlorin X FIG. 28, where R1-R6 and R8 are CH.sub.2 CH.sub.3, and R14 is CH.sub.3Purpurin FIG. 33, where R1-R6 and R8 are CH.sub.2 CH.sub.3,XXVII R10 and R12 are H, R14 is CH.sub.3, and R15 is CO.sub.2 CH.sub.2 CH.sub.3______________________________________
It will be appreciated that the purpurins that can be produced by the methods of the foregoing examples, and the chlorins that can be produced from those purpurins by the hydrogenation method of Example 5, have seven substituents which are present in the porphyrin starting materials from which the purpurins are produced. These are seven of the eight R1-R8 substituents, all but R6 in the purpurins of FIG. 34, all but R4 in the purpurins of FIG. 35, etc. The identities of these substituents depend on the identities of their pyrrole-, dipyrromethane-, and porphyrin precursors. At least as initially produced, each purpurin also has an R9 substituent, an R15 substituent, or both; as is explained above, the identities of these substituents are determined by the identities of their porphyrin precursors. In addition, the purpurins of FIGS. 29-33 have the potential for substitution at R14, which is formyl when the purpurins are first produced, as described above, and can be reduced to methyl, as also described above, or to any other desired substituent by the reactions about to be described to which a formyl group introduced as R10, R11, R12, R13 or R16 can be subjected. Finally, each purpurin has additional sites for potential substitution, a plurality of R10, R11, R12, R13 and R16 (see FIGS. 34-38) substitutions being possible, which ones depending on the position of an exocyclic ring to which R15 and R16 are attached; as the purpurins are produced, there is hydrogen in each of these positions. A formyl group can be introduced by reaction with the Vilsmeier reagent as the lowest of R10, R11 and R12, as R13 and as R16 (in the compounds of FIGS. 34-38), or the unsaturated exocyclic ring or rings can be saturated by hydrogenation (see procedure of Example 5, supra) and the formyl group can then be introduced as the lowest of R10, R11 and R12. The formyl group, after separation of isomers, if necessary, can be reduced to CH.sub.3, or can be reduced to CH.sub.2 OH or converted to an oxime group, which can then be converted to a cyano group, which, in turn, can be converted to an amide. The formyl group can also be reacted with Wittig reagents to give alkyl, alkenyl or carboxy side chains or to introduce the previously identified substituents which have an amine or an alcoholic OH function as the lowest of R10, R11 and R12, as R13 or as R16. After the desired group has been introduced as the lowest of R10, R11 and R12, as R13, as R16, or as some combination, the purpurin can be reacted in the same way to introduce a desired group as R11, if present. Finally, the chemistry can be used to introduce a desired group as R12, if present.
As has been indicated above, the instant invention, in one aspect, is a purpurin, a chlorin or a metal complex which has a structure that has been enriched in an atom that can be detected by nuclear magnetic resonance. Such purpurins are produced by repeating the procedure of Example 1, but producing Pyrrole I from a saturated aqueous solution containing one gram equivalent of sodium nitrite enriched in .sup.15 N, a 5 percent w/w solution in glacial acetic acid containing one gram equivalent of benzyl propionylacetate, a suspension in glacial acetic acid of one gram equivalent of ethyl acetoacetate and four gram equivalents of zinc dust, and producing Pyrrole III from a saturated aqueous solution containing one gram equivalent of sodium nitrite enriched in .sup.15 N, a 5 percent w/w solution in glacial acetic acid containing one gram equivalent of benzyl propionylacetate, a suspension in glacial acetic acid of one gram equivalent of 2,4-pentanedione and four gram equivalents of zinc dust. A 10 percent enrichment of the sodium nitrite in .sup.15 N is adequate to produce Purpurin I, Purpurin II, Purpurin III, Purpurin IV and Purpurin V enriched in .sup.15 N to such an extent that, after they have been administered intravenously as described above, their location in the patient to whom they were administered can be monitored by nuclear magnetic resonance.
Purpurins that can be detected by nuclear magnetic resonance are also produced by repeating Example 1, but producing Porphyrin Complex VII from a solution in 50 ml xylene of 506 mg Porphyrin Complex V and 1.024 g (carbethoxymethylene)triphenylphosphorane in which the carbethoxymethylene moiety is enriched in .sup.13 C. A 10 percent enrichment of the carbethoxymethylene moiety in .sup.13 C is adequate to produce Purpurin I, Purpurin II, Purpurin III, Purpurin IV and Purpurin V enriched in .sup.13 C to such an extent that, after they have been administered intravenously as described above, their location in the patient in whom they were administered can be monitored by nuclear magnetic resonance.
