JPS59231087A - Novel nucleoside - Google Patents

Abstract

NEW MATERIAL:The compound of formula I (X and Y are amino, OH, thiol or H). EXAMPLE:9-(2-Deoxy-2-amino-B-D-arabinofuranosyl)-adenine. USE:A carcinostatic agent. PREPARATION:The compound of formula I can be prepared by reacting the 2'- sulfonyloxy compound of formula II (R is alkyl or aryl) with a hydrazoic acid salt preferably in a solvent such as DMF at 130-150 deg.C for 1-24hr. The starting compound of formula II is obtained by reacting the ribonucleoside of formula IIIwith di-n-butyltin oxide, and then with a sulfonylation agent in the presence of a base.

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 is not known that the substituent at the 2' position is an amino group. Surprisingly, by reducing the compound represented by the general formula [■] It was possible to obtain a novel nucleoside in which the substituent at the 2' position is an amine group, and this nucleoside was also found to exhibit anticancer activity. 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.

Note that specification H of Japanese Patent Application No. 48-26061 (Japanese Unexamined Patent Publication No. 49-110) No. 391 filed by the present applicant contains the formula (X
) H 2 , which appears to literally include the compounds of the present invention. However, the special request Sho 4+3
According to U.S. Patent No. 3,987,030, which was filed by claiming priority to No. 26061, in the compound described in the above patent publication, the amine group at the 2'-position of the pentose ring of formula (X) is at the α-position. I understand that there is something. Since the compound of the present invention is of the arabinofuranosyl type and the amine group at the 2' position is at the β position, 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. It is clear from Experimental Example 2 below that the compound of the present invention is superior in antitumor activity compared to the compound described in JP-A-49-11.0891.

Sixth nucleoside general formula [I] provided by the present invention
It is characterized by being shown in

50 (wherein X and Y have the same meanings as above) formula [I
] Examples of the physical and chemical properties of nucleosides are as follows.

(Il 9-(2-deoxy-2-amino-β-D-
Arabinofuranosyl)-guanine (see Example 2) Thin layer chromatography: Rf value, o, os developing solvent n-butanol:acetic acid:water (4:1:2) thin layer Cellulose (Azecel 19F) elemental analysis value
As Cl0H14N604 (molecular weight 282.26), c q N total value (chil 42.55 5.00 29.7
8 Actual value (ch) 42.33 4.99 29.51
Nuclear magnetic resonance spectrum (Figure 4) Infrared absorption spectrum (Figure 9) (9-(2-thioxy-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 60F254 Art 5719 (
Merck) Elemental analysis value Cyu0H14N6Q3 (molecular weight 266
．． 26) CHN Calculated value (@45, 11 5.30 31.57 Actual value (
Chil 45.35 5.47 31.02 Nuclear magnetic resonance spectrum (Figure 2), infrared absorption spectrum
(Figure 7) The nucleosides represented by the formula [I] are new, and in the case of (A), the nucleosides represented by the formula [I]
where X is a hydroxyl group and Y is an amine group, that is, 9-(2-deoxy-2-amino-β-D-arabinofuranosyl)-guanine, and in the case of (b), the formula [■
] in which X is an amine group and Y is a hydrogen atom,
That is, it was confirmed to be 9-(2-deoxy-2-amino-β-D-arabinofuranosyl)-adenine.

Next, an object of the present invention is to provide a method for producing the above-mentioned novel nucleoside [■]. According to the invention, the above-mentioned nucleosides are obtained by reducing the compound of formula [II] (where X and Y have the same meanings as above).

Catalytic reduction using the compound of formula (n) and hydrogen in the presence of a catalyst is an ineffective method for converting an azide group into an amine group. As a catalyst, palladium on carbon,
Platinum oxide, Raney nickel, etc. are used, but reducing agents commonly used for reducing the oxidation group such as hydrogen sulfite, sodium sulfide, etc. 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 another time, 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 a mineral acid (e.g. hydrochloric acid) or an organic acid (e.g. acetic acid) in an amount equivalent to or more than the raw material (preferably 1 to 10 times equivalent), the reaction product is converted into an addition salt of the corresponding acid. Obtainable.

