PHEOPHORBIDE DERIVATIVES AND PHARMACEUTICAL PREPARATIONS CONTAINING THEM

Abstract: The subject matter of the invention are pheophorbide derivatives and their alkali metal salt of the general formulas I, Ia and II according to the patent claims. The compounds of the invention are useful in cancer treatment.

Description: Pheophorbide Derivatives and Pharmaceutical Preparations Containing Them The present invention relates to novel pheophorbide derivatives which are useful in cancer treatment. The com- pounds according to the present invention are novel and are extremely useful in cancer treatment because of their pharmacological effects such as photo-sensitivity and accumulative and destructive effects in cancer cells. Porphyrin compounds, particularly porphine derivatives obtained from hemoglobin, are known for their binding property with cancer tissues and photo-dynamic characteristics. Their use in cancer treatment is now being studied. However, production of porphyrin compounds requires animal blood which is limited in supply and extensive production steps. Supply of the material animal blood, moveover, is not stable and porphine derivatives such as hematoporphyrin which are said to be the active constituent are unstable and it is extremely difficult to obtain them with high purity. It has heretofore been known that porphine derivatives of hemoglobin origin, i.e. hematoporphyrin and diacetyl hematoporphyrin, are likely to be conjugated with cancerous tissues and are photo sensitive. Endo et al have recently found that 10-hydroxy-pheophorbide is carcinostatic (Jap. Pat. Appln. Laid-open No. Sho 57-185220). This application discloses that pheophorbide and pyropheophorbide also have the same activities. Although these compounds are photo-sensitive (because they are porphyrin related compounds), they have no selectively accumulative effect in cancer cells. No data on their affinity to cancer cells are found in references nor is there any description concerning the differences in selective accumulation in cancer cells among the listed compounds other than the calculated results on the tumor suppression rate after administration of the substance and laser irradiation. Yori et al reported on the bactericidal effect of ethylenediamine hydrochloride of pheophorbide (Jap. Pat. Appln. Laid-open No. Sho 58-981). This application relates to an antibacterial agent comprising water soluble phorbine derived from pheophorbide by converting the same into ethylenediamine hydrochloride. Processes for synthesizing chlorophyll dimers are disclosed in the literature (M.R. Wasielewski et al, C.A. 86, 68532f(1977), C.A. 88 148988rC1978) and S.G. Boxer et al, J.A.C.S., 1976,,5406). Also disclosed in references is the synthesis of compounds such as 9-desoxo-9-hydroxy-pheophytin (M.R. Wasielewski et aL, Tetrahedron Letters, 1043 (1978)) and 9-desoxo- 9-hydroxy- 1 O-methoxy-methylpheophorbide (H. Scheer et awl., Tetrahedron, 28, 5839 (1972)). However these substances are different from the compounds of the invention. Moreover, the aim of the authors of said articles was to synthesize the compounds as a part of their study in elucidating the meachanism of photo-synthesis in order to obtain compounds having an W absorption profile more Similar to that of chlorophyll P-700. The latter substance is the result of the study in which various related derivatives were synthesized to determine their absolute configuration, to investigate the influence of reaction conditions on yield and to publish data obtained from analyses and measurements by various devices. These references do not discuss physiological activities of the compounds on cancerous tissues