JPS6221795B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention provides antibiotic neothramycin A or/
The present invention also relates to a sulfite adduct, which is a new, useful and stable water-soluble derivative of neothramycin B, and a method for producing the same. Neothramycin A and neothramycin B have only a weak antibacterial effect on various types of bacteria, but they strongly suppress the growth of mouse leukemia L-1210 cells and certain types of cancer cells, and are used as anticancer agents. (Japanese Patent Application No. 1972-27674, filed on March 12, 1970; Japanese Patent Application No. 123256, filed on October 15, 1975; Japanese Patent Application No. 1972-259, filed on January 1, 1978) Application: Patent application 1977
-No. 110722, filed on September 17, 1975; patent application 1977-
No. 110723, filed on September 17, 1975; Journal
Journal of Antibiotics, Vol. 29, January issue, pp. 93-96 (1976) and Journal of Antibiotics, Vol. 30, April issue, pp. 340-343 (1977)). Neothramycin A and neothramycin B are unstable not only in aqueous solution but also in powdered form, exhibiting the property of being easily oxidized and colored, and their relatively low solubility in water, limiting their use as injections. . In view of these circumstances, the present inventors conducted various studies aimed at producing useful and stable water-soluble derivatives of neothramycin A and neothramycin B. We have discovered that sulfite adducts can be obtained as new derivatives by reacting sulfites. Moreover, 1 the new derivative is highly soluble in water (solubility 1 g/ml at room temperature), 2 retains the antibacterial and antitumor activity of neothramycin, and 3 reduces pain and local damage when injected as an aqueous solution. 4. It has better stability than neothramycin A and neothramycin B, both in powder form and in aqueous solution state, and can effectively prevent coloring, and 5. Advantageous new findings were obtained, including lower acute toxicity and increased safety. The present invention was completed based on these new findings. That is, the gist of the present invention is a derivative of neothramycin A or neothramycin B, in which a sulfite group or a sulfite group is added and bonded to the azomethine group of neothramycin A or neothramycin B, and a salt thereof. . Specifically, the present invention provides structural formula [wherein R is a hydrogen atom, an alkali metal atom, an alkaline earth metal atom, or an ammonium group] A sulfite adduct of neothramycin A or neothramycin B and a pharmaceutically usable salt thereof This is the summary. The object of the present invention is neothramycin A or/and neothramycin B having an azomethine group.
It can be produced by reacting sulfite with sulfite. Any sulfite that can be used in this method may be one that can generate sulfite ions in solution; examples include acidic sulfites, such as sodium acid sulfite (NaHSO ₃ ) and potassium acid sulfite (KHSO ₃ ). Acidic alkali metal sulfites such as acidic ammonium sulfite (NH ₄ HSO ₃ ); and sulfites such as sodium sulfite (Na ₂ SO ₃ ), potassium sulfite (K ₂ SO ₃ ); ₃ ), alkaline earth metal salts of sulfite, such as barium sulfite (BaSO ₃ ); and metabisulfites, such as sodium metabisulfite (Na ₂ S ₂ O ₅ ), potassium metabisulfite (K ₂ S ₂ O ₅ ) and alkali metal salts of metabisulfite. Sulfite per one azomethine group of neothramycin A or/and neothramycin B,
1 to 1.5 molar equivalents may be used, but most preferably 1 molar equivalent is used. This method is usually carried out in an aqueous solution, but a water-containing solution of a water-containing organic solvent such as acetone, dioxane, ethyl acetate, etc. can be used as the solvent. The reaction temperature generally proceeds easily at around room temperature, but is preferably about 5°C. After completion of the reaction, the object of the present invention produced by the above method can be obtained by conventional collection means from the reaction mixture, concentration drying, freeze drying, precipitation by adding an insoluble solvent,
