JPH093091A - Nucleoside derivative substance, its production and antitumor agent - Google Patents

Abstract

PURPOSE: To obtain the subject new substance from a culture product prepared by culturing an actinomyces RK-16 belonging to the genus Streptomyces, having a returning action of a cell transformed with a temperature sensitive cancer gene v-src to a normal state, useful as an antitumor agent, etc. CONSTITUTION: This new nucleoside derivative substance is shown by the formula (R1 is H or methyl; R2 is H or pyrrolidine-2-carbonyl; with the proviso that a case in which R1 is methyl and R2 is pyrrolidine-2-carbonyl is omitted) (e.g. phosmidosine-A, etc.). The substance has an action of returning a srcts-NRK cell obtained by transforming a normal rat nephrocyte with a temperature sensitive cancer gene v-src to a normal state and is useful as an antitumor agent, etc. The nucleoside substance is obtained by inoculating an actinomyces RK-16 strain (FERM P-9196) belonging to the genus Streptomyces into a medium, culturing and collecting the product from the culture substance.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel nucleoside derivative, a method for producing the nucleoside derivative, and an antitumor agent containing the nucleoside derivative as an active ingredient. More specifically, a method for producing a nucleoside derivative substance, comprising culturing an actinomycete RK-16 belonging to the genus Streptomyces and isolating and collecting a novel nucleoside derivative substance from the culture, and a normal rat kidney cell ( NRK cells) with the temperature-sensitive oncogene vs.
returning the src ^ts -NRK cells transformed with rc in normal form, relates to an antitumor agent containing the nucleoside derivative substance as an active ingredient with that new mechanism of action.

2. Description of the Related Art Many non-specific DNA and RNA synthesis inhibitors have been known as antitumor agents. Inhibitors which target a specific oncogene and suppress its expression, Little is known about specific inhibitors for proteins. Therefore, the development of inhibitors having a new mechanism of action as described above has been desired in the field of antitumor agents, and more specifically, the expression of highly specific and more effective oncogenes. There is a demand for the development of inhibitors that suppress such proteins and specific inhibitors for expressed proteins.

[0002]

The present invention relates to a novel nucleoside derivative, a method for producing the nucleoside derivative substance,
And an antitumor agent containing the nucleoside derivative substance as an active ingredient.

Means for Solving the Problems The present inventor has disclosed that actinomycete RK-16 belonging to the genus Streptomyces isolated from soil collected in Wakayama Prefecture is a nucleoside derivative represented by the following structural formula (I). And that the substance is capable of transforming normal rat kidney cells into the temperature-sensitive oncogene v-src.
In returning the src ^ts -NRK cells and transformed into normal form, with the finding that, the present invention has been completed.

[0003]

Embedded image

[0004] The above nucleoside derivatives are used as phosmidosine, phosmidosine-A, phosmidosine-
B and fosmidosine-C, respectively. this house,
Fosmidosine-A, fosmidosine-B, fosmidosine-
C is a novel nucleoside derivative. Also, fosmidosine is a substance which is in agreement with the antibiotic RK-16 disclosed in Japanese Patent Publication No. 11556/1991,
It can be manufactured according to the method disclosed in US Pat. Hereinafter, fosmidosine-A, fosmidosine-B, and fosmidosine-C, which are novel nucleoside derivative substances, will be described.
A method of manufacturing the device will be described. First, the characteristics of the mycological properties of the actinomycete RK-16 strain belonging to the genus Streptomyces, which produces the above nucleoside derivative substance, are summarized as follows. a. Aerial mycelia form well, and the color changes from yellowish white to light gray. Ten or more spores are chain threads, and spore chains are linear. The spores are cylindrical and the surface is smooth. b. The back side of the colony of the fungus mainly shows a light brown to black brown color. c. Form a melanin-like pigment. d. Use various sugars except fructose sorbose and sorbitol. e. With LL-diaminopimelic acid. In addition, the strain Streptomyces sp. (Streptomyc
es- sp.) RK-16 has been deposited with the National Institute of Bioscience and Human-Technology, National Institute of Advanced Industrial Science and Technology as Microbial Laboratory No. 9196 (FERM P-9196). Further, more detailed mycological properties of the RK-16 strain are described in Japanese Patent Publication No. 4-1556.