As has also been indicated above, the instant invention, in another aspect, is a purpurin, a chlorin or a metal complex which has a structure that has been enriched in an atom that is radioactive to such an extent that its presence can be detected by an instrument that measures the level of ionizing radiation. Such purpurins are produced by repeating the procedure of Example 1, but producing Porphyrin Complex VII from a solution in 50 ml xylene of 506 mg Porphyrin Complex V and 1.024 g (carbethoxymethylene)triphenyl-phosphorane in which the carbethoxymethylene moiety is enriched in .sup.14 C. A 10 percent enrichment of the carbethoxymethylene moiety in .sup.14 C is adequate to produce Purpurin I, Purpurin II, Purpurin III, Purpurin IV and Purpurin V enriched in .sup.14 C to such an extent that, after they have been administered intravenously as described above, their location in the patient to whom they were administered can be monitored by an instrument which measures the level of ionizing radiation. .sup.14 C has an extremely long half life; it will be appreciated, therefore, that a purpurin, chlorin or complex that has a structure which is enriched in .sup.14 C should not be administered to a human, but that such a compound can be administered to a laboratory animal and that monitoring its location in the body of the laboratory animal can then provide extremely valuable information which has application in the treatment of humans.
Purpurins, chlorins and complexes having structures which are enriched in an atom that emits ionizing radiation and which are suitable for administration to humans can also be produced. For example, any of the purpurins according to the invention where at least one of R10 through R14 is hydrogen can be reacted in sunlight with elemental .sup.131 I or with .sup.131 ICl, and chlorins and complexes can be produced as described above from the iodinated purpurin which is produced. Further, purpurin and chlorin complexes can be produced as described above from a gallium, indium or technetium chelate of pentane, 2,4-dione where the gallium is .sup.67 Ga, the indium is rain or the technetium is .sup.99m Tc, or from Tl(CF.sub.3 CO.sub.2).sub.3 where the thallium is 201Tl. Like .sup.67 Ca and .sup.131 I, rain, .sup.99m Tc and 201Tl all have sufficiently short half-lives to be physiologically acceptable for use as tracers in human patients. Indeed, its short half life (6.049 hours, Lange's Handbook of Chemistry, 11th edition, edited by John A. Dean, McGraw-Hill Book Company, New York et al., page 3-39) makes .sup.99m Tc less desirable than some other nuclides for use to detect dimers according to the instant invention in vivo. Because of their longer half lives, .sup.131 I, .sup.67 Ca, .sup.111 In and .sup.201 Tl are preferred nuclides; their half lives, according to the same reference, are 8.05 days, 77.9 hours, 2.81 days and 74 hours, respectively. The foregoing and other nuclides and the required starting compounds thereof are all known, and can be purchased or produced by known methods (see, for example, Principles of Nuclear Medicine, Edited by Henry N. Wagner, Jr., W. B. Saunders Company, Philadelphia, London, Toronto, 1968, pages 259-298 and cited references).
It will be appreciated that Purpurins I through V produced as described above, and enriched in .sup.15 N, in .sup.13 C, in .sup.14 C, in .sup.131 I or in .sup.99m Tc can be used as also described above to produce other purpurins which are so enriched and that the methods of the examples hereof can be varied as described above to produce purpurins having the structures of FIGS. 7, 9-18, 29-33 and 34-38 which are enriched in .sup.15 N, in .sup.13 C, in .sup.14 C, in .sup.131 I or in .sup.99m Tc and wherein each of R1 through R8 has the meaning set forth above. Similarly, the method described above can be used to produce purpurins that are so enriched where each of R9 through R16 has the meaning set forth above. Likewise, chlorins and purpurin metal complexes can be produced from those purpurins as described above, and metal complexes can be produced from the chlorins as so described.
As is indicated above, there are indications that the purpurins, chlorins and metal complexes are X ray sensitizers which increase the therapeutic ratio of X rays. Accordingly, in one aspect, the instant invention involves administering, for example, as described above, a purpurin having the structure of one of FIGS. 7, 9-18 and 29-38, a corresponding chlorin or a chlorin or purpurin metal complex and, after the purpurin, chlorin or complex has localized, treating the affected region with X rays or other ionizing radiation.
As is also indicated above, the purpurins, chlorins and complexes can be administered topically, for example as dilute, e.g., 1 percent w/w solutions in DMSO or ethanol to non-malignant lesions, e.g., of the vagina or bladder, or to such cutaneous lesions as are involved in psoriasis, followed by illumination of the area involved with light of a wavelength at which the purpurin, chlorin or complex has an absorbance peak. The purpurin or the like solution should be applied only to the lesions to prevent damage to healthy tissue adjacent the lesions. Illumination of the lesions, for example, for from 15 to 30 minutes then completes the treatment. It is to be understood, however, that purpurins, chlorins and complexes according to the invention can also be administered systemically, i.e., intravenously, intramuscularly or subcutaneously, in the treatment of non-malignant lesions.