which are the main object of the present invention. The present inventors have long been engaged in the study of chlorophyll related compounds and established a highly economical process for separating pheophorbide using photo-synthesized organisms which are available in a stable and inexpensive supply. (Jap. Pat. Appln. Laidopen No. Sho 58-69884). It is the object of the present invention to provide novel pheophorbide-derivatives having more potent physiological activities -t-han said porphines derived from blood. This object is achieved by the invention. The subject matter of the invention are novel pheophorbide derivatives having various functional groups expressed by general formula (I) and their dimers expressed by general formula (II) (hereinafter referred to as the present invention substances), excluding pheophorbide and pyropheophorbide: EMI3.1 wherein R1 represents EMI4.1 EMI4.2 R2 represents CH3, CHO or CH2OH, R3 represents C=O, OH or OCOCH3, R4 represents H,COOCH3, COOCH2CH2OH COOCH2CH2OCOCH3 or COONH residue of amino acid and R5 represents COOH, COONa or COOCH3. Preferred compounds of the invention are 9-desoxo-9hydroxy-pheophorbide derivatives and alkaline salts thereof represented by the general formula (Ia): EMI4.3 wherein R2 represents CH3 or CH2OH, R6 represents H or Ac, and R7 represents H or an alkali metal such as Na and K. Results of elemental analysis and nuclear magnetic resonance absorption of 9-desoxo-9-hydroxy-pheophorbide which is one of the preferred compounds of the invention are as follows: Formula: C35H38N405 Elementary analysis: calcd. C, 70.69%: H, 6.44%:N, 9.42% Found: C, 70.64%: H, 6.57%:N, 9.16% Nuclear magnetic resonance absorption ( H-NMR) (# ppm) EMI5.1 &alpha; - H : 9.4 9 7 - H : 4.1 ss - H : 9.6 8 7a - CH2 : 2 ¯ 3 # - H : 8.8 2 7b - CH2 : 2 ¯ 3 1 - Me : 3.4 4 8 - II : 4.3 5 2 - Vinyl : 8.0 0 8 - Me : 1.7 5 : 5.8 9 9 - H : 6.4 8 : 6.0 3 10 - H : 6.2 6 3 - Me : 3.2 3 10b - COOMe : 3.7 0 4b - Me : 1.5 9 N - H : 0.3 3 5 - Me : 3.8 1 :-0.2 8 The compounds of the invention have excellent pharmacological properties such as marked affinity to cancerous tissues, high photo-sensitivity and destructive effect on cancerous tissues that are superior to those observed in porphines. It has so far been unknown that the phorbine related compounds had such strong physiological activities. The inventors often observed this fact during the course of their study, which is surprising as, for example, no specific activities against cancer were found in the raw material of the invention substances, that is, pheophorbide and its alkaline salts and methyl pheophorbide. The invention will now be further explained with respect to the drawing in which Fig. 1 and 2 are graphs showing the affinity of compounds according to the invention to cancer cells as measured by N2 pulsed laser spectrofluoro metry; Fig. 3 and 4 are graphs showing the destructive effect of said compounds on cancer cells as measured by biochemiluminescence in a single photon counter system. Pharmacological activities of the present invention substances will now be explained. The present invention substances accumulate selectively in cancer tissues and only slowly excrete therefrom. When irradiated with light, they react and produce singlet oxygen having a strong oxidizing effect to thereby destroy cancer cells. As the present invention substances are readily excreted from normal organs and cells, cancer can thus be treated without damage to normal cells. It is possible to treat not only the cancer projecting on the surface but also small cancer lesion in the mucous epithelium which is difficult to find and abnormal cells in precancer