JPS6312066B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel derivative of geldanamycin, a method for producing the same, and an antitumor agent containing the same as an active ingredient. The novel compound of the present invention is represented by the following formula. In this formula, R is a residue

[Formula] or

[Formula], R ¹ , R ² and R ³ represent a hydrogen atom or a methyl group, in which case R ¹ , R ² and
At least one of R ³ is a methyl group. Geldanamycin is known to be an antiprotozoal antibiotic produced by Streptomyces hygroscopicus val geldanus val nova (Journal of Antibiotics Vol.
23, p. 442, 1970), and has the chemical structure of the following formula (see Journal of the American Chemical Society, vol. 92, p. 7591, 1970). The present inventors synthesized geldanamycin derivatives and tested various effects on living organisms. As a result, the new compound of the formula has excellent antitumor activity and is useful as an intermediate for synthesizing other derivatives. I found that. New compounds of formula can be prepared by methylating compounds of formula. As the methylating agent, the usual ones can be used, but methyl halides such as methyl chloride, methyl bromide or especially methyl iodide are preferred. This reaction can be carried out, for example, as follows.
A compound of the formula is dissolved in an organic solvent such as chloroform, methanol, etc. or a mixture thereof, and an excess amount, preferably 4 to 6 times the amount of silver oxide is added thereto and suspended, and then an excess amount, preferably 8 to 10 times the amount of silver oxide is added and suspended. Add methyl halide and stir. Generally at room temperature 3
The reaction is complete after ~24 hours. Under these reaction conditions, compounds of several formulas are usually produced simultaneously. Each target substance can be isolated and purified by conventional methods. For example, after separating the lower solution of silver iodide and excess silver oxide from the reaction mixture, the liquid is evaporated to dryness under reduced pressure, and the residue can be purified by chromatography, recrystallization, or the like. The compound of the formula is generally a yellow to yellow-orange substance that is sparingly soluble in water, such as methanol, ethanol, acetone, ethyl acetate, halogenated hydrocarbons, ethyl ether, tetrahydrofuran, dioxane, dimethyl sulfoxide, dimethoxyformamide, Easily soluble in organic solvents such as benzene and pyridine. The novel compound of the present invention has a remarkable growth inhibiting effect on experimental tumor cell W-2K-11 cells, which are widely recognized as a model of cancer cells, and is useful as an antitumor agent. Therefore, the present invention further provides an antitumor agent containing a compound of the formula as an active ingredient. Although compounds of one or several formulas may be used alone as antitumor agents of the present invention, they are usually combined with common excipients or adjuvants to form preparations suitable for oral as well as parenteral administration. It is preferable to use the form. As the excipient or auxiliary agent, for example, the following organic or inorganic solid or liquid substances can be used. Suitable excipients are, for example, water, gelatin, lactose, starch, cellulose calcium glycolate, microcrystalline cellulose, stearyl alcohol, magnesium stearate, talc, vegetable oil, benzyl alcohol, propylene glycol, gums, polyalkylene glycols, These include white petroleum, jelly, and cholesterol. Examples of auxiliary agents used include preservatives, wetting agents, emulsifiers, solubility promoters, salts for adjusting osmotic pressure, buffers, binders, suspending and dispersing agents, and the like. Examples of the preparation include powders, granules, capsules, pills, tablets, sugar-coated tablets, injections, suppositories, and ointments, and these preparations can be manufactured by conventional methods. The antitumor agent of the present invention can be used not only for human treatment, but also as a veterinary medicine in the form described above. When using the antitumor agent of the present invention for treatment, the dosage of the active ingredient is generally 0.5 to 80 mg/Kg per adult per day, preferably 1 to 80 mg/Kg in the case of parenteral administration.
