United States Patent (19) 11 Patent Number: 4,977,177 Bommer et al. 45 Date of Patent: * Dec. 11, 1990

54. TETRAPYRROLE and Zinc-Tetra(p-sulfophenyl)porphine Res. Comm. POLYAMNOMONOCARBOXYLCACD Chem. Path. & Parm. 30(2) 317-327, (1980). THERAPEUTICAGENTS Jackson, Anthony H., Kenner, George W., Smith, Kevin M., and Suckling, Calvin, J. Synthetic Ap 75 Inventors: Jerry C. Bommer, Ogden; Bruce F. proaches To Versatile Hemoprotein Model Com Burnham, Logan, both of Utah pounds, Built from Porphyrins and Peptides, J. Chem. 73 Assignee: Nippon Petrochemicals Company, Soc. Perkin Trans. I, 1441-1448, (1982). Ltd., Tokyo, Japan Chemical Berischet, 90, 470-481, 1957 by Lautsch et al., Hoppe-Seyler's Ztschr. Phy Chem. 327, 205-216, 1962, * Notice: The portion of the term of this patent Lossee and Miller. subsequent to Apr. 7, 2004 has been Chimia, 13, 129-180, 1959, By Karrer Tetrahedron disclaimed. Letters, 23, 2017-2020, 1978, by Pelter, et al. 21 Appl. No.: 728,752 Current Microbiology 8, 195-199, 1983, by Gauthier, et al., Zhurnal Organicheshoi K111 M11, 15, 828-835, 22 Filed: Apr. 30, 1985 1979 by Bacunbee, et al. 51) Int. Cl...... A61K 31/40; A61K 49/00; CO7D 487/00 Primary Examiner-Douglas W. Robinson 52 U.S. C...... 514/410; 540/145; Assistant Examiner-Kevin Weddington 424/9 Attorney, Agent, or Firm-Scully, Scott, Murphy & 58) Field of Search ...... 540/145; 560/19, 125; Presser 372/81; 514/427, 41 (57 ABSTRACT 56 References Cited This invention relates to new therapeutic compositions which are useful in photodiagnosis and phototherapy, U.S. PATENT DOCUMENTS especially in the detection and treatment of tumors and 4,393,071 7/1983 Fujii et al...... 514/10 cancerous tissues in the human or animal body. The 4,649,151 3/1987 Dougherty et al. 540/145 compounds of the present new therapeutic composition 4,656,186 4/1987 Bommer et al. .... 514/410 are mono-, di or polyamides of an amino monocarbox 4,675,338 6/1987 Bommer et al. .... 514/410 ylic acid and a tetrapyrrole containing at least one car 4,693,885 9/1987 Bommer et al...... 540/145 boxyl group of the structure FOREIGN PATENT DOCUMENTS 01 18913 9/1984 European Pat. Off...... 540/145 O168831 7/1985 European Pat. Off. . O168832 7/1985 European Pat. Off. . 2809093 9/1978 Fed. Rep. of Germany . 0077280 6/1981 Japan ...... 540/145 0000981 1/1983 Japan ...... 540/145 8401382 4/1984 PCT Int'l Appl. . wherein Z is the aminomonocarboxylic acid residue less OTHER PUBLICATIONS the amino group, X is the tetrapyrrole residue less the Lautsch et al., Chem. Ber. 90(4): 470-481, 1957. carboxy group, and 'n' is an integer from 1 to 4 inclu Grenan, Marie, Tsutsui, Minoru and Wysor, Michael sive. Phototoxicity of the Chemotherapeutic Agents, Hema toporphyrin D, Meso-Tetra(p-sulfophenyl) Porphine 46 Claims, No Drawings 4,977,177 1. 2 pounds in which one or more double bonds are absent. TETRAPYRROLE There are present in the ring system four pyrrole rings POLYAMNOMONOCARBOXYLIC ACID joined through the alpha positions of the respective THERAPEUTICAGENTS pyrrole rings by a methine group, i.e., -CH-. The compounds of the present invention are designated as FIELD OF THE INVENTION derivatives of the tetrapyrroles for convenience in the This invention relates to new compounds which are disclosure and the appended claims and it will be under useful in photodiagnosis and phototherapy, especially in stood that the term "tetrapyrrole' will designated com the detection and treatment of tumors and cancerous pounds of the characteristic ring structure designated tissues in the human or animal body. 10 hereinbefore as well as the corresponding perhydro derivatives, and the corresponding non-cyclic pyrroles, DESCRIPTION OF THE PRIOR ART i.e., the linear tetrapyrroles, commonly known as the It is known to irradiate tumors and cancerous tissues bile pigments. in the human body with intensive light following ad The tetrapyrroles employed in the present invention ministration of a hematoporphyrin derivative in the 15 are all derived by various means and various alteration wavelength range of 626 to 636 namometers to reduce procedures from natural tetrapyrroles. The naturally and, at times, destroy the cancerous cells (see PCT occurring tetrapyrroles have as their common ancestor published specification No. WO 83/00811). It is also uroporphyrinogen III, a hexahydroporphyrin reduced known that porphyrins, especially the sodium salt of at the bridge positions. For example, synthetic or bio protoporphyrins, can maintain or promote the normal 20 synthetic derivatives or products of protoporphyrins IX functions of cells and are useful for preventing the gene or protoporphyrinogen IX are well-known in the art sis, growth, metastasis, and relapse of malignant tumors. (see, for example, Porphyrins and Metalloporphyrins, Japanese Published Patent Application No. 125737/76 describes the use of porphyrins as tumor.inhibiting K. Smith Elsivier; The Porphyrins (Vols. 1-7) D. Dol agents, exemplifying etioporphyrin, mesoporphyrin, 25 phin, Academic Press; and Biosynthetic Pathways, Vol. protoporphyrin, deuteroporphyrin, hematoporphyrin, III, Chapter by B. Burnham, editor D. M. Greenberg, coproporphyrin, and uroporphyrin. Academic Press). In Tetrahedron Letters No. 23, pp. 2017-2020 (1978), The non-cyclic tetrapyrroles are commonly known there is described an amino monocarboxylic acid ad as bile pigments and include, for example, bilirubin and duct of the pigment bonellin obtained by extraction of 30 biliverdin. These tetrapyrroles are also derived from principally the body wall of the marine echuroid B. protoporphyrin, e.g., as metabolic products in animals. viridis. The structure of these adducts is presumed to be A further characteristic of the present new com an amide formed through either of the free carboxy pounds is the presence of at least one amide linkage in a groups of bonellin and the amino mono-carboxylic acid. substituent at any of the numbered positions of the ring Hydrolysis of the adduct yielded a mixture of valine, 35 structure. These are present in the instant new com isoleucine, leucine and alloisoleucine. No use for these pounds together with other substituents as defined here amino acid adducts is described in this reference. inafter. That the tetrapyrroles cause intense photosensitivity Thus, the present invention contemplates amino acid in animals in well known and has been documented in or peptide derivatives of compounds which contain the numerous articles in literature, e.g., J. Intr. Sci. Vitami chromophore of porphyrins, chlorins or bacteriochlo nol, 27, 521-527 (1981); Agric. Biol. Chem, 46(9), rins, as well as related porphyrin compounds. The pep 2183-2193 (1982); Chem. Abst. 98, 276 (1983) and 88 tide linkage involves a carboxy group of the chromo 6976m (1928). phore-bearing compound and the amino group of the SUMMARY OF THE INVENTION specified amino acid. The present new compounds em 45 brace, inter alia, derivatives of the tetrapyrroles which The products contemplated by this invention are contain a free carboxy group. These derivatives include cyclic and acyclic tetrapyrroles derived by various the major classes of tetrapyrroles: carboxy-containing procedures from naturally-occurring tetrapyrroles. The porphyrins, chlorins, and bacteriochlorins, which are cyclic tetrapyrroles have as their common parent tetra well-known to those skilled in this art. pyrrole, uroporphyrinogen, and possess the following 50 The amino acid employed in the present invention to ring structure: form the aforesaid peptide linkage are aminomonocar

boxylic acids in which the amino group, of course, is located on a carbon atom of the monocarboxylic acid. The specific position of the amino group in the carbon 55 atom chain is not critical, the only requirement that the amino group be available to form the requisite peptide linkage with the carboxyl group of the selected porphy rin. Thus, a variety of amino monocarboxylic acids are useful in the present invention, including serine, gly 6 cine, a-aminoalanine, (3-aminoalanine, e-amino-n- caproic acid, piperidine-2-carboxylic acid, piperidine-6- carboxylic acid, pyrrole-2-carboxylic acid, pyrrole-5- carboxylic acid, piperidine-6-propionic acid, pyrrole-5- acetic acid, and similar such acids. These amino acids in which the positions in the molecule are numbered 65 may be substituted with angular alkyl groups, such as 1-20, and the rings identified by letters A, B, C and D, methyl and ethyl groups, as well as other groups which and also include perhydro-, e.g., dihydro- and tetrahy do not adversely affect the capability of the amino dro-, derivatives of the said ring structure, e.g., con group to form the peptide linkage, e.g., alkoxy groups, 4,977,177 3 4. or acyloxy groups, and may also include additional amino groups. The preferred amino acids are the natu rally occurring a-amino acids, serine, alanine, and gly- O cine, which are readily available and up to the present, have provided the best results. 5 z--c X Exemplary compounds of the tetrapyrrole classes are H illustrated in Table I in which the numbered positions of the tetrapyrrole ring structure are used to designate the position of the indicated substituent. The absence of wherein Z is the aminomonocarboxylic acid residue less double bonds in the ring system is designated under 10 the amino group and X is the tetrapyrrole residue less "dihydro' with each set of numbers (ring position) the carboxy group and "n' is an integer from 1 to 4 indicating the absence of a double bond between the inclusive. designated positions. The particularly preferred compounds are fluores TABLE I Ring Position - A - - B - - - - P - PORPHYRIN 2 6 7 11 12 14 16 17 Dihydro Coproporphyrin III Me Pr Me Pr Me Pr H Pr Me ww. Deuteroporphyrin IX Me H Me H Me Pr H P Me Hematoporphyrin IX Me Me Me Me Me Pr H Pr Me -h -h OH OH

Protoporphyrin IX Me w Me W Me Pir H Pr Me w Photoprotoporphyrin IX Me V on Me CHCHO Me Pir H Pr Me 6,7 (one of two isoners shown) --OH Mesoporphryin IX Me Et Me Et Me Pr H Pr Me H H Transmesochlorin X Me Et Me Et Me Pir H Pr Me 12 Transmesochlorin IX Me Et ( H ( H Me Pir H Pr Me 6,7 Me Et Chlorine Me w Me Et Me CO2H Me H H 16, 17 Pr Me Chlorin e6 ye w Me Et Me CO2H Ac ( H ( H 16, 17 Pr Me Mesochlorine Me Et Me Et Me CO2H Me ( H ( H 16, 17 Pr Me Isochlorin e4. Me W Me Et Me H Ac ( H H 16, 17 Pr Me Mesoisochlorin e4. Me Et Me Et Me H Ac ( H H 16, 17 Pr Me Mesochlorines Me Et Me Et Me CO2H Ac H H 16, 17 Pr Me Bacteriochlorines Me ACL H H Me CO2H Ac H H 6,7 Me Et Pr Me 16, Bacteriochlorine Me ACL H Me CO2H Me H H 6,7 Me Et Pr Me 16, 17 Bacterioisochlorin e4 Me ACL H H Me H Ac H H 6,7 Me Et Pr Me 16, 17 Notes: Me: -CH (Methyl group) Pr: CHCHCOOH (Propionic acid group) V: CHFCH (Vinyi group) Et: -CHCH (Ethyl group) Ac: CH3COOH (Acetic acid group) ACL: CH-CO-(Acetyl group) The present new compounds are mono, di- or poly- 65 anides of an aminomonocarboxylic and a tetrapyrrole cent mono, di, tri, or polyamides of an aminomonocar containing at least one carboxyl group of the structure boxylic acid and a tetrapyrrole compound of the for mula: 4,977,177 6 substituents or are divalent and attached to the same carbon, the respective pyrrole ring to which attached is a dihydropyrrole; R is lower alkyl or benzyl; with the proviso that at least two of R1-R9 include a free carboxyl group; and salts thereof. Another preferred embodiment of the present inven tion is the fluorescent mono, di, tri or polyamide of an aminomonocarboxylic acid and a tetrapyrrole con O pound of the formula: or the corresponding di- or tetrahydrotetrapyrroles wherein 5 R1 is methyl;