The reaction of a monoclonal antibody with Purpurin II is described in Example 2. The monoclonal antibody, when it is one which localizes in tumors, can enhance the ability of the purpurin, or of a chlorin or complex produced therefrom, to localize in tumors, as discussed above. However, the monoclonal antibody can also be of a different type, for example one which localizes in a particular kind of lymphocyte, in a leukemia cell, in a lymphoma cell, or the like; a product of the reaction of Purpurin II or the like with such an antibody which localizes in a particular kind of lymphocyte can be used to modulate lymphocyte populations in the treatment of immune diseases, e.g., arthritis, or to re-establish a lymphocyte balance in transplant patients. Some of the blood is removed from the patient's body, and such a purpurin, chlorin or complex according to the invention, i.e., one where at least one of the substituents is a monoclonal antibody directed against the lymphocyte or lymphocytes present in excess, is mixed with the blood in a suitable amount; after the purpurin or the like localizes in the lymphocyte or lymphocytes present in excess, the blood is exposed to light of a wave length at which the purpurin or the like has an absorbance peak, destroying the lymphocyte or lymphocytes where localization had occurred. The blood is then returned to the patient's body. This technique can be carried out repeatedly as required to modulate lymphocyte populations in treating immune diseases and transplant patients who develop the lymphocyte imbalance that is associated with the rejection of a transplanted organ. Since the treatment is entirely outside the patient's body, there is no opportunity for the development of a natural resistance to the treatment which is characteristic of prior attempts to modulate lymphocyte populations. Purpurins or the like according to the invention where one of the substituents is an antibody against leukemia cells or against lymphoma cells can be used in a similar manner in the treatment of leukemia and lymphoma.
It will be appreciated that purpurins and chlorins according to the invention where R10 through R13 and R16 are hydrogen are preferred, other factors being equal, because the production of the compounds with other groups in these positions is complicated, time consuming and expensive. R9 and R15 in purpurins, chlorins and purpurin and chlorin complexes according to the invention are preferably CO.sub.2 R' where R' is an alkyl group other than t-butyl having from 1 to 4 carbon atoms, other factors being equal, because these groups are present at the end of the ring closure reaction which produces the purpurins (see Examples 1 and 3). However, the esters of these R9 and R15 substituents can be reduced to formyl groups and reacted as discussed above to introduce any of the R1 to R8, R10 to R14 and R16 substituents.
The production of purpurin solutions in the specific non-ionic solubilizer that is available under the designation CREMOPHOR EL, and the production of emulsions of such solutions with 1,2-propanediol and saline solution is described above, as is the use of such solutions to detect and treat tumors. It will be appreciated that purpurins, chlorins and their metal complexes can be dissolved in other non-ionic solubilizers and that the solutions can be used to produced emulsions that can be administrated intravenously. For example, other reaction products of ethylene oxide and castor oil can be so used, as can reaction products of ethylene, propylene and other similar oxides with other fatty acids and the reaction products of propylene and other similar oxides with castor oil. Similarly, glycols other than 1,2-propanediol can be used in producing the emulsions for intravenous administration, or the glycol can be omitted, particularly if the solubilizer is prepared to have a lower viscosity and greater compatibility with water, by comparison with the solubilizer that is available under the designation CREMOPHOR EL. It is necessary only that the solution or emulsion be one which is physiologically acceptable and of a suitable concentration, or dilutable to a suitable concentration, for intravenous administration. An indefinitely large number of such solutions and emulsions will be apparent to those skilled in the relevant art from the foregoing specific disclosure. Similarly, the aqueous phase need not be 0.9 percent w/w or any other concentration of sodium chloride. Such saline is presently favored for intravenous administration, but other aqueous phases can also be used, so long as the entire composition is physiologically acceptable for intravenous administration and, in fact, other aqueous phases may subsequently be favored
Dosages of 4 and 10 mg per kg of body weight were used in the in vivo procedures described above. It has not been determined that 4 mg per kg is the minimum dosage or that 10 mg per kg is the maximum. Both dosages caused the biological consequences described above. It will be appreciated, therefore, that it is necessary only to use an effective amount of a purpurin or chlorin according to the invention in the detection and treatment of tumors, preferably as small a dosage as possible, and that the exact dosage can be determined by routine experimentation. Both systemic administration, specifically intravenous, intramuscular and subcutaneous, and local administration, i.e., as a lotion or a solution in dimethyl sulfoxide or ethanol, have been described above; however, it will also be appreciated that other methods of administration will be suitable, at least in some instances. In addition, the dimers can be used in vitro to eradicate infectious pathogenic biological contaminants from blood, semen and other body fluids or from tissue, e.g., skin, removed from a human or animal patient or donor. Pathogenic contaminants that can be eradicated include envelope-containing viruses, bacteria, malarial, trypanosomes and other parasites. A purpurin, chlorin or metal complex according to the invention, e.g., at a dosage of 5 or 10 mg per kg of body weight, as described above, can be administered to a patient or a donor and, after a suitable time, a blood, semen, skin or the like sample can be taken and irradiated with light of a suitable wavelength, either as taken in the case of a fluid, or suspended in a physiologically acceptable saline solution in the case of a tissue. Instead, a blood, semen, skin or the like sample can be taken and a purpurin or the like according to the invention added thereto, either as taken in the case of a fluid, or suspended in a physiologically acceptable saline solution in the case of a tissue and, after a suitable time for the dimer to associate itself with the pathogenic contaminants, the sample can be irradiated with light of a suitable wavelength. When a purpurin or the like according to the invention is added to a fluid sample, whether a body fluid or a suspension of a tissue in a saline solution, the dosage, in either case, should be sufficient for there to be enough of the dimer to associate with all of the pathogenic contaminants in the sample, and usually ranges from about 0.1 to 50 mg per L of sample, preferably from about 2 to 50 mg per L. The wavelength of the irradiating light should be or should include one at which the purpurin or the like has an absorbance peak. The density of radiation used with the sample can range from 0.1 to 50 Joules per cm.sup.2, preferably from 1 to 20 Joules per cm.sup.2, and most desirably about 5 Joules per cm.sup.2.