The present invention further provides a novel compound represented by the formula [nl (X and Y have the same meanings as above) and a method for producing the same. According to the present invention, this compound of formula [■]
■] 2 represented by HO0602R (X and Y have the same meanings as above, and R represents an alkyl group or an aryl group)
It can be obtained by reacting a '-sulfonyloxy compound with hydroazide.

In formula [m], R is methyl, ethyl, p −
) Ryl p-nitrophenyl & etc. are included in the JL body.

This reaction is a substitution reaction for an azide group that involves rearrangement of the sulfonyl group of the compound represented by the general formula [m], and the solvent used is not particularly limited, but examples include dimethylformamide, dimethylacetamide, and dimethylsulfoxide. Preferably, the reaction is carried out in a solvent such as hexamethylphosphoric triamine P. There is no particular limitation on the oxidizing agent as long as it carries out a nucleophilic substitution reaction, but it is preferable to use a hydroazide salt such as sodium azide or lithium hydride. The reaction temperature is about 100-180°C, preferably 130-150°C to minimize side reactions. The reaction time varies depending on the reaction temperature and solvent, but is usually 1 hour to u hours.

Among the 2'-sulfonyloxy compounds represented by the above group [uI:), there are those in which X is a hydroxyl group, Y is a hydrogen atom, and R is a p-)lyl group, and those in which X is an amino group and Y is Hydrogen atoms in which H is a p-) IJ group are known. The compound represented by the formula [III] is obtained by reacting the ribonucleoside represented by the formula [■] (in the formula, X and Yid: have the same meanings as above) with di-n-butyltin oxide, and then reacting the compound represented by the formula [■] with di-n-butyltin oxide. Obtained by reacting with a sulfonylating agent.

Regarding the reaction with di-n-butyltin oxide, the solvent used in the reaction is not particularly limited, but aliphatic lower alcohols such as methanol, ethanol, n-furopanol, and isopronominol are suitable. Although the reaction temperature is not particularly limited, 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. The 2/, 3/-〇-dibutyl stanylene compound produced in this reaction is generally used as it is in the next sulfonylation reaction without being isolated. That is, in the next sulfonylation reaction, an organic base (for example, triethylamine) is added to the above reaction solution.

A tertiary amine such as trimethylamine, tripropylamine, tributylamine, triamylamine or a heterocyclic amine such as pyridine is added as a catalyst to react with the sulfonylation reagent.

Examples of the sulfonylation reagent include methanesulfonyl chloride, ethanesulfonyl chloride', 1)-) toluenesulfonyl chloride, p-)-toluenesulfonyl bromide, p-
-Alkyl or arylsulfonyl leno-1 rides such as nitrobenzenesulfonyl chloride,
Generally p-)luenesulfonyl chloride is 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.

Example HeLa-83 cells were used as human cancer cells. Y
LK medium with small serum and penicillin/streptomycin at 100 f/l and 100 u/m, respectively.
Measurement medium (pH 7
, 2) were adjusted.

HeLa-83 cells are cultured at 37°C in assay medium.

The test compound, i.e., 9-(2
-deoxy-2-amino-β-D-arabinofuranosyl)guanine is still 9-(2-deoxy-2-amino-β
A solution of -D-arabinofuranosyl) adenine in phosphate buffered saline (-PBr3) (pH 7,2) is added to the concentration shown in Table 1, and the culture is continued.

After 96 hours of culture, the oral medium is removed by centrifugation. Crystal violet was added to the obtained HeLa-83 cells at 0.2? Add a 0.1M citric acid solution containing / and count the number of cell nuclei using a microscope. As a control, phosphate buffered saline (pH 7.2) containing no test compound is added to the inlet U hours after the start of culture, and the number of cell nuclei is counted in the same manner.

Calculate the value obtained by dividing the number of cell nuclei in the case of the test compound by the number of cell nuclei in the control and multiplying it by 100 as Etc (inhibition 1) [), and calculate the concentration of the compound that makes Etchi 50% as Ec50 ( 50% growth inhibition concentration).

The results are shown in Table 1.