It can be collected by methods such as filtration, centrifugation, and column chromatography. New derivative according to the present invention, neothramycin A
Or/and a sodium sulfite adduct of neothramycin B was obtained by the reaction shown in the following formula, and its structure was confirmed by the physicochemical and spectral analysis shown below. The sulfite adduct of neothramycin A and B equilibrium mixture is a white powder with no clear melting point.
Foaming decomposes at 220℃. The specific optical rotation was [α] ²² _D =34.7° (c1, water). The actual values of elemental analysis are C40.47%, H4.75%,
Showing N7.44%, O32.78%, S8.96%,
The calculated value for C ₁₃ H ₁₅ N ₂ O ₄・SO ₃ Na・H ₂ O is
C40.62%, H4.45%, N7.28%, O33.30%, S8.34
%. The sulfite adduct of neothramycin A and B equilibrium mixture is highly soluble in water, slightly soluble in lower alkanols such as methanol and ethanol, and insoluble in ethyl ether and n-hexane. Figure 1 shows the infrared absorption spectrum curves measured with potassium bromide tablets of sulfite adducts of neothramycin A and B equilibrium mixtures.
Fig. 2 shows the ultraviolet absorption spectrum curve measured at 100 g/ml), and Figs. 3 and 4 show the hydrogen nuclear and carbon nuclear magnetic resonance spectra measured in a heavy aqueous solution. The sulfite adducts of neothramycin A and B equilibrium mixtures are Lydon-Smith reaction, red tetrazolium reaction, FastBlue-B reaction, silver nitrate-
The fluorescein reaction is positive, the ninhydrin reaction is slightly positive, and the Ehrlich reaction is negative. This substance was analyzed by silica gel thin layer chromatography (developing system n-butanol/methanol-water 8:
1:4 volume) gave an R _f value of 0.38, which was distinguishable from the R _f value of neothramycin of 0.80. In addition, this substance can be used in cellulose thin layer chromatography (development system n-
propanol-water (7:3 vol) gave an R _f value of 0.45, while neothramycin gave an R _f value of 0.88. In high-pressure paper migration (3000V, 20 minutes) using formic acid-acetic acid-water (25:75:900 volume), when the movement of alanine from the origin to the pole is +1.0, the equilibrium mixture of neothramycin A and B that of the sulfite adduct showed −0.53. The sulfite adduct of neothramycin A is a white powder with no definite melting point and decomposes at 230-240°C. The specific optical rotation was [α] ²⁴ _D = 16.4° (c1, water). Figure 5 shows the infrared absorption spectrum curve of this substance measured with potassium bromide tablets, and the aqueous solution (10mcg/ml)
The ultraviolet absorption spectrum curve A measured in FIG. 6 is shown in FIG. 6, and the hydrogen nuclear magnetic resonance spectrum measured in a heavy water solution is shown in FIG. The sulfite adduct of neothramycin B is a white powder with no clear melting point and decomposes at 220-230°C. The specific optical rotation was [α] ²⁴ _D =+12.6° (c1, water). Figure 8 shows the infrared absorption spectrum curve measured with potassium bromide tablets of this substance. Figure 6 shows the ultraviolet absorption spectrum curve B measured in an aqueous solution (10 mcg/ml), and hydrogen nuclear magnetic field measured in a heavy aqueous solution. The resonance spectrum is shown in FIG. The minimum inhibitory concentrations of neothramycin A and B equilibrium mixtures and their sulfite adducts on nutrient agar plates are shown in Table 1, and both exhibit only weak antibacterial activity.