40mg/Kg, generally 1-100 for oral administration
mg/Kg, preferably 2 to 50 mg/Kg. Example 1 5 g of geldanamycin is dissolved in a mixed solvent of 200 ml of chloroform and 200 ml of methanol, 20 g of silver oxide is added thereto, and 40 g of methyl iodide is added while stirring. After stirring at room temperature for 5 hours, insoluble matter was removed by filtration, and the liquid was evaporated to dryness under reduced pressure at a bath temperature of 40°C. The resulting red-orange oily residue is subjected to silica gel column chromatography using 2.5% methanol-chloroform. Fractions Nos. 42 to 60 are collected, evaporated to dryness, and then recrystallized from ether-n-hexane to obtain 600 mg of the compound of the following formula as yellow-orange crystals. Melting point: 213-215℃ Elemental analysis value: C ₃₀ H ₄₂ N ₂ O ₉・1/2H ₂ O C H N Calculated value (%) 61.73 7.42 4.80 Actual value (%) 62.09 7.37 4.73 NMR spectrum (100MHz, CDCl ₃ middle): δ (ppm) 3.85 (imino methyl ether) Ultraviolet absorption spectrum: λ ^CH3OH _nax (nm) 262, 305, 390 (shoulder) Molecular weight by mass spectrometry: m/e547 (M ⁺ ) Example 2 Example In the same manner as in 1, the fractions No. 77 to 128 in silica gel column chromatography were collected and evaporated to dryness, and then recrystallized from chloroform-n-hexane to obtain the compound of the following formula.
2.42 g are obtained as yellow powder crystals. Melting point: 141-143℃ Elemental analysis value: C ₃₀ H ₄₂ N ₂ O ₉・1/2H ₂ O Calculated value (%) 61.73 7.42 4.80 Actual value (%) 61.39 7.28 4.80 NMR spectrum (100MHz, CDCl ₃ ): δ (ppm) 3.12 (N-methyl) Ultraviolet absorption spectrum: λ ^CH3OH _nax (nm) 254, 316, 390 (shoulder) Molecular weight by mass spectrometry: m/e574 (M ⁺ ) Example 3 Implementation Following the same procedure as in Example 1, fractions No. 13 to 34 in silica gel column chromatography were collected and evaporated to dryness, and the residue was purified by re-chromatography using 2% methanol-chloroform. Chloroform-n
-Crystallization from hexane yields compound 452 of the following formula:
mg as yellow crystals. Melting point: 130-132℃ _Elemental analysis _{: C31H44N2O9} Calculated value ₍ %) 63.25 7.53 4.77 Actual value (%) 63.01 7.48 4.83 NMR spectrum (100MHz, in _CDCl3 ): δ( ppm) 3.12 (N-methyl), 2.77 (N-methyl carbamate) Ultraviolet absorption spectrum: λ ^CH3OH _nax (nm) 252, 310, 410 (shoulder) Molecular weight by mass spectrometry: m/e 588 (M ⁺ ) Conducted Example 4 In the same manner as in Example 1, fractions No. 3 to 12 in silica gel column chromatography were collected, evaporated to dryness, and purified by rechromatography using 1.25% methanol-chloroform. Fractions No. 1 to No. 40 are collected and crystallized from acetone-n-hexane to obtain 192 mg of the compound of the following formula as yellow needle crystals. Melting point: 230-232℃ Elemental analysis value: C ₃₀ H ₄₂ N ₂ O ₉・1/2H ₂ O C H N Calculated value (%) 61.73 7.42 4.80 Actual value (%) 62.01 7.35 4.73 NMR spectrum (100 MHz, CDCl ₃ ): δ (ppm) 2.80 (N-methyl carbamate),
8.75 (NH) Ultraviolet absorption spectrum: λ ^CH3OH _nax (nm) 258, 303, 404 Molecular weight by mass spectrometry: m/e 574 (M ⁺ ) Example 5 Re-chromatographed in the same manner as in Example 4. Fractions Nos. 61 to 82 in Graphi were collected and crystallized from chloroform-n-hexane to obtain 334 mg of the compound of the following formula as yellow powder crystals. Melting point: 119-121℃ Elemental analysis value: C ₃₁ H ₄₄ N ₂ O ₉ H ₂ O Calculated value (%) 61.36 7.64 4.62 Actual value (%) 61.10 7.58 4.50 NMR spectrum (100 MHz, in CDCl ₃ ): δ (ppm) 2.75 (N-methylcabamate),