It is clear from Table 1 that the compounds of the present invention have anticancer activity.

Example Anti-Sarcoma 180 Test Sarcoma-180 5xlO' cells were subcutaneously transplanted into the right armpit of 1 group of 5 DD mouse mice, and after a period of cooling, 5xlO' cells of Sarcoma-180 were transplanted subcutaneously into the right armpit of 1 group of 5 mice.
Compound 1000 obtained in Publication No. 110891 Example 1
#/k17. Compound 500m1i described in Example 1 of the present invention
9 is administered intraperitoneally once. One week after transplantation, the ratio of tumor volume between the drug-treated group (T) and the untreated control group (C) (
The anticancer effect is expressed as T/C).

The present invention will be explained below using examples.

Example 1 (a) Method for producing 9-(2-deoxy-2-azido-β-D-arabinofuranosyl)-adenine 9-(2-deoxy-2-0-tosyl-β-ri1lipo7 600 μl of (lanosyl)-adenine and 560 μl of sodium azide are heated in hexamethylphosphoric triamide 6− at 130° (:’) for 6 hours. The solvent is distilled off under reduced pressure, leaving a residue e of 50%.
Dowex 1x2 (OH) dissolved in aqueous methanol
After passing through the column (100d) and washing the column with water, twice the volume of 30% and 60% aqueous methanol was sequentially passed through the column, and the fraction having an absorbance of 5 or more at 260 mμ eluted with 99% methanol was collected. After concentration under reduced pressure, recrystallization from 100'n ethanol yields 190cm white crystals.

Melting point: 204-205°C Thin layer chromatography: Rf value 0.61 developing solvent
Chloroform:methanol (3:1) thin 1- Kieselgel 60F254 Art 5719 (Merck) Elemental analysis value Cl0H12N803 (molecular weight 2cn,
z6) as CHN calculated value (@41.09 4.14 38.34 actual measured value (
%i 40,87 4.06 38.12 FIG. 1 shows the nuclear magnetic resonance spectrum of this compound, and FIG. 6 shows the infrared absorption spectrum.

Based on these physical property values, this compound was identified as 9-(2-deoxy-2-azido-β-D-arabinofuranosyl)-adenine. Yield 45.7%.

(b) 9-(2-deoxy-2-amino-B-D −
Arabinofuranosyl)-Adenine production method 9-(2-deoxy-2-azido-β-D-arabinofuranosyl)-
Adenine 1.01 in 67-line aqueous methanol 150 m7
! Hydrogen gas was introduced into the solution for 5 hours with continuous stirring using 500 μm of 10% Nonoradium carbon as a catalyst. After almost all methanol has been distilled off under reduced pressure, 100 g of hot water is added, filtered using a filter aid (Celite 545), the filtrate is concentrated to dryness, and reconsolidated from ethanol to obtain 390 q of powder.

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 60F254 Art 5719 (
Merck) Elemental analysis value Cl0H14N603 (Molecular H266,
26) Calculated value (CHN) 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 nuclear magnetic resonance spectrum of this compound. Infrared absorption spectrum is shown.

Based on these physical property values, this compound is identified as 9-(2-deoxy-2-amino-β-D-arabinofuranosyl)-adenine. Yield 68.5%.

Example 2 (a) Method for producing 9-(2-thioxy-2-azido-β-D-arabinofuranosyl)-guanine 9-(2-deoxy-2-0-tosyl-β-D-ribonofuranosyl) - 3,6f guanine and 3,2r sodium azide in 4511 Ll of total hexamethylphosphoric triamide at 140°C
Heat for 7 hours. The solvent was distilled off under reduced pressure, and the residue was thoroughly washed in ether and then thoroughly triturated. Add 200 d of 33 hydrophilic methanol to the solid after filtering off the ether,
After stirring while hot to remove insoluble matter, the filtrate is partially distilled under reduced pressure to the extent that no precipitate is formed. Dowex 1
Pass through a x2(OH) column (200d). After washing the column with water, sequentially add 50% and 80% aqueous methanol.
After passing about 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,
After adjusting the pH to 6.8 with dilute hydrochloric acid, concentrate to 30 ml and add Diaion HPIO resin (Mitsubishi Kasei) (30 ml).
0d). After washing the column with water, 10 qb of aqueous methanol was passed through the column, approximately twice the volume of the column, and elution was performed with 20 qb of aqueous methanol. After elution, fractionate into 40 fractions. The 14th to 21st fractions were collected and concentrated under reduced pressure to yield 220
(2) A thin, single, pale yellow powder is obtained.

