PRODRUG COMPOUNDS, PROCESS FOR THE PREPARATION THEREOF AND SUSTAINED RELEASE PREPARATION COMPRISING THE SAME

Abstract: A Prodrug compound of the formula : A-CO(CH₂) m (NHCO) n -R wherein A is a residue of pharmaceutical compound having NH or -NH₂ group in the molecule, R is a residue of steroid compound, m is an integer of l to 5 and n is 0 or l, and its salts, process for its preparation and a sustained release preparation comprising it.

Description: The present invention relates to new pharmaceutical prodrug compounds, processes for the preparation thereof and sustained release preparations comprising them. The expression "prodrugs" as used here means spacers linking a drug to a liposome soluble steroid compound. They have the effect to provide a sustained release of a drug in the body, and they have been developed by chemical modification of a drug molecule. For example, the prodrug which is the combined form of 5-fluorouracil and cholesterol through the spacer [-CONH(CH₂)₅CO-] has been synthesized and the fact that the prodrug, which is entrapped in liposomes, could be gradually converted to the parent compound, 5-fluorouracil, in buffer solution is known (see e.g. Proceedings of the 105th Annual Conference of Pharmaceutical Society of Japan (1985), Title: Synthesis of 5-FU prodrugs having cholesterol moiety as a carrier and application of the prodrugs to lipid dispersion preparations; and C.A. 96, 1982, ref. 143180 b). From "Journal of Supramolecular Structure " 11 , 295-309 (1979), corresponding to C.A. 93 , No. 3, 1980, ref. 21121a prodrugs are known, in which aminosugars are coupled to cholesterol chloroformates by means of [-CO-] spacers. Further prodrugs are known from GB-A-2 028 336, wherein spacers [-R'CO-], wherein R' is a C₁-C₄ alkylene group are disclosed, which are used to couple various pharmaceutical compounds to various steroids. Furthermore, from "Chemical and Pharmaceutical Bulletin" 31 (11), 4083-4090 (1983), it has been already known to couple Mitomycin C to cholesteryl moiety through the spacer [-CO-] for the synthesis of the Mitomycin C derivative, cholesteryloxycarbonyl Mitomycin C However, there were some problems: i.e. the above-mentioned Mitomycin C derivative could hardly be converted to the parent compound, Mitomycin C, because of the stability of the linkage between Mitomycin C and cholesteryl moiety against chemical and enzymatic hydrolysis. Therefore, the object of the present invention is to provide novel prodrugs including spacers linking a drug (an antitumor substance) to cholesterol which are able to gradually release the drug when the prodrug is administered to the body. The inventors of the present invention have carried out extensive studies in order to accomplish the above object. They have synthesized the derivatives of Mitomycin C by introducing cholesterol moiety though various kinds of new spacers, and have found that the obtained new prodrugs could be gradually converted to Mitomycin C which is a parent compound, when the prodrugs were administered into the body. Further, they have prepared liposome preparations wherein the prodrugs were entrapped into liposomes, and have found that the entrapment of the derivative into liposomes decreased the conversion rate to Mitomycin C and the blood concentration of Mitomycin C was maintained for a long time and the toxicity was reduced when compared with that of the parent compound. Further, they have found that similar effects could be obtained when this invention was applied to 4-formyl-6,9-dihydroxy-14-oxa-1 ,11-diazatetracyclo-[7.4.1.0 2,7 .0 10,12 ] tetradeca-2,4,6-trien-8-ylmethylcarbamate (hereinafter referred to as FR900482 substance) and bis-(2-chloroethyl) amine (hereinafter referred to as Nitrogen mustard), and the anti tumor activity was extensively improved in addition to the above-mentioned effects when this invention was applied to 1-ß-D-arabinofuranosylcytosine (hereinafter referred to as Cytarabine). Subject-matter of the present invention are novel prodrug compounds represented by the following general formula (I): wherein, A is a residue of an antitumor substance having group in the molecule, R is a residue of cholesterol, m is an integer of 1 to 2 and n is O or 1 and their salts. In the prodrug compounds (I), antitumor substances in the term "a residue of an antitumor substance having group in the molecule" represented by A may be e.g. Mitomycin C, Nitrogen mustard, and FR900482 substance. Antitumor substances in the term "a residue of an antitumor substance having -NH₂ group in the molecule" may be e.g. Cytarabine, Aminopterin, Azacitidine, Doxorubicin, and Daunorubicin. These residues of antitumor substances are intended to mean residues wherein a hydrogen atom is removed from group in the molecule of the antitumor substance. The