The use of HPD and a compound which is said to contain about 90 percent of DHE (dihematoporphyrin ether) to treat blood, semen and the like body fluids and skin and other body tissues is disclosed in detail in "Judy et al." (U.S. Pat. No. 4,878,891, issued Nov. 7, 1989). Dimers according to the present invention "bind" in the same way as the two materials disclosed by Judy et al., differing in that they are more effective as photosensitizers. Accordingly, the disclosure of Judy et al. is applicable to the treatment of blood and other body fluids and skin and other body tissues with purpurins and the like according to the instant invention, except that the dosages and light intensity required are somewhat less with the purpurins and the like.
Illumination of tumors containing a purpurin, a chlorin or a metal complex in accordance with the instant invention can be a surface illumination with a conventional light source, as described above, or can be a surface illumination with a laser. The illumination can also be into the body of a tumor, for example through optical fibers inserted thereinto.
Various changes and modification can be made from the specific details of the invention as described above without departing from the spirit and scope thereof as defined in the appended claims.

Claims

1. A method for detecting diseased tissue which comprises administering intravenously, intramuscularly, subcutaneously or topically to a human or animal patient an effective amount of a purpurin or a purpurin metal complex, or a chlorin or a chlorin metal complex, and, after sufficient time for healthy tissue to reject the purpurin or the like, examining the patient under illumination which causes residual purpurin or the like to fluoresce, wherein the purpurin has the structure of one of FIGS. 7, 14-18 and 29-38, below: ##STR4## the purpurin metal complex has the structure of one of FIGS. 1, 44-48 and 54-58, below: ##STR5## the chlorin has the structure of one of FIGS. 8, 24-28, and 39.gtoreq.43, below: ##STR6## and the chlorin metal complex has the structure of one of FIGS. 2 and 49 through 53, below: ##STR7## and wherein each of R10 through R13 and R16 is hydrogen, and each of R1 through R9, R14 and R15 is:
H or CHO,
an alkyl group having from 1 to 4 carbon atoms, excluding tertiary butyl,
an alkylene group having from 2 to 4 carbon atoms,
a group having the formula R.sub.2 N(R.sub.3).sub.2 where R.sub.2 is a bivalent aliphatic hydrocarbon radical having from 1 to 4 carbon atoms, wherein any carbon to carbon bond is either a single or a double bond, and not more than one is a double bond; R.sub.3 is hydrogen or an alkyl group having from 1 to 2 carbon atoms and the two R.sub.3 groups can be the same or different,
a group having the formula R.sub.2 N(R.sub.4).sub.3 A where R.sub.2 is a bivalent aliphatic hydrocarbon radical having from 1 to 4 carbon atoms, wherein any carbon to carbon bond is either a single or a double bond, and not more than one is a double bond; A is a physiologically acceptable anion; and R.sub.4 is an alkyl group having from 1 to 2 carbon atoms and the three R.sub.4 groups can be the same or different,
a group having the formula R.sub.2 OH were R.sub.2 is a bivalent aliphatic hydrocarbon radical having from 1 to 4 carbon atoms, wherein any carbon to carbon bond is either a single or a double bond, and not more than one is a double bond,
CO.sub.2 R', CH.sub.2 CO.sub.2 R' or CH.sub.2 CH.sub.2 CO.sub.2 R', where R' is hydrogen or an alkyl group having from 1 to 4 carbon atoms, excluding tertiary butyl,
an amino acid moiety which is attached to the purpurin or chlorin moiety through a carbonyl which is a part of an amide produced by reaction between an amine function of lysine, glutamic acid, histidine, cystine, arginine or aspartic acid and a CO.sub.2 R', CH.sub.2 CO.sub.2 R' or CH.sub.2 CH.sub.2 CO.sub.2 R' group of the purpurin or chlorin, or
in the purpurins and purpurin metal complexes of FIGS. 1, 7, 14-18, 34-38 and 54-58 and in the chlorins and chlorin metal complexes of FIGS. 2 and 8 R1 can be a bivalent aliphatic hydrocarbon radical having from 2 to 4 carbon atoms wherein both of the valences of the radical are attached to the same carbon atom thereof and to a carbon atom of the purpurin, chlorin, or metal complex, and in the purpurins and purpurin metal complexes of FIGS. 29-33 and of FIGS. 44-48 and in the chlorins and chlorin metal complexes of FIGS. 24-28, 39-43 and 49-53, both R1 and R2 can be bivalent aliphatic hydrocarbon radicals having from 2 to 4 carbon atoms wherein both of the valences of the radical are attached to the same carbon atom thereof and to a carbon atom of the purpurin or metal complex, and
M comprises a metal cation that is complexed with two of the nitrogens of the purpurin or chlorin and is Ag, Al, Ce, Co, Cr, Dy, Er, Eu, Fe, Ga, Gd, Hf, Ho, In, La, Lu, Mn, Mo, Nd, Pb, Pd, Pr, Pt, Rh, Sb, Sc, Sin, Sn, Tb, .sup.99m Tc, Th, Ti, TI, Tm, U, V, Y, Yb, Zn or Zr, with the proviso that not more than one of R1 through R9, R14 and R15 is CHO, a group having the formula R.sub.2 N(R.sub.3).sub.2, a group having the formula R.sub.2 N(R.sub.4).sub.3 A, or an amino acid moiety.