-H -OH -CH39 -CH3; R2 is H, vinyl, ethyl,

-HCH, 25 OH or the corresponding di- or tetrahydratetrapyrroles wherein R1, R2, R3, R4, R5, R6, R7, R8 and R9 are as acetyl, defined heretofore. The preferred tetrapyrrole carboxylic acids are those 30 wherein at least three carboxylic acid groups are pres H ent in the tetrapyrrole, preferably asymmetrically at -ethyl, -C=O, tached to the porphyrin ring system, e.g., the carboxylic acid groups are present on the rings A and B side of the CH2CH2CO2H, or -CHCHO; molecule or on the rings D and C side of the molecule. R3 is methyl 35 The particularly preferred compounds of this inven tion are those represented by the formula:

or -CH3 or R4 is H, vinyl, ethyl,

-CHCH, OH 45 COOH CH2CH2CO2H, =CHCHO; or

H -ethyl; 50

R5 is methyl; wherein; R6 is H, CH2CH2CO2H, CH2CH2CO2R or CO2H; X = H, vinyl, ethyl, acetyl or formyl; R7 is CH2CH2CO2H, CH2CH2CO2R, or 55 Y= methyl or formyl; M= methyl; and E=ethyl -CH2CH2CO2H; and pharmaceutically-acceptable salts thereof. -H The present new compounds form salts with either acids or bases. The acid salts are particularly useful for R8 is methyl or purification and/or separation of the final amide prod ucts as are the salts formed with bases. The base salts, however, are particularly preferred for diagnostic and -CH3 therapeutic use as hereindescribed. -H 65 The acid salts are formed with a variety of acids such as the mineral acids, hydrochloric, hydrobromic, nitric R9 is H, COOH, CH2COOH or methyl; provided that and sulfuric acids, organic acids such as tolunesulfonic when R, R2, R3, R4, R7 and R8 represent two and benzenesulfonic acids. 4,977, 177 8 The base salts include, for example, sodium, potas sium, calcium, magnesium, ammonium, triethyl Photodiagnosis and Phototherapy ammonium, trimethylammonium, morpholine and pi The compounds of the present invention are useful peridine salts and similar such salts. for the photodiagnosis and phototherapy of tumor, The acid and base salts are formed by the simple 5 cancer and malignant tissue (hereinafter referred to as expediency of dissolving the selected amino acid tetra “tumor'). pyrrole amide in an aqueous solution of the acid or base When a man or animal having tumor is treated with and evaporation of the solution to dryness. The use of a doses of a compound of the present invention and when water-miscible solvent for the amide can assist in dis appropriate light rays or electromagnetic waves are Solving the amide. 10 applied, the compound emits light, i.e., fluorescence. The final amide products can also be converted to Thereby the existence, position and size of tumor can be metal complexes for example by reaction with metal detected, i.e., photodiagnosis. salts. The magnesium complexes may be useful for the When the tumor is irradiated with light of proper same purpose as the adduct product. Other metal com wavelength and intensity, the compound is activated to 15 exert a cell killing effect against the tumor. This is called plexes, as well as the magnesium complex, including, "phototherapy'. for example, iron and zinc, are useful to preclude con Compounds intended for photodiagnosis and photo tamination during processing of the adduct product by therapy ideally should have the following properties: metals such as nickel, cobalt and copper, which are (a) non-toxic at normal therapeutic dosage unless and difficult to remove. Zinc and magnesium are readily until activated by light; removed from the final adduct product after processing (b) should be selectively photoactive; is completed. (c) when light rays or electromagnetic waves are Since many of the aminomonocarboxylic acids exist applied, they should emit characteristic and detectable in both the D- and L-forms, and also are employed in fluorescence; mixtures of these forms as well as the D, L-forn, the 25 (d) when irradiated with light rays or electromag selection of the starting amino acid will, of course, re netic waves are applied, they are activated to an extent sult in products in which the respective isomer or mix to exert a cell killing effect against tumor; and ture of isomers exist. The present invention contem (e) easily metabolized or excreted after treatment. plates the use of all such isomers, but the L-form is In accordance with testing up to the present, the particularly preferred. 30 present new compounds have the foregoing properties The present new compounds are prepared by the and are also characterized by reasonable solubility in usual peptide synthetic routes which generally include saline at physiological pH. any amide-forming reaction between the selected amino The present new compounds possess greater fluores acid and the specific tetrapyrrole. Thus, any amide cence in tumors than do the corresponding basic tet forming derivative of the tetrapyrrole carboxylic acid 35 rapyrroles. Their use provides the best contrast in tu can be employed in producing the present new peptides, mors compared to normal tissue around the tumor. The e.g., lower alkyl esters, anhydrides and mixed anhy instant compounds absorb activating energy for photo drides. therapy in the convenient range of 600 to 800 nanome The preferred preparative methods use mixed anhy ters, with the preferred compounds absorbing in the drides of the carboxylic acid or carbodiimides. The 620-760 nanometer range, i.e., light of longer wave reactants are merely contacted in a suitable solvent lengths which more readily permits penetration of en therefor and allowed to react. Temperatures up to the ergy into the tumor for phototherapeutic purpose. reflux temperature can be used, with the higher temper In present experience, the present compounds more atures merely reducing the reaction time. However, uniformly distribute throughout the tumor than the excessively high temperatures are usually not preferred 45 basic tetrapyrrole permitting the use of considerably So as to avoid unwanted secondary reactions. lower dosage (to about 1/10th of the required normal The procedures for forming the instant peptides are dose of the basic tetrapyrrole) which lessens, if not well known in this art and are provided in detail in the eliminates, photosensitization in the host. They also accompanying examples. possess a more consistent fluorescence whereas some of SO the corresponding tetrapyrroles show inconsistent fluo Since the selected tetrapyrrole contains more than rescence or the fluorescence varies from day to day in one carboxyl group, mixtures of products can be the host. formed including isomeric di- and even tri- or higher A particularly advantageous property of the present peptide products, depending on the number of carboxyl compounds resides in the ease with which they are groups and depending on the selected stoichiometry. 55 excreted by the host. Generally, within 48 to 72 hours of Thus, when equivalent mixtures of amino acid and tet intravenous or intraperitoneal administration, there are rapyrrole are reacted, the product may contain some little or no detectable amounts in normal muscle tissue. monopeptides, but also present will be di- or polypep The present compounds which are excreted with their tides. It is generally possible to separate the monopep chromophore intact are recovered from the feces of the tides and higher peptides using known chromato host within 48-72 hours of injection. Under equivalent graphic techniques. However, such separations are not circumstances, substantial amounts of the correspond necessary since the mixed peptides are usually compara ing tetrapyrroles remain, as compared with only minor ble to the separated products in their ultimate use. Thus, amounts of peptides formed with the aminocarboxylic mixtures of mono, di, and tri-peptides of the same tetra acids remain in the host, e.g., up to about 20%. This pyrrole can be used. property is extremely important in that it contributes to Usually, unreacted tetrapyrrole is separated from the minimization of photosensitization of the host. peptide products of the invention during purification as, The instant compounds can be used for diagnosis and for example, by chromatographic techniques. therapeutic treatment of a broad range of tumors. Ex 4,977,177 10 amples of tumors are gastric cancer, enteric cancer, For both diagnosis and treatment, the present con lung cancer, breast cancer, uterine cancer, esophageal pounds can be administered by the oral, intravenous, or cancer, ovarian cancer, pancreatic cancer, pharyngeal intramuscular routes. They can be formulated as lyophi cancer, sarcomas, hepatic cancer, cancer of the urinary lized sterile, pyrogen-free compounds, preferably in the bladder, cancer of the upper jaw, cancer of the bile form of basic salts, e.g., sodium salt The preferred dos duct, cancer of the tongue, cerebral tumor, skin cancer, age forms are provided as injectable solutions (isotonic). malignant goiter, prostatic cancer, cancer of the parotid The irradiation source used in treatment of tumors gland, Hodgkin's disease, multiple myeloma, renal can containing compounds of this invention is a filtered, cer, leukemia, and malignant lymphocytoma. For diag highintensity, continuous source or pumped dye, or nosis, the solc requirement is that the tumor be capable 10 other laser and light delivery system, which is capable of selectivity fluorescing when exposed to proper light. of performing within the following limits power inten For treatment, the tumor must be penetrable by the sity 20–500 mw/cm2 at wavelengths between 620 and activation energy. For diagnosis, light of shorter wave 760 nm. and a total output of at least 500 m.w.. or greater. length is used whereas for therapeutic purposes light of Several currently commercially available lasers meet longer wavelength is used to permit ready penetration 15 these criteria. of the tumor tissue. Thus, for diagnosis, light of from The tetrapyrroles can be prepared by various syn 360-760 nanometers can be used, and for treatment, thetic methods which are found in the literature, e.g., from 620-760, depending on the individual characteris tics of the tetrapyrrole. The absorption characteristics Pheophorbides of the present new compounds are substantially the 20 Willstatter, R., Stoll, A.; Investigations on Chlorophyll, same as the tetrapyrrole from which