state. As for effects on cancer, most of porphines,except for a very few substances, show no selective affinity to cancer cells although they are photo-sensitive. Absence of selectivity leads to destruction of normal cells as well as cancer cells. In view of the above facts, there has been proposed a combined use of several porphines having selective affinity. The present invention substances are of great utility as they are provided with all the properties mentioned above (i.e., affinity to cancer cells, photo-sensitivity, destructive effect on cancer). These substances can be used not only singly but in combination. The process for preparing the compounds according to the present invention will now be described. Roughly, there are two ways of preparing the invention compounds having the general formula I. According to one process pheophobide (1) is treated with hydrobromic acid to obtain 2-desethenyl---2- (1-hydroxyethyl) -pheophorbide (2), which is then reduced to obtain 2-desethenyl-2-(1-hydroxy ethyl)-9-desoxo-9-hydroxy-pheophorbide (3). (3) is acetylated to obtain diacetylate (4). The reactions can be expressed by the following chemical formula: EMI8.1 In partical application of the present invention, (1) is dissolved in hydrobromic acid/acetic acid, reacted and neutralized to obtain (2). (2) is reacted using a suitable reducing agent such as NaBH4 to obtain the product (3). Diacetylated product (4) can be obtained from (3) by reaction with acetic anhydride/pyridine in the usual way. According to another process for preparing the compounds of the general formula I, methylpheophorbide (5) is esterexchanged with ethylene glycol to obtain ethylene glycol mono-10b-methyl pheophorbate (6) , and (5) is ester-exchanged with phenylalanine t-butyl ester to obtain N-phenylalanine t-butyl ester 1Ob-methylpheo- phorbidamide (7). (6) and (7) are subjected to acid hydrolysis to obtain ethylene glycol mono-lOb-methyl pheophorbate (8) and N-phenylalanine lOb-methyl pheophorbidamide (9). The reactions can be expressed by the following chemical formula: EMI10.1 Hydroxides, reduced derivatives or acetylates of (8) and (9) may be produced by subjecting said esters of pheo- phorbide to hydrobromic acid treatment, reduction or acetylation as mentioned above. In practical application of the present invention, (5) is dissolved in a suitable solvent such as xylene or DMF, ethylene glycol is added and the mixture is heated and reacted to obtain (6). Alternatively, phenylalanine t-butyl ester is added in place of ethylene glycol, heated and reacted to obtain (7). The resultant product (6) is reacted with 50% HzSO4 and the product (7) with CF3COOH respectively to obtain (8) and (9). The dimers of the present invention having the general formula II are obtained by condensating (8) with (1) through ester-exchange to obtain ethylene glycol di-lOb- pheophorbate (10). The reaction can be expressed by the following formula: EMI11.1 Said pheophorbide dimer (k) can be subjected to hydrobromic acid treatment, reduction or acetylation in the same manner as mentioned above to produce hydroxides, reduced derivatives or acetylates of X, respectively. In practical application of the present invention, (8) may be dissolved in a suitable solvent such as DMF or DMA. Then (1) is added and the mixture is heated and reacted to obtain (10). As a still another process or preparing the present invention substances, it is possible to form the hydroxide or reduced derivatives of methyl pheophorbide (5) prior to condensation by ester-exchange, and obtain preferable results. Any combination of reactions other than those mentioned above may also be employed so long as they