prodrug compound [I] of this invention or its salt can be prepared by the following process. wherein A, R, m and n are each as defined above. Namely, the prodrug compound [I] or its salt can be prepared by reacting the compound [II] or its reactive derivative at the carboxy group or its salt with the compound [III] or its salt. Suitable pharmaceutically acceptable salts of the compounds [I], [II] and [III] may include an acid addition salt such as an organic acid salt [e.g. acetate, maleate, tartrate, methanesulfonate, benzenesulfonate, and toluenesulfonate] or an inorganic acid salt [e.g. hydrochloride, hydrobromide, and sulfate, phosphate,], a metal salt [e.g. sodium salt, potassium salt, calcium salt, and magnesium salt,], an ammonium salt, an organic amine salt [e.g. trimethylamine salt, triethylamine salt, and dicyclohexylamine salt,], . Suitable reactive derivative at the carboxy group of the compound [II] may include an acid halide, an acid anhydride, an activated amide, and an activated ester, The suitable example may be an acid chloride, an acid azide; a mixed acid anhydride with an acid such as substituted phosphoric acid (e.g. dialkylphosphoric acid, phenylphosphoric acid or halogenated phosphoric acid), aliphatic carboxylic acid (e.g. pivalic acid or pentanoic acid), or aromatic carboxylic acid (e.g. benzoic acid); a symmetrical acid anhydride; an activated amide with imidazole, 4-substituted imidazole, dimethylpyrazole, triazole or tetrazole; or an activated ester (e.g. cyanomethyl ester or methoxymethyl ester) or an ester with N,N-dimethylhydroxylamine, l-hydroxy-2-(lH)-pyridone, N-hydroxysuccinimide, N-hydroxyphthalimide or l-hydroxy-6-chloro-lH-benzotriazole. These reactive derivatives can be optionally selected from them according to the kind of the compound [II] to be used. The reaction is usually carried out in a conventional solvent such as water, methanol, ethanol, acetone, dioxane, acetonitrile, chloroform, methylene chloride, ethylene chloride, tetrahydrofuran, ethyl acetate, N,N-dimethyl formamide, pyridine or any other solvent which does not adversely influence to the reaction. These conventional solvents may be used in a mixture with water. When the compound [II] is used in free acid form or its salt form in the reaction, the reaction is preferably carried out in the presence of a conventional condensing agent such as N,N′-dicyclohexylcarbodiimide, N-cyclohexyl-N′-morpholinoethylcarbodiimide, N-cyclohexyl-N′-(4-diethylaminocyclohexyl)carbodiimide, N,N′-diethylcarbodiimide, N,N′-diisopropylcarbodiimide, N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide, l-ethyl-3-(3-dimethyl)carbodiimide hydrochloride, N,N-carbonylbis(2-methylimidazole), trialkyl phosphite, ethyl polyphosphate, phosphorus oxychloride (phosphoryl chloride), thionyl chloride, oxalyl chloride, 2-ethyl-7-hydroxybenzisoxazolium salt, l-(p-chlorobenzenesulfonyloxy)-6-chloro-lH-benzotriazole, a Vilsmeier reagent which is, for example, prepared by the reaction of dimethylformamide with thionyl chloride, phosgen or oxalyl chloride. The reaction may be also carried out in the presence of an inorganic or organic base such as an alkali metal bicarbonate, tri(lower)alkylamine (e.g. trimethylamine or triethylamine), pyridine, N-(lower)alkylmorphorine, or N,N-di(lower)alkylbenzylamine. The reaction temperature is not critical, and the reaction is usually carried out under cooling or at room temperature. It is to be noted that each of the object compound [I] and the starting compounds [II] and [III] may include one or more stereoisomers such as optical isomers or geometrical isomers due to asymmetric carbon atom or double bond in the molecule, and all of such isomers of the compounds [I], [II] and [III] are included within the scope of this invention. The prodrug compound [I] of this invention or its salt is converted to the parent compound such as Mitomycin C, FR900482 substance, Nitrogen mustard or Cytarabine in blood, and all of these parent compounds have anti tumor activity and are useful for the treatment of various cancers (e.g. stomach cancer, lung cancer, lung adenocarcinoma, liver cancer, rectum cancer, pancreas cancer, breast carcinoma, and hystero carcinoma,) and leukemia (e.g. lymphocytic leukemia, and myeloid leukemia,). The prodrug compound [I] of this invention can be prepared as an aqueous solution, an emulsion, or a liposome suspension, according to the conventional methods, and then these preparations are administered to the body, for example, by parenteral administration (e.g. intravenous injection, intramuscular injection, intratumor injection, rectal administration,), or oral administration. The prodrug compound thus administered is gradually converted to the parent compound (e.g. Mitomycin C, FR900482 substance, Nitrogen mustard or