JPS6261598B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel nucleoside whose pentose moiety has an arabinose structure and a method for producing the same. Nucleosides of this type are known, and some of them, such as 9-β-D-arabinofuranosyl adenine and 9-β-D-arabinofuranosylcytosine, have anticancer effects. However, it was not known that the substituent at the 2' position was an amino group. Well, surprisingly general formula〔〕 (In the formula, X and Y may be the same or different,
By reducing a compound represented by an amino group, a hydroxyl group, a thiol group, or a hydrogen group, a novel nucleoside in which the pentose moiety is an arabinose structure and the substituent at the 2' position is an amino group can be obtained. Moreover, this nucleoside was found to have anticancer effects. Note that the compound of the above formula [] is also a new compound. The present invention is based on the above findings. An object of the present invention is to provide a novel nucleoside whose pentose moiety has an arabinose structure and a method for producing the same. Please note that the patent application filed by the applicant in 1973-26061
No. (Japanese Unexamined Patent Publication No. 49-110891), the formula (X) There is a disclosure of a compound represented by the following, which appears to literally include the compound of the present invention. however,
According to U.S. Patent No. 3,987,030, which was filed by claiming priority from Japanese Patent Application No. 48-26061, the compound described in the above-mentioned patent publication has an amino group at the 2'-position of the pentose ring of formula (X) at the α-position. I understand that there is something. Since the compound of the present invention is of the arabinofuranosyl type, the amino group at the 2'-position is at the β-position, so it is a different compound from the compounds described in the above-mentioned patent publications and the compounds described in the above-mentioned US patents. The compound of the present invention is disclosed in JP-A-49-
It is clear from Experimental Example 2 described later that the antitumor activity is superior to that of the compound described in No. 110891. The nucleoside provided by the present invention is characterized by being represented by the following general formula []. (In the formula, X and Y have the same meanings as above.) Examples of the physical and chemical properties of the nucleosides represented by the formula [] are as follows. (a) 9-(2-deoxy-2-amino-β-D-
Arabinofuranosyl)-guanine (see Example 2) Thin layer chromatography: Rf value, 0.08 Developing solvent n-butanol:acetic acid:water (4:1:2) Thin layer Cellulose (Azecel SF) Elemental analysis value C ₁₀ H ₁₄ N ₆ O ₄ (molecular weight 282.26) C H N Calculated value (%) 42.55 5.00 29.78 Actual value (%) 42.33 4.99 29.51 Nuclear magnetic resonance spectrum (Figure 4) Infrared absorption spectrum (Figure 9) ( b) 9-(2-deoxy-2-amino-β-D-
arabinofuranosyl)-adenine (see Example 1) Melting point: 215-218°C (decomposition) Thin layer chromatography: Rf value, 0.18 Developing solvent n-butanol:acetic acid:water (4:1:2) thin Layer Kieselgel 60F ₂₅₄ Art 5719 (Merck) Elemental analysis value C ₁₀ H ₁₄ N ₆ O ₃ (Molecular weight 266.26)
As C H N Calculated value (%) 45.11 5.30 31.57 Actual value (%) 45.35 5.47 31.02 Nuclear magnetic resonance spectrum (Figure 2) Infrared absorption spectrum (Figure 7) The above shows the nucleosides represented by the formula [] is new, and in the case of (a), in the formula [], X is a hydroxyl group and Y is an amino group, that is, 9-(2-deoxy-2-amino-β-D-arabinofuranosyl)- In the case of (b), X is an amino group and Y is a hydrogen atom, that is, 9-(2-deoxy-2
-amino-β-D-arabinofuranosyl)-adenine. Next, the present invention provides the above-mentioned novel nucleoside []
The purpose is to provide a manufacturing method for According to the present invention, the above-mentioned nucleosides are obtained by reducing a compound of formula [] (X and Y have the same meanings as above). Catalytic reduction of a compound of formula [] with hydrogen in the presence of a catalyst is an advantageous method for converting an azide group into an amino group. As the catalyst, palladium on carbon, platinum oxide, Raney nickel, etc. are used, but reducing agents commonly used for reducing azide groups, such as sodium bisulfite and sodium sulfide, may also be used. The hydrogen pressure during the reaction may generally be normal pressure. The reaction temperature may generally be room temperature, but it can also be carried out in the range of 10°C to 80°C. The reaction time is 30 minutes to 24 hours, and varies depending on conditions such as reaction temperature and hydrogen pressure. The solvent used in the reaction is not particularly limited as long as it can dissolve the raw materials and does not participate in the reaction, and for example, aqueous methanol, aqueous ethanol, etc. are used. When the reaction is carried out in a solvent containing an equivalent amount or more (preferably 