JPH0560478B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a novel nucleoside compound, a pharmaceutically acceptable salt thereof, a method for producing the same, and a pharmaceutical composition containing the compound as an active ingredient.

(Prior Art) Treatment techniques for various diseases are rapidly progressing, but malignant tumors are still in the early stages of research. Currently, only a few types of nucleoside compounds, such as thionosine and cytarabine, are used as medicines as antitumor substances, and research is being conducted to obtain more effective antitumor substances.

For example, as one of the nucleoside antitumor substances, cadeguomycin (2-amino-
3,4-dihydro-4-oxo-7-β-D-ribofuranosyl-7H-pyrrolo[2,3-d]pyrimidine-5-carboxylic acid) is disclosed in JP-A-58-92693. Cadeguomycin is produced naturally in very small amounts, so a method for mass synthesis is desired.
An example of a synthetic method is disclosed in Tetrahedron Letters, 24 3647 (1983).

(Problems to be Solved by the Invention) An object of the present invention is to provide a novel nucleoside compound, a pharmaceutically acceptable salt thereof, a method for producing the same, and a pharmaceutical composition containing the compound as an active ingredient. be.

(Means for Solving the Problems) The present inventors conducted research on nucleoside compounds, their antitumor properties, and synthesis methods, and found that the nucleoside compound of the present invention, in which the carboxyl group at the 5-position of cadeguomycin was replaced with an aldehyde group, was superior. The present invention was completed based on the discovery that it has an antitumor effect and is useful as an antitumor agent.

The compound of the present invention is a novel nucleoside compound represented by the following general formula ().

(In the formula, the broken line represents that both ribofuranose and arabinofuranose are included.) The nucleoside compound of the present invention includes a pharmaceutically acceptable salt of the compound represented by the general formula (), and includes, for example, , acid addition salts of inorganic acids such as hydrochloric acid and sulfuric acid, and organic acids such as acetic acid and citric acid.

Next, a method for producing the compound of the present invention will be described.

(1) For example, the compound of the present invention can be produced using a compound represented by the following general formula () as a starting material.

(In the formula, R ₁ is an alkoxyalkyl group, R ₂ is an acyl group, R ₃ and R ₄ are an acyl group, alkylidene group, or aralkyl group, R ₅ is an acyl group or an aralkyl group, and X is a halogen. Broken line represents that it includes both ribofuranose and arabinofuranose) As a specific example in the above general formula (),
R ₁ is an alkoxyalkyl group such as methoxymethyl group, R ₂ is an acyl group such as acetyl or benzoyl group, R ₃ and R ₄ are any protecting groups used in general nucleoside chemistry, for example,
Similarly, R ₅ is an acyl group such as acetyl or benzoyl group, an aralkyl group such as trityl or benzyl group, or an alkylidene group such as isopropylidene or ethylidene group; Examples of the aralkyl group and X include halogens such as chlorine and bromine.

Among the compounds represented by the general formula (), for example, 6-bromo-2-diacetylamino-3,
4-dihydro-5-hydroxymethyl-3-methoxymethyl-7-(5-O-acetyl-2,
3-O-isopropylidene-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidin-4-one is a synthetic intermediate for cadeguomycin, as described by T. Kondo et al., Tetrahedron.
Letters, 24 3647 (1983), it can be easily obtained by known methods.

This manufacturing method can be performed as follows.

The halogen group at the 6-position of the compound represented by the general formula () is reduced under a catalyst such as palladium-carbon. Next, it is treated with an oxidizing agent such as activated manganese dioxide in a suitable solvent that does not inhibit the reaction, such as acetonitrile, to oxidize the 5-position hydroxylmethyl group and convert it into an aldehyde group. Finally, the general formula The nucleoside compound of the present invention represented by () can be obtained.

(2) The compound of the present invention can also be produced using cadeguomycin represented by the following formula () or its arabinose derivative as a starting material.

(In the formula, the broken line indicates that both ribofuranose and arabinofuranose are included.) In this production method, first, cadeguomycin or its arabinose compound represented by the above formula () is dissolved in methanol under a hydrochloric acid catalyst. Heat to reflux to esterify the carboxyl group at position 5,
Subsequently, the alcohol is obtained by reacting with lithium borohydride under reflux in tetrahydrofuran. Finally, the 5-position methoxymethyl group is oxidized with activated manganese dioxide in dimethyl sulfoxide to obtain the compound of the present invention represented by the general formula ().