Cytarabine,), and can maintain the concentration of the parent compound for a long time, improve the anti tumor effect, and reduce the toxicity when compared with the administration of the parent compound per se. Among above-mentioned preparations, the most sustained blood concentration of the parent compound can be obtained when the prodrug compound is administered as a liposome suspension. Phospholipid to be used as film substance for preparing the liposome preparation may be phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, and sphingomyelin, which are derived from egg, soybean or other animal tissue, egg york lecithin or soybean lecithin which are the mixture of the above-mentioned phospholipids, and synthetic lecithin such as dipalmitoyl lecithin, and distearoyl lecithin. Conventional additives [e.g. cholesterols, dicetyl phosphate, and α-tocopherol,] may be optionally added to the phospholipids. The liposomes can be prepared according to the conventional methods. That is prodrug compound [I] or its salt and phospholipid(s) are dissolved in a suitable organic solvent such as chloroform methanol , and ethanol, and the solution is poured into a suitable container and is evaporated under reduced pressure. Then, surfactant (e.g. sodium cholate,) and aqueous solution (e.g. phosphate buffered saline,) are added to the residue and agitated. After the residue is dissolved, the surfactant is removed from this solution by a detergent removal apparatus to give the liposome preparation. Another method is that phospholipid(s) is dissolved in organic solvent such as chloroform, or ethanol, and the solution is poured into a suitable container and is evaporated to give thin layer of phospholipid(s) under reduced pressure. Then, a solution of prodrug compound [I] or its salt is added thereto and shaked or sonicated. Liposomes can also be prepared by other conventional methods (e.g. ether injection method,) and accordingly the method for preparing liposomes is not limited to the aforesaid methods. Prodrug compound [I] of this invention or its salt may usually be administered with a unit dose of l mg to l000 mg, l to 4 times a day. However, the above dosage may be increased or decreased according to kinds of parent compound, age, weight and conditions of the patient or the administering method, or in combination with other anti tumor substances. In the case of parenteral administration, said dosage may be administered l to 2 times a week or with an interval of l to more than 2 weeks. To illustrate the effect of the present invention, test examples are shown in the following. Conversion test l Chloroform solution containing egg yolk lecithin (84 µmole), egg yolk sphingomyelin (36 µmole) and Mitomycin C prodrug ( 6 µmole) obtained in the below-mentioned Example l or 2 was poured into a round-bottom flask and evaporated to dryness. To this were added sodium cholate (258 mg) and pH 7.4 phosphate buffered saline (hereinafter referred to as PBS) (4.8 ml) and the mixture was agitated to give a clear purple solution. Sodium cholate was removed from the solution by the Lipoprep dialyzer (detergent removal apparatus prepared by Diachema Co., Ltd.) to give a liposome suspension containing Mitomycin C prodrug. Chloroform solution containing egg yolk lecithin (70 µ mole), egg yolk sphingomyelin (30 µmole) and FR900482 substance prodrug (Example 6)(5 µmole) was poured into a round-bottom flask and a liposome suspension containing FR900482 substance prodrug was obtained in a similar manner to that of above-mentioned method. Above-mentioned two liposome suspensions containing Mitomycin C prodrug were each diluted with PBS at 2.5 times. The liposome suspension containing FR900482 substance prodrug was diluted with PBS to obtain the drug concentration of 0.5 µmole/ml and was filtered through millex-GV (trademark: prepared by Millipore Co., Ltd.) filter (0.22 µ). After the liposome suspension and ethanol solution of each prodrug (0.5 ml) were added to mouse, rat or human serum (4.5 ml, 37°C), samples were taken periodically and the concentration of each prodrug was determined by high-performance liquid chromotography. Conversion half lives (t l/2) of each prodrug to each parent compound in various kinds of serum were calculated by the concentration of each prodrug remaining at each time. Test result Conversion half lives (t l/2) of each prodrug to each parent compound in various kinds of serum are shown in Table 1. From the above test results, it is found that the prodrugs of this invention can be converted to the parent compounds in blood in vitro and entrapment of each prodrug into liposomes increased its conversion half-life. Conversion Test 2 The mixture of chloroform and methanol (8:2 v/v) mixture containing egg yolk lecithin (70 µmole), egg yolk sphingomyelin (30 