derived. (Transl. Schertz, FM.M., Merz, A. R.) p. 249. Sci It is necessary that the light rays be so intense as to ence Printing Press, Lancaster, Pa., 1928. cause the compounds to emit fluorescence for diagnosis Pennington, F. C. Strain, H. H., Svec, W. A., Katz, J. J.; and to exert a cell killing effect for therapy. J. Amer. Chem. Soc., 86, 1418 (1964). The source of irradiation for photodiagnosis and 25 phototherapy is not restricted, however, but the laser Chlorin e6 beam is preferable because intensive light rays in a de Willstatter, R. Stoll, A.; Investigations on Chlorophyll, sired wavelength range can be selectively applied For (Trans., Schertz, F. M., Merz, A. R.) p. 176. Science example, in photodiagnosis, the compound of the inven Printing Press, Lancaster, Pa., 1928. tion is administered to a human or animal body, and 30 Willstatter, R., Isler, M.; Ann. Chem., 390, 269 (1912). after a certain period of time, light rays are applied to Fisher, H., Baumler, R.; Ann Chem, 474, 65 (1929). the part to be examined. When an endoscope can be Fisher, H., Siebel, H.; Ann. Chenn., 499, 84 (1932). used for the affected part, such as lungs, gullet, stom Conant, J. B., Mayer, W. W.; J. Amer. Chem. Soc, 52, ach, womb, urinary bladder or rectum, it is irradiated 3013 (1930). using the endoscope, and the tumor portion selectively 35 emits fluorescence. This portion is observed visually, or Chlorin e4 observed through an adapted fiber scope by eye or on a Fisher, H., Heckmaier, J., Plotz, E.; Justus Leibigs Ann. CRT Screen. Chem, 500,215 (1933). In phototherapy, after administration of the dosage, Chlorin e6, e4, isochlorin e4, mesocchlorin e6, bacterio the irradiation is carried out by laser beams from the tip 40 pheophorbide, bacteriochlorin e6 of quartz fibers. Besides the irradiation of the surface of Fischer and Orth, “Des Chemie des Pyrrole' Akademische tumor, the internal part of the tumor can be irradiated Verlazsgesellschaft, Leipzio, 1940, Vol II, Part 2. by inserting the tip of quartz fibers into the tumor. The irradiation can be visually observed or imaged on a General Reference for Porphyrins CRT Screen. 45 “Porphyrins and Metalloporphyrins' ed. Kevin M. For photodiagnosis, light of wavelengths between Smith, Elsevier 1975 N.Y. 360 and 760 nm. is suitable for activating the present The compounds of the present invention can be ad tetrapyrrole compounds. Of course, each compound ministered to the host in a variety of forms adapted to has a specific optimal wavelength of activation. A long the chosen route of administration, i.e., orally, intrave wavelength ultraviolet lamp is particularly suitable for SO nously, intramuscularly or subcutaneous routes. photodiagnosis. Similar methods for viewing of the The active compound may be orally administered, for treated tumor can be used as already described for pho example, with an inert diluent or with an assimilable totherapy. edible carrier, or it may be enclosed in hard or soft shell The dosages of the present new compounds will vary gelatin capsule, or it may be compressed into tablets, or depending on the desired effect, whether for diagnosis 55 or for treatment. For diagnosis, doses of as little as 1 mg it may be incorporated directly with the food of the /kg will be effective, and up to about 20 mg/kg can be diet. For oral therapeutic administration, the active used. For treatment, the dose will usually approximate compound may be incorporated with excipients and about 0.5 mg/kg. Of course, the dosage for either diag used in the form of ingestible tablets, buccal tablets, nosis or treatment can be varied widely in view of 60 troches, capsules, elixirs, suspensions, syrups, wafers, aforesaid advantageous properties of the present con and the like. Such compositions and preparations should pounds, e.g., the ease of elimination from the host, for contain at least 0.1% of active compound. The percent Oe age of the compositions and preparations may, of The present compounds are apparently non-toxic at course, be varied and may conveniently be between the dosage levels employed for diagnosis or treatment. 65 about 2 to about 60% of the weight of the unit. The No mortality of test animals due the present compounds amount of active compound in such therapeutically has been noted in studies employing dosage levels up to useful compositions is such that a suitable dosage will be 20 mg/kg. obtained. Preferred compositions or preparations ac 4,977,177 11 12 cording to the present invention are prepared so that an medium and the required other ingredients from those oral dosage unit form contains between about 50 and enumerated above. In the case of sterile powders for the 300 mg of active compound. preparation of sterile injectable solutions, the preferred The tablets, troches, pills, capsules and the like may methods of preparation are vacuum drying and the also contain the following: A binder such as gum traga freeze-drying technique which yield a powder of the canth, acacia, corn starch or gelatin; excipients such as active ingredient plus any additional desired ingredient dicalcium phosphate; a disintegrating agent such as from previously sterile-filtered solution thereof. corn starch, potato starch, alginic acid and the like; a The present new compounds may also be applied lubricant such as sucrose, lactose or saccharin may be directly to tumors, whether internal or external, in the added or a flavoring agent such as peppermint, oil of O host in topical compositions. Exemplary compositions wintergreen, or cherry flavoring. When the dosage unit include solutions of the new compounds in solvents, form is a capsule, it may contain, in addition to materials particularly aqueous solvents, most preferably water. of the above type, a liquid carrier. Various other materi Alternatively, for topical application particularly to als may be present as coatings or to otherwise modify skin tumors, the present new compounds may be dis the physical form of the dosage unit. For instance, tab 15 persed in the usual cream or salve formulations com lets, pills, or capsules may be coated with shellac, sugar monly used for this purpose or may be provided in the or both. A syrup or elixir may contain the active com pound, Sucrose as a Sweetening agent, methyl and pro form of spray solutions or suspensions which may in pylparabens as preservatives, a dye and flavoring such clude a propellant usually employed in aerosol prepara as cherry or orange flavor. Of course, any material used tions. in preparing any dosage unit form should be pharma As used herein, "pharmaceutically acceptable car ceutically pure and substantially non-toxic in the rier' includes any and all solvents, dispersion media, amounts employed. In addition, the active compound coatings, antibacterial and antifungal agents, isotonic may be incorporated into sustained-release preparations and absorption delaying agents and the like. The use of and formulations. 25 such media and agents for pharmaceutical active sub The active compound may also be administered par stances is well known in the art. Except insofar as any enterally or intraperitoneally. Solutions of the active conventional media or agent is incompatible with the compound as a free base or pharmacologically accept active ingredient, its use in the therapeutic compositions able salt can be prepared in water suitably mixed with a is contemplated. Supplementary active ingredients can surfactant such as hydroxypropylcellulose. Dispersions also be incorporated into the compositions. can also be prepared in glycerol, liquid polyethylene It is especially advantageous to formulate parenteral glycols, and mixtures thereof and in oils. Under ordi compositions in dosage unit form for ease of administra nary conditions of storage and use, these preparations tion and uniformity of dosage. Dosage unit form as used contain a preservative to prevent the growth of micro herein refers to physically discrete units suited as uni organisms. 35 tary dosages for the mammalian Subjects to be treated; The pharmaceutical forms suitable for injectable use each unit containing a predetermined quantity of active include sterile aqueous solutions or dispersions and . material calculated to produce the desired therapeutic sterile powders for the extemporanous preparation of effect in association with the required pharmaceutical sterile injectable solutions or dispersions. In all cases the carrier. The specification for the novel dosage unit form must be sterile and must be fluid to the extent that 40 forms of the invention are dictated by and directly de easy syringability exists. It must be stable under the pendent on (a) the unique characteristics of the active conditions of manufacture and storage and must be material and the particular therapeutic effect to be preserved against the contaminating action of microor achieved, and (b) the limitations inherent in the art of ganisms such as bacteria and fungi. The carrier can be a compounding such an active material for the treatment Solvent or dispersion medium containing, for example, 45 of tumors in living subjects. water, ethanol, polyol (for example, glycerol, propy The following examples further illustrate the inven lene glycol, and liquid polyethylene glycol, and the Ol. like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the Mono-, di and Trianides use of a coating such as lecithin, by the maintenance of 50 EXAMPLE 1. the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of Di and mono(DL) serinyl mesoporphyrin IX (mixed microorganisms can be brought about by various anti anhydrides method) bacterial and antifungal agents, for example, parabens, 400 mg (0.0007 moles) of mesoporphyrin IX was chlorobutanol, phenol, Sorbic acid, thimerosal, and the 55 suspended in 50 ml of tetrahydrofuran (THF). 