The obtained compound of the present invention was purified by conventional means such as distillation, chromatography, and recrystallization, and identified by melting point measurement, IR, NMR, UV, mass spectrum, optical rotation, etc.

(Example) An example of a method for producing the compound of the present invention is shown below.

Example 1 (1) 6-bromo-2-diacetylamino-3,4
--dihydro-5-hydroxymethyl-3-methoxymethyl-7-(5-O-acetyl-2,
545 mg of 3-O-isopropylidene-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidin-4-one and 10% palladium-carbon 300
300 mg of potassium acetate and 20 ml of methanol/water were added, the mixture was replaced with hydrogen gas, and the mixture was stirred at room temperature for 30 minutes. After removing the catalyst by filtration and washing, the filtrate was concentrated, water was added to the residue, and the mixture was extracted with ethyl acetate. The organic layer was washed with water and saturated sodium chloride, dried over anhydrous sodium sulfate, and then the catalyst was distilled off. The obtained crude product was purified with a silica gel column and recrystallized from n-hexane to give 2-diacetylamino-
3,4-dihydro-5-hydroxymethyl-3
-methoxymethyl-7-(5-O-acetyl-
390 mg of white crystals of 2,3-O-isopropylidene-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidin-4-one were obtained.

(Yield 82%) Melting point: 57-59℃ UV (MeOH): λmax=300nm (ε=8770) λmax=265nm (ε=5414) IR (KBr): 3450, 1740, 1690, 1675, 1572,
1372, 1224, 1090 cm ^-1 NMR (CDCl ₃ ): δ = 1.35 (s, 3H), 1.60 (s,
3H), 2.11 (s, 3H), 2.37 (s, 3H), 2.38
(s, 3H), 3.42 (s, 3H), 4.20 (dd, 1H,
J ₁ = 6.6Hz, J ₂ = 12.7Hz), 4.33 (dd, 1H, J ₁ =
4.2Hz, J ₂ = 12.7Hz), 4.36 (ddd, 1H, J ₁ = 3.7
Hz, J ₂ = 4.2Hz, J ₃ = 6.6Hz), 4.3−4.5(br.s,
1H), 4.75 (br.s, 2H), 4.80 (dd, 1H, J ₁ =
3.7Hz, J ₂ = 6.3Hz), 4.93 (dd, 1H, J ₁ = 2.9
Hz, J ₂ = 6.3Hz), 5.35 (s, 2H), 6.18 (d,
1H, J = 2.9Hz), 6.94 (s, 1H) [α] ¹² _D = -47.9° (C = 0.16, CHCl ₃ ) (2) Add 390mg of the product obtained in (1) above to 25ml of acetonitrile. After dissolving, 3 g of activated manganese dioxide was added while stirring at room temperature. After stirring for 1 hour, it was filtered and washed with acetone, and the filtrate was concentrated. 15 ml of methanol and 15 ml of 28% aqueous ammonia were added to the residue, and after stirring at room temperature for 15 hours, the mixture was concentrated under reduced pressure. The obtained crude product was purified with a silica gel column and recrystallized from n-hexane to give 2-amino-3,4-dihydro-5-formyl-3-
208 mg of yellow crystals of methoxymethyl-7-(2,3-O-isopropylidene-β-D-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidin-4-one were obtained. (Yield 71%) Melting point: 89-91℃ UV (MeOH): λmax=317nm (ε=6663) λmax=290nm (ε=5149) λmax=249nm (ε=21303) IR (KBr): 3440, 1695, 1662, 1630, 1530,
1205, 1153, 1077 cm ^-1 NMR (CDCl ₃ ): δ = 1.37 (s, 3H), 1.61 (s,
3H), 3.44 (s, 3H), 3.77 (d, 1H, J=
12.2Hz), 3.95 (dd, 1H, J ₁ = 2.0Hz, J ₂ = 12.2
Hz), 4.43 (dd, 1H, J ₁ = 2.0Hz, J ₂ = 2.0Hz),
5.02 (dd, 1H, J ₁ = 2.0Hz, J ₂ = 5.9Hz), 5.13
(dd, 1H, J ₁ = 4.4Hz, J ₂ = 5.9Hz),
5.50and5.61 (ABquartet, 2H, J=10.8Hz),
5.60 (br.s, 2H), 5.75 (d, 1H, J=4.4Hz),
7.49 (s, 1H), 10.25 (s, 1H) [α] ¹¹ _D = -77.4° (C = 0.11, CHCl ₃ ) FAB Mass: m/z 395 (M+1) (3) Obtained in (2) above 15 ml of trifluoroacetic acid/water (2/1) was added to 200 mg of the obtained product, and the mixture was stirred at 70°C for 1 hour. After concentration under reduced pressure, the residue was dissolved in water, passed through an Amberlite IR-45 column, and eluted with water/acetonitrile. The eluate was concentrated to dryness to give the yellow color of 2-amino-3,4-dihydro-5-formyl-7-β-D-ribofuranosyl-7H-pyrrolo[2,3-d]pyrimidin-4-one (compound 1). 133 mg of crystals were obtained. (Yield 85%) Melting point: 205-213℃ (decomposition) UV (H ₂ O): λmax = 311nm (ε = 7452) λmax = 245nm (ε = 19110) IR (KBr): 3410, 3320, 1701, 1685 ,1635,
1598, 1406, 1185, 1140, 1045 cm ^-1 NMR (D ₂ O, t-BuOH = 1.23 ppm): δ = 3.79 (dd, 1H, J ₁ = 4.2 Hz, J ₂ = 12.7
Hz), 3.89 (dd, 1H, J ₁ = 3.1Hz, J ₂ =
12.7Hz), 4.19(ddd, 1H, J ₁ = 3.1Hz, J ₂
= 4.2Hz, J ₃ = 4.6Hz), 4.35 (dd, 1H, J ₁
= 4.6Hz, J ₂ = 5.1Hz), 4.57 (dd, 1H, J ₁
=5.1Hz, J ₂ =5.4Hz), 5.98(d, 1H, J
=5.4Hz), 7.92(s, 1H), 9.68(s,
1H) [α] ³⁰ _D = -47.7° (C = 0.10, H ₂ O) [α] ³⁰ ₄₃₅ = -111.6° (C = 0.10, H ₂ O) FAB Mass: m/z 311 (M + 1) Example 2 6-bromo-2-diacetylamino-3,4-
Dihydro-5-hydroxymethyl-3-methoxymethyl-7-(2,3,5-tri-O-acetyl-β-D-arabinofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine-4 Using 290 mg of -one as a starting material, the arabinose derivative of Compound 1, 2-amino-3,4-dihydro-5-formyl-7-β-D-arabinofuranosyl-7H-, was prepared in the same manner as in Example 1. pyrrolo[2,3-d]pyrimidine-
30 mg of 4-one (compound 2) was obtained.