µmole) and Cytarabine prodrug (30 µmole) obtained in the below-mentioned Example 8 was poured into a round-bottom flask and evaporated to dryness. To this were added sodium cholate (53.8 mg) and PBS (5.0 ml) and the mixture was agitated to give a clear colorless solution. Sodium cholate was removed from the solution by the Lipoprep dialyzer to give liposome suspension containing Cytarabine prodrug. The liposome suspension was diluted with PBS to obtain the drug concentration of 5 µmole/ml and was filtered successively through polycarbonate membranes(prepared by Nuclepore Co., Ltd.) (0.2, 0.l and 0.08µ respectively) and then filtered through a millex-GV filter (0.22 µ). After the above-mentioned liposome suspension and ethanol solution of Cytarabine prodrug (0.08 ml) were added to rat serum (4.0 ml, 37°C), samples were taken periodically and the concentration of Cytarabine prodrug was determined by high-performance liquid chromatography. Conversion half lives (t l/2) of the prodrug to the parent compound were calculated by the concentration of prodrug remaining at each time. Test result Conversion half lives (t l/2) of the prodrug to the parent compound are shown in Table 2. From the above test result, it is found that the prodrug of this invention can be converted to the parent compound in blood in vitro and entrapment of the prodrug into liposomes increased its conversion half-life. Bioavailability Test l Chloroform solution (l4.3 ml) containing egg yolk lecithin (l26 µmole), egg yolk sphingomyelin (54 µmole) and Mitomycin C prodrug (Example l) (24 µmole) was poured into a round-bottom flask and evaporated at one hour. To this were added sodium cholate (l94 mg) and PBS (6 ml) and agitated. A liposome suspension containing Mitomycin C prodrug was obtained according to a similar manner to that of above-mentioned method. The liposome suspension thus prepared was diluted with PBS to obtain the drug concentration of 3.0 µmole/ml and was filtered through a polycarbonate membrane (0.l µ). l.0 ml of PBS solution of Mitomycin C or the liposome suspension containing Mitomycin C prodrug prepared by the above-mentioned method was administered intravenously to SD male rats (7 weeks, body weight : about 300 g, 4 per one group) and blood samples were withdrawn at appropriate intervals from subclavian vein. The plasma samples were deproteinzed and the concentration of Mitomycin C was determined by high-performance liquid chromatography. While Mitomycin C prodrug in the blood was determined by high-performance liquid chromatography after extraction with ethanol from the freeze-dried blood samples. Test result The blood concentrations of Mitomycin C and Mitomycin C prodrug are shown in Table 3 as mean value ± standard deviation of four rats. From the above test results, it is found that the liposome suspension containing prodrug can maintain the concentration of the parent compound Mitomycin C) for a long time. Bioavailability Test 2 The liposome suspension containing Mitomycin C prodrug (Example l), which was prepared in the above Bioavailability Test 1, was diluted with PBS at the concentration of Mitomycin C prodrug of 2.5 µmole/ml and was filtered through a polycarbonate membrane (0.1 µ). 0.2 ml of PBS solution of Mitomycin C or the liposome suspension containing Mitomycin C prodrug was administered intravenously to ICR female mice (8 weeks, body weight : about 25 g, 3 per one group) respectively, and the blood samples were withdrawn by cardiac puncture under anesthesia. The blood concentrations of Mitomycin C and Mitomycin C prodrug were determined in accordance with the same procedures as described in Bioavailability Test 1. Test result The blood concentrations of Mitomycin C and Mitomycin C prodrug are shown in Table 4 as mean value ± standard deviation of three mice. From the above test result, it is found that the liposome suspension containing prodrug can maintain the blood concentration of the parent compound (Mitomycin C) for a long time. Bioavailability Test 3 Each chloroform solution of Mitomycin C prodrug (Example l) (4 µmole), Panacete 800 (trademark, prepared by Nihon Yushi Co., Ltd.) (40 mg), egg yolk lecithin (l4 mg) and HCO-60 (trademark, prepared by Nikko Chemicals Co., Ltd.) (8 mg) was combined in a round-bottom flask and evaporated to dryness. To this was added PBS (l ml) and emulsified with a sonicator to give an emulsion containing Mitomycin C prodrug. Each chloroform solution of Mitomycin C prodrug (Example l) (4 µmole) and HCO-60 (24 mg) was combined in a round-bottom flask and evaporated to dryness. The residue was dispersed in l ml of PBS and agitated vigorously to yield a clear solution. 