include hydroxidization, hydrolysis and ester-exchange reactions. It is also preferable but not essential to add a suitable antioxidant or solvent and to heat the reaction system. Although pheophorbide is used as the starting material, pheophorbide a,b,c, pyropheophorbide and pheophytin may also be used. The invention is, however, not limited to those so long -as -the material is obtained from a photo-synthesized substance. The preferred compounds of general formula Ia can be obtained by reducing pheophorbide (1) to produce 9-desoxo9-hydroxy-pheophorbide (11). The reaction can be expressed by the following chemical formula: EMI12.1 EMI13.1 In practical application of the present invention, (1) is dissolved in a suitable solvent, i.e. methanol, and reacted with a suitable reducing agent such as Nabs4. A preferable result can be. obtained by another preparation method of the present invention substances, wherein pheophytin or methyl pheophorbide is reduced in advance in the first step and the reaction product, 9-desoxo9-hydroxy-pheophytin or 9-desoxo-9-hydroxy-methyl pheophorbide is hydrolyzed in the second step. A similarly preferable result can be obtained by sill another process; pheophorbide obtained through acid hydrolysis of pheophytin is similarly reduced ot obtain (11), or (11) is acetylated produce 9-desoxo-9-acetyloxy-pheophorbide (12) because when (12) is an alkaline salt, it is released prior to (11). Any combination of reactions other than those mentioned above may also be employed so long as they include hydrolysis, reduction and acetylation. It is also preferable but not essential to add a suitable antioxidant or solvent and to heat the reaction system. Although methanol, ethanol, acetonitrile, pyridine,etc. as solvents and Na2S203, Na2S204, ascorbic acid, etc. as reducing agents may be employed in preparing the present invention substances, the invention is not restricted to these solvents and reducing agents. Table I illustrates data on Nuclear Magnetic Resonance absorption spectrum of the present invention substances. EMI14.1 EMI15.1 <SEP> Table <SEP> I <SEP> (# <SEP> ppm) <tb> <SEP> I <SEP> II <SEP> III <SEP> V <SEP> VI <SEP> VII <SEP> VIII <SEP> IX <SEP> X <tb> Mw <SEP> 592 <SEP> 610 <SEP> 612 <SEP> 606 <SEP> 636 <SEP> 795 <SEP> 622 <SEP> 739 <SEP> 1182 <tb> &alpha; ;-H <SEP> 9.05 <SEP> 8.85 <SEP> 8.70 <SEP> 9.16 <SEP> 9.00 <SEP> 9.07 <SEP> 9.02 <SEP> 9.08 <SEP> 9.12 <tb> ss <SEP> -H <SEP> 9.22 <SEP> 9.25 <SEP> 9.00 <SEP> 9.36 <SEP> 9.31 <SEP> 9.30 <SEP> 9.29 <SEP> 9.32 <SEP> 9.19 <tb> # <SEP> -H <SEP> 8.43 <SEP> 8.29 <SEP> 8.32 <SEP> 8.53 <SEP> 8.42 <SEP> 8.50 <SEP> 8.43 <SEP> 8.48 <SEP> 8.40 <tb> 1 <SEP> -CH3 <SEP> 3.26 <SEP> 3.25 <SEP> 3.15 <SEP> 3.30 <SEP> 3.22 <SEP> 3.25 <SEP> 3.38 <SEP> 3.26 <SEP> 3.37 <tb> 2 <SEP> -CH=CH2 <SEP> 7.72 <SEP> - <SEP> - <SEP> 7.83 <SEP> 7.70 <SEP> 7.73 <SEP> 7.71 <SEP> 7.72 <SEP> 7.80 <tb> <SEP> 6.05 <SEP> - <SEP> - <SEP> 6.15 <SEP> 6.10 <SEP> 6.10 <SEP> 6.10 <SEP> 6.10 <SEP> 6.15 <tb> <SEP> 5.95 <SEP> - <SEP> - <SEP> 6.06 <SEP> 5.93 <SEP> 5.98 <SEP> 6.01 <SEP> 5.95 <SEP> 5.99 <tb> 3 <SEP> -CH3 <SEP> 3.00 <SEP> 3.05 <SEP> 3.00 <SEP> 3.04 <SEP> 2.95 <SEP> 2.99 <SEP> 3.17 <SEP> 2.98 <SEP> 3.14 <tb> 4b <SEP> -CH3 <SEP> 1.62 <SEP> 1.63 <SEP> 1.68 <SEP> 1.60 <SEP> 1.53 <SEP> 1.58 <SEP> 1.66 <SEP> 1.58 <SEP> 1.68 <tb> 5 <SEP> -CH3 <SEP> 3.57 <SEP> 3.43 <SEP> 3.22 <SEP> 3.60 <SEP> 3.52 <SEP> 3.52 <SEP> 3.62 <SEP> 3.52 <SEP> 3.59 <tb> 7 <SEP> -H <SEP> 4.13 <SEP> 4.2 <SEP> 4.1 <SEP> 4.16 <SEP> 4.2 <SEP> 4.4 <SEP> 4.2 <SEP> 4.3 <SEP> 4.2 <tb> 7a,b <SEP> -CH2CH2- <SEP> 2¯3 <SEP> 2¯3 <SEP> 2¯3 <SEP> 2¯3 <SEP> 2¯3 <SEP> 2¯3 <SEP> 2¯3 <SEP> 2¯3 <SEP> 2¯3 <tb> 7c <SEP> -CH3 <SEP> - <SEP> - <SEP> - <SEP> 3.56 <SEP> 3.60 <SEP> 3.61 <SEP> - <SEP> 3.60 <SEP> 8 <SEP> -H <SEP> 4.40 <SEP> 4.7 <SEP> 4.4 <SEP> 4.44 <SEP> 4.5 <SEP> 4.55 <SEP> 4.4 <SEP> 4.53 <SEP> 4.43 <tb> 8 <SEP> -CH3 <SEP> 1.86 <SEP> 1.90 <SEP> 1.71 <SEP> 1.81 <SEP> 1.80 <SEP> 1.84 <SEP> 1.85 <SEP> 1.82 <SEP> 1.85 <tb> 9 <SEP> -H <SEP> - <SEP> - <SEP> 6.69 <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> 10 <SEP> -H <SEP> 6.17 <SEP> 6.11 <SEP> 6.13 <SEP> 6.25 <SEP> 6.21 <SEP> 6.13 <SEP> 6.22 <SEP> 6.15 <SEP> 6.24 <tb> 10b <SEP> -CH3 <SEP> 3.85 <SEP> 3.88 <SEP> 3.42 <SEP> 3.88 <SEP> - <SEP> - <SEP> - <SEP> - <SEP> 10b <SEP> -CH2CH2- <SEP> - <SEP> - <SEP> - <SEP> - <SEP> 3.93 <SEP> - <SEP> 4.15 <SEP> - <SEP> 10b <SEP> -CH2- <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> 4.2 <tb> 10b <SEP> -OBut <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> 1.40 <SEP> - <SEP> - <SEP> 10b <SEP> -#CH2- <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> 7.2 <SEP> - <SEP> 7.2 <SEP> As mentioned above, the present invention utilizes photosynthesized substances as the raw material which are inexpensive and stably supplied. The process involves extremely simple operations which can be conducted in a short period of time to achieve isolation and synthesis. The present invention substances have physiological activities which are unique to phorbine related compounds and can be used per se as medical preparations, or can be formulated to medical preparations in a usual manner. Unlike porphines derived from hemoglobin, they can be mass produced at higher. purity and thus are extremely useful. The preparation of the compounds of the present invention will now be described by way of examples. Example 1 Three grams of pheophorbide (1) were dissolved in 12 ml of pyridine and 60 ml of methanol into which 5% NaBH4 solution was dropped under stirring. After the reaction was completed, crystals of 9-desoxo-9-hydroxy-pheophorbide (11) were.precipitated after addition of an aqueous solution of 5% citric acid. Crystals were collected, washed with water and dried. (Yield 3g). The yield was 98%. The product obtained was dissolved in acetone and neutralized by adding 5% Na2CO3 aqueous solution to obtain sodium 9--desoxo-9-hydroxy pheophorbide. The yield was 98%. Example 2 Three grams of methyl pheophorbide (5) was dissolved in 70 ml of acetonitrile, an aqueous solution of NaBH4 was added dropwise under stirring to obtain crude 9-desoxo 9-hydroxy-methyl pheophorbide. The product obtained was suspended and dissolved in 40% H2 SO2 aqueous solution and reacted for 1 hour under agitation. After the reaction was completed, 5% citric acid aqueous solution was added to the reaction liquid by a similar process as in Example 1 to collect the product (11), which was washed with water and dried. The yield was 69%. Example 3 Fifty grams of crude chlorophyll (chlorophyll content 10%) was suspended and dissolved in 3-fold 508 H2SO4 aqueous solution, 0.5 gof Na2S203 was added,and the mixture was reacted-for-1 hour under agitation. Similar process as in Example 2-was conducted to obtain (1), which was further treated as in Example 1 to obtain (11) (yield -2. D g). -The yield (in proportion to crude chlorophyll used) was 7.6%. Example 4 One gram of (11) was acetylated with acetic anhydridepyridine by a -conventional method to obtain 9-desoxo9-actyloxy-pheophorbide (yield ig). The yield was 99%: 0.5 g of this product was dissolved in ether and neutralized with 5% Na2CO3 aqueous solution to obtain the sodium salt of 9-desoxo-acetyloxy-pheophorbide. The aqueous solution of this sodium salt was left standing for reaction to obtain 0.2 g of (11). The yield was 40%. Example 