2. A method for treating blood and other body fluids and tissues which comprises in vitro irradiation of the body fluid or tissue and an effective amount of a purpurin or a purpurin metal complex, or a chlorin or a chlorin metal complex, the irradiation being with light of a wavelength at which the purpurin, purpurin metal complex, chlorin or chlorin metal complex has an absorbance peak, wherein the purpurin has the structure of one of FIGS. 7, 14-18 and 29-38, below: ##STR8## the purpurin metal complex has the structure of one of FIGS. 1, 44-48 and 54-58, below: ##STR9## the chlorin has the structure of one of FIGS. 8, 24-28, and 39-43, below: ##STR10## and the chlorin metal complex has the structure of one of FIGS. 2 and 49 through 53, below: ##STR11## and wherein each of R10 through R13 and R16 is hydrogen, and each of R1 through R9, R14 and R15 is:
H or CHO,
an alkyl group having from 1 to 4 carbon atoms, excluding tertiary butyl,
an alkylene group having from 2 to 4 carbon atoms,
a group having the formula R.sub.2 N(R.sub.3).sub.2 where R.sub.2 is a bivalent aliphatic hydrocarbon radical having from 1 to 4 carbon atoms, wherein any carbon to carbon bond is either a single or a double bond, and not more than one is a double bond; R.sub.3 is hydrogen or an alkyl group having from 1 to 2 carbon atoms and the two R.sub.3 groups can be the same or different,
a group having the formula R.sub.2 N(R.sub.4).sub.3 A where R.sub.2 is a bivalent aliphatic hydrocarbon radical having from 1 to 4 carbon atoms, wherein any carbon to carbon bond is either a single or a double bond, and not more than one is a double bond; A is a physiologically acceptable anion; and R.sub.4 is an alkyl group having from 1 to 2 carbon atoms and the three R.sub.4 groups can be the same or different,
a group having the formula R.sub.2 OH were R.sub.2 is a bivalent aliphatic hydrocarbon radical having from 1 to 4 carbon atoms, wherein any carbon to carbon bond is either a single or a double bond, and not more than one is a double bond,
CO.sub.2 R', CH.sub.2 CO.sub.2 R' or CH.sub.2 CH.sub.2 CO.sub.2 R', where R' is hydrogen or an alkyl group having from 1 to 4 carbon atoms, excluding tertiary butyl,
a monoclonal antibody moiety which is attached to the purpurin or chlorin moiety through a carbonyl which is a part of an amide produced by reaction between an amine function of a monoclonal antibody and a CO.sub.2 R' CH.sub.2 CO.sub.2 R' or CH.sub.2 CH.sub.2 CO.sub.2 R' group of the purpurin or chlorin, and wherein the moiety is of a monoclonal antibody which selectively binds to malignant tumors,
an amino acid moiety which is attached to the purpurin or chlorin moiety through a carbonyl which is a part of an amide produced by reaction between an amine function of lysine, glutamic acid, histidine, cystine, arginine or aspartic acid and a CO.sub.2 R', CH.sub.2 CO.sub.2 R' or CH.sub.2 CH.sub.2 CO.sub.2 R' group of the purpurin or chlorin, or
in the purpurins and purpurin metal complexes of FIGS. 1, 7, 14-18, 34-38 and 54-58 and in the chlorins and chlorin metal complexes of FIGS. 2 and 8 R1 can be a bivalent aliphatic hydrocarbon radical having from 2 to 4 carbon atoms wherein both of the valences of the radical are attached to the same carbon atom thereof and to a carbon atom of the purpurin, chlorin, or metal complex, and in the purpurins and purpurin metal complexes of FIGS. 29-33 and of FIGS. 44-48 and in the chlorins and chlorin metal complexes of FIGS. 19-28, 39-43 and 49-53, both R1 and R2 can be bivalent aliphatic hydrocarbon radicals having from 2 to 4 carbon atoms wherein both of the valences of the radical are attached to the same carbon atom thereof and to a carbon atom of the purpurin or metal complex, and
M comprises a metal cation that is complexed with two of the nitrogens of the purpurin or chlorin and is Ag, Al, Ce, Co, Cr, Dy, Er, Eu, Fe, Ga, Gd, Hf, Ho, In, La, Lu, Mn, Mo, Nd, Pb, Pd, Pr, Pt, Rh, Sb, Sc, Sm, Sn, Tb, .sup.99m Tc, Th, Ti, TI, Tin, U, V, Y, Yb, Zn or Zr, with the proviso that not more than one of R1 through R9, R14 and R15 is CHO, a group having the formula R.sub.2 N(R.sub.3).sub.2, a group having the formula R.sub.2 N(R.sub.4).sub.3 A, or an amino acid moiety.
