JPH02138292A - 2'-methylidenepyrimidine nucleoside compound and drug use thereof - Google Patents

Abstract

NEW MATERIAL:A compound shown by formula I (R<1> is amino or OH; when R<1> is amino, R<2> is halogen, 2-4C alkynyl or lower alkyl and when R<1> is OH, R<2> is 2-4C alkyl, 2-4C alkynyl or haloalkyl; R3 is H or phosphoric acid residue). EXAMPLE:2'-Deoxy-2'-methylidene-5-fluorocytiaine. USE:A carcinostatic agent. PREPARATION:Hydroxyl groups at saccharide part 3' position and 5' position of a compound shown by formula II (R is halogen, alkyl, haloalkyl or 2-4C alkynyl) is protected and then hydroxyl group at saccharide 2' position is oxidized. Then the resulting substance is reacted with Wittig reagent to give a saccharide 2' position methylidene derivative, then protecting groups at saccharide 3' position and 5' position are removed and optionally hydroxyl group at saccharide 5' position is phoshorylated.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a novel 2°-methylidenepyrimidine nucleoside compound having excellent antitumor activity and its pharmaceutical use.

(Problems to be solved by conventional techniques and inventions) Under the circumstances where cancer deaths are increasing, chemotherapy and immunotherapy are widely used in conjunction with surgical therapy. Cytosine arabinoside and 5-fluorouracil are clinically used as anti-cancer 117i agents as they are effective against acute leukemia.However, conventional anticancer agents still do not have satisfactory curative effects. However, there are various problems such as side effects, etc., and the development of excellent anticancer drugs is required from various fields.

Under these circumstances, the present inventors invented 2'-deoxy-2'-methylidenecytidine or a pharmaceutically acceptable salt thereof.

However, since 2°-deoxy-2°-methylidenecytidine has poor solubility and its acid addition salts are unstable, they have not been satisfactory from a pharmaceutical standpoint.

An object of the present invention is to provide a novel compound having excellent anti-II tumor activity and its medical use.

[Means to solve the problem]

As a result of repeated research to develop a new compound that is free from formulation problems and is useful as an anticancer agent, the present inventors have found that 2.
-Methylidenepyrimidine nucleoside compounds are excellent anti-II! It was discovered that it has an ITgj effect, and the present invention was completed.

The present invention c; t, ax,.

(In the formula, R1 represents an amino or hydroxyl group; when R1 is amino, R2 represents a halogen, alkynyl or lower alkyl having 2 to 4 carbon atoms; when R' is a hydroxyl group, R1 represents a carbon number of 2 to 4 alkyl, alkynyl or haloalkyl having 2 to 4 carbon atoms, and R3 represents hydrogen or a phosphoric acid residue) or a salt thereof.

Furthermore, the present invention relates to an anticancer agent characterized by containing the compound of general formula (1) or a salt thereof as an active ingredient.

In the maximum (1) above, halogen represented by R2 means fluorine, chlorine, bromine, and iodine, and lower alkyl means methyl,
Ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, etc., and alkyl with 2 to 4 carbon atoms include ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, etc., and have 2 to 4 carbon atoms. Alkynyl means ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, and the like.

Further, haloalkyl means trifluoromethyl, chloroethyl, etc.

When R1 is hydrogen, the salts of the compound of general formula (1) include hydrochloride, sulfate, hydrobromide, phosphate, maleate, fumarate, tartrate, and succinate. , citrate, p-toluenesulfonate, etc., and when R'' is a phosphoric acid residue, metal salts such as sodium salt, potassium salt, lithium salt, calcium salt, or ammonium salt, etc. The present invention also includes a hydrate (monohydrate, monohydrate) of the compound of general formula (I) or a salt thereof.
/dihydrate, 1/4 hydrate, 3/2 hydrate) or solvates.

In the compound of general formula (1), when R1 is a hydroxyl group, the general formula % formula % as its tautomer is ten-5-ethynyl uridine or 2″-deoxy-2
°-Methylidene-5-fluorocytidine-5°-phosphoric acid is mentioned.