5 One gram of pheophorbide (1) was dissolved in 25 g of hydrobromic acid/acetic- acid- and reacted for 15 hours under stirring. After reaction was completed2 50 ml of water was added under stirring and then neutralized by adding 20 ml of 33 % NaOH aqueous solution, whereby crystals of 2-desethenyl-2-(1-hydroxyethyl)-pheophorbide (2) were precipitated. Crystals were then collected, washed with water and dried (yield: 1 g). The yield was 97 %. The resultant product :(2) was dissolved in aceton, added with 5% Na2CO3 aqueous solution for neutralization to obtain sodium 2-desethenyl-2-(1-hydroxyethyl) pheophorbide. The yield was 98%. Example 6 One gram of (2) was dissolved in 6 ml of pyridine and 30ml-of methanol; 5% NaBH4-aqueous solution was dropped into the mixture under stirring and reacted. After the reaction was completed, 5% aqueous solution of citric acid was added¯to separate excess NaBH4. Water was then added to the reaction product and crystals of 2-desethenyl-2-(1-hydroxyethyl)-9-desoxo-9-hydroxy-phenophorbide (3) were precipitated. Crystals were collected, washed with water and dried. -(yield 1 g). The yield was 98%. Example 7 One gram of (3) was acetylated using acetic anhydride/ pyridine by the conventional method to obtain diacetylate (4) of (3) (yield 1.lg). The yield was 99%. Example 8 One gram of methyl pheophorbide (5) and 2 g of ethylene glycol were dissolved in 20 ml of DMF and reacted for 2 hours under heating and stirring. After the reaction was completed, water was added to precipitate crystals of ethylene glycol mono-lOb-methyl pheophorbate (6). Crystals were collected, washed with water and dried (yield 0.7 g). The yield was 70%. The product (0.7g) thus obtained was dissolved in 10 ml of 50% H > SO4 and reacted for 1 hour at room temperature under stirring. After the reaction was completed, water was added to precipitate¯crystals of ethylene glycol mono-109-pheophorbate (8)-. Crystals were collected washed with water and dried (yield 0.65 g). The yield was 95%. Example 9 One gram of (5) and 2 g of phenylalanine t-butyl ester were dissolved in -30 ml of xylene and reacted for 2 hours under heating and stirring. After the reaction was completed, the reaction solution was subjected to column chromatography (silicic acid column, n-hexane: ethyl acetate = 3:1) to obtain N-phenylalanine-t-butyl ester 1Ob-methylpheophorbid amide (7) (yield 0.9 g). The yield was 68%. The resultant product (0.9 g) was dissolved in 10 ml of trifluoroacetic acid and reacted for 2 hours at room temperature under stirring. After the reaction was completed, water was added to precipitate crystals of N-phenylalanine 1Ob-methylpheophorbidamide (9). Crystals were collected, washed with water and dried (yield 0.8 g). The yield was 95%. E x a m p 1 e 10 One gram of (8) and 1 g of (1)were dissolved in 20 ml of DMF and reacted for 2 hours under heating and stirring. After the reaction was completed1 water was added to precipitate crystals of ethylene glycol di 1Ob-pheophorbate (10). Crystals were collected,washed with water and dried (yield 1.5 g). The yield was 80%. The pharmacological effect of the compounds of the invention is shown by the following tests of laser irradiation on a removed organ in vitro. A) N2 Pulsed Laser Spectro-Fluorometry Experiment 1 Five mg of the test drug sodium 9-desoxo-9-hydroxy pheophorbide diluted in physiological saline (1 ml) was administered (1 ml) to golden hamsters (5 each for a group) 14 - 21 days after transplatation of nitrosoamineinduced cancer cells of the pancreas, after which cancer cells and other organs were removed. N2-pulsed laser (N2 337 nm, 2 ns 400 - 1000 nm)- was irradiated to each