1 to 10 times equivalent) of a mineral acid (e.g., hydrochloric acid) or an organic acid (e.g., acetic acid) as the raw material, the reaction product is obtained as an addition salt of the corresponding acid. be able to. The present invention further provides a novel compound represented by the formula [] (X and Y have the same meanings as above) and a method for producing the same. According to the present invention, the compound of the formula [] (However, X and Y represent the same meanings as above, and R represents an alkyl group or an aryl group.) Obtained by reacting a 2'-sulfonyloxy compound represented by the following with a hydrogen azide salt. Can be done. In formula [], specific examples of R include methyl, ethyl, p-tolyl, p-nitrophenyl, and the like. This reaction is a substitution reaction with an azide group that involves rearrangement of the sulfonyl group of the compound represented by the general formula [], and the solvent used is not particularly limited, but examples include dimethylformamide, dimethylacetamide, dimethylsulfoxide, hexa Preferably, the reaction is carried out in a solvent such as methylphosphoric triamide. The azidating agent is not particularly limited as long as it performs a nucleophilic substitution reaction, but it is preferable to use a hydroazide salt such as sodium azide, lithium azide, or the like. Reaction temperature is 100-180
℃, but in order to minimize side reactions,
The preferred temperature is 130-150°C. The reaction time is
Although it varies depending on the reaction temperature and solvent, it is usually 1 hour to 24 hours. Among the 2'-sulfonyloxy compounds represented by the above group [], there are those in which X is a hydroxyl group, Y is a hydrogen atom, and R is a p-tolyl group, and those in which X is an amino group and Y is a hydrogen atom. , R is a p-tolyl group is known. The compound represented by the formula [] is the compound represented by the formula [] (wherein X and Y have the same meanings as above) It is obtained by reacting a ribonucleoside represented by the following with di-n-butyltin oxide, and then reacting it with a sulfonylating agent in the presence of a base.
Regarding the reaction with di-n-butyltin oxide, the solvent used in the reaction is not particularly limited, but for example, methanol, ethanol, n-
Propanol Aliphatic lower alcohols such as isopropanol are preferred. The reaction temperature is not particularly limited, but it is usually preferable to heat the reaction to reflux near the boiling point of the solvent used, and the reaction time is about 1 to 10 hours. 2′ produced in this reaction,
The 3'-o-dibutylstanylene compound is generally used as it is in the next sulfonylation reaction without being isolated. That is, the following sulfonylation reaction is
An organic base (such as triethylamine, trimethylamine, tripropylamine, tributylamine, triamylamine, etc.) is added to the reaction solution.
amine or a heterocyclic amine such as pyridine) is added as a catalyst and reacted with a sulfonylation reagent. Sulfonylation reagents include methanesulfonyl chloride, ethanesulfonyl chloride, p
Examples include alkyl or arylsulfonyl halides such as -toluenesulfonyl chloride, p-toluenesulfonyl bromide, and p-nitrobenzenesulfonyl chloride, but p-toluenesulfonyl chloride is generally preferred. The reaction temperature is usually around room temperature. The reaction time varies mainly depending on the reaction temperature, reaction materials, solvent, etc., but is usually about 5 minutes to 5 hours. The aminonucleosides provided by the present invention are novel and have anticancer effects as shown in the following experimental examples. Furthermore, as mentioned above, the compound of formula [] is not only useful as an intermediate for producing the compound of formula [], but also has antitumor activity itself, as shown in the experimental examples below. I found out. Experimental Example 1 HeLa-S ₃ cells were used as human cancer cells.
100g/100g/and 100u/penicillin/streptomycin of calf serum and penicillin/streptomycin in YLE medium.
A measurement medium (PH7.2) was prepared by adding ml of the solution. HeLa-S ₃ cells are cultured at 37°C in assay medium. 24 hours after the start of culture, the test compound, i.e., 9-(2-deoxy-2-amino-β-D-
arabinofuranosyl)guanine or 9-(2-
Deoxy-2-amino-β-D-arabinofuranosyl) adenine in phosphate-buffered saline (-PBS)
(PH7.2) solution is added to the concentration shown in Table 1 and culture is continued. At 96 hours of culture, the medium is removed by centrifugation. A 0.1M citric acid solution containing 0.2 g of crystal violet is added to the obtained HeLa-S ₃ cells, and the number of cell nuclei is counted using a microscope. As a control, phosphate buffered saline (PH7.2) containing no test compound is added 24 hours after the start of culture, and the number of cell nuclei is counted in the same manner. I% is the value obtained by dividing the number of cell nuclei for the test compound by the number of cell nuclei for the control, multiplied by 100.