360 ul like. In many cases, it will be preferable to include iso (0.0035 moles) of triethylamine was added with stirring. tonic agents, for example, sugars or sodium chloride. After 10 minutes, 340 ul (0.0031 moles) ethyl chlorofor Prolonged absorption of the injectable compositions mate was added. After stirring 10 minutes, 10 ml (0.01 can be brought about by the use in the compositions of moles) of 1M KOH containing 761 mg (0.0072 moles) of agents delaying absorption, for example, aluminum 60 DL serine was added to the THF solution. This mixture monostearate and gelatin. was stirred 60 minutes at room temperature. Sterile injectable solutions are prepared by incorpo The organic solvent was flashed off and the reaction rating the active compound in the required amount in mixture was checked by silica TLC for product. Ben the appropriate solvent with various of the other ingre zene/methanol/88% formic acid (8.5/1.5/0.13) was dients enumerated above, as required, followed by fil 65 used to develop the chromatrogram. tered sterilization. Generally, dispersions are prepared After checking for product, the solution was adjusted by incorporating the various sterilized active ingredient to pH 7.5-8.0 and placed on a reverse phase (C-18 silica) into a sterile vehicle which contains the basic dispersion column 2.5X30cm. The reaction mixture was resolved 4,977,177 13 14 using a linear gradient of 20-70% methanol in 0.01M mesoporphyrin IX and unsubstituted mesoporphyrin KPO4 buffer pH 6.85 (1 liter total volume). IX. The column effluent was collected via fraction col The methanol was flashed off and the material was lector and the tube contents were pooled according to precipitated at pH 2.5-3.0. The ppt. was washed 3 times individual components. The order of elution was di with dilute acetic acid in water. The product was dried DL-serinyl mesoporphyrin IX, mono-DL-serinyl meso under vacuum. porphyrin IX and unsubstituted mesoporphyrin IX. The methanol was flashed off and the material was EXAMPLE 4 precipitated at pH 2.5-3.0. The ppt. was washed 3 times Di and mono (3alanyl mesoporphyrin IX (mixed with dilute acetic acid in water. The product was dried O anhydride method) under vacuum. The yield of di (DL) serinyl mesopor phyrin IX was 95.6 mg. 400 mg (0.0007 moles) of mesoporphyrin IX was suspended in 100 ml of tetrahydrofuran (THF). 360 ul EXAMPLE 2 (0.0035 moles) of triethylamine was added with stirring. Di and mono glycyl mesoporphyrin IX (mixed 15 After 10 minutes, 340 ul (0.0031 moles) ethyl chlorofor anhydride method) mate was added. After stirring 10 minutes, 10 ml (0.01 100 mg (0.000175 moles) of mesoporphyrin IX was moles) of 1M KOH containing 400 mg (0.0044 moles) of suspended in 100 ml of tetrahydrofuran (THF). 360 ul (3alanine was added to the THF solution. This mixture (0.0035 moles) of triethylamine was added with stirring. was stirred 60 minutes at room temperature. After 10 minutes, 340 ul (0.0031 moles) of ethylchloro 20 The organic solvent was flashed off and the reaction formate was added. After stirring 10 minutes, 10 ml mixture was checked by silica TLC for product. Ben (0.01 moles) of 1M KOH containing 500 mg (0.0066 zene/methanol/88% formic acid (8.5/1.5/0.13) was moles) of glycine was added to the THF solution. This used to develop the chromatogram. mixture was stirred 60 minutes at room temperature. After checking for product, the solution was adjusted The organic solvent was flashed off and the reaction 25 to pH 7.5-8.0 and placed on a reverse phase (C-18 silica) mixture was checked by silica TLC for product. Ben column 2.530 cm. The reaction mixture was resolved zene/methanol/88% formic acid (8.5/1.5/0/13) was using a linear gradient of 40–80% methanol in 0.01M used to develop the chromatogram. KPO4 buffer pH 6.85 (1 1 total volume). After checking for product, the solution was adjusted The column effluent was collected via fraction col to pH 7.5-8.0 and placed on a reverse phase (C-18 silica) 30 lector and the tube contents were pooled according to column 2.5X30cm. The reaction mixture was resolved individual components. The order of elution was di-6- using a linear gradient of zero to 50% methanol in alanyl mesoporphyrin IX, mono-6-alanyl mesoporphy 0.01M KPO4 buffer pH 6.85 (1 1 total volume). rin IX, and unsubstituted mesoporphyrin IX. The column effluent was collected in a fraction col The methanol was flashed off and the material was lector and the contents were sorted according to indi 35 precipitated at pH 2.5-3.0. The ppt. was washed 3 times vidual components. The order of elution was diglycyl with dilute acetic acid in water. The product was dried mesoporphyrin IX, monoglycyl mesoporphyrin IX and under vacuum. The yield for di-g-alanyl mesoporphy unsubstituted mesoporphyrin IX. rin IX was 40 mg; the yield for mono-6-alanyl mesopor The methanol was flashed off and the material was phyrin IX was 23 mg. precipitated at pH 2.5-3.0. The ppt. was washed 3 times with dilute acetic acid in water. The product was dried EXAMPLE 5 under vacuum. Di and mono e amino-n-caproyl mesoporphyrin IX EXAMPLE 3 (mixed anhydride method) Di and Mono a CDL) alanyl mesoporphyrin IX (mixed 45 400 mg (0.0007 moles) of mesoporphyrin IX was anhydride method) suspended in 50 ml of tetrahydrofuran (THF). 360 ul 100 mg (0.0007 moles) of mesoporphyrin IX was (0.0035 moles) of triethylamine was added with stirring. suspended in 100 ml of tetrahydrofuran (THF). 210 ul After 10 minutes, 340 ul (0.0031 moles) of ethylchloro (0.002 moles) of triethylamine was added with stirring. formate was added. After stirring 10 minutes, 10 ml After 10 minutes 195ul (0.00177 moles) of ethylchloro SO (0.01 moles) of 1M KOH containing 543 mg (0.00414 formate was added. After stirring 10 minutes, 10 ml moles) of e-amino-n-caproic acid was added to the THF (0.01 moles) of 1M KOH containing 500 mg (0.0056 solution. This mixture was stirred 60 minutes at room moles) of a(DL) alanine was added to the THF solu temperature. tion. This mixture was stirred 60 minutes at room tem The organic solvent was flashed off and the reaction perature. 55 mixture was checked by silica TLC for product. Ben The organic solvent was flashed off and the reaction zene/methanol/88% formic acid (8.5/1 5/0.13) was mixture was checked by silica TLC for product. Ben used to develop the chromatogram. zene/methanol/88% formic acid (8.5/1.5/0.13) was After checking for product, the solution was adjusted used to develop the chromatogram. to pH 7.5-8.0 and placed on a reverse phase (C-18 silica) After checking for product, the solution was adjusted column 2.5X30cm. The reaction mixture was resolved to pH 7.5-8.0 and placed on a reverse phase (C-18 silica) using a linear gradient of 20-70% methanol in 0.01M column 2.5X30cm. The reaction mixture was resolved KPO4 buffer pH 6.85 (11 total volume). using a linear gradient of 20-70% methanol in 0.01M The column effluent was collected via fraction col KPO4 buffer pH 6.85 (1 1 total volume). lector and the tube contents were pooled according to The column effluent was collected via fraction col 65 individual components. The order of elution was di-e- lection and the tube contents were sorted according to amino-n-caproyl mesoporphyrin IX, mono-e-amino-n- individual components. The order of elution was di caproyl mesoporphyrin IX, and unsubstituted mesopor a(DL) alanyl mesoporphyrin IX, mono-o (DL)-alanyl phyrin IX. 4,977,177 15 16 The methanol was flashed off and the material was tions of pure di-L-a-serinyl chlorin e6 were pooled. The precipitated at pH 2.5-3.0. The ppt. was washed three methanol was flashed off and the product was precipi times with dilute acetic acid in water. The product was tated at pH 2.5-3.0. The precipitate was centrifuged dried under vacuum. The yield of di-e-amino-n-caproyl down, washed three times with dilute acetic acid, and mesoporphyrin IX was 237 mg. dried under vacuum. The yield was 200 mg of di-L-a- EXAMPLE 6 Serinyl chlorin e6. Utilizing the aforementioned carbodiimide or the Di and mono-f3-alanyl Hematoporphyrin IX (mixed mixed anhydride methods, the following preferred anhydride method) compounds of this invention can be synthesized: 400 mg (0.00059 moles) Hematoporphyrin IX dihyd rochloride was suspended in 50 ml of tetrahydrofuran Chlorin Derivatives (THF). Di - (DL)-serinyl-trans-mesochlorin IX 360 ul (0.0035 moles) of triethylamine was added Di - glycyl-trans-mesochlorin IX with stirring. After 10 minutes, 340 ul (0.0031 moles) of Di - a-(DL)-alanyl-trans-mesochlorin IX ethyl chloroformate was added. After stirring 10 min Di -g-alanyl-trans-mesochlorin IX utes, 10 ml (0.01 moles) of 1M KOH containing 400 mg Di -e-amino-n-caproyl-mesochlorin IX (0.0044 moles) of galanine was added to the THF solu tion. This mixture was stirred 60 minutes at room tem Di, tri- (D,L)-serinyl chlorin e6 perature. Di, tri- (D,L)-serinyl mesochlorin e6 The organic solvent was removed by flash evapora Di, tri-glycyl chlorin e6 tion, keeping the temperature below 50° C. The reac Di, tri-glycyl mesochlorin e6 tion mixture was checked for product by silica TLC Di, tri- a-(D,L)-alanyl chlorin e6 using Benzene/methanol/88% formic acid Di, tri-a-(D,L)-alanyl mesochlorin e6 (8.5/1.5/0.13) as solvent to develop the chromatogram. Di, tri- (3-alanyl chlorin e6 The solution was adjusted to pH 7.5-8.0 with HCl Di, tri- 3-alanyl mesochlorin e6 and placed on a reverse phase (C-18 silica) column Di, tri- e-amino-n-caproyl chlorin e6 2.5X30 cm. Di, tri-e-amino-n-caproyl mesochlorin e6 The mixture was resolved using a linear gradient of Di- (D,L)-serinyl chlorin e4 40-80% methanol in 0.01M KPO4 buffer pH 6.85 (1 liter Di- (D,L)-serinyl mesochlorin e4. total volume). The individual components were col 30 Di- (D,L)-serinyl isochlorines lected as they came off the column in the order di-g-ala Di- (D,L)-serinyl mesoisochlorin e4 nyl hematoporphyrin IX, mono-6-alanyl hematopor Di-glycyl chlorin e4 phyrin IX and hematoporphyrin IX. Di-glycyl mesochlorin e4 The methanol was removed from each component by Di- glycyl isochlorin ea. flash evaporation and the material was precipitated by 35 Di- glycy mesoisochlorin e4. adjusting the pH to 2.5-3.0 using HCl. The precipitate Di- a-(DL)-alanyl chlorin ea. was washed three times with dilute acetic acid in water Di- a-(DL)-alanyl mesochlorin e4. at the centrifuge and the products dried under vacuum. Di- a-(DL)-alanyl isochlorin e4 The yield of di-6-alanyl hematoporphyrin IX was 52 Di- a-(DL)-alanyl mesoisochlorin e4. mg and of mono-6-alanyl hematoporphyrin was 30 mg. Di-g-alanyl chlorin e4 Di-g-alanyl mesochlorin e4. EXAMPLE 7 Di-6-alanyl isochlorin e4 Di-L-a-Serinyl chlorin e6 (mixed anhydride method) Di-g-alanyl mesoisochlorin e4 650 mg chlorin e6 was dissolved in 30 ml of dimethyl Di- e-amino-n-caproyl chlorin ed formamide (DMF). 