organ thus obtained to measure N2 pulsed-laser spectro-fluorometry. The result is shown in Fig. 1. N2 pulsed laser spectro-fluorometryof each removed organ was measured 24 hours and -48hours¯afterwards to investigate the wavelengths of 600 to 900 nm based on the peak wavelength of NADH of 470 nm. Fig. 1 shows that though sodium 9-desoxo-9-hydroxy pheophorbide was generally found in organs after 24 hours, it had markedly accummulated in cancer cells alone but not in other cells after 48 hours. As -is obvious from the above result, sodium 9-desoxo-9hydroxy pheophorbide was proved-to have remarkable selective affinity to cancer cells. Experiment 2 Golden hamsters (5 per one group) 14 - 21 days after transplantation of nitrosoamine-induced cancer cells of the pancreas were administered 5 mg each of sodium 2 desethenyl-2- (1-hydroxyethyl) pheophorbide (sodium salt of (2)), sodium 2-desethynyl-2- (1-hydroxyethyl) -9-desoxo -9-hydroxy pheophorbide (sodium salt (3)), sodium 2-desethenyl-2- {1-acetyloxyethyl) -9-desoxo-9-acetyloxy pheophorbide (sodium salt of (4)), sodium ethylene glycol mono-lOb-pheophorbate (sodium salt of (8)), disodium ethylene glycol di-l0b-pheophorbate (sodium salt of (10)) and sodium N-phenylalanine 10b-methyl pheophorbid amide (sodium salt of (9)) respectively diluted with physiological saline (1 ml). Cancer cells and other organs were removed, irradiated with N2-pulsed laser (N2, 337 nm, 2 ns 400 - 1000 nm) to measure N2 pulsed laser spectro fluorometry. Using the NADH peak wavelength of 470 nm as the reference, wavelengths in the range of from 600 - 900 nm were studied. Fig. 2 shows that these products showed marked accumulation in cancer cells 24 hours after administration, but left no trace in other cells. Similar tests were conducted on other products and the results are shown in ableII.--Various organs were removed 24 hours after administration and their N2 pulsed laser spectro-fluorometry was measured. Using the NADH peak wavelength of 470 nm as the reference, the wavelengths were calculated and shown in Table II.. As is evident from the result above, these phorbine related compounds have a- marked selective affinity to cancer cells B) Biochemical luminescence by single photoncounter Experiment 3 Sodium 9-desoxo-9-hydroxy pheophorbide dissolved in physiological saline of respective concentrations (25 g/2 ml, 50 g/2 ml, 100 g/2 ml) was added to leukemic Molt 4 Cell (1 x 107 Cells) and the resultant product was incubated for 24 hours at 37 OC. Each incubated cell thus obtained was irradiated with laser (He-Ne gas, 630 nm, 10 min, 20 mW) and trace biochemiluminescence was measured by single photoncounter system. Intensity of biochemiluminescene substantially corresponds to the degree of destructive effect on cancer cells and thus by studying the biochemicluminescene by single photoncounter system, it is possible to predict the destructive effect on cancer cells. Fig. 3 shows the intensity of biochemiluminescene and the destructive effect on cancer cells after laser irra dilation. It is seen from Fig. 3 that more than 95% of the cancer cells were destroyed when the subject substance in the amount of 100 ssg/2 ml was used. The foregoing result shows that sodium 9-desoxo-9-hydroxy pheophorbide has intense photo-sensitivity and remarkable destructive effect on cancer cells. Experiment 4 7 Leukemic Molt 4 cell (1 x 10 cells) was added with physiological saline containing sodium 2-desethenyl2-(1-hydroxyethyl) pheophorbide (sodium salt of (2)) in respective concentrations (25 ssg/2-ml, 50 pug/2 ml, 100 pg/ 2 ml) and then incubated (370C, 24 hours). Incubated cells each were irradiated with laser (He-He gas, 