(inhibition %), and the concentration of the compound that makes I% 50% is calculated as IC ₅₀ (50% growth inhibition concentration). The results are shown in Table 1.

[Table] It is clear from Table 1 that the compounds of the present invention have anticancer activity. Experimental example 2 Anti-Sarucomer 180 test 5 × 10 ⁶ cells of Sarcomer 180 were added to DD mouse 1
It was subcutaneously transplanted into the right armpit of 5 animals in the group, and 24 hours later, the
Compound 1000 obtained in Example 1 of Publication No. 49-110891
mg/Kg, 500 mg/Kg of the compound described in Example 1 of the present invention
Administer once intraperitoneally. The anticancer effect is expressed as the tumor volume ratio (T/C) between the drug-treated group (T) and the untreated control group (C) one week after transplantation.

[Table] Experimental Example 3 An antitumor experiment using 9-(2-deoxy-2-azido-β-D-arabinofuranosyl)-adenine was conducted as follows. Ascites cells were collected from L1210 tumor-bearing mice on the 7th day after transplantation, and diluted with sterile physiological saline to ⁵ x 10 cells/ml.
A cell suspension was prepared. Add this 0.2ml to your body weight 25±
2 g (6 weeks old) of CDF was implanted intraperitoneally into male mice. One dose was administered 24 hours after transplantation, and continuous administration was administered once a day for 6 days from 24 hours after transplantation, 5 times per group.
Drugs were administered intraperitoneally to each mouse. The effect of the drug is determined by the increase of life span (increase of life span) in the drug administration group (T) compared to the control group (C).
ILS) was calculated and expressed using the following formula.

[Table] The present invention will be explained below with reference to Examples. Example 1 (a) 9-(2-deoxy-2-azido-β-D-
Method for producing arabinofuranosyl)-adenine 9-(2-deoxy-2-o-tosyl-β-D
600 mg of -ribofuranosyl)-adenine and 560 mg of sodium azide are heated in 6 ml of hexamethylphosphoric triamide at 130 DEG C. for 6 hours. The solvent was removed under reduced pressure and the residue was dissolved in 50% aqueous methanol.
After passing through a Dowex 1×2 (OH) column (100ml) and washing the column with water, twice the volume of 30% and 60% aqueous methanol was sequentially passed through the column, and the absorbance at 260 mμ was eluted with 99% methanol. Collect 5 or more fractions, concentrate under reduced pressure, and recrystallize from 100% ethanol to obtain 190 mg of white crystals. Melting point: 204-205℃ Thin layer chromatography: Rf value 0.61 Developing solvent Chloroform: methanol (3:1) Thin layer Kieselgel 60F ₂₅₄ Art 5719 (Merck) Elemental analysis value C ₁₀ H ₁₂ N ₈ O ₃ (Molecular weight 292.26 )
As C H N Calculated value (%) 41.09 4.14 38.34 Actual value (%) 40.87 4.06 38.12 Figure 1 shows the nuclear magnetic resonance spectrum of this compound, and Figure 6 shows the infrared absorption spectrum. From these physical property values, this compound has 9-(2-
It is identified as deoxy-2-azido-β-D-arabinofuranosyl)-adenine. Yield 45.7%. (b) 9-(2-deoxy-2-amino-B-D-
Method for producing 9-(2-deoxy-2-azido-β-D-arabinofuranosyl)-adenine Dissolve 1.0 g of 9-(2-deoxy-2-azido-β-D-arabinofuranosyl)-adenine in 150 ml of 67% aqueous methanol and dissolve 10% palladium on carbon 500.