3. A method as claimed in claim 1 which comprises administering a chlorin having the structure of FIG. 8, a chlorin metal complex having the structure of FIG. 2, a purpurin having the structure of FIG. 7, or a purpurin metal complex having the structure of FIG. 1, wherein each of R10 through R12 and R13 is hydrogen, each of R1 through R8 is an alkyl group other than t-butyl having from 1 to 4 carbon atoms, R9 is CO.sub.2 R', CH.sub.2 CO.sub.2 R' or CH.sub.2 CH.sub.2 CO.sub.2 R', where R' is hydrogen or an alkyl group other than t-butyl having from 1 to 4 carbon atoms, and M comprises a metal cation that is complexed with two of the nitrogens of the purpurin or chlorin and is Sn or Zn.
4. A method as claimed in claim 1 which comprises administering a chlorin having the structure of FIG. 8, a chlorin metal complex having the structure of FIG. 2 or a purpurin metal complex having the structure of FIG. 1, wherein each of R10 through R12 and R13 is hydrogen, each of R1 through R8 is an alkyl group other than t-butyl having from 1 to 4 carbon atoms, R9 is CO.sub.2 R', CH.sub.2 CO.sub.2 R' or CH.sub.2 CH.sub.2 CO.sub.2 R', where R' is hydrogen or an alkyl group other than t-butyl having from 1 to 4 carbon atoms, and M comprises a metal cation that is complexed with two of the nitrogens of the purpurin or chlorin and is Sn or Zn.
5. A method as claimed in claim 1 which comprises administering a purpurin having the structure of FIG. 7 of the attached drawings wherein each of R10 through R12 and R13 is hydrogen, each of R1 through R8 is an alkyl group other than t-butyl having from 1 to 4 carbon atoms, and R9 is CO.sub.2 R', CH.sub.2 CO.sub.2 R' or CH.sub.2 CH.sub.2 CO.sub.2 R', where R' is hydrogen or an alkyl group other than t-butyl having from 1 to 4 carbon atoms.
6. A method as claimed in claim i which comprises administering a purpurin having the structure of FIG. 1 of the attached drawings where each of R1, R3, R5 and R7 is methyl, each of R2, R4, R6 and R8 is ethyl, R9 is CH.sub.2 CH.sub.2 CO.sub.2 R', and each of R10, R11, R12 and R13 is H.
7. A method as claimed in claim 2 which comprises irradiating the body fluid or tissue and a chlorin having the structure of FIG. 8, a chlorin metal complex having the structure of FIG. 2, a purpurin having the structure of FIG. 7, or a purpurin metal complex having the structure of FIG. 1, wherein each of R10 through R12 and R13 is hydrogen, each of R1 through R8 is an alkyl group other than t-butyl having from 1 to 4 carbon atoms,
R9 is CO.sub.2 R', CH.sub.2 CO.sub.2 R' or CH.sub.2 CH.sub.2 CO.sub.2 R', where R' is hydrogen or an alkyl group other than t-butyl having from 1 to 4 carbon atoms, and M comprises a metal cation that is complexed with two of the nitrogens of the purpurin or chlorin and is Sn or Zn.
8. A method as claimed in claim 2 which comprises irradiating the body fluid or tissue and a chlorin having the structure of FIG. 8, a chlorin metal complex having the structure of FIG. 2 or a purpurin metal complex having the structure of FIG. 1 of the attached drawings, wherein each of R10 through R12 and R13 is hydrogen, each of R1 through R8 is an alkyl group other than t-butyl having from 1 to 4 carbon atoms, R9 is CO.sub.2 R', CH.sub.2 CO.sub.2 R' or CH.sub.2 CH.sub.2 CO.sub.2 R', where R' is hydrogen or an alkyl group other than t-butyl having from 1 to 4 carbon atoms, and M comprises a metal cation that is complexed with two of the nitrogens of the purpurin or chlorin and is Sn or Zn.
9. A method as claimed in claim 2 which comprises irradiating a body fluid or tissue and a purpurin having the structure of FIG. 7 of the attached drawings wherein each of R10 through R12 and R13 is hydrogen, each of R1 through R8 is an alkyl group other than t-butyl having from 1 to 4 carbon atoms, and R9 is CO.sub.2 R', CH.sub.2 CO.sub.2 R' or CH.sub.2 CH.sub.2 CO.sub.2 R', where R' is hydrogen or an alkyl group other than t-butyl having from 1 to 4 carbon atoms.