0.2 ml of each preparations thus obtained which contained the prodrug at 4 µmole/ml, was administered to mice and the blood concentrations of Mitomycin C and Mitomycin C prodrug were determined according to the same procedures as described in Bioavailability Test 2. Test result The blood concentrations of Mitomycin C and Mitomycin C prodrug when two kinds of preparations containing Mitomycin C prodrug (Example l) were administered intravenously to mouse are shown in Table 5 as mean value ± standard deviation of three mice. From the above test results, the emulsion and the aqueous soltuion of the prodrug can maintain the concentration of the parent compound (Mitomycin C) for a long time. Bioavailability Test 4 The mixture of chloroform and methanol (8:2 V/V, l2 ml) containing egg yolk lecithin (l47 µmole), egg yolk sphingomyelin (63 µmole) and Cytarabine prodrug (Example 8) (42 µmole) was poured into a round-bottom flask and evaporated at 37°C for l0 minutes. To this were added sodium cholate (262.5 µmole) and PBS (7 ml) and agitated. A liposome suspension containing Cytarabine prodrug (concentration : 5 µmole/ml) was prepared according to a similar manner to that of above-mentioned method. The aqueous solution of Cytarabine or the liposome suspension containing Cytarabine prodrug prepared by the above-mentioned method (l.0 ml, each contained 5.0µmole of drug/ml) was administered intravenously to SD male rat (7 weeks, body weight : about 250 g, 4 per one group) respectively, and blood samples were taken periodically from subclavian vein. The blood concentration of Cytarabine and Cytarabine prodrug were determined by high-performance liquid chromatography. Test result The blood concentrations of Cytarabine and Cytarabine prodrug are shown in Table 6 as mean value ± standard deviation of four rats. From the above test result, it is found that the liposome suspension containing prodrug can maintain the blood concentration of the parent compound (Cytarabine) for a long time. Toxicity Test Each chloroform solution of egg yolk lecithin (l26 µmole),egg yolk sphingomyelin (54 µmole) and Mitomycin C prodrug (Example l) (3.75, 24 and 37.5 µmole respectively) was combined in a round-bottom flask and evaporated to dryness. Liposome suspensions containing Mitomycin C prodrug were obtained according to the same procedures as described above. The liposome suspensions were diluted with PBS to obtain the desired drug concentration (0.l5, 0.96 and l.50 mg/ml as Mitomycin C) and were filtered through polycarbonate membranes (0.l µ). PBS solution of Mitomycin C (dose : l.0 and 6.4 mg/kg/day respectively) or above-mentioned liposome suspension containing Mitomycin C prodrug (dose : l.0, 6.4 and l0 mg/kg/day as Mitomycin C) were administered intravenously to ICR male mice (6 week) at Day l, 3 and 5 and the body weight change was recorded from Day l to l5. Test result The body weight change when the each preparation was administered intravenously are shown in Table 7 as mean value ± standard deviation of five mice. From the test results, it is found that toxicity of the liposome suspension containing the prodrug is lower than that of the aqueous solution of the parent compound. Anti tumor Test l The mixture of chloroform and methanol (8:2 V/V) containing egg yolk lecithin (245 µmole), egg yolk sphingomyelin (l05 µmole) and Cytarabine prodrug (Example 8) (87.5 and 262.5 µmole respectively) was taken in a round-bottom flask. Liposome suspensions containing Cytarabine prodrug (Concentration : l0 and 20 µmole/ml respectively) were obtained in a similar manner to that of the above-mentioned method. Mouse leukemia cell L l2l0 (l0⁵ cells) were implanted intraperitoneally into BDF female mice (7 weeks). After 24 hours after inoculation,Cytarabine aqueous solution (dose : l000 and 3000 µmole/kg respectively), above-mentioned liposome suspension containing Cytarabine prodrug (dose: l00 and 300 µmole/kg respectively) and aqueous solution of N⁴-behenoyl-l-β-arabinofuranosylcytosine (hereinafter referred to as BHAC) which is a known compound as Cytarabine prodrug and is shown by the following structure (dose : l00 and 300 µmole/kg respectively) were administered intravenously to the mouse at a volume of 0.2 ml or 0.3 ml (in case the administration at the dose of 300 µmole/kg of Cytarabine prodrug) per 20 g of mouse body weight. After the treatment, survival of the mice was recorded. Test result Mean survival times and percent of T/C, which was evaluated by the mean survival time of treated groups as compared to that of the control group when each preparation was administered intravenously are shown in Table 8. It is found that the anti tumor effect of the liposome suspension containing the prodrug of this invention was superior to that of the aqueous solution of the parent compound or BHAC. Anti tumor Test 2 The mixture of chloroform and methanol (3:l V/V) containing egg yolk lecithin (245 µmole), egg yolk sphingomyelin (l05 µmole) and Cytarabine prodrug (Example 8) (87.5 and l57.5 µmole respectively) was taken in a round-bottom flask. Liposome suspensions containing Cytarabine prodrug (Concentration : l0 and l8 µmole/ml respectively) were obtained in a similar manner to that of the above-mentioned method. A fragment of human lung adenocarcinoma A 549 was implanted under the subrenal capsule of BDF female mouse (8 weeks). Cytarabine aqueous solution (dose : l000 µmole/kg) and above-mentioned liposome suspension containing Cytarabine prodrug (dose : l00 and l80 µmole/kg respectively) were administered intravenously to the mice at a volume of 0.2 ml per mouse body weight 20 g at Day l, 5 and 9 after implantation. The tumor volume at Day l4 after implantation was measured and the growth inhibition percent was calculated. (In this test, to clarify the anti tumor effect of each preparation, l7-allyl-l,l4-dihydroxy-l2-[2-(4-hydroxy-3-methoxycyclohexyl)-l-methylvinyl]-23,25-dimethoxy-l3,l9,2l,27-tetramethyl-ll,28-dioxa-4-azatricyclo[22.3.l.0 4,9 ]octacos-l8-ene-2,3,l0,l6-tetraone as immunosuppressive substance(dose : 32 mg/kg) was administered subcutaneously at Day l, 2, 5, 7, 9 and l2 after implanation.) Test result The tumor volume at Day l4 after implantation and the growth inhibition percent of tumor when each preparation was administered intravenously are shown in Table 9. From the test results, it is found that the anti tumor effect of the liposome suspension containing the prodrug of this invention is superior to that of the aqueous solution of the parent compound. The following Preparations and Examples are shown for the purpose of illustrating the present invention in more detail. Preparation l To a suspension of glycine (l.50 g) in water (l00 ml) was added triethylamine (4.04 g). Dioxane (200 ml) and cholesterol chlorocarbonate (8.98 g) were added to the resulting clear solution at 0°C and the mixture was stirred for 3 hours. The reaction mixture was concentrated in vacuo and to the residue were added lN-hydrochloric acid (23 ml) and chloroform (l00 ml). Organic layer was separated, washed with water and dried over magnesium sulfate. Evaporation of the solvent gave crude crystal, which was washed with ethanol and collected by filtration to afford N-(cholest-5-en-3β-oxycarbonyl)glycine (6.64 g) as a white crystal. mp : l75-l77°C (dec.) IR (Nujol) : 3320, l750, l665, l570 cm⁻¹ Preparation 2 To a solution of N-(cholest-5-en-3β-oxycarbonyl)glycine (2.92 g) in a mixture of dioxane (40 ml) and chloroform (6 ml) were added successively N-hydroxysuccinimide (0.69 g) and dicyclohexylcarbodiimide (l.236 g) at 0°C and the reaction mixture was stood for l6 hours in a refrigerator. The resulting precipitate was collected by filtration and the filtrate was concentrated in vacuo to give succinimidyl N-(cholest-5-en-3β-oxycarbonyl)glycinate (3.23 g). mp : l59-l64°C IR (CHCl₃) : 3440, l820, l785, l740, l720 cm⁻¹ Preparation 3 To a solution of β-alanine (56 mg) and sodium bicarbonate (84 mg) in water (l0 ml) were added tetrahydrofuran (20 ml) and cholesterol chlorocarbonate (449 mg) at 0°C. The mixture was stirred for l hour. The reaction mixture was concentrated in vacuo and to the residue were added 0.lN hydrochloric acid (l0 ml) and chloroform (40 ml). Organic layer was separated, washed with water and dried over magnesium sulfate. Evaporation of the solvent gave a crude product, which was dissolved in chloroform and applied to a silica gel column (40 g). Elution with a mixture of chloroform and methanol gave N-(cholest-5-en-3β-oxycarbonyl)-β-alanine (l87 mg) as a white powder. mp : l27-l30°C IR (CHCl₃) 3450, l705 cm⁻¹ Preparation 4 To a solution of 6-amino-n-caproic acid (l3l mg) and sodium bicarbonate (84 mg) in a mixture of tetrahydrofuran (l0 ml) and water (l0 ml) were added tetrahydrofuran (l0 ml) and cholesterol chlorocarbonate (449 mg) at 0°C. The mixture was treated in a similar manner to that of Preparation 3 to give N-(cholest-5-en-3β-oxycarbonyl)-6-amino-n-caproic acid (l26 mg) as a white powder. IR (CHCl₃) 3440, l700 cm⁻¹ Example l To a solution of Mitomycin C (l.00 g) in N,N-dimethylformamide (20 ml) were added triethylamine (0.33 g) and succinimidyl N-(cholest-5-en-3β-oxycarbonyl)glycinate (l.75 g) at room temperature and the reaction mixture was stirred for 4 hours at the same temperature. The solvent was evaporated off and to the residue was added chloroform and water. The organic layer was separated, washed with water and dried over magnesium sulfate. Evaporation of the solvent gave a crude product, which was dissolved in chloroform and applied to a silica gel column (50 g). Elution with chloroform gave [laS-(laα, 8β, 8aα, 8bα)]-6-amino-8-carbamoyloxymethyl-N′-[N-(cholest-5-en-3β-oxycarbonyl)glycyl]-l,la,2,8,8a,8b-hexahydro-8a-methoxy-5-methy-aziridino[2′,3′:3,4]pyrrolo[l,2-a]indole-4,7-dione (l.74 g) as a dark purple powder. mp : >250°C Mass m/z 804 (M⁺) Example 2 To a solution of cholest-5-en-3β-oxyacetic acid (667 mg), which was prepared according to the literature [Australian J. Chem., 24 ,l43-5l (l97l)], in chloroform (l5 ml) were added N-hydroxysuccinimide (l72 mg) and dicyclohexylcarbodiimide (324 mg) at 0°C and the mixture was stirred overnight at room temperature. The precipitate was filtered off and the filtrate was concentrated in vacuo to give an active ester, which was used for the next reaction without further purification. To a solution of Mitomycin C (502 mg) in N,N-dimethylformamide (l0 ml) were added active ester, triethylamine (l52 mg) and 4-dimethylaminopyridine (20 mg). The reaction mixture was stirred overnight at 60°C and concentrated in vacuo. To the residue were added chloroform and water. The organic layer was separated and dried over magnesium sulfate. Evaporation of the solvent gave a crude product, which was dissolved in a small amount of chloroform and applied to a silica gel column. Elution with chloroform gave [laS-(laα, 8β, 8aα, 8bα)]-6-amino-8-carbamoyloxymethyl-N-(cholest-5-en-38-oxyacetyl)-l,la,2,8,8a,8b-hexahydro-8a-methoxy-5-methyl-aziridino-[2′,3′:3,4]pyrrolo[l,2-a]indole-4,7-dione (695 mg) as a dark purple powder. mp : l45° - l50°C (dec.) IR (Nujol) : 3430, 3320, 3200, l7l0, l650, l600, l550 cm⁻¹ Example 3 In a similar manner to that of Example 2, there was obtained [laS-(laα,8β,8aα,8bα)]-6-amino-8-carbamocyloxymethyl-N-(cholest-5-en-3α-oxyacetyl)-l,la,2,8,8a,8b-hexahydro-8a-methoxy-5-methyl-aziridino[2′,3′:3,4]pyrrolo[l,2-a]indole-4,7-dione, which is α-isomer of 3-position of cholesterol in Example 2. mp : ll8-l20°C IR (Nujol) : 3420, 3320, 3200, l700, l600, l550 cm⁻¹ Example 4 To a solution of N-(cholest-5-en-3β-oxycarbonvl)-β-alanine (56 mg) in a mixture of tetrahydrofuran (l5 ml) and chloroform (5 ml) were added Mitomycin C (37 mg), triethylamine (l3 mg) and l-Ethyl-3-(3-dimethyl)carbodiimide hydrochloride (24 mg). The reaction mixture was stirred overnight at room temperature and concentrated in vacuo. To the residue were added chloroform and water. The organic layer was separated and dried over magnesium sulfate. Evaporation of the solvent gave a crude product, which was dissolved in a small amount of chloroform and applied to a silica gel column. Elution with a mixture of chloroform and methanol gave [laS-(laα, 8β,8aα,8bα)]-6-amino-8-carbamoyloxymethyl-N′-[N-(cholest-5-en-3β-oxycarbonyl)-β-alanyl]-l,la,2,8,8a,8b-hexahydro-8a-methoxy-5-methyl-aziridino[2′,3′:3,4]pyrrolo-[l,2-a]indole-4,7-dione (22 mg) as a dark purple powder. IR (CHCl₃) 3500, 3440, 3380, l695, l600, l565 cm⁻¹ Example 5 To a solution of N-(cholest-5-en-3β-oxycarbonyl)-6-amino-n-caproic acid (63 mg) in a mixture of tetrahydrofuran (20 ml) and chlorofrm (5 ml) were added Mitomycin C (39 mg), triethylamine (l3 mg) and l-Ethyl-3-(3-dimethyl)carbodiimide hydrochloride (24 mg). The reaction mixture was stirred overnight at room temperature and concentrated in vacuo. To the residue were added chloroform and water. The organic layer was separated and dried over magnesium sulfate. Evaporation of the solvent gave a crude product, which was dissolved in a small amount of chloroform and applied to a silica gel column. Elution with a mixture of chloroform and methanol gave [laS-(laα,8β,8aα,8bα)]-6-amino-8-carbamoyloxymethyl-N′-[N-(cholest-5-en-3β-oxycarbonyl)-6-amino-n-caproyl]-l,la,2,8,8a,8b-hexahydro-8a-methoxy-5-methylaziridino[2′,3′:3,4]pyrrolo[l,2-a]indole-4,7-dione (3l mg) as a dark purple powder. IR (CHCl₃) : 3500, 3440, 3370, l700, l600, l560 cm⁻¹ Example 6 4-Formyl-6,9-dihydro-l4-oxa-l,ll-diazatetracyclo[7.4.l.0 2,7 .0 l0,l2 ]tetradeca-2,4,6-triene-8-ylmethylcarbamate (32 mg) was dissolved in N,N-dimethylformamide (l.0 ml). To the solution was added triethylamine (l4 µl) and succinimidyl N-(cholest-5-en-3β-oxycarbonvl)glycinate (60 mg). The mixture was stirred for 6 hours at ambient temperature and evaporated in vacuo. The residue was subjected to a preparative thin layer chromatography, developing with a mixture of chloroform and methanol to afford 2 isomers at C-9 of ll-[N-(cholest-5-en-3β-oxycarbonyl)glycyl]-4-formyl-6,9-dihydroxy-l4-oxa-l,ll-diazatetracyclo[7.4.l.0 2,7 .0 l0,l2 ] tetradeca-2,4,6-triene-8-ylmethylcarbamate (9-α-OH isomer 3l mg and 9-β-OH isomer l5 mg). mp : l68-l70°C (dec.) (a mixture of 9-α-OH isomer and 9-β-OH isomer) 9α-OH isomer IR (CHCl₃) : 3340, 2950, l695, l586, l355 cm⁻¹ SIMS : 79l [M+H]⁺, 8l3 [M+Na]⁺ 9β-OH isomer IR (CHCl₃) : 3340, 2945, l695, l583, l350 cm⁻¹ SIMS : 79l [M+H]⁺, 829 [M+K]⁺ Example 7 To bis(2-chloroethyl)amine hydrochloride (l.347 g) in 25% sodium hydroxide solution (l00 ml) was added methylene chloride (l00 ml) and the mixture was