Melting point: 203-210℃ (decomposition) UV (H ₂ O): λmax = 311nm (ε = 10845) λmax = 247nm (ε = 26895) IR (KBr): 3400, 3330, 3200, 1685, 1645,
1612, 1552, 1498, 1380, 1067 cm ^-1 NMR (D ₂ O, t-BuOH = 1.23 ppm): δ = 3.87 (dd, 1H, J ₁ = 5.1 Hz, J ₂ = 12.5
Hz), 3.93 (dd, 1H, J ₁ = 3.2Hz, J ₂ =
12.5Hz), 4.01(ddd, 1H, J ₁ = 3.2Hz, J ₂
=5.1Hz, J ₃ =5.6Hz), 4.27(t, 1H, J
= 5.6Hz), 4.48 (t, 1H, J = 5.6Hz),
6.32 (d, 1H, J=5.6Hz), 7.97 (s,
1H), 9.71 (s, 1H) [α] ²⁵ _D = +35.2° (C = 0.1, H ₂ O) [α] ²⁵ ₄₃₅ = +36.0° (C = 0.1, H ₂ O) Example 3 (1) 20 mg of cadeguomycin and 5 ml of anhydrous methanol
and concentrated sulfuric acid were added, and the mixture was refluxed on an oil bath for 4 hours. Concentrate under reduced pressure and dissolve the residue in water to obtain Amberlite.
Passed through an IR-45 column and eluted with methanol/water. The eluate was concentrated to dryness and methyl 2-amino-3,4-dihydro-4-oxo-7-β-D
-ribofuranosyl-7H-pyrrolo[2,3-d]
19 mg of pale yellow crystals of pyrimidine-5-carboxylate were obtained. (Yield 91%) Melting point: 240-242℃ (decomposition) UV (H ₂ O): λmax=296nm (ε=8840) λmax=270nm (ε=7782) λmax=232nm (ε=19253) IR (KBr) :3400, 1711, 1645, 1600, 1550,
1512, 1442, 1398, 1300, 1212, 1092 cm ^-1 NMR (D ₂ O, t-BuOH = 1.23 ppm): δ = 3.81 (s, 3H), 3.65-3.96 (m,
2H), 4.16 (q, 1H, J=3.2Hz), 4.34
(t, 1H, J=4.6Hz), 4.55 (t, 1H,
J = 5.0Hz), 5.94 (d, 1H, J = 5.1Hz),
7.72 (s, 1H) [α] ¹² _D = -49.2° (C = 0.12, H ₂ O) (2) Add 3 ml of dry tetrahydrofuran and 10 ml of lithium borohydride to 15 mg of the product obtained in (1) above.
After adding 50 mg of nitrogen and purging with nitrogen, the mixture was refluxed on an oil bath for 46 hours.
After concentration under reduced pressure, the residue was dissolved in water and purified by preparative high performance liquid chromatography to obtain 2-amino-
3,4-dihydro-5-hydroxymethyl-7
-β-D-ribofuranosyl-7H-pyrrolo[2,
3-d] 6 mg of white crystals of pyrimidin-4-one
I got it. (Yield 44%) Melting point: 230-235°C (decomposition) UV (H ₂ O): λmax=283nm (ε=6771) λmax=261nm (ε=10142) λmax=221nm (ε=
17823) IR (KBr): 3460, 3370, 1652, 1632, 1610,
1450, 1198, 1130, 1042, 1014, 860 cm ^-1 NMR (DMSO-d ₆ /D ₂ O=1/1, t-
BuOH = 1.23ppm): δ = 3.73 (dd, 1H, J ₁ = 4.1Hz, J ₂ = 12.3
Hz), 3.75 (dd, 1H, J ₁ = 3.5Hz, J ₂ =
12.3Hz), 4.08(ddd, 1H, J ₁ = 3.0Hz, J ₂
= 3.5Hz, J ₃ = 4.1Hz), 4.25 (dd, 1H, J ₁
= 3.0Hz, J ₂ = 5.5Hz), 4.51 (dd, 1H, J ₁
=5.5Hz, _J2 =6.8Hz), 4.68(s, 2H),
5.91 (d, 1H, J=6.8Hz), 6.94 (s,
1H) [α] ¹³ _D = -48.0° (C = 0.06, DMSO) (3) After dissolving 5 mg of the product obtained in (2) above in 2 ml of dimethylformamide, 50 mg of activated manganese dioxide was added and the mixture was heated at room temperature. Stirred for 15 hours. After filtering through Celite and washing with water/methanol, the filtrate was concentrated. The residue was purified by preparative high performance liquid chromatography to obtain 2.5 mg of Compound 1. (Yield 50%) Furthermore, the melting point of the compound obtained in Example 3 above,
Physical property values such as IR, NMR, UV, mass spectrum, and optical rotation matched those of Compound 1 obtained in Example 1, so it was confirmed that the same substance was produced by both production methods.