[0005] In order to produce the novel nucleoside derivative of the present invention, RK-16 belonging to the above Streptomyces genus is used.
The strain may be cultured in a medium, and the target nucleoside derivative may be separated and collected from the culture. The culture conditions and culture method of the RK-16 strain when producing the novel nucleoside derivative of the present invention generally follow the culture method of bacteria belonging to the genus Streptomyces, but the conditions disclosed in Japanese Patent Publication No. 11556/1992 and It can be done according to the method. RK-1
After completion of the cultivation of the six strains, the nucleoside derivative of the present invention can be purified and isolated from the culture broth by appropriately using a method generally used for collecting a metabolite of a microorganism. For example, various ion-exchange resins, non-ionic adsorption resins, gel filtration chromatography, activated carbon, alumina, silica gel and other adsorbents and high-performance liquid chromatography, or crystallization, concentration under reduced pressure, freeze drying and other means. Each of them can be used alone or in combination as appropriate, or repeatedly.
The novel nucleoside derivative represented by the structural formula (I) is obtained by culturing the above actinomycete strain RK-16 under suitable culture conditions and methods, and performing isolation and purification from the culture using the above-mentioned means. Fosmidosine-A, fosmidosine-B, fosmidosine-C, and fosmidosine can be obtained.

The novel nucleoside derivative substances fosmidosine-A and fosmidosine-B produced as described above,
Fosmidosine-C and fosmidosine, which is a known substance as described above, cause normal rat kidney cells to express the temperature-sensitive oncogene v-
The form of the src ^ts -NRK cells transformed with src showing the effect of successfully restored. This can be said to indicate an inhibitory effect of suppressing the expression of a specific oncogene or a specific inhibitory effect on the expressed protein. Therefore, an antitumor agent containing at least one of the above nucleoside derivative substances as an active ingredient, unlike an antitumor agent which is a known nonspecific DNA and RNA synthesis inhibitor, has a new mechanism of action. It can be used as an antitumor agent. The dosage form of the antitumor agent containing the nucleoside derivative substance as an active ingredient may be oral administration or parenteral administration. As a dosage form, for example,
Examples include oral preparations such as tablets, powders, capsules, granules, extracts, and syrups, and parenteral preparations such as injections and suppositories. These formulations contain excipients,
It is manufactured by a known method using a pharmaceutically acceptable additive such as a binder. Further, the clinical dose of an antitumor agent containing the above nucleoside derivative substance as an active ingredient,
Depending on the age, body weight, patient sensitivity, and the degree of symptoms, the effective amount is usually about 0.1 mg to 1 g per day for an adult, and it can be administered once or several times a day. is there. If necessary, an amount outside the above range can be used. Hereinafter, the present invention will be described in more detail with reference to examples.

[0007]

EXAMPLE Production Example of Nucleoside Derivative Substance Glucose 2%, soluble starch 1%, meat extract 0.1%, yeast 0.4%, salt 0.2%, dipotassium phosphate 0.005
% And soy flour 2.5% in a medium (pH 7.0) containing RK
-16 strain (FERM P-9196) and inoculated at 28 ° C for 48
Shaking culture was performed for hours. 350 ml of this culture solution was inoculated into 72 L of a medium having the same composition, and agitated culture at 350 rpm was performed at 28 ° C. for 72 hours with aeration at 18 L / min.

[0008] The above culture solution is subjected to press filtration,
Divide into cells and filtrate. The filtrate (40 L) is passed through a column of a cation exchange resin: Dowex 50w × 8 (20 L), the column is washed with 80 L of water, and the active substance is eluted with 0.5N aqueous ammonia. The eluate (80 L) was neutralized with 3N hydrochloric acid, and the solution was Diaion HP-2.
Pass through a 0 (20 L) column and wash the column with 40 L of water. The active ingredient is then eluted with 40 L of 70% aqueous MeOH. The eluate is concentrated under reduced pressure and a small amount (600
ml) and lyophilized to give 16 g of coarse powder. This powder was dissolved in 4 ml of water, and a column of Sephadex LH-20 (manufactured by Pharmacia) (26 × 700) was used.
mm). The column is developed with water and fractionated in 10 ml fractions by a fraction collector. src ^ts -NRK
Return the cell morphology to normal, collect fractions 31 to 40, 41 to 50, 51 to 60, and 61 to 70, concentrate by freeze-drying, and concentrate from fractions 31 to 40. Is 10
mg of fosmidosine-A was obtained as a pure product. Also, 4
For the fractions 1 to 50, 51 to 60, and 61 to 70, fosmidosine-
C, fosmidosine-B, fosmidosine white coarse powder 1.2
mg, 17 mg and 4 mg were obtained. Finally, fractionation was performed by preparative high-performance liquid chromatography using an ODS column (diameter 2 cm, length 25 cm, Senshu Pack, Senshu Science). The separation conditions were as follows: solvent (methanol: water = 7:93, flow rate 9.99 ml / min, column temperature 4
At 0 ° C, retention times were 11.8 minutes and 9.
6 minutes and 14.4 minutes. As a result, 1 mg, 15 mg, and 3 mg of purified fosmidosine-C, fosmidosine-B, and fosmidosine were obtained as white powders, respectively. Table 1 shows the physical properties and the like of the purified sample.