In the compound of general formula (1) of the present invention, a compound in which R' is a hydroxyl group (hereinafter referred to as a uridine compound) is, for example, represented by -max (in the formula, R1 and R3 have the same meanings as above). Also included in the present invention are uridine compounds.

As the compound of general formula (1), 2°-deoxy-2'
-Methylidene-5-fluorocytidine, 2'-deoxy-2'-methylidene-5-chlorocytidine, 2'-deoxy-2'-methylidene-5-bromocytidine, 2'-deoxy-2'-methylidene-5-iodocytidine , 2°
-deoxy-2”-methylidene-5-methylcytidine,
2゜-deoxy-2゛-methylidene-5-ethylcytidine, 2゛-deoxy-2゛-methylidene-5-ethyluridine, 2゜-deoxy-2''-methyl (wherein R is halogen, alkyl, haloargyl or After protecting the hydroxyl groups at the 3° and 5′ positions of the sugar moiety of the compound represented by (2) representing alkynyl having 2 to 4 carbon atoms, the hydroxyl group at the 2° position of the sugar moiety is oxidized, and then
It is produced by reacting a reagent to form a methylidene at the 2'-position of the sugar moiety, then removing the protecting groups for the hydroxyl groups at the 3'- and 5'-positions of the sugar moiety, and phosphorylating the hydroxyl group at the 5'-position of the sugar moiety, if necessary. Ru.

In the above, the protecting groups for the hydroxyl groups at the 3° and 5'' positions of the sugar moiety may be those commonly used as protecting groups for hydroxyl groups, such as acyl groups such as acetyl, propionyl, butyryl, benzoyl, and naphthoyl; Acetal or ketal type groups such as ethylidene, propylidene, isopropylidene, benzylidene, cyclohexylidene, cyclopentylidene, methoxymethylidene, ethoxymethylidene, dimethoxymethylidene, benzyl, p-methoxybenzyl, 3.4- Aralkyl groups such as dimethoxybenzyl, diphenylmethyl, triphenylmethyl, α- or β-naphthylmethyl, α-naphthyldiphenylmethyl, trimethylsilyl, t-butyldimethylsilyl, methyldiisopropylsilyl, triisopropylsilyl, tetraisopropyldisiloxyl ( Examples include silyl groups such as T I PDS).

As a method for oxidizing the 2-position hydroxyl group, chromate pyridine-
Chromic acid oxidation using a complex of acetic anhydride (Method A) or activated dimethyl sulfoxide oxidation using activated dimethyl sulfoxide produced from oxalyl chloride-dimethyl sulfoxide (Method B) can be employed.

The oxidation reaction is carried out in the presence of an oxidizing agent in an amount of 1 to 10 times the mole of the compound, in the case of method A at -10°C to room temperature, and in the case of method B at -10 to -80°C. This can be carried out by reacting for 10 hours.

The Witzteig reagent used in the methylidene reaction is specifically triphenylphosphine methylene, and immediately before use, it is mixed with a triphenylphosphonium compound (methyltriphenylphosphonium bromide, methyltriphenylphosphonium iodide, etc.) and a strong alkali ( potassium hydride,
It is preferable to use a reagent prepared from sodium hydride, n-butyllithium, sodium methoxide, potassium L-butoxide, sodium amide, etc. according to a conventional method.The amount of Witzteig reagent used is 1 per mole of the compound. It can be appropriately selected from 3 to 3 times the mole.

Methylidene reaction using such a Wittich reagent can be carried out by reacting in a solvent (single or mixed solvent such as tetrahydrofuran, dioxane, ether, benzene, dimethyl sulfoxide, etc.) at -30 to 30°C for 0.5 to 20 hours. It can be implemented.

The reaction for removing the protecting group from the hydroxyl group on the sugar moiety may be carried out by appropriately selecting a conventional treatment such as acidic hydrolysis, alkaline hydrolysis, ammonium fluoride treatment, or catalytic reduction depending on the protecting group used. When a silyl group is used as a protecting group, the silyl group can be removed by ammonium fluoride treatment or acidic or alkaline hydrolysis.