(Action) The pharmacological action of the compound of the present invention will be described below.

(1) Growth inhibitory effect on mouse lymphoma L5178Y cells Compound 1 and cadeguomycin were added to the culture medium of mouse lymphoma L5178Y cells and incubated at 37°C.
After culturing for 72 hours, the cell number was measured using an electric cell count (manufactured by Coulter), and the tumor cell growth inhibiting effect of the test compound was investigated. mouse lymphoma
L5178Y fat was cultured in Fitscher medium supplemented with 10% horse serum.

The results are shown in Figure 1.

(2) Effect of promoting the uptake of [ ^3H ]thymidine in human leukemia K562 cells Compounds 1, 2, and cadeguomycin were added to human leukemia K562 cells, and the mixture was incubated at 37°C for 18 hours under a flow of 5% carbon dioxide and 95% air. After culturing [ ^3H ]
Thymidine was added and cultured for an additional 6 hours. cells
Human leukemia was detected by collecting radioactivity using Cell-Harvester and measuring the radioactivity incorporated into the cells.
The effect of the test compound on promoting [ ³ H]thymidine uptake on K562 cells was investigated. human leukemia K562
Cells were cultured in PRMI-1640 medium supplemented with 10% fetal bovine serum.

The results are shown in Figure 2.

(Efficacy) As shown in Figure 1, the compound of the present invention was significantly more effective against mouse lymphoma than cadeguomycin.
Blocks the development of L5178Y cells. The IC ₅₀ of the tumor cell growth inhibiting effect of Compound 1 is a very low concentration of 10 μg/ml, and the compound of the present invention exhibits antitumor activity at a trace amount.