Thin layer chromatography: Rf value 0.46 Developing solvent Inbutyric acid: Ethyl acetate: IN ammonia (10:5:1) Thin layer Kieselgel 60F "264 Art 571.
9 (Merck) Elemental analysis value Cxo H12N804 (molecular weight 308
．． 26) Total CHN Total 1-value (%) 38.96 3.92 36.35
Actual value (@ 38.26 4.11 36.07 3rd
The figure shows the nuclear magnetic resonance spectrum of this compound, and FIG. 8 shows the infrared absorption spectrum.

Based on these physical property values, this compound! It is identified as J-(2-deoxy-2-azido-β-D-arabinofuranosyl)-guanine. Yield io, ss.

(1) Method for producing 9-(2-deoxy-2-amino-β-ri-arabinofuranosyl)-guanine 9-(2-deoxy-2-azido-β-D-arabinofuranosyl)-
130 μg of guanine in 33% aqueous methanol 45 m/'
10%/R radium on carbon was added as a catalyst, and hydrogen gas was introduced for 3 hours while stirring. After most of the methanol is distilled off under reduced pressure, 50 ml of hot water is added and filtered using a filter aid (Celai 545'). The filtrate is concentrated to dryness and reconsolidated from water to obtain white powder 90hIli+. 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
CHN calculated value (fission 1 42.55 5.00 29.78 actual value (@ 42.33 4.99 29.51) as l0H14N604 (molecular weight 282.26) Figure 4 shows the nuclear magnetic resonance spectrum of this compound, Figure 9 shows the infrared absorption spectrum.

Based on these physical property values, this compound is identified as 9-(2-deoxy-2-amino-β-D-arabinofuranosyl)-guanine. Yield 75.6%.

Reference Example 1 Method for producing 9-(2-deoxy-2-0-)yl-β-syribofuranosyl)-guanine Guanosine 5.66 f
! and 1.1 t of 9 n-butyltin oxide 5.0 Or
of methanol and heated under reflux for 3 hours. After cooling the reaction solution to 5°C, triethylamine 42rnl p-
) Add 57.2 f of luenesulfonyl chloride and stir at room temperature for 45 minutes. After methanol was distilled off under reduced pressure, 600 d of water was added to the residue, and the aqueous layer was washed three times with 600 d of ether. After concentrating the aqueous layer to about 300 rILe, the precipitate insoluble in the aqueous layer and the ether layer was combined with the crystals that had precipitated by leaving it in the refrigerator. After that, the solvent was concentrated under reduced pressure, and the precipitate obtained by cooling and storing at 0°C was collected by filtration to obtain white crystals 4.27.

Thin layer chromatography: Rf value 0.58 Developing solvent Water saturated n-butanol Thin layer Kieselgel 6 (l F254 Art 571
9 (Merck) Elemental analysis value C17H19N, 0.8 (molecular weight 437
．． 42) as CHN B1 calculated value (CH) 46.68 4.38 16.01
Actual 1mf chill 46.63 4.50 15.93
FIG. 5 shows the nuclear magnetic resonance spectrum of this compound, and FIG. 10 shows the infrared absorption spectrum.

From these physical property values, this compound is identified as 9-(2-deoxy-2-0-)yl-β-siribofuranosyl)-guanine. Yield 48.0%.