In addition, compounds in which R3 is a phosphoric acid residue can be obtained by reacting with a phosphorylating agent used for selective phosphorylation of the 5'' position of ordinary nucleosides, such as phosphorus oxychloride and tetrachloropyrophosphoric acid. .

Further, in the compound of the general formula (1) of the present invention, a compound in which R1 is amino can be obtained by subjecting the hydroxyl group at the 4-position of the base moiety to an ordinary amination reaction to convert it into an amino group. For example, a compound obtained by the above method in which the hydroxyl groups at the 3' and 5' positions of the sugar moiety are protected and the 2' position of the sugar moiety is methylidene and 1.2.4-triazole are mixed in the presence of phosphorus oxychloride and triethylamine. to form the 4-(1,2,4-)riazol-1-yl) form, then pass through ammonia gas to form the 4-amino form, and finally remove the protecting group from the hydroxyl group by the same method as above. , obtained by phosphorylation if necessary.

1.2.The reaction with 4-triazole is carried out at O″C in a solvent such as acetonitrile under a stream of inert gas such as argon.
The process proceeds from about 30 minutes to 5 hours at room temperature. The ammonia gas used in the amination reaction is preferably passed through sodium hydroxide in advance. The amination reaction using ammonia gas is usually carried out for about 1 to 5 hours.

Among the compounds of general formula (■) used as starting materials in the present invention, compounds in which R is alkynyl having 2 to 4 carbon atoms can be produced, for example, by the method shown below.

(III) (IV) (
X'+ (In the above reaction formula, R'' represents a silyl-protected alkynyl having 2 to 4 carbon atoms, and R'' represents an alkynyl having 2 to 4 carbon atoms.) l) and a silyl-protected alkynyl metal compound to obtain the compound (■), which is then desilylated to obtain the desired compound (■
°) can be obtained.

Furthermore, among the compounds with -maximum (n), the compounds in which R is alkyl having 2 to 4 carbon atoms are the above compounds (■°
) by protecting the hydroxyl groups at the 3′ and 5′ positions of the sugar moiety according to the method described above, subjecting it to a reduction reaction, and then removing the protecting groups for the hydroxyl groups at the 3′ and 5′ positions of the sugar moiety. Can be done. However, when producing a compound in which R2 is alkyl having 2 to 4 carbon atoms among the compounds of the general formula (1) of the present invention, the sugar moiety at the 3' position and the 5' position of the compound obtained by the above-mentioned reduction reaction is Without removing the protecting group for the hydroxyl group at the `` position, the methylidene form at the 2° position of the sugar moiety is obtained by the same reaction method as described above, and then the protecting group is removed, and if necessary, the hydroxyl group at the 5° position of the sugar moiety is phosphorylated. good.

The target compounds and synthetic intermediates of the present invention can be isolated and purified by appropriately combining conventional isolation and purification methods (for example, various chromatography methods such as ion exchange and adsorption, recrystallization methods, etc.). Specifically, for example, in the case of a nucleoside, after distilling off the solvent, it may be subjected to column chromatography as necessary and crystallized with an appropriate organic solvent.
In the case of a nucleotide (R' is a phosphate residue), it is purified by ion exchange column chromatography using ion exchange resin, adsorption column chromatography using activated carbon, etc., and the free acid form is obtained by freeze-drying or crystallization. If necessary, it can also be obtained in the form of a salt.

The compound of general formula (1) thus obtained can be converted into the above-mentioned acid addition salt, metal salt or ammonium salt by a conventional method.

[For J]

Since the compounds of the present invention exhibit remarkable growth-inhibiting effects on various cultured tumor cell lines and have low toxicity, the pharmaceuticals of the present invention containing these as active ingredients are useful as anticancer agents. The anticancer agent can be prepared by mixing an effective amount of compound (1) with a pharmaceutically acceptable carrier, excipient, diluent, etc., and preparing it as a powder, granule, tablet, sugar-coated tablet, capsule, pill, or suppository. It can take the form of external preparations, injections, drip preparations, etc.