mg as a catalyst and hydrogen gas was introduced for 5 hours with stirring. After almost all methanol has been distilled off under reduced pressure, hot water 100%
ml, filtered using a filter aid (Celite 545), concentrated the filtrate to dryness, and reconsolidated from ethanol to obtain 390 mg of powder. Melting point: 215-218℃ (decomposition) Thin layer chromatography: Rf value 0.18 Developing solvent n-butanol:acetic acid:water (4:1:2) Thin layer Kieselgel 60F ₂₅₄ Art 5719 (Merck) Elemental analysis value C ₁₀ H ₁₄ N ₆ O ₃ (molecular weight 266.26)
As C H N Calculated value (%) 45.11 5.30 31.57 Actual value (%) 45.35 5.47 31.02 Figure 2 shows the nuclear magnetic resonance spectrum of this compound, and Figure 7 shows the infrared absorption spectrum. From these physical property values, this compound has 9-(2-
It is identified as deoxy-2-amino-β-D-arabinofuranosyl)-adenine. Yield 68.5%. Example 2 (a) 9-(2-deoxy-2-azido-β-D-
Method for producing arabinofuranosyl)-guanine 9-(2-deoxy-2-o-tosyl-β-D
3.6 g of -ribofuranosyl)-guanine and 3.2 g of sodium azide are heated at 140 DEG C. for 7 hours in 45 ml of hexamethylphosphoric triamide. The solvent was distilled off under reduced pressure, and the residue was thoroughly washed in ether and then thoroughly triturated. 200 to the solid after filtering the ether
Add ml of 33% aqueous methanol and stir while hot.
After removing the insoluble matter, the filtrate is partially distilled under reduced pressure to an extent that no precipitate is formed. Dowex 1x
Pass through a 2(OH) column (200ml). After washing the column with water, sequentially pass 50% and 80% aqueous methanol through the column approximately 4 times the volume of the column, collect the fraction with absorbance of 5 or more at 260 mμ eluted with 0.3M lithium chloride solution, and dilute with dilute hydrochloric acid to pH 6.8. After that, concentrate it to 30 ml and pass it through a column of Diaion HP10 resin (Mitsubishi Kasei) (300 ml). After washing the column with water, 10
After passing approximately twice the volume of the column with 20% aqueous methanol,
Elution is carried out with % aqueous methanol. 40ml after elution
Fractionate each. The 14th to 21st fractions were collected and concentrated under reduced pressure to obtain 220 mg of a thin, single, pale yellow powder. Thin layer chromatography: Rf value 0.46 Developing solvent Isobutyric acid: Ethyl acetate: 1N ammonia (10:5:1) Thin layer Kieselgel 60F ₂₅₄ Art 5719 (Merck) Elemental analysis value C ₁₀ H ₁₂ N ₈ O ₄ (Molecular weight 308.26 )
As C H N Calculated value (%) 38.96 3.92 36.35 Actual value (%) 38.26 4.11 36.07 Figure 3 shows the nuclear magnetic resonance spectrum of this compound, and Figure 8 shows the infrared absorption spectrum. From these physical property values, this compound has 9-(2-
It is identified as deoxy-2-azido-β-D-arabinofuranosyl)-guanine. Yield 10.8%. (b) 9-(2-deoxy-2-amino-β-D-
Method for producing 9-(2-deoxy-2-azido-β-D-arabinofuranosyl)-guanine Dissolve 130 mg of 9-(2-deoxy-2-azido-β-D-arabinofuranosyl) guanine in 45 ml of 33% aqueous methanol, and dissolve 60 mg of 10% palladium on carbon.
was added as a catalyst, and hydrogen gas was introduced for 3 hours while stirring. After almost all methanol has been distilled off under reduced pressure, hot water
Add 50 ml, filter using a filter aid (Celite 545), concentrate the filtrate to dryness, and reconstitute from water to obtain 90 mg of white powder. The powder did not exhibit a clear melting point. Thin layer chromatography: Rf value 0.08 Developing solvent n-butanol:acetic acid:water (4:1:2) Thin layer Cellulose (Avicel SF) Elemental analysis value C ₁₀ H ₁₄ N ₆ O ₄ (molecular weight 282.26)
As C H N Calculated value (%) 42.55 5.00 29.78 Actual value (%) 42.33 4.99 29.51 Figure 4 shows the nuclear magnetic resonance spectrum of this compound, and Figure 9 shows its infrared absorption spectrum. From these physical property values, this compound has 9-(2-
It is identified as deoxy-2-amino-β-D-arabinofuranosyl)-guanine. Yield 75.6%. Reference example 1 9-(2-deoxy-2-o-tosyl-β-D
-Ribofuranosyl)-Production method of guanine 5.66g of guanosine and di-n-butyltin oxide
Suspend 5.00 g in 1.1 methanol and heat under reflux for 3 hours. After cooling the reaction solution to 5°C, 42 ml of triethylamine and p-toluenesulfonyl chloride were added.