277 ul (0.002 moles) of triethylam 45 Di- e-amino-n-caproyl mesochlorin e4. ine was added to the DMF solution. After stirring for Di-e-amino-n-caproyl isochlorin e4 five minutes, 201 ul (0.002 moles) of ethyl chlorofor Di- e-amino-n-caproyl mesoisochlorin e4 mate was added and stirring was continued for an addi Di- (D,L)-serinylphotoprotoporphyrin IX tional 30 minutes. 0.95 g (0.009 moles) of L-a-serine was Di-glycylphotoprotoporphyrin IX added to the DMF solution and allowed to stir for one 50 Di-a-(D,L)-alanylphotoprotoporphyrin IX hour at 50-60 C. Di-6-alanylphotoprotoporphyrin IX The DMF solution was checked for product forma Di-e-amino-n-caproylphotoprotoporphyrin IX tion by reverse phase (C-18 silica) TLC using me thanol/0.01M sodium phosphate buffer, pH 6.85, Porphyrin Derivatives (7.0/3.0) to develop the chromatogram. The DMF solu 55 Di- (D,L)-serinylmesoporphyrin IX tion was flash evaporated to near dryness and the reac Di-glycylmesoporphyrin IX tion mixture was then taken up in dilute NaOH and the Di-a-(DL)-alanylmesoporphyrin IX pH was adjusted to 2.5-3.0 to precipitate out the mix Di-g-alanylmesoporphyrin IX ture. The precipitate was then centrifuged down and Di-e-amino-n-caproylmesoporphyrin IX washed twice with diluted acetic acid in water. The Di-(D,L)-serinylprotoporphyrin IX precipitate was then centrifuged down and washed Di-glycylprotoporphyrin IX twice with diluted acetic acid in water. The precipitate Di-a-(D,L)-alanylprotoporphyrin IX was then redissolved in dilute NaOH and the pH ad Di-6-alanylprotoporphyrin IX justed to 7.0. This was applied to a reverse phase (C-18 Di-e-amino-n-caproylprotoporphyrin IX silica) column 3.7 cm x 45 cm. 65 The product was eluted from the column with a solu tion of 0.01M sodium phosphate buffer, pH 6.85/me thanol (7.0/3.0). Fractions were collected and the frac 4,977,177 17 18 Di-(D,L)-serinyldeuteroporphyrin IX Di-Cysteinyl bacterioisochlorin e4 Di-glycyldeuteroporphyrin IX Di, tri-Cysteinyl bacteriochlorin e6 Di-O-(D,L)-alanyldeuteroporphyrin IX Di-Tyrosyl trans-mesochlorin IX Di-6-alanyldeuteroporphyrin IX Di, tri-Tyrosyl chlorin e6 Di-e-amino-n-caproyldeuteroporphyrin IX 5 Di, tri-Tyrosyl mesochlorines Di, tri, tetra- (D,L)-serinylcoproporphyin III Di-Tyrosyl chlorin e4. Di, tri, tetra-glycylcoproporphyrin III Di-Tyrosyl mesochlorin e4 Di, tri, tetra-a-(D,L)-alanylcoproporphyrin III Di-Tyrosyl isochlorin e4 Di, tri, tetra-g-alanylcoproporphyrin III Di-Tyrosyl mesoisochlorin ea. Di, tri, tetra-e-amino-n-caproylcoproporphyrin III 10 Di-Tyrosyl photoprotoporphryin IX Di-(D,L)-serinylhematoporphyrin IX Di-Tyrosyl mesoporphyrin IX Di-glycylhematoporphyrin IX Di-Tyrosyl protoporphyrin IX Di-a-(D,L)-alanylhematoporphyrin IX Di-Tyrosyl deuteroporphyrin IX Di-6-alanylhematoporphyrin IX Di,tri, tetra-Tyrosyl coproporphyrin III Di-e-amino-n-caproylhematoporphyrin IX 5 Di-Tyrosyl hematoporphryin IX Di-Tyrosyl bacteriochlorin e4. Bacteriochlorin Derivatives Di-Tyrosy bacterioisochlorin e4. Di-(D,L)-serinylbacteriochlorin e4 Di,tri-Tyrosyl bacteriochlorin e6 Di-glycylbacteriochlorin e4 Di-Valyl trans-mesochlorin IX Di-a-(DL)-alanylbacteriochlorin e4 20 Di, tri-Valyl chlorin e6 Di-3-alanylbacteriochlorin e4 Di, tri-Valyl mesochlorin e6 Di-e-amino-n-caproylbacteriochlorin e4 Di-Valyl chlorin ea. Di-(D,L)-serinylbacterioisochlorin e4 Di-Valyl mesochlorin e4. Di-glycylbacterioisochlorin e4. Di-Valyl isochlorin e4 Di- a-(D,L)-alanylbacterioisochlorin e4 25 Di-Valyl mesoisochlorin e4 Di- (3-alanylbacterioisochlorin e4 Di-Valyl photoprotoporphyrin IX Di-e-amino-n-caproylbacterioisochlorin e4 Di-Valyl mesoporphyrin IX Di, tri- (D,L)-serinylbacteriochlorin e6 Di-Valyl protoporphyrin IX Di, tri-glycylbacteriochlorin e6 Di-Valyl deuteroporphyrin IX Di, tri-a-(D,L)-alanylbacteriochlorin e6 Di,tri, tetra-Valyl coproporphyrin III Di, tri- 3-alanylbacteriochlorin e6 Di-Valyl hematoporphyrin IX Di, tri-e-amino-n-caproylbacteriochlorin e6 Di-Valyl bacteriochlorin e4. Similarly, by utilizing other amino acids, peptides Di-Valyl bacterioisochlorin e4 which further illustrate embodiments of, but do not Di, tri-Valyl bacteriochlorin e6 limit the present invention, can be employed: 35 Di-Luccyl trans-mesochlorin IX Di-Threoninyl trans-mesochlorin IX Di,tri-Leucyl chlorin e6 Di,tri-Threoninyl chlorin e6 Di, tri-Leucyl mesochlorin e6 Di,tri-Threoninyl mesochlorin e6 Di-Leucyl chlorin e4 Di-Threoninyl chlorin e4 Di-Leucyl mesochlorin e4 Di-Threoninyl mesochlorin e4 Di-Leucyl isochlorin e4 Di-Threoninyl isochlorin e4 Di-Leucyl mesoisochlorin e4 Di-Threoninyl mesoisochlorin e4 Di-Leucyl photoprotoporphyrin IX Di-Threoninyl photoprotoporphyrin IX Di-Leucyl mesoporphyrin IX Di-Threoninyl mesoporphyrin IX Di-Leucyl protoporphyrin IX Di-Threoninyl protoporphyrin IX 45 Di-Leucyl deuteroporphyrin IX Di-Threoninyl deuteroporphyrin IX Di,tri, tetra-Leucyl coproporphyrin III Di,tri, tetra-Threoninyl coproporphyrin III Di-Leucyl hematoporphyrin IX Di-Threoninyl hematoporphyrin IX Di-Leucyl bacteriochlorin e4 Di-Threoninyl bacteriochlorin e4 50 Di-Leucyl bacterioisochlorin e4 Di-Threoninyl bacterioisochlorin e4 Di, tri-Leucyl bacteriochlorin e6 Di, tri-Threoninyl bacteriochlorin e6 Di-Isoleucyl trans-mesochlorin IX Di-Cysteinyl trans-mesochlorin IX Di, tri-Isoleucyl chlorin e6 Di, tri-Cysteinyl chlorin e6 Di, tri-Isoleucyl mesochlorin e6 Di, tri-Cysteinyl mesochlorin e6 55 Di-Isoleucyl chlorin e4 Di-Cysteinyl chlorin e4. Di-Isoleucyl mesochlorin e4. Di-Cysteinyl isochlorin e4 Di-Isoleucyl isochlorin e4 Di-Cysteinyl mesoisochlorin e4 Di-Isoleucyl mesoisochlorin e4. Di-Cysteinyl photoprotoporphyrin IX Di-Isoleucyl photoprotoporphyrin IX Di-Cysteinyl mesoporphyrin IX Di-Isoleucyl mesoporphyrin IX Di-Cysteinyl protoporphyrin IX Di-Isoleucyl protoporphyrin IX Di-Cysteinyl deuteroporphyrin IX Di-Isoleucyl deuteroporphyrin IX Di,tri, tetra-Cysteinyl-coproporphyrin III Di,tri, tetra-Isoleucyl coproporphyrin III Di-Cysteinyl hematoporphyrin IX Di-Isoleucyl hematoporphyrin IX Di-Cysteinyl bacteriochlorin e4. 65 4,977, 177 19 20 Di-Isoleucyl bacteriochlorin e.g. Di-Methionyl hematoporphryin IX Di-Isoleucyl bacterioisochlorin e4. Di-Methionyl bacteriochlorin e4 Di, tri-Isoleucyl bacteriochlorin e6 Di-Methionyl bacterioisochlorin e4 Di-Prolyl trans-mesochlorin IX Di, tri-Methionyl bacteriochlorin e6 Di, tri-Prolyl chlorin e6 Di-Histidyl trans-mesochlorin IX Di, tri-Prolyl mesochlorin e6 Di, tri-Histidyl Chlorin e6 Di-Prolyl chlorine Di, tri-Histidyl mesochlorines Di-Prolyl mesochlorin e4 Di-Histidyl chlorin e4. Di-Prolyl isochlorine Di-Histidyl mesochlorin e4 Di-Prolyl mesoisochlorin e4 10 Di-Histidyl isochlorin e4 Di-Prolyl photoprotoporphyrin IX Di-Histidyl mesoisochlorin e4. Di-Prolyl mesoporphryin IX Di-Histidyl photoprotoporphyrin IX Di-Prolyi protoporphyrin IX Di-Histidyl mesoporphyrin IX Di-Prolyl deuteroporphyrin IX Di-Histidyl protoporphyrin IX Di,tri, tetra-Prolyl coproporphyrin III 5 Di-Histidyl deuteroporphyrin IX Di-Prolyl hematoporphyrin IX Di,tri, tetra-Histidyl coproporphyrin III Di-Prolyi bacteriochlorin e4 Di-Histidyl hematoporphyrin IX Di-Prolyl bacterisochlorin e. Di-Histidyl bacteriochlorin e4 Di, tri-Prolyl bacteriochlorin e6 Di-Histidyl bacterioisochlorin e4. Di-Phenylalanyl trans-mesochlorin IX Di, tri-Histidyl bacteriochlorin e6 Di, tri-Phenylalanyl chlorin e6 Di-Arginyl trans-mesochlorin IX Di, tri-Phenylalanyl mesochlorin e6 Di, tri-Arginyl chlorin e6 Di-Phenylalanyl chlorin e4 Di,tri-Arginyl mesochlorin e6 Di-Phenylalanyl mesochlorin e4. Di-Arginyl chlorin ea. Di-Phenylalanyl isochlorin e4 25 Di-Arginyl mesochlorin e. Di-Phenylalanyl mesoisochlorin e4 Di-Arginyl isochlorin e4. Di-Phenylalanyl photoprotoporphyrin IX Di-Arginyl mesoisochlorin e4. Di-Phenylalanyl mesoporphyrin IX Di-Arginyl photoprotoporphyrin IX Di-Phenylalanyl protoporphyrin IX Di-Arginyl mesoporphryin IX Di-Phenylalanyl deuteroporphyrin IX 30 Di-Arginyl protoporphyrin IX Di,tri, tetra-Phenylalanyl coproporphyrin III Di-Arginyl deuteroporphyrin IX Di-Phenylalanyl hematoporphyrin IX Di,tri, tetra-Arginyl coproporphyrin III Di-Phenylalanyl bacteriochlorin e4 Di-Arginyl hematoporphyrin IX Di-Phenylalanyl bacterioisochlorin e4 Di-Arginyl bacteriochlorin e. Di,tri-Phenylalanyl bacteriochlorin e6 35 Di-Arginyl bacterioisochlorin e4 Di-Tryptophyl trans-mesochlorin IX Di, tri-Arginyl bacteriochlorin e6 Di, tri-Tryptophyl chlorin e6 Di-Lysyl trans-mesochlorin IX Di, tri-Tryptophyl mesochlorin e6 Di,tri-Lysyl chlorin e6 Di-Tryptophy chlorin ea. Di, tri-Lysyl mesochlorin e6 Di-Tryptophyl mesochlorin ed Di-Lysyl chlorin e4 Di-Tryptophyl isochlorin e. Di-Lysyl mesochlorin e4. Di-Tryptophyl mesoisochlorin ea. Di-Lysyl isochlorin e4 Di-Tryptophyl photoprotoporphyrin IX Di-Lysyl mesoisochlorin e4. Di-Tryptophyl mesoporphyrin IX Di-Lysyl photoprotoporphyrin IX Di-Tryptophyl protoporphyrin IX 45 Di-Lysyl mesoporphyrin IX Di-Tryptophyl deuteroporphyrin IX Di-Lysyl protoporphyrin IX Di,tri, tetra-Tryptophyl coproporphyrin III Di-Lysyl deuteroporphyrin IX Di-Tryptophyl hematoporphyrin IX Di, tri-tetra-Lysyl coproporphyrin III Di-Tryptophyl bacteriochlorine Di-Lysyl hematoporphyrin IX Di-Tryptophy bacterioisochlorin e. 50 Di-Lysyl bacteriochlorin ea. Di, tri-Tryptophyll bacteriochlorin e6 Di-Lysyl bacterioisochlorin e4. Di-Methionyl trans-mesochlorin IX Di, tri-Lysyl bacteriochlorin e6 Di, tri-Methionyl chlorin e6 Di-Glutaminyl trans-mesochlorin IX Di, tri-Methionyl mesochlorines Di, tri-Glutaminyl chlorin e6 Di-Methionyl chlorin e4 55 Di, tri-Glutaminyl mesochlorin e6 Di-Methionyl mesochlorines Di-Glutaminyl chlorines Di-Methionyl isochlorin e4 Di-Glutaminyl mesochlorin ei Di-Methionyl mesoisochlorines Di-Glutaminyl isochlorin e4. Di-Methionyl photoprotoporphyrin IX Di-Glutaminyl mesoisochlorin e4 Di-Methionyl mesoporphyrin IX Di-Glutaminyl photoprotoporphyrin IX Di-Methionyl protoporphyrin IX Di-Glutaminyl mesoporphyrin IX Di-Methionyl deuteroporphyrin IX Di-Glutaminyl protoporphyrin IX Di,tri, tetra-Methionyl coproporphyrin III Di-Glutaminyl deuteroporphyrin IX 65 4,977,177 21 22 Di,tri, tetra-Glutaminyl coproporphyrin III zene/methanol/88% formic acid (8.5/1.5/0/13) was Di-Glutaminyl hematoporphyrin IX used to develop the chromatogram. Di-Glutaminyl bacteriochlorin e4 After checking for product, the solution was adjusted Di-Glutaminyl bacterioisochlorin e4. to pH 7.5-8.0 and placed on a reverse phase (C-18 silica) Di, tri-Glutaminyl bacteriochlorin e6 column 2.5X30cm. The reaction mixture was resolved Di-Asparginyl trans-mesochlorin IX using a linear gradient of zero to 50% methanol in Di, tri-Asparginyl chlorin e6 0.01M KPO4 buffer pH 6.85 (1 1 total volume). Di, tri-Asparginyl mesochlorin e6 The column effluent was collected in a fraction col Di-Asparginyl chlorin e4 lector and the contents were sorted according to indi Di-Asparginyl mesochlorin e4 10 vidual components. The order of elution was diglycyl Di-Asparginyl isochlorin e4 mesoporphyrin IX, monoglycyl mesoporphyrin IX and Di-Asparginyl mesoisochlorin e4 unsubstituted mesoporphyrin IX. Di-Asparginyl photoprotoporphyrin IX The methanol was flashed off and the material was Di-Asparginyl mesoporphyrin IX precipitated at pH 2.5-3.0. The ppt. was washed 3 times Di-Asparginyl protoporphyrin IX 15 Di-Asparginyl deuteroporphyrin IX with dilute acetic acid in water. The product was dried Di,tri, tetra-Asparginyl coproporphyrin III under vacuum. Di-Asparginyl hematoporphyrin IX EXAMPLE 10 Di-Asparginyl bacteriochlorin e4. Mono a(DL) alanyl mesoporphyrin IX (Mixed Di-Asparginyl bacterioisochlorin e4 20 Di, tri-Asparginyl bacteriochlorin e6 anhydride method) 100 mg (0.000175 moles) of mesoporphyrin IX was Monoamides suspended in 100 ml of tetrahydrofuran (THF). 