2'-deoxy-2'-methylidenecytidine is 25 μ/mi in physiological saline at room temperature! The compound of Example 1 (2°-deoxy-2°-methylidene-5-fluorocytidine) did not dissolve under similar conditions.
It is melting.

The anticancer agent of the present invention is administered orally or parenterally to mammals including humans, but oral administration is particularly preferable, and the dose may be any amount that effectively inhibits the target cancer. The dosage may vary depending on the animal to be treated, the type of cancer, the route of administration, the dosage form, etc., but in general, in the case of oral drugs, the dosage is 10 to 10 times a day.
400■/kg body weight, preferably 50-200■/kg
The body weight is 1 to 20 .mu./kg body weight, preferably 1 to 5 .mu./kg body weight per day for injections. The number of administrations is once a day.
The number of times can be selected as appropriate within the range of 4 times.

The anticancer agent of the present invention can be used in combination with other anticancer agents, immunostimulants, or other acceptable drugs.

[Examples] Hereinafter, the present invention will be specifically explained with reference to Examples, but the present invention is not limited thereto.

Example 1 (1) 2.62 g of 5-fluorouridine was mixed with 3 g of pyridine.
1. 3-dichloro-1,1
, 3,3-tetraisopropyldisiloxane was added thereto for 2 hours, and the mixture was returned to room temperature and stirred for 1.5 hours. 5 liters of water
1 was added, and the solvent was distilled off under reduced pressure. ethyl acetate 100-1,
1,001 liters of water was added, the layers were separated, and the organic layer was washed twice with water, dried over sodium sulfate, and the solvent was distilled off under reduced pressure. The residual fragrance was collected using a silica gel column (φ2.4×23 cm). 25%
Elution with ethyl acetate/hexane yields 3',5'-0-
Tetraisoprobyldisiloxanyl-5-fluorouridine was obtained as a candy-like substance. Yield 4.24g, yield 8
3.9%.

Mass (II/Z): 504 (M”), 46
1 (M"-isopropyl) (2) (+) 5 g of the compound obtained in 10-1 methylene chloride was dissolved, and 4 equivalents of chromic acid complex (4 g of chromium oxide in 1001 methylene chloride, 6.67 ml of pyridine. Acetic anhydride 4-1
) and stirred at room temperature for 1 hour. ethyl acetate 500
el, and filtered through φ6, OXl, 5C11 silica gel. The filtrate was distilled off under reduced pressure, and the residual aroma was dissolved in ethyl acetate.
The liquid was separated with sIl and 200 ml of water. After washing the organic layer with water, it was dried over sodium sulfate, the solvent was distilled off under reduced pressure, and the residual aroma was collected using a silica gel column (φ2.4 x 2 BC 1 m) to give 20%
Elution with ethyl acetate/hexane and crystallization from hexane gave 3",5"-〇-tetraisoprobyl disiloxanyl-2°-oxo-5-fluorouridine.

Yield: 14.3 g, yield: 86%, melting point: 183-186°C.

Mass (m/z): 502 (M.).

Elemental analysis value Cz+H3sNzOyFS iz and shite:
Calculated value: C: 50.17 H; 7.02 N
; 5.57 actual measurement (!!: C, 50, 29H, 7
, 33N, 5.58 (3) Tetrahydrofuran 80
8.57 g of methyltriphenylphosphonium bromide was suspended in nl under an argon atmosphere, and 15.1 m of n-butyllithium (], 58 mol/l, hexane solution) was suspended at 0°C.
1 was added dropwise, the temperature was returned to room temperature, and the mixture was stirred for 1 hour. 3.02 g of the compound obtained in (2) and 25 ml of tetrahydrofuran
The solution was added dropwise at 0°C under an argon stream. After 20 minutes, the mixture was returned to room temperature and stirred for 3 hours. Neutralized with IN ammonium bromide and concentrated the solution. ethyl acetate 1ocal, water 100%
The organic layer was washed with water, dried over sodium sulfate, and evaporated under reduced pressure. The residual fragrance was removed through a silica gel column (φ3.
5 x 1 Bcm) and eluted with 20% ethyl acetate/hexane to give 3',5'-0-tetraisoprobyl disiloxanyl-2'-deoxy-2'-methylitin-5.
-Fluorouridine was obtained as a candy-like substance. Yield 2
．． 43g, yield 81%, Mass (m/z): 50
0 (M”).