10. A method as claimed in claim 2 which comprises irradiating a body fluid or a tissue and a purpurin having the structure of FIG. 1 where each of R1, R3, R5 and R7 is methyl, each of R2, R4, R6 and R8 is ethyl, R9 is CH.sub.3 CH.sub.2 O.sub.2 C-, and each of R10, R11, R12 and R13 is H.
11. A method as claimed in claim 1 wherein the purpurin, chlorin or complex which is administered has a structure that is enriched in an atom which emits ionizing radiation, said atom being present in the purpurin, chlorin or complex in a sufficient proportion to enable the use of an instrument which detects the presence of ionizing radiation to detect small quantities of the purpurin, chlorin or metal complex.
12. A method as claimed in claim 1 wherein the purpurin, chlorin or complex which is administered has a structure that is enriched in an atom which can be detected by NMR, said atom being present in the purpurin, chlorin or complex in a sufficient proportion to enable the use of NMR to detect small quantities of the purpurin, chlorin or metal complex.
13. A method as claimed in claim 1 wherein the purpurin, chlorin or complex which is administered has a structure that is enriched in an atom which can be detected by NMR.
14. A method as claimed in claim 1 wherein the purpurin, chlorin or complex which is administered has a structure that is enriched in an atom which emits ionizing radiation.
15. A method for treating a human or animal patient who has diseased tissue, which method comprises administering intravenously, intramuscularly, subcutaneously or topically an effective amount of a purpurin or a purpurin metal complex, or a chlorin or a chlorin metal complex, and, after sufficient time for healthy tissue to reject the purpurin or the like, irradiating the diseased tissue with light of a wave length which causes a reaction which destroys the diseased tissue, wherein the purpurin has the structure of one of FIGS. 7, 14-18 and 29-38, below: ##STR12## the purpurin metal complex has the structure of one of FIGS. 1, 44-48 and 54-58, below: ##STR13## the chlorin has the structure of one of FIGS. 8, 24-28, and 39-43, below: ##STR14## and the chlorin metal complex has the structure of one of FIGS. 2 and 49 through 53, below: ##STR15## and wherein each of R10 through R13 and R16 is hydrogen, and each of R1 through R9, R14 and R15 is:
H or CHO,
an alkyl group having from 1 to 4 carbon atoms, excluding tertiary butyl,
an alkylene group having from 2 to 4 carbon atoms,
a group having the formula R.sub.2 N(R.sub.3).sub.2 where R.sub.2 is a bivalent aliphatic hydrocarbon radical having from 1 to 4 carbon atoms, wherein any carbon to carbon bond is either a single or a double bond, and not more than one is a double bond; R.sub.3 is hydrogen or an alkyl group having from 1 to 2 carbon atoms and the two R.sub.3 groups can be the same or different,
a group having the formula R.sub.2 N(R.sub.4).sub.3 A where R.sub.2 is a bivalent aliphatic hydrocarbon radical having from 1 to 4 carbon atoms, wherein any carbon to carbon bond is either a single or a double bond, and not more than one is a double bond; A is a physiologically acceptable anion; and R.sub.4 is an alkyl group having from 1 to 2 carbon atoms and the three R.sub.4 groups can be the same or different,
a group having the formula R.sub.2 OH were R.sub.2 is a bivalent aliphatic hydrocarbon radical having from 1 to 4 carbon atoms, wherein any carbon to carbon bond is either a single or a double bond, and not more than one is a double bond,
CO.sub.2 R', CH.sub.2 CO.sub.2 R' or CH.sub.2 CH.sub.2 CO.sub.2 R', where R' is hydrogen or an alkyl group having from 1 to 4 carbon atoms, excluding tertiary butyl,
an amino acid moiety which is attached to the purpurin or chlorin moiety through a carbonyl which is a part of an amide produced by reaction between an amine function of lysine, glutamic acid, histidine, cystine, arginine or aspartic acid and a CO.sub.2 R', CH.sub.2 CO.sub.2 R' or CH.sub.2 CH.sub.2 CO.sub.2 R' group of the purpurin or chlorin, or
in the purpurins and purpurin metal complexes of FIGS. 1, 7, 14-18, 34-38 and 54-58 and in the chlorins and chlorin metal complexes of FIGS. 2 and 8 R1 can be a bivalent aliphatic hydrocarbon radical having from 2 to 4 carbon atoms wherein both of the valences of the radical are attached to the same carbon atom thereof and to a carbon atom of the purpurin, chlorin, or metal complex, and in the purpurins and purpurin metal complexes of FIGS. 29-33 and of FIGS. 44-48 and in the chlorins and chlorin metal complexes of FIGS. 24-28, 39-43 and 49-53, both R1 and R2 can be bivalent aliphatic hydrocarbon radicals having from 2 to 4 carbon atoms wherein both of the valences of the radical are attached to the same carbon atom thereof and to a carbon atom of the purpurin or metal complex, and
M comprises a metal cation that is complexed with two of the nitrogens of the purpurin or chlorin and is Ag, Al, Ce, Co, Cr, Dy, Er, Eu, Fe, Ga, Gd, Hf, Ho, In, La, Lu, Mn, Mo, Nd, Pb, Pd, Pr, Pt, Rh, Sb, Sc, Sin, Sn, Tb, .sup.99m Tc, Th, Ti, Tl, Tin, U, V, Y, Yb, Zn or Zr, with the proviso that not more than one of R1 through R9, R14 and R15 is CHO, a group having the formula R.sub.2 N(R.sub.3).sub.2, a group having the formula R.sub.2 N(R.sub.4).sub.3 A, or an amino acid moiety.