3.85 (iminomethyl ether) Ultraviolet absorption spectrum: λ ^CH3OH _nax (nm) 263, 295 (shoulder), 410 (shoulder) Molecular weight by mass spectrometry: m/e 588 (M ⁺ ) Formulation example 2500 g of the compound of formula, lactose 1375g, microcrystalline cellulose 775g and cellulose calcium glycolate
After sieving 375g using a 16-mesh sieve and mixing it uniformly, it was put into a kneading machine, and to this was added 3% hydroxypropyl cellulose solution (in a mixture of isopropyl alcohol/water = 3:7). Practice. The mixture is granulated using an extrusion granulator and dried with air at 50°C for 6 hours. Next, after sizing the granules to a size in the range of 16 to 60 meshes, the granules are mixed with 0.3% magnesium stearate and compressed into tablets. Test Example Cancer cells W-2K-11, which are C3H-2K clones of mouse kidney-derived fibroblasts transformed into cancers using the SV40 oncogenic virus, were used as test cells, and were cultured according to the method described below. (1) Preparation of growth medium solution: Dissolve 9.4 g of Eagle MEM medium (manufactured by Nissui Pharmaceutical) in 900 ml of distilled water, autoclave at 120°C for 15 minutes, cool, and add 100 ml of calf serum and sterilized 10% carbon dioxide. Add 3-5 ml of sodium hydrogen solution to pH 7.1-7,
Adjust to 2. L-Glutamine (2.92g/100ml) passed through a Millipore filter just before using the medium
100ml of solution was added. (2) Preparation of transplant cell solution: The test cells stored in a Jeep freezer (-80℃) were lysed at room temperature, centrifuged at 670 x g for 5 minutes, and the precipitated cells were added to the growth culture solution in (1). After suspending in 50 ml, the cells are transferred to a Roux flask and cultured at 37°C. The cells begin to proliferate and fully proliferate in 3 to 4 days. The culture solution is decanted, then 10 ml of 0.2% trypsin solution is added, and after standing at room temperature for 2 to 3 minutes, the trypsin solution is decanted. Furthermore, 50% of the growth culture solution of (1)
ml to make a cell suspension. (3) Cell culture and administration of test compound: Dispense 1.8 ml of the cell suspension obtained in (2) into a shear dish, and place it in a carbon dioxide incubator (5%
Incubate at 37°C in CO ₂ , 95% air). 0.2ml of ethanol solution of test compound after 24 hours of culture
and continue culturing. After 48 hours of culture, the number of surviving cells was counted under a microscope for cell proliferation, and the inhibition rate of the test cell proliferation was determined using the following formula. Suppression rate (%) = (Number of cells in non-administered shear) - (Number of cells in administered shear) / (Number of cells in non-administered shear)
×100 The results obtained are shown in the table below.

【table】

[Table] Acute toxicity The 50% lethal dose (LD ₅₀ ) for mice was 120 mg/Kg for the compound of Example 2 when administered intraperitoneally.
The LD ₅₀ of geldanamycin is 15 mg/Kg, which shows significantly reduced toxicity.

Claims (1)

[Claims] 1. General formula (wherein R means a residue [formula] or [formula], R ¹ , R ² and R ³ mean hydrogen or a methyl group, in which case at least one of R ¹ , R ² and R ³ is A compound represented by a methyl group. Quadratic equation A general formula characterized by methylating a compound represented by (wherein R means a residue [formula] or [formula], R ¹ , R ² and R ³ mean hydrogen or a methyl group, in which case at least one of R ¹ , R ² and R ³ is A method for producing a compound represented by a methyl group. 3 General formula (wherein R means a residue [formula] or [formula], R ¹ , R ² and R ³ mean hydrogen or a methyl group, in which case at least one of R ¹ , R ² and R ³ is An antitumor agent whose active ingredient is a compound represented by a methyl group.