210 ul EXAMPLE 8 (0.002 moles) of triethylamine was added with stirring. Mono (DL) serinyl mesoporphyrin IX (mixed 25 After 10 minutes 195ul (0.00177 moles)of ethylchloro anhydride method) formate was added. After stirring 10 minutes, 10 ml (0.01 moles) of 1M KOH containing 500 mg (0.0056 moles) of 400 mg (0.0007 moles) of mesoporphyrin IX was suspended in 50 ml of tetrahydrofuran (THF). 360 ul a(DL) alanine was added to the THF solution. This (0.0035 moles) of triethylamine was added with stirring. mixture was stirred 60 minutes at room temperature. After 10 minutes, 340 ul (0.0031 moles) ethyl chlorofor 30 The organic solvent was flashed off and the reaction mate was added. After stirring 10 minutes, 10 ml (0.01 mixture was checked by silica TLC for product. Ben moles) of 1M KOH containing 761 mg (0.0072 moles) of Zene/methanol/88% formic acid (8.5/1.5/0.13) was DL serine was added to the THF solution. This mixture used to develop the chromatogram. was stirred 60 minutes at room temperature. After checking for product, the solution was adjusted The organic solvent was flashed off and the reaction 35 to pH 7.5-8.0 and placed on a reverse phase (C-18 silica) mixture was checked by silica TLC for product. Ben column 2.5X30cm. The reaction mixture was resolved zene/methanol/88% formic acid (8.5/1.5/0.13) was using a linear gradient of 20-70% methanol in 0.01M used to develop the chromatrogram. KPO4 buffer pH 6.85 (1 1 total volume). After checking for product, the solution was adjusted The column effluent was collected via fraction col to pH 7.5-8.0 and placed on a reverse phase (C-18 silica) lection and the tube contents were sorted according to column 2.5X30 cm. The reaction mixture resolved individual components. The order of elution was di using a linear gradient of 20-70% methanol was in a(DL) alanyl mesoporphyrin IX, mono- d.(DL)-alanyl 0.01M KPO4 buffer pH 6.85 (1 liter total volume). mesoporphyrin IX and unsubstituted mesoporphyrin The column effluent was collected via fraction col IX. lector and the tube contents were pooled according to 45 The methanol was flashed off and the material was individual components. The order of elution was diseri precipitated at pH 2.5-3.0. The ppt. was washed 3 times nyl mesoporphyrin IX, monoserinyl mesoporphyrin IX with dilute acetic acid in water. The product was dried and unsubstituted mesoporphyrin IX. under vacuum. The methanol was flashed off and the material was precipitated at pH 2.5-3.0. The ppt. was washed 3 times 50 EXAMPLE 11 with dilute acetic acid in water. The product was dried Mono (3 alanyl mesoporphyrin IX (mixed anhydride under vacuum. method) EXAMPLE 9 400 mg (0.0007 moles) of mesoporphyrin IX was 55 suspended in 100 ml of tetrahydrofuran (THF). 360 ul Mono qlycyl mesoporphyrin IX (mixed anhydride (0.0035 moles) of triethylamine was added with stirring. method) After 10 minutes, 340 ul (0.0031 moles) ethyl chlorofor 100 mg (0.000175 moles) of mesoporphyrin IX was mate was added. After stirring 10 minutes, 10 ml (0.01) suspended in 100 ml of tetrahydrofuran (THF). 360 ul moles) of 1M KOH containing 400 mg (0.0044 moles) of (0.0035 moles) of triethylamine was added with stirring. galanine was added to the THF solution. This mixture After 10 minutes, 340 ul (0.0031 moles) of ethylchloro was stirred 60 minutes at room temperature. formate was added. After stirring 10 minutes, 10 ml The organic solvent was flashed off and the reaction (0.01 moles) of 1M KOH containing 500 mg (0.0066 mixture was checked by silica TLC for product. Ben moles) of glycine was added to the THF solution. This zene/methanol/88% formic acid (8.5/1.5/0.13) was mixture was stirred 60 minutes at room temperature. 65 used to develop the chromatogram. The organic solvent was flashed off and the reaction After checking for product, the solution was adjusted mixture was checked by silica TLC for product. Ben to pH 7.5-8.0 and placed on a reverse phase (C-18 silica) column 2.5X30cm. The reaction mixture was resolved 4,977,177 23 24 using a linear gradient of 40-80% methanol in 0.01 M were collected as they came off the column in the order KPO4 buffer pH 6.85 (1 1 total volume). di-6-alanyl hematoporphyrin IX, mono-6-alanyl hema The column effluent was collected via fraction col toporphyrin IX and hematoporphyrin IX. lector and the tube contents were pooled according to The methanol was removed from each component by individual components. The order of elution was di-g- flash evaporation and the material was precipitated by alanyl mesoporphyrin IX, mono-6-alanyl mesoporphy adjusting the pH to 2.5-3.0 using HCl. The precipitate rin IX, and unsubstituted mesoporphyrin IX. was washed three times with dilute acetic acid in water The methanol was flashed off and the material was at the centrifuge and the products dried under vacuum. precipitated at pH 2.5-3.0. The ppt. was washed 3 times The yield of di-3-alanyl hematoporphyrin IX was 52mg with dilute acetic acid in water. The product was dried 10 under vacuum. The yield for mono-6-alanyl mesopor and of mono-g-alanyl hematoporphyrin was 30 mg. phyrin IX was 23 mg. EXAMPLE 4 EXAMPLE 12 Mono Glycyl Chlorin e6 (Mixed Anhydride Method) Mono e amino-n-caproyl mesoporphyrin IX (mixed 15 625 mg of chlorin e5 was dissolved in 300 ml of di anhydride method) methyl formamide (DMF) and 277 ul (0.002 moles) of 400 g (0.0007 moles) of mesoporphyrin IX was sus triethylamine (TEA) was added to the DMF solution. pended in 50 ml of tetrahydrofuran (THF). 360 ul After stirring for five minutes, 201 ul (0.002 moles) of (0.0035 moles) of triethylamine was added with stirring. ethylchloroformate (EC) was added and stirred for 1. After 10 minutes, 340 ul (0.00414 moles) of ethylchloro hours at room temperature. formate was added. After stirring 10 minutes, 10 ml 75 mg (0.0009 moles) of glycine (ammonia free) was (0.01 moles) of 1M KOH containing 543 mg (0.00369 added to the DMF solution and allowed to stir three moles) of e-amino-n-caproic acid was added to the THF hours at 50-60' C. solution. This mixture was stirred 60 minutes at room The DMF solution was tested for product by reverse temperature. 25 phase (C-18 silica) TLC using methanol/0.01M sodium The organic solvent was flashed off and the reaction phosphate buffer, pH 6.85, 70/30, to develop the chro mixture was checked by silica TLC for product. Ben matogram. zene/methanol/88% formic acid (8.5/1.5/0.13) was The DMF solution was flashed to near dryness, then used to develop the chromatogram. dissolved in dilute NaOH and the pH adjusted to 2.5-3 After checking for product, the solution was adjusted 30 to precipitate the solid. The precipitate was then placed to pH 7.5-8.0 and placed on a reverse phase (C-18 silica) on a reverse phase (C-18 silica) column 3.7 cm x 45 cm. column 2.5X30cm. The reaction mixture was resolved Fractions were eluted, using 20-40% methanol in using a linear gradient of 20–70% methanol in 0.01M 0.01M sodium phosphate buffer, pH 6.85. The fractions KPO4 buffer pH 6.85 (1 l total volume). were pooled according to individual components. The column effluent was collected via fraction col 35 lector and the tube contents were pooled according to The methanol was flashed off and the material was individual components. The order of elution was di-e- precipitated at pH 2.5-3.0. The precipitate was washed amino-n-caproyl mesoporphyrin IX, mono-e-amino-n- and centrifuged 3 times in dilute acetic acid in water. caproyl mesoporphyrin IX, and unsubstituted mesopor The product was dried under vacuum. The yield of phyrin IX. mono glycyl chlorin e6 was 87.5 mg. The methanol was flashed off and the material was Utilizing the aforementioned carbodiimide or the precipitated at pH 2.5-3.0. The ppt. was washed three mixed anhydride methods of Examples 8-14, the fol times with dilute acetic acid in water. The product was lowing preferred monoamide compounds of this inven dried under vacuum. tion are synthesized: EXAMPLE 13 45 Chlorin Derivatives Mono-6-alanyl Hematoporphyrin IX (mixed anhydride (DL)-Serinyl-trans-mesochlorin IX method) Glycyl-trans-mesochlorin IX 400 mg (0.00059 moles) Hematoporphyrin IX dihyd a-(DL)-Alanyl-trans-mesochlorin IX rochloride was suspended in 50 ml of tetrahydrofuran (3-Alanyl-trans-mesochlorin IX (THF). e-Amino-n-caproyl-mesochlorin IX 36 ul (0.0035 moles) of triethylamine was added with (D,L)-Serinyl chlorines stirring. After 10 minutes, 340 ul (0.0031 moles) of ethyl (D,L)-Serinyl mesochlorin e6 chloroformate was added. After stirring 10 minutes, 10 Glycyl chlorin e6 ml (0.01 moles) of 1M KOH containing 400 mg (0.0044 55 Glycyl mesochlorin e6 moles) of 3-alanine was added to the THF solution. a-(D,L)-Alanyl chlorin e6 This mixture was stirred 60 minutes at room tempera a-(D,L) Alanyl mesochlorin e6 ture. f3-Alanyl chlorin e6 The organic solvent was removed by flash evapora £3-Alanyl mesochlorin e6 tion, keeping the temperature below 50° C. The reac e-Amino-n-caproyl chlorin e6 tion mixture was checked for product by silica TLC e-Amino-n-caproyl mesochlorin e6 using Benzene/methanol 88% formic acid (D,L)-Serinyl chlorines (8.5/1.5/0.13) as solvent to develop the chromatogram. (D,L)-Serinyl mesochlorines The solution was adjusted to pH 7.5-8.0 with HCl (D,L)-Serinyl isochlorin e. and placed on reverse phase (C-18 silica) column 65 (D,L)-Serinyl mesoisochlorin e4 2.5X30 cm. The mixture was resolved using a linear Glycyl chlorin e. gradient of 40-80% methanol in 0.01M KPO4 buffer pH 6.85 (1 liter total volume). The individual components 4,977, 177 25 26 Glycyl mesochlorin e4. Glycylbacterioisochlorin e4 Glycyl isochlorin e4 a-(DL)-Alanylbacterioisochlorin e4 Glycyl mesoisochlorin e4 g-Alanylbacterioisochlorin e4 a-(DL)-Alanyl chlorin e4 e-Amino-n-caproylbacterioisochlorin e4 C-(DL)-Alanyl mesochlorin e4 (D,L)-Serinylbacteriochlorin e6 a-(DL)-Alanyl isochlorin ea. Glycylbacteriochlorin e6 a-(DL)-Alanyl mesoisochlorin e4. a-(DL)-Alanylbacteriochlorin e6 g-Alanyl chlorin e4. g-Alanylbacteriochlorin e6 B-Alanyl mesochlorin e4. e-Amino-n-caproylbacteriochlorin e6 3-Alanyl isochlorin e4. 