16. A method as claimed in claim 15 which comprises administering a chlorin having the structure of FIG. 8, a chlorin metal complex having the structure of FIG. 2, a purpurin having the structure of FIG. 7, or a purpurin metal complex having the structure of FIG. 1, wherein each of R10 through R12 and R13 is hydrogen, each of R1 through R8 is an alkyl group other than t-butyl having from 1 to 4 carbon atoms, R9 is CO.sub.2 R', CH.sub.2 CO.sub.2 R' or CH.sub.2 CH.sub.2 CO.sub.2 R', where R' is hydrogen or an alkyl group other than t-butyl having from 1 to 4 carbon atoms, and M comprises a metal cation that is complexed with two of the nitrogens of the purpurin or chlorin and is Sn or Zn.
17. A method as claimed in claim 15 which comprises administering a chlorin having the structure of FIG. 8, a chlorin metal complex having the structure of FIG. 2 or a purpurin metal complex having the structure of FIG. 1, wherein each of R10 through R12 and R13 is hydrogen, each of R1 through R8 is an alkyl group other than t-butyl having from 1 to 4 carbon atoms, R9 is CO.sub.2 R', CH.sub.2 CO.sub.2 R'or CH.sub.2 CH.sub.2 CO.sub.2 R', where R' is hydrogen or an alkyl group other than t-butyl having from 1 to 4 carbon atoms, and M comprises a metal cation that is complexed with two of the nitrogens of the purpurin or chlorin and is Sn or Zn.
18. A method as claimed in claim 15 which comprises administering a purpurin having the structure of FIG. 7 of the attached drawings wherein each of R10 through R12 and R13 is hydrogen, each of R1 through R8 is an alkyl group other than t-butyl having from 1 to 4 carbon atoms, and R9 is CO.sub.2 R', CH.sub.2 CO.sub.2 R' or CH.sub.2 CH.sub.2 CO.sub.2 R', where R' is hydrogen or an alkyl group other than t-butyl having from 1 to 4 carbon atoms.
19. A method as claimed in claim 1.5 which comprises administering a purpurin having the structure of FIG. 1 of the attached drawings where each of R1, R3, R5 and R7 is methyl, each of R2, R4, R6 and R8 is ethyl, R9 is CH.sub.2 CH.sub.2 CO.sub.2 R', and each of R10, R11, R12 and R13 is H.
20. A method as claimed in claim 15 wherein the purpurin, chlorin or complex which is administered has a structure that is enriched in an atom which emits ionizing radiation, said atom being present in the purpurin, chlorin or complex in a sufficient proportion to enable the use of an instrument which detects the presence of ionizing radiation to detect small quantities of the purpurin, chlorin or metal complex.
21. A method as claimed in claim 15 for detecting a tumor or treating a patient wherein the purpurin, chlorin or complex which is administered has a structure that is enriched in an atom which can be detected by NMR, said atom being present in the purpurin, chlorin or complex in a sufficient proportion to enable the use of NMR to detect small quantities of the purpurin, chlorin or metal complex.
22. A method as claimed in claim 15 wherein the purpurin, chlorin or complex which is administered has a structure that is enriched in an atom which can be detected by NMR.
23. A method as claimed in claim 15 wherein the purpurin, chlorin or complex which is administered has a structure that is enriched in an atom which emits ionizing radiation.

REFERENCE TO RELATED APPLICATIONS

This is a continuation of application Ser. No. 08/070,105, filed May 28, 1993 (now abandoned which is a continuation in part of application Ser. No. 07/763,916, filed Sep. 23, 1991 (now U.S. Pat. No. 5,216,012) which is a continuation in part of application Ser. No. 07/388,643, filed Aug. 2, 1989 (now U.S. Pat. No. 5,051,415), which is a continuation in part of application Ser. No. 06/874,097, filed Jun. 13, 1986, (now abandoned) and a continuation in part of application Ser. No. 06/842,125, filed Mar. 18, 1986, (now abandoned) which is a continuation in part of application Ser. No. 06/815,714, filed Jan. 2, 1986. (now abandoned).

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Continuations (1)

	Number	Date	Country
Parent	70105	May 1993

Continuation in Parts (4)

	Number	Date
Parent	763916	Sep 1991
Parent	388643	Aug 1989
Parent	874097	Jun 1986
Parent	815714	Jan 1986

Use of purpurins, chlorins and purpurin- and chlorin-containing compositions

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Abstract