[0009] src ^ts -NRK cells in pairs of the present invention compounds
Src ^ts -NRK cells 96-well plates (Nu was transformed with a temperature-sensitive v-src normal mode restoration test normal rat kidney cells
nc) (5 × 10 ² cells / well).
Then, at a limited temperature (32 ° C.), a measurement target at a constant dilution rate was added, and 16 hours after the addition of the measurement target, the number of cells returned to a normal form with respect to the total number of cells in the visual field under an inverted microscope was determined. measure. This was used as an index for determining the presence or absence of the activity of the compound of the present invention. Table 2 shows the results
Shown in

[0010]

Table 1 Fosmidosine-A Fosmidosine-B mp. > 230 ° C> 230 ° C Molecular formula C ₁₅ H ₂₂ N ₇ O ₈ PC ₁₁ H ₁₈ N ₆ O ₇ P Molecular weight 459 376 HRFAB-MS (m / z) Calculated 460.1345 (MH ⁺ ) 377.0992 (MH ⁺ ) Obtained 460.1301 (MH ⁺ ) 377.0975 (MH ⁺ ) UV (nm, H ₂ O) (log ε) 267 (3.90) 267 (3.93) [α] _D ²⁰ (C: 0.1, H ₂ O) +18.6 ゜ -11 ° ¹ H-NMR (400 MHz, D ₂ O) δ: 1.81 (4 H, m) 2.20 (1 H, m) 3.17 (2 H, m) 3.48 (3 H, d, J = 11 Hz, P-OMe) 3.92 (2 H, m , C-5 ') 4.08 (2H, m, C-4') 4.00 (1H, m, C-4 ') 4.12 (1H, m, C-4') 4.34 (1H, m, C-3 ') 4.47 (1H, m, C-3 ') 4.92 (1H, m, C-2') 5.01 (1H, m, C-2 ') 5.66 (1H, d, J = 4.9Hz, C-1') 5.72 (1H, d, J = 4.9Hz, C-1 ') 7.91 (1H, s, base H-2) 7.94 (1H, s, base H-2)

[0011]

[Table 2]

[0012]

[Table 3] Table 2 率 Reversion rate to normal (%) Sample concentration Fosmidosine-A fosmidosine-B fosmidosine-C fosmidosine-C (μg / ml) ─────────────────────────────────── 5 60 72 60 72 17 96 64 52 69 50 95 77 77 47 71 170 92 92 92 43 67 As a result, fosmidosine, fosmidosine-A, fosmidosine-B, and fosmidosine-C can convert normal rat kidney cells into the temperature-sensitive oncogene v-src. Transformed by
thereby returning the rc ^ts -NRK cells to normal morphology revealed

Industrial Applicability According to the present invention, there are provided novel nucleoside derivative substances fosmidosine-A, fosmidosine-B, fosmidosine-C and a method for producing the same. The novel nucleoside substance and the known substance fosmidosine provide temperature-sensitive normal rat kidney cells. since showing a normal morphology return movement against src ^ts -NRK cells transformed with the oncogene v-src, it proves useful as an antitumor agent.

Claims (3)

[Claims] 1. A nucleoside derivative substance represented by the following structural formula (I). Embedded image (R ₁ represents a hydrogen atom or a methyl group, R ₂ represents a hydrogen atom or a pyrrolidine-2-carbonyl group, provided that R ₁ is a methyl group, and R ₂ is a pyrrolidine-2
-Except when it is a carbonyl group. ) 2. An actinomycete RK belonging to the genus Streptomyces.
A method for producing a nucleoside derivative substance, comprising culturing -16 and separating and collecting the nucleoside derivative substance represented by the structural formula (I) according to claim 1 from the culture. 3. An antitumor agent comprising a nucleoside derivative represented by the following structural formula (I) as an active ingredient. Embedded image (R ₁ represents a hydrogen atom or a methyl group, and R ₂ represents a hydrogen atom or a pyrrolidine-2-carbonyl group.)