10 (D,L)-Serinylpyrobacteriopheophorbide a (3-Alanyl mesoisochlorin e4 Glycylpyrobacteriopheophorbide a e-Amino-n-caproyl chlorin e4 a-(D.L.) Alanylpyrobacteriopheophorbide a e-Amino-n-caproyl mesochlorin e4 g-Alanylpyrobacteriopheophorbide a e-Amino-n-caproyl isochlorin e4 e-Amino-n-caproylpyrobacteriopheophorbide a e-Amino-n-caproyl mesoisochlorin e4. 15 (D,L)-Serinylbacteriopheophorbide a (D,L)-Serinyl pyropheophorbide a Glycylbacteriopheophorbide a Glycyl pyropheophorbide a a-(D,L)-Alanylbacteriopheophorbide a a-(D,L)-Alanyl pyropheophorbide a (3-Alanylbacteriopheophorbide a (3-Alanyl pyropheophorbide a e-Amino-n-caproylbacteriopheophorbide a e-Amino-n-caproyl pyropheophorbide a 20 Other amino acid derivatives of the tetrapyrroles can (D,L)-Serinylpheophorbide a also be prepared. The following amino acids can also be Glycyl pheophorbide a used to prepare the mono- di-, tri-, or where appropri a-(D,L)-Alanylpheophorbide a ate, the tetra-amino acid derivatives of the chlorins, p3-Alanylpheophorbide a porphyrins, or bacteriochlorins, employing the proce e-Amino-n-caproylpheophorbide a 25 dures of one of the aforementioned methods: Piperi (D,L)-Serinylphotoprotoporphyrin IX dine-2-carboxylic acid, Piperidine-6-carboxylic acid, Glycylphotoprotoporphyrin IX Pyrrole-2-carboxylic acid, Pyrrole-5-carboxylic acid, a-(D,L)-Alanylphotoprotoporphyrin IX Piperidine-6-propionic acid, and Pyrrole-5-acetic acid £8-Alanylphotoprotoporphyrin IX Mixed amino acid derivatives of the tetrapyrroles can e-Amino-n-caproylphotoprotoporphyrin IX 30 also be prepared. The various chlorin derivatives, por phyrin derivatives and bacteriochlorin derivatives can Porphyrin Derivatives include any two or three of the following amino acids: (D,L)-Serinylmesoporphyrin IX Glycine, Serine, Threonine, Cysteine, Tyrosine, Aspar Glycymesoporphyrin IX agine, Glutamine, Lysine, Arginine, Histidine, a-Ala a-(DL)-Alanylmesoporphyrin IX 35 nine, (3-Alanine, Valine, Leucine, Isoleucine, Proline, g-Alanylmesoporphyrin IX a-Phenylalanine, g-Phenylalanine, Tryptophan, Methi e-Amino-n-caproylmesoporphyrin IX online, e-Amino-n-caproic acid, Piperidine-2-carboxylic (D,L)-Serinylprotoporphyrin IX acid, Pyrrole-5-carboxylic acid, Piperidine-6-propionic Glycylprotoporphyrin IX acid, Pyrrole-5-acetic acid. a-(D,L)-Alanylprotoporphyrin IX Physical characteristics of the compounds (relative (3-Alanylprotoporphyrin IX polarity) is measured by a standard chromatographic e-Amino-n-caproylprotoporphyrin IX system. The chromatographic data (Rf values) were (D,L)-Serinyldeuteroporphyrin IX measured on Baker silica gel-Cl8 thin layer chromato Glycyldeuteroporphyrin IX graphic plates, the particle size of which is 20 LM, and a-(D,L)-Alanyldeuteroporphyrin IX 45 the coating thickness of which is 200 uM. The solvent (3-Alanyldeuteroporphyrin IX system for these chromatographic runs consisted of e-Amino-n-caproyldeuteroporphyrin IX 75% methanol, and 25% 0.01 M potassium phosphate tetra- (D,L)-Serinylcoproporphyrin III buffer, pH 6.85. The Rf values for the various deriva tetra- Glycycoproporphyrin III tives are tabulated in TABLE 1. Spectroscopic data are tetra-a-(D,L)- Alanylcoproporphyrin III 50 indicated in TABLE 2. tetra-g-Alanylcoproporphyrin III: TABLE 1. tetra-e-Amino-n-caproylcoproporphyrin III (D,L)-Serinylhematoporphyrin IX Rf VALUES Glycylhematoporphyrin IX Compounds Derivative Rf 55 Mesoporphyrin IX .32 a-(D,L)-Alanylhematoporphyrin IX Mesoporphyrin IX mono-o-alanyl g-Alanylhematoporphyrin IX Mesoporphyrin IX mono-g-alanyl e-Amino-n-caproylhematoporphyrin IX Mesoporphyrin IX di-a-alanyl 51 Mesoporphyrin IX di-g-alanyl 49 Bacteriochlorin Derivatives Mesoporphyrin IX mono-glycycl 47 Mesoporphyrin IX di-seryl 58 (D,L)-Serinylbacteriochlorin e4 Mesoporphyrin IX di-glycyl 54 Glycylbacteriochlorin e4 Mesoporphyrin IX di-e-aminocaproyl .34 a-(DL)-Alanylbacteriochlorin e4. Hematoporphyrin IX 8 Hematoporphyrin IX mono-g-alanyl 83 £3-Alanylbacteriochlorin e4 Hematoporphyrin IX di-8-alanyl 83 e-Amino-n-caproylbacteriochlorin e4 65 Chlorin e6 66 (D,L)-Serinylbacterioisochlorin e4 Chlorines di-L-a-serinyl 78 4,977, 177 27 28 TABLE II mined using a Yellow Springs Instrument, Model 65A, radiometer. Spectroscopic Absorption Data Absorption The micro lens was positioned at such a distance from Maxina (nm) mM Extinction Soret the skin of the animal so as to provide an illumination in Visible Coefficient Band diameter of 1.5cm, and the light flux was varied by Compounds Region (ErnM) - 10% M control of the laser output. Photoprotoporphyrin IX 668 38 5 Subsequent to illumination, the animal was returned isoner mixture to its cage and, 24 hours later, it was treated intrave Pheophorbide a 667 35 408.6 Pyropheophorbide a 668 38 41.2 nously in the external jugular vein with 14 mg of Evans Trans-nesochlorin IX 643 60 388 10 Blue dye, dissolved in 250 ul of 0.9% NaCl. Two hours Chlorin e6 665.6 42 402 after injection, the rat was sacrificed and the tumor Bacteriopheophorbide a 753.5 44.7 359 cross-sectioned. The extent of tumor necrosis was as Hematoporphyrin deriv- 626 2.9 399 sessed by the lack of dye uptake (), and the depth of the ative (HPD) necrotic cross section of the tumor was recorded in Solvent in all cases is p-dioxane. 5 millimeters. (1) M. C. Berenbaum, Br. J. Cancer 45: 571 (1982) The following protocol describes the procedure for Table III summarizes the effects of these drugs on the utilization of these new compounds of the present tumors and includes a range of wavelengths, dosages, invention in the treatment of rat tumors. intensities, and time intervals for treatment. This has 20 been necessary, in order to attempt to establish the PROCEDURE optimal conditions for phototherapy utilizing this new The photodynamic therapy experiments have been drug. The conditions described result in measurable and carried out on Buffalo rats, using the transplantable significant damage to the tumors. tumor, Morris Hepatoma 7777. The tumors were trans In all cases except where noted, tissue damage oc planted subcutaneously on the outside of the thigh. 25 curred selectively to the tumor tissue as assayed by the During treatment, the tumors ranges in size between 1 Evans Blue method, even though, in nearly all cases, and 2.5 cm in diameter. normal skin overlayed the tumor and the treatment area The general treatment regime is as follows. The rats overlapped significant areas of normal muscle tissue. are injected with a solution of the chlorin prepared as The photodynamic therapy date is presented in tabu follows: 20 mg of the sodium salt of the chlorin was 30 lar form. Column No. 2 is the total light dose adminis dissolved in 1 ml of 0.9% NaCl. The chlorin solution tered in terms of Joules per square centimeter. Column was then injected intravenously through the external No. 3 is the dose of chlorin administered in terms of mg jugular while the rat was anesthetized with ether. The of drug per kilogram of rat body weight. Column No. 4 Volume of solution injected was calculated based upon is the time lapse between administration of drug and the weight of the animal and the dosage, on a weight to 35 treatment with laser light. Column No. 5 is the wave weight basis, for the particular experiment. A specified length of treatment light in nanometers. Column No. 6 time interval was then allowed to elapse before light is the intensity of the treatment light in milliwatts per treatment was instigated. square centimeter. In Column No. 7, x is the mean depth Light treatment of the rats was without anesthesia. of necrosis in millimeters of the tumor tissue, i.e., the The rats were restrained, the hair removed in the treat 40 distance from the necrotic top of the tumor next to the ment area and treated with laser light from a Cooper skin to the necrotic edge of the tumor most distant from Aurora argon pumped, tunable dye laser. the skin. The laser was equipped with a fiber optic light deliv S.D. is the standard deviation of x. ery system coupled to a microlens system developed by (N) is the number of tumors or legs involved in the Dr. Daniel Doiron, D.R.D. Consulting, Santa Barbara, 45 experiment. Calif. Column No. 8 is the range of depth of necrosis in The lens disperses the laser beam, providing a circu millimeters within the group. TABLE III Time in hrs Drug bitwin Wave Joules/ dose drugs & inth intensity Range Tumor cm mg/kg light nm mW/cm X s.d. (n) Mono Glycyl chlorines 7777 2O 20 24 665 OO 3.9 - 3.0 (5) 2-9 Mono-L-a-alanyl chlorin e6 7777 20 2O 24 665 OO 3.8 - 1.8 (2) 2.5-5 Mono-L-a-serinyl chlorines 7777 20 2O 24 665 100 6.3 - 2.7 (6) 3-0 Diglycyl chlorin e6 7777 20 20 24 665 100 4.7 it 1.0 (3) 3.5-5.5 "3 of 8 tumors showed no necrosis due to drug and light. lar distribution of light with homogeneous light inten- 65 What is claimed is: sity throughout the area of the incident light beam. The 1. A therapeutic composition for the detection and wavelength of light was adjusted using a Hartridge treatment of tumors sensitive thereto comprising a ther reversion spectroscope. The light intensity was deter apeutically effective amount of a fluorescent mono, di 4,977,177 29 30 or polyamide of a aminomonocarboxylic acid and a tetrapyrrole compound of the formula: -CH3, -H