630 nm, 10 min, 20 mW) to investigate generation of biochemiluminescene by single photoncounter system by measuring biochemiluminescence. The intensity of biochemiluminescence by singel photoncounter system substantially corresponds to the degree of destruction of the cancer cells. Therefore, by observing generation of biochemiluminescence by single photoncounter system, destruction of cancer cells by be predicted. Fig. 4 shows the degree of biochemiluminescence and the destruction of cancer cells after laser irradiation. The result indicates that when 100 g/2 ml of a test substance was used, more than 95 % of the cancer cells were destroyed. As is evident from the results shown in Fig. 4, phorbine related compounds are highly photo-sensitive and display a marked destructive effect on cancer cells. The foregoing exper-iments A and B together prove that the present invention substances have a marked affinity to cancer cells, remarkable photo-sensitivity, and destructive effect on cancer cells. Table II (Sodium compounds) Comparison Pheophorbide (1) 0.78 Pyropheophorbide 0.25 Application 9-Desoxo-9-hydroxy-pheophorbide+ethylene glycol mono-10b-methylpheophorbate (6) 2.00 9-Desoxo-9-hydroxy-pyropheophorbide 0.98 9-Desoxo-9-hydroxy-pheophorbide C 0.32 9-Desoxo-9-acetyloxy-pheophorbide 0.89 N-Phenylalanine 10b-methylpheophorbidamide (9) 0.20 2-Desethenyl-2-(1-acetyloxyethyl)-pheophorbide 0.48 2-Desethenyl-2-(1-hydroxyethyl)-9-desoxo-9-hydroxy-pheophorbide (3) 2.20 2-Desethenyl-2-(1-acetyloxyethyl)-9-desoxo-9-acetyloxypheophorbide (4) 0.38 2-Desethenyl-2-(1-acetyloxyethyl)-9-desoxo-9-hydroxy-pheophorbide 0.95 N-Phenylalanine 10b-9-desoxo-9-hydroxy-methylpheophorbidamide 0.73 Ethylene glycol di-10b-pheophorbate (10) 3.30 Ethylene glycol di-10b-9-desoxo-9-hydroxypheophorbate 2.50 Ethylene glycol mono-10b-pheophorbate (8) 1.00 Monoacetylethylene glycol mono-10b-pheophorbate 1.30 Ethylene glycol mono-10b-9-desoxo-9-hydroxy-pheophorbate 2.10 Monoacetylethylene glycol mono-10b-9-desoxo-9-acetyloxy-pheophorbate 0.85 Monoacetylethylene glycol mono-10b-9-desoxo-9-hydroxy-pheophorbate 1.20 2-Desethenyl-2-(1-hydroxyethyl)-pheophorbide (2) 1.90 Ethylene glycol di-10b-2-desethenyl-2-(1-hydroxyethyl)-pheophorbate 2.50 Ethylene glycol di-10b-2-desethenyl-2-(1-hydroxyethyl)-9-desoxo-9-hydroxy-pheophorbate 1.80 9-Desoxo-9-hydroxy-pheophorbide (11) 0.93

Claims: Patent Claims 1. Pheophorbide derivatives andalkalimetal salts thereof represented by the general formula I and their dimers and-alkali metals saltsthereof represented hy the ge- EMI26.1 wherein R1 represents EMI26.2 EMI26.3 R2 represents CH3, CHO or CH2OH, R3 represents C=O, OH or OCOCH3, R4 represents H, COOCH3, COOCH2-CH2OH COOCH2CH2OCOCH3 or COONH residue of amino acid,and R5 represents COOH, COONa or COOCH3; privided, however, said derivatives and alkaline salts exclude pheophorbide and pyropheophorbide. 2. 9-Desoxo-9-hydroxy-pheophorbide derivatives and alkali salts thareof represented by the general formula Ia EMI27.1 wherein R2 represents CH3 or CH2OH, R6 represents H or Ac, and R7 represents-H-or an alkali metal such as Na and K. 3. Pheophorbide derivatives having the general formula I, Iaor-II -as -claimed-in- claims-- 1 or 2 for use in the treatment of cancer. 4. Pharmaceutical -compositions containing a pheophorbide derivative having the general formula I, Ia or II as claimed in claims 1 or 2 and a pharmaceutically ac ceptable carrier.

Inventors: INOHARA, KAZUMI, KOSHIMIZU, KOICHI, NAKAJIMA, SUSUMU, SAKATA, ISAO, SAMEJIMA, NATSUKI, TAKATA, HIROYUKI
Applicants: TOYO HAKKA KOGYO KABUSHIKI KAISHA
Application year: 1984
Main classification: C07D487
IPC codes: C07D519, C07D487