Furthermore, as shown in FIG. 2, the compound of the present invention significantly promotes the uptake of [ ³ H]thymidine by human leukemia K562 cells. In particular, the ribose form of the compound of the present invention (compound 1) exhibits a remarkable effect even at low concentrations, as it exhibits an effect of promoting [ ^3H ]thymidine uptake approximately 2.5 times as much as cadeguomycin at a concentration of 0.5 μg/ml.

As is clear from the above experimental results, the nucleoside compound of the present invention exhibits excellent antitumor effects and is useful as an antitumor agent. Furthermore, the compound of the present invention can be expected to have an effect of increasing blood cells and an immune-enhancing effect of activating lymphocytes.

The compound of the present invention can be combined with a suitable pharmaceutical carrier or diluent to form a medicine, and can be formulated by any conventional method to form a solid, semisolid, or liquid formulation for oral or parenteral administration. Or it can be formulated into a gaseous dosage form.

For formulation, the compounds of the present invention may be used in the form of their pharmaceutically acceptable salts, and the compounds of the present invention may be used alone or in appropriate combinations, and other antitumor substances, etc. It may also be used in combination with other pharmaceutically active ingredients.

Orally administered preparations can be used as such or with appropriate additives, such as conventional excipients such as lactose, mannitrate, corn starch, and potato starch, as well as binders such as crystalline cellulose, cellulose derivatives, gum arabic, corn starch, and gelatin. , corn starch, potato starch, disintegrants such as sodium carboxymethylcellulose, lubricants such as talc and magnesium stearate, other fillers, wetting agents, buffers,
Tablets, powders, etc. are prepared by appropriately combining preservatives, fragrances, etc.
It can be made into granules or capsules.

Furthermore, the compound of the present invention can be mixed with various bases, such as oily bases such as cacao butter, emulsion bases, water-soluble bases such as macrogol, hydrophilic bases, etc., to form suppositories. I can do it.

The injection may be a solution or suspension in an aqueous or non-aqueous solvent, such as distilled water for injection, physiological saline, Ringer's solution, vegetable oil, synthetic fatty acid glyceride, higher fatty acid ester, or propylene glycol.

Furthermore, depending on the site of tumor occurrence, it can be formulated into dosage forms other than those mentioned above that are most suitable for the treatment, such as inhalants, aerosols, eye drops, ointments, poultices, etc.

The preferred dosage of the compound of the present invention is to
Although it varies depending on the dosage form, administration method, administration period, etc.
To obtain the desired effect, an adult can generally be administered 1 to 3000 mg, preferably 5 to 1500 mg, of the compound of the present invention per day, and a unit dosage containing an appropriate amount of the compound of the present invention can be administered in one dose. One to several units can be administered per day.

(Example) Examples of formulations of pharmaceutical compositions containing the compound of the present invention as an active ingredient are shown below, but the present invention is not limited thereto.

Formulation example 1 (tablet) Ingredients per capsule (mg) Compound of the invention 50 Lactose 180 Corn starch 40 Magnesium stearate 10 Total 280 mg Formulation example 2 (capsule) Ingredients per capsule (mg) Compound of the invention 50 Lactose 250 total 300 mg Prescription example 3 (injection) Ingredients per ampoule (mg) Compound of the present invention 10 Sodium chloride Appropriate amount Distilled water for injection Appropriate amount 1 ml Prescription example 4 (Suppositories) Ingredients per unit (mg ) Compound of the present invention 100 Cocoa butter 1900 Total 2000 mg

[Brief explanation of the drawing]

Figure 1 shows murine lymphoma treated with the compound of the present invention.
It is a graph showing the growth inhibiting effect of L5178Y cells,
FIG. 2 is a graph showing the effect of the compound of the present invention on promoting the uptake of [ ³ H]thymidine in human leukemia K562 cells.

Claims (1)

[Claims] 1. A novel nucleoside compound represented by the general formula () and a pharmaceutically acceptable salt thereof. 2 General formula () (In the formula, R ₁ is an alkoxyalkyl group, R ₂ is an acyl group, R ₃ and R ₄ are an acyl group, an alkylidene group,
After oxidizing the hydroxymethyl group of a compound represented by an aralkyl group, _R5 is an acyl group or an aralkyl group, and X is a halogen to convert it into an aldehyde group, the protecting group is removed and the general formula () A method for producing a novel nucleoside compound represented by 3 General formula () An antitumor agent containing a novel nucleoside compound represented by: or a pharmaceutically acceptable salt thereof as an active ingredient.