shaked. Organic layer was separated and dried with potassium carbonate and filtered. Methylene chloride was evaporated under reduced pressure to obtain bis(2-chloroethyl)amine (0.52 g) as an oily substance. To a solution of N-(cholest-5-en-3β-oxycarbonyl)glycine (l60.8 mg) in dry tetrahydrofuran (50 ml) was added said bis(2-chloroethyl)amine (85.2 mg) and dicyclohexylcarbodiimide (6l.9 mg) at 0°C under blowing nitrogen gas and the mixture was stirred for 6 hours. After standing overnight (l7 hours) at refrigerator, the reaction mixture was filtered and the solvent was evaporated under reduced pressure. The residue was solved in a mixture of chloroform and methanol (50:l) and was subjected to preparation column chromatography (PLC) eluting with a mixture of chloroform and methanol (50:l). The desired parts were gathered, extracted with a mixture of chloroform and methanol (4:l), filtered and concentrated under reduced pressure to give N-[N-(cholest-5-en-3β-oxycarbonyl)glycyl]bis(2-chloroethyl)amine (ll0 mg). mp : 98-l00°C Example 8 To a solution of N-(cholest-5-en-3β-oxycarbonyl)glycine (2.74 g) and triethylamine (0.78 ml) in tetrahydrofuran (40 ml) was added dropwise ethyl chlorocarbonate (0.6l g) at -l5°C, and the reaction mixture was stirred for 20 minutes at -l5°C. To the reaction mixture a solution of l-β-D-arabinofuranosylcytosine (l.37 g) in dimethylformamide (30 ml) was added dropwise at -l5°C to -l0°C. After the addition, the mixture was stirred for one hour at 5°C and then for 30 minutes at room temperature. The reaction mixture was concentrated in vacuo. The residue was washed with methanol and dried to afford a crude reaction product, which was purified by silica gel column chromatography to yield N⁴-[N-(cholest-5-en-3β-oxycarbonyl)glycyl]-l-β-D-arabinofuranosylcytosine (l.8) as a white powder. mp : l59-l6l°C (dec.) IR (Nujol) : 3330, 2850, l690, l640, l570 cm⁻¹ SIMS (m/z) : 7l3 [M+H]⁺, 75l [M+K]⁺

Claims: A Prodrug compound of the formula: A-CO(CH₂) m (NHCO) n -R wherein A is a residue of an antitumor substance having group in the molecule, R is a residue of cholesterol, m is an integer of 1 or 2 and n is 0 or 1, and its salts. A Prodrug compound according to claim 1, wherein the antitumor substance is Mitomycin C. A Prodrug compound according to claim 1, wherein the antitumor substance is Nitrogen mustard. A Prodrug compound according to claim 1, wherein the antitumor substance is 4-formyl-6,9-dihydroxy-14-oxa-1,11-diazatetracyclo-[7.4.1.0 2,7 .0 10,12 ] tetradeca-2,4,6-trien-8-yl-methylcarbamate. A Prodrug coumpound according to claim 1, wherein the antitumor substance is Cytarabine. A process for preparing a Prodrug compound of the formula: A-CO(CH₂) m (NHCO) n -R wherein A is a residue of antitumor substance having group in the molecule, R is a residue of cholesterol, m is an integer of 1 or 2 and n is 0 or 1 or its salt, which comprises reacting a compound of the formula: HOOC(CH₂) m (NHCO) n -R wherein R, m and n are each as defined above, or its reactive derivative at the carboxy group or it salt, with a compound of the formula: A-H wherein A is as defined above, or its salt. A sustained release antitumor preparation comprising, as an active ingredient, a Prodrug compound of claim 1 or salts thereof in association with pharmaceutically acceptable carrier or excipient. A Prodrug compound of claim 1 for use as a medicament. Claims for the following Contracting States : AT, ES, GR A process for preparing a prodrug compound of the formula: wherein A is a residue of an antitumor substance having group in the molecule, R is a residue of cholesterol, m is an integer of 1 or 2 and n is 0 or 1, and its salts, which comprises reacting a compound of the formula: wherein R, m and n are each as defined above, or its reactive derivative at the carboxy group or its salt, with a compound of the formula: wherein A is as defined above, or its salt. The process according to claim 1, wherein as antitumor substance Mitomycin C is used. The process according to claim 1, wherein as antitumor substance Nitrogen mustard is used. The process according to claim 1, wherein as antitumor substance 4-formyl-6,9-dihydroxy-14-oxa-1,11-diazatetracyclo-[7.4.1.0 2,7 .0 10,12 ] tetradeca-2,4,6-trien-8-yl-methylcarbamate is used. The process according to claim 1, wherein as antitumor substance Cytarabine is used. A process for preparing a sustained release antitumor preparation wherein as an active ingredient a prodrug compound of claims 1 to 5 or salt thereof is associated with a pharmaceutically acceptable carrier or excipient.

Inventors: HASHIMOTO, MASASHI, IWASA, TOMOAKI, KAGAYAMA, AKIRA, KAYAKIRI, HIROSHI, TAKENO, HIDEKAZU, TOKUNAGA, YUJI
Applicants: FUJISAWA PHARMACEUTICAL CO., LTD.
Application year: 1987
Main classification: C07J43
IPC codes: C07J43, C07H21