Reference Example 2 Production method of 9-(2-deoxy-2-0-)yl-β-syribofuranosyl)-adenine 7.01 of adenosine and 5.07 of n-butyltin oxide were suspended in 500 methanol. Let it become cloudy and heat under reflux for 30 minutes. After cooling the resulting clear solution to 5 °C, triethylamine 42
．． 0ml and p-toluenesulfonyl chloride 57.2r
and stirred at room temperature for 10 minutes. After distilling off methanol under reduced pressure, 600 d of water was added to the residue, and 500 d of ether was added.
Wash the aqueous layer three times with d. After concentrating the aqueous layer to about 300 ml, the precipitate that is insoluble in the aqueous layer and the ether layer is combined with the precipitated crystals left in the refrigerator, and dissolved in 300 ml of 80% hydrophilic ethanol while hot, and after removing the insoluble matter. , solvent 3
After concentrating under reduced pressure to 0mff1, the resulting precipitate was stored under cooling at 0°C and collected by filtration to obtain white crystals 6.32.

Melting point: 228-230°C Thin layer chromatography: Rf value 0.71 Developing solvent Chlorotherm: methanol (2+1) thin layer Kieselgel 60 F2+54 Art 5719 (Merck) Elemental analysis value C17H19NaOaS (molecular weight 421
．． 42) as CHN word 1n1 direct (%) 48.54 4.54
16.62 Actual value f%l 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 properties, this compound is equivalent to 9-(2=deoxy-2-0-1-yl-β-D-ribofuranosyl)-adenine. Yield 57.0%.

[Brief explanation of drawings]

Figures 1 and 6 show the nuclear magnetic resonance spectrum and infrared absorption spectrum of 9-(2-deoxy-2-azido-β-D-arabinofuranosyl)-adenine, 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. FIGS. 3 and 8 show the nuclear magnetic resonance spectrum and infrared absorption spectrum of 9-(2-deoxy-2-azito 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. Figures 5 and 10 show 9-(2-deoxy-2-〇~
Figure 2 shows the nuclear magnetic resonance spectrum and infrared absorption spectrum of tosyl-β-D-ribonlanosyl)-guanine, respectively. Figures 11 and 12 show the nuclear magnetic resonance spectrum and infrared absorption spectrum of 9-(2-deoxy-2-0-tosyl-β-D-ribofuranosyl)-adenine, respectively. (Voluntary) Procedural Amendment Commissioner Kazuo Wakasugi, Director of the Patent Office1, Indication of the case, 1988 Patent No. 2495002, Title of the invention: New nucleoside3, Relationship with the person making the amendment Patent applicant address Tokyo Chiyoda-ku Otemachi-chome 6-1 Mr.
Name) (102) Kyowa Hakko Kogyo Co., Ltd. Representative Ikube Kinoshita 4 353-5521 Address 11 Shinanomachi, Shinjuku-ku, Tokyo Name (6739) Patent attorney Shunji Nonami 5
, Date of amendment order6, Number of inventions increased by amendment None'-i, h<
8, Contents of amendment Dalian ], it was found that it is not only useful as an intermediate for the production of the compound, but also has antitumor activity itself, as shown in the experimental example below.'' (2) Same page 14, bottom 7th line, before "hereinafter referred to as the present invention", the following addition is made: Example 9 - Antitumor experiment of (2-deoxy-2-azido-β-D-arabinofuranosyl)-adenine The procedure was as follows: Ascites cells were collected from L1210 tumor-bearing mice 7 days after transplantation, and a suspension of 5 x 10 cells/cell in sterile physiological saline was prepared. Weight 25±2
I (6 weeks old) male mice were implanted intraperitoneally. The drug was intraperitoneally administered to 5 mice in each group, with a single dose being administered 24 hours after transplantation and continuous administration being administered 24 hours post-transplant once a day for 6 days. The effect of the drug was determined by comparing the drug administration group (C) with the control group (C).
Increase of LifeS
pan = ILS) was determined and expressed using the following formula. Dosage frequency Dose (m9/Ky/day) IL
L (%) 1 100
256
100 61928-

Claims (3)

[Claims] (1) A compound represented by the general formula [II] O (wherein X and Y may be the same or different and represent an amine group, a hydroxyl group, a thiol group, or hydrogen). (2) A compound according to claim 1, wherein in formula [■], X is an amine group and Y is hydrogen. (3) A compound according to claim 1, wherein in formula (III), X is a hydroxyl group and Y is an amino group.