R9 is H, COOH, CH2COOH or methyl; provided that when R, R2, R3, R4, R7 and R8 represent two substituents or are divalent and attached to the same carbon, the respective pyrrole ring to which at O tached is a dihydropyrrole; R is lower alkyl or benzyl; R6 and R9, taken together are

5

with the proviso that at least one of R1-R9 includes a or the corresponding di- or tetrahydrotetrapyrroles free carboxyl group, or salts thereof, and a pharmaceuti wherein cally acceptable carrier therefor. R1 is methyl; 20 2. The composition according to claim 1 wherein the tetrapyrrole is a porphyrin. 3. Composition according to claim 1 wherein the mH -OH tetrapyrrole is a bacteriochlorin, di- or triamide. -CH39 -CH3; 4. Composition according to claim 1 wherein the 25 amide-containing substituents are asymmetrically ar R2 is H, vinyl, ethyl, ranged on the tetrapyrrole molecule. 5. Composition according to claim 1 wherein the amide-containing substituents are asymmetrically ar ranged on the tetrapyrrole molecule. -HCH, 30 OH 6. Composition according to claim 1 wherein the amide is diserinyl mesoporphyrin IX. acetyl, 7. Composition according to claim 1 wherein the amide is diglycyl mesoporphyrin IX. 35 8. Composition according to claim 1 wherein the H amide is di-a-(DL)-alanyl mesoporphyrin IX. 9. Composition according to claim 1 wherein the -ethyl, -C=O, amide is di-3-alanyl mesoporphyrin IX. 10. Composition according to claim 1 wherein the CH2CH2CO2H, or -CHCHO; amide is di-e-amino-n-caproyl mesoporphyrin IX. R3 is methyl 11. Composition according to claim 1 wherein the amide is diglycyl trans-mesochlorin IX. 12. Composition according to claim 1 wherein the -H amide is diglycyl trans-mesochlorin e6. -CH O 45 13. Composition according to claim 1 wherein the amide is diglycyl mesochlorin e4. R4 is H, vinyl, ethyl, 14. Composition according to claim 1 wherein the amide is diglycyl hematoporphyrin IX. 15. Composition according to claim 1 wherein the -CHCH3, 50 amide is diglycyl chlorin e6. OH 16. Composition according to claim 1 wherein the amide is diglycyl protoporphyrin IX. CH2CH2CO2H, =CHCHO; or 17. Composition according to claim 1 wherein the amide is diglycyl deuteroporphyrin. 55 18. Composition according to claim 1 wherein the amide is di-a-(DL)-alanyl trans-mesochlorin IX. -ethyl; 19. Composition according to claim 1 wherein the amide is di-a-(DL)-alanyl mesochlorin e6. 20. Composition according to claim 1 wherein the R5 is methyl; amide is di-a-(DL)-alanyl mesochlorin e4. R6 is H, CH2CH2CO2H, CH2CH2CO2R or CO2H.; 21. Composition according to claim 1 wherein the R7 is CH2CH2CO2H, CH2CH2CO2R, or amide is di-a-(DL)-alanyl hematoporphyrin IX. 22. Composition according to claim 1 wherein the amide is di-a-(DL)-alanyl chlorin e6. 65 23. Composition according to claim 1 wherein the amide is di-a-(DL)-alanyl protoporphyrin IX. 24. Composition according to claim 1 wherein the R8 is methyl or amide di-a-(DL)-alanyl deuteroporphyrin. is 4,977,177 31 32 25. Composition according to claim 1 wherein the amide is di-3-DL)-alanyl trans-mesochlorin IX. -CHCH3, 26. Composition according to claim 1 wherein the amide is di-3-DL)-alanyl mesochlorin e6. OH 27. Composition according to claim 1 wherein the amide is di-p3-(DL)-alanyl mesochlorin ei. acetyl, 28. Composition according to claim 1 wherein the amide is di-6-(DL)-alanyl hematoporphyrin IX. H 29. Composition according to claim 1 wherein the -H amide is di-g-(DL)-chlorin e6. O -ethyl, -CO, 30. Composition according to claim 1 wherein the CH2CH2CO2H, or =CHCHO; amide is di-3-(DL)-alanyl protoporphyrin IX. R3 is methyl 31. Composition according to claim 1 wherein the amide is di-6-(DL)-alanyl deuteroporphyrin. 15 32. Composition according to claim 1 wherein the amide is di-L-a-serinyl chlorin e6. 33. Composition according to claim 1 wherein the amide is di-L-a-serinyl trans-mesochlorines. R4 is H, vinyl, ethyl, 34. Composition according to claim 1 wherein the 20 amide is di-L-a-serinyl trans-mesochlorin IX. 35. Composition according to claim 1 wherein the -CHCH3, amide is di-L-a-serinyl trans-mesochlorin e.g. OH 36. Composition according to claim 1 wherein the amide is di-L-a-serinyl hematoporphyrin IX. 25 CH2CH2CO2H, -CHCHO; or 37. Composition according to claim 1 wherein the amide is di-L-a-serinyl protoporphyrin IX. 38. Composition according to claim 1 wherein the amide is di-L- a-serinyl deuteroporphyrin. 39. Composition according to claim 1 wherein the amide is di-e-amino-n-caproyl-hematoporphyrin IX. 40. Composition according to claim 1 wherein the R5 is methyl; amide is di-e-amino-n-caproyl-chlorin e6. R6 is H, CH2CH2CO2H, CH2CH2CO2R or CO2H; 41. Composition according to claim 1 wherein the 35 R7 is CH2CH2CO2H, CH2CH2CO2R, or amide is di-e-amino-n-caproyl-protoporphyrin IX. 42. Composition according to claim 1 wherein the -CH2CH2CO2H; amide is di-e-amino-n-caproyl-deuteroporphyrin. -H 43. A method for detecting tumors sensitive thereto in a mammal comprising administering to said mammal R8 is methyl or a therapeutically effective amount of fluorescent mono, di or polyamide of an aminomonocarboxylic acid and a tetrapyrrole compound of the formula: -CH3, 45 -H

R9 is H, COOH, CH2COOH or methyl; provided that when R, R2, R3, R4, R7 and R8 represent two substituents or are divalent and attached to the 50 same carbon, the respective pyrrole ring to which attached is a dihydropyrrole; R is lower alkyl or benzyl; R6 and R9, taken together are 55 --o Or -=o -CH2 -CHCOCHs or the corresponding di- or tetrahydrotetrapyrroles 60 with the proviso that at least one of R1-R9 includes a wherein free carboxyl group, or salts thereof; applying light R1 is methyl; of sufficient wavelength to activate said mono-, di or polyamide to the area of the mammal to be ex amined; and observing the emitted fluorescence -H 65 from said tumor. -CH39 -CHs; 44. A method for detecting tumors according to claim 43 wherein light from 360-760 nanometers is used R2 is H, vinyl, ethyl, for the diagnosis of the tumor. 4,977, 177 33 34 45. A method for treating tumors sensitive thereto in R4 is H, vinyl, ethyl, a mammal comprising administering to said mammal a therapeutically effective amount of a fluorescent mono, -CHCH3, di or polyamide of an aminomonocarboxylic acid and a tetrapyrrole compound of the formula: OH

CH2CH2CO2H, -CHCHO; or

10 -ethyl;

R5 is methyl; R6 is H, CH2CH2CO2H, CH2CH2CO2R or CO2H; 15 R is CH2CH2CO2H, CH2CH2CO2R, or

20 or the corresponding di- or tetrahydrotetrapyrroles R8 is methyl or wherein R1 is methyl; 25 -H arOH -CH39 -CH3; R9 is H, COOH, CH2COOH or methyl; provided that when R1, R2, R3, R4, R7 and R8 represent two R2 is H, vinyl, ethyl, substituents or are divalent and attached to the 30 same carbon, the respective pyrrole ring to which attached is a dihydropyrrole; -CHCH3, R is lower alkyl or benzyl; OH Re and R9, taken together are acetyl, 35

with the proviso that at least one of R1-Rg includes a free carboxyl group, or salts thereof; and apply ing light of sufficient wavelength and intensity to CH2CH2CO2H, or =CHCHO; activate said mono-, di- or polyamide, whereby R3 is methyl, said mono-, di- or polyamide exerts a cell-killing effect on such tumor. 45 46. A method for treating tumors according to claim 45 whereby light from 620-760 nanometers is used for the treatment of the tumor. x: sk k

SO

55

65 - S UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION PATENT NO. : 4,977, 177 Page 1 of 3 DATED : December ll l99 O INVENTOR(S) : Jerry C. Bommer, et al. It is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below:

Column 1 line 24: "tumor. inhibiting" should read as --tumor inhibiting-- Column 9 line lO: "solic" should read as -- sole-- Column 9, line 28: "applied For" should read as --applied. For-- Column 9 line 63: "one" should read as --one. -- Column lO line 5: "salt. The " should read as --salt. The-- Column lO, line 9: "high intensity" should read as --high-intensity-- Column l0, line ll: "limits power" should read as --limits: power-- Column lO line 27: Chlorophvill" should read as Chlorophyll Column O line 39: "mesocchlorin" should read as --Mesocchlorin-- Column lo, line 63: "and . " should read as --and-- Column l3, line 33: "(l. 1 total" should read as --(l & total.-- Column l3 line 64: "(l. l total" should read as -- (l 9. total.--

UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION PATENT NO. : 4,977, 77 Page 2 of 3 DATED : December ll l990 INVENTOR(S) : Jerry C. Bommer, et al. it is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below:

Column l4, line 26: "2.5' 30" should read as --2.5 x 3O-- Column l4, line 28 : " (l. 1 total" should read as --l R total.-- Column l4, line 56: "l 5" should read as --l. 5-- Column l4, line 62 : " (l l total" should read as -- (l s total.-- Column 2l, line 55: "qlycyl" should read as --glycyl-- Column 22 lines 7 & 38: "(l. l total" should read as -- (l 2 total.-- Column 23 line 2 : " (l l total" should read as --(l. Q total.-- Column 23, line l7: "400 pug" should read as -- 400mg -- Column 23 line 34 : " (l l total" should read as -- (l 3 total.-- Column 23, line 52: "36 ul" should read as --36Oul-- Column 29 line l: "of a " should read as -- of an--

UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATF OF CORRECTION PATENT N.E. : 4,977, 177 Page 3 of 3 DATED : December lil, l990 INVENTOR{S} : Jerry C. Bommer, et al. it is certified that error appears in the above-identified patent and that said Lette:S Patent is hereby corrected as shown below: Column 29, line 66, Claim l; "-H" should read as - - - H -- Column 3 O, line 68, Claim 24 : "amide" should read as - -amide is -- and delete "is" at the end of line Column 3l, lines 2 & 4: "DL)" should read as -- (DL) -- Column 32, lines lé & 17: " -CH; " should read aS - - -CH -OH -OH ; Column 32, line 38: "-H" should read as -- -H;-- Column 33, lines 36 & 37: " -CH;" should read as -- -CH -OH Column 34, lines l8 & 19: " -CH2CH2CO2H;" should read as - - -CHCH CO. H. " -H 2 - 2 - 2 -H;

- Signed and Sealed this Twenty-fifth Day of August, 1992

d Attest:

DOUGLAS B. COMER

Attesting Officer Acting Commissioner of Patents and Trademarks