Add 57.2 g and stir at room temperature for 45 minutes. After distilling off methanol under reduced pressure, add 600ml of water to the residue.
Wash the aqueous layer three times with 600 ml of ether. Approximately 300 ml of the aqueous layer and the insoluble precipitate in the aqueous and ether layers.
After concentrating to
The precipitate obtained by cooling and storing at 0°C is collected by filtration to obtain 4.2 g of white crystals. Thin layer chromatography: Rf value 0.58 Developing solvent Water saturated n-butanol Thin layer Kieselgel 60F ₂₅₄ Art 5719 (Merck) Elemental analysis value C ₁₇ H ₁₉ N ₅ O ₇ S (molecular weight 437.42)
As C H N Calculated value (%) 46.68 4.38 16.01 Actual value (%) 46.63 4.50 15.93 Figure 5 shows the nuclear magnetic resonance spectrum of this compound, and Figure 10 shows the infrared absorption spectrum. From these physical property values, this compound has 9-(2-
It is identified as deoxy-2-o-tosyl-β-D-ribofuranosyl)-guanine. Yield 48.0%. Reference example 2 9-(2-deoxy-2-o-tosyl-β-D
-Ribofuranosyl)-adenine production method Adenosine 7.0g and di-n-butyltin oxide
Suspend 5.0 g in 500 ml of methanol and heat under reflux for 30 minutes. After cooling the resulting clear solution to 5°C, 42.0 ml of triethylamine and 57.2 g of p-toluenesulfonyl chloride were added and the solution was stirred for 10 min at room temperature.
Stir for a minute. After distilling off methanol under reduced pressure, add 600 ml of water to the residue and wash the aqueous layer three times with 500 ml of ether. After concentrating the aqueous layer to about 300 ml, the insoluble precipitate in the aqueous layer and the ether layer was combined with the precipitated crystals left in the refrigerator, and dissolved in 300 ml of 80% aqueous ethanol while hot. After removing the insoluble matter, 30% solvent
After concentrating under reduced pressure to 1.0 ml, the resulting precipitate was stored cooled at 0° C. and collected by filtration to obtain 6.3 g of white crystals. Melting point: 228-230℃ Thin layer chromatography: Rf value 0.71 Developing solvent Chloroform: methanol (2:
1) Thin layer Kieselgel 60F ₂₅₄ Art 5719 (Merck) Elemental analysis value C ₁₇ H ₁₉ N ₅ O ₆ S (molecular weight 421.42)
As C H N Calculated value (%) 48.54 4.54 16.62 Actual value (%) 48.19 4.56 16.39 Figure 11 shows the nuclear magnetic resonance spectrum of this compound, and Figure 12 shows the infrared absorption spectrum. From these physical property values, this compound has 9-(2-
It is identified as deoxy-2-o-tosyl-β-D-ribofuranosyl)-adenine. Yield 57.0%.

[Brief explanation of the drawing]

Figures 1 and 6 show 9-(2-deoxy-2
-Azide-β-D-arabinofuranosyl)-adenine nuclear magnetic resonance spectrum and infrared absorption spectrum, respectively. FIGS. 2 and 7 show the nuclear magnetic resonance spectrum and infrared absorption spectrum of 9-(2-deoxy-2-amino-β-D-arabinofuranosyl)-adenine, respectively. Figures 3 and 8 show 9-(2-deoxy-
2 shows the nuclear magnetic resonance spectrum and infrared absorption spectrum of 2-azido-β-D-arabinofuranosyl)-guanine, respectively. FIGS. 4 and 9 show the nuclear magnetic resonance spectrum and infrared absorption spectrum of 9-(2-deoxy-2-amino-β-D-arabinofuranosyl)-guanine, respectively. 5 and 10 are 9-(2-deoxy-2-o-tosyl-β-D-ribofuranosyl)-
The nuclear magnetic resonance spectrum and infrared absorption spectrum of guanine are shown respectively. Figures 11 and 12 show 9-(2-deoxy-2-o-tosyl-
Figure 3 shows the nuclear magnetic resonance spectrum and infrared absorption spectrum of β-D-ribofuranosyl)-adenine, respectively.

Claims (1)

[Claims] 1. General formula [] (However, X and Y may be the same or different and represent an amino group, a hydroxyl group, a thiol group, or hydrogen.) 2. A compound according to claim 1, wherein in formula [], X is an amino group and Y is hydrogen. 3. A compound according to claim 1, wherein in formula [], X is a hydroxyl group and Y is an amino group.