[Table] Neothramycin A and B equilibrium mixture and its sulfite adduct for murine leukemia Leukemia P
-388, when neothramycin (an equilibrium mixture of A and B) or its sulfite adduct was intraperitoneally administered for 3 consecutive days after leukemia cells were transplanted into the peritoneal cavity of a mouse at a rate of 10 ⁶ cells/mouse. The life-prolonging effect shown in Table 2 was observed.

[Table] Survival days of untreated mice Acute toxicity of neothramycin sulfite adduct was administered intravenously to mice, LD ₅₀ 46 mg/Kg (male);
46.5 mg/Kg (female). In contrast, the acute toxicity of neothramycin was LD ₅₀ 23.1 mg/Kg under the same conditions.
(male) and 27.0 mg/Kg (female), and the toxicity of neothuramycin sulfite addition salt is reduced compared to neothuramycin. Since the neothramycin sulfite adduct exhibits the above-mentioned usefulness, it can be used for the purpose of an anticancer agent like the raw material antibiotic neothramycin. Example 1 1 g of neothramycin A, B equilibrium mixture and 397 mg of sodium bisulfite were dissolved in 10 ml of water.
After reacting at room temperature, the mixture was placed in a column (inner diameter 21 mm) packed with Cephadex G-10 (200 ml) and developed with water at 5°C. Fractionate into 20ml and collect the active fraction (No. 7-
11) was collected and lyophilized to obtain 1214 mg of white powder. This powder was placed in the Cephadex tower and the same operation was repeated twice to obtain Neothramycin A,
White powder of sulfite adduct of B equilibrium mixture (934
mg, yield 66.8%). Example 2 1 g of neothramycin A, B equilibrium mixture and 0.97 g of sodium sulfite heptahydrate were dissolved in 10 ml of water.
After the reaction, a column (inner diameter 21 mm) packed with Cephadex G-10 (200 ml) was applied and the same operation as in Example 1 was carried out to obtain a pale yellow powder (1.30 g, yield 87.5) of the sulfite adduct of neothramycin A and B equilibrium mixture. %) was obtained. Example 3 1 g of neothramycin A, B equilibrium mixture and 0.37 g of sodium metabisulfite were dissolved in 10 ml of water,
After the reaction, a column (inner diameter 21 mm) packed with Cephadex G-10 (200 ml) was operated in the same manner as in Examples 1 and 2 to obtain a white powder (1.19 g, yield) of the sulfite adduct of neothramycin A and B equilibrium mixture. rate 85
%) was obtained. Example 4 Neothramycin A, B equilibrium mixture 10 mg in 1 ml
After aseptic filtration, an aqueous solution containing 4 mg of sodium bisulfite (1 molar ratio to 1 mol of neothramycin) was filled into 5 ml ampoules and lyophilized to form a sodium sulfite adduct of the neothramycin A, B equilibrium mixture. A white powder is obtained. After replacing the air in the ampoule space with nitrogen gas, the ampoule is sealed. Example 5 Neothramycin A (Neothramycin A95.6
%) 200 mg and sodium bisulfite 79.4
mg (1 molar ratio to 1 molar of neothramycin A) was dissolved in 4 mg of water and then lyophilized to obtain 250 mg of a white powder of neothramycin A sulfite adduct. Example 6 Neothramycin B (Neothramycin B91.2
%) 200 mg and sodium bisulfite 79.4
mg (1 molar ratio to 1 mol of neothramycin B) was dissolved in 4 ml of water and lyophilized to obtain 214 mg of neothramycin B sulfite adduct as a white powder.

[Brief explanation of the drawing]

Figure 1 shows the infrared absorption spectral curve of the sulfite adduct of the neothramycin A, B equilibrium mixture of the present invention (product of Example 1), and Figure 2 shows the ultraviolet absorption spectral curve of the same sulfite adduct (aqueous solution). ,
(in 0.1N hydrochloric acid and 0.1N sodium hydroxide aqueous solution), Figure 3 is the hydrogen nuclear magnetic resonance spectrum curve of the same sulfite adduct, Figure 4 is the carbon nuclear magnetic resonance spectrum curve of the same sulfite adduct, Figure 5 6 is an infrared absorption spectrum curve of neothramycin A sulfite adduct (product of Example 5), and FIG. 6 shows neothramycin A sulfite adduct and neothramycin B.
Ultraviolet absorption spectrum curve with sulfite adduct (product of Example 6), Figure 7 is Neothramycin A
The hydrogen nuclear magnetic resonance spectrum curve of the sulfite adduct, Figure 8 is the infrared absorption spectrum curve of the neothuramycin B sulfite adduct, and Figure 9 is the hydrogen nuclear magnetic resonance spectrum curve of the neothuramycin B sulfite adduct. shows.

Claims (1)

[Claims] 1. Structural formula Neothramycin A or
A sulfite adduct of neothramycin B and a pharmaceutically usable salt thereof. 2 Structural formula The compound according to claim 1, which is a sulfite adduct of neothramycin A represented by the formula [wherein R is a hydrogen atom or an alkali metal atom] and a pharmaceutically usable salt thereof. 3 Structural formula The compound according to claim 1, which is a sulfite adduct of neothramycin B represented by the formula [wherein R is a hydrogen atom or an alkali metal atom] and a pharmaceutically usable salt thereof. 4 Structural formula Neothramycin A or/and Neothramycin B having the following structural formula is reacted with a sulfite in an aqueous solution or in a water-containing organic solvent. Neothramycin A or/in which R is a hydrogen atom or an alkali metal atom
and a method for producing a sulfite adduct of neothramycin B.