(4) 1.4 parts triethylamine to 10 parts acetonitrile
684 ml of 1.2.4-triazole was melted and cooled to θ°C under an argon stream. Phosphorus oxychloride 28
02a was added dropwise, the temperature was returned to room temperature, and the mixture was stirred for 1 hour. The precipitate was filtered off, and a solution of 500 μl of compound (3) in 5 ml of acetonitrile was added dropwise to the filtrate. After 3 hours at room temperature, the mixture was stirred at 50°C for 1 hour, and ammonia gas passed through sodium hydroxide was passed through the mixture for 4 hours. Chloroform 100-1, water 1ocal
The aqueous layer was washed twice with chloroform, the chloroform layers were combined, dried over sodium sulfate, and evaporated under reduced pressure. Remove the residual fragrance using a silica gel column (φ3.5X14c+
When fractionated in step a) and eluted with 5% ethanol/chloroform, 3'',5'-0-tetraisoprobyldisiloxanyl-2'-deoxy-2'-methylidene-5-fluorocytidine was obtained as a candy-like substance. Yield: 435 cm, yield: 87%, Mass (m/z) = 499
(11”).

(5) Compound 4001Ig obtained in (4) was dissolved in 10 ml of tetrahydrofuran, 1.8 ml of tetra-n-butylammonium fluoride was added, and the mixture was stirred at room temperature for 30 minutes.

The mixture was neutralized with acetic acid, and the solvent was distilled off under reduced pressure. The residual fragrance was collected using a silica gel column (φ2.4×18c), and ethanol/
Elution with chloroform and crystallization from ethanol-ethyl acetate yielded 2'-deoxy-2°-methylidene-5-
Fluorocytidine was obtained. Yield 1 it 177 m, yield 86
．． 2%, melting point 185-187'C, Mass (m/z
): 25701”).

Elemental analysis value as C1゜H1x N'x O4F:
Calculated value: C, 46,70H; 4.70 N i
16.34 Actual value: C; 46.06 II
; 4.67 N i 16.00 Example 2 (1) 684 μ of triethylamine 1.4-1.1,2.4-triazole was dissolved in 10 ml of acetonitrile and cooled to 0° C. under an argon stream. Phosphorus oxychloride 2
80μ was added dropwise, the temperature was returned to room temperature, and the mixture was stirred for 1 hour. The precipitate was filtered off, and the filtrate was mixed with thymidine from Example 1 (1) to (3).
A solution of 498 ml of 3°,5'-0-tetraisopropyldisiloxanyl-2°-deoxy-2'-methylidenethymidine obtained as above in a7tonitrile 5-1 was added dropwise. After 3 hours, ammonia gas was passed through sodium hydroxide. After 3 hours, 100 parts of chloroform, 10 parts of water
Add 0ml and separate the layers, wash the aqueous layer twice with chloroform,
The chloroform layer was collected, dried over sodium sulfate, and then evaporated under reduced pressure. Remove the residual fragrance using a silica gel column (φ3, 5×12
C1) and eluted with 5% ethanol/chloroform at 3°.

5゛-0-tetraisopropyldisiloxanyl-2゛-
Deoxy-2゛-methylidene-5-methylcytidine,
Obtained as a candy-like substance. Yield 315■, Yield 63.3
%, Mass (m/z): 495 (M
”).

(2) 300 ml of the compound obtained in (1) was dissolved in 10 ml of tetrahydrofuran, 1.4 ml of tetra-n-butylammonium fluoride was added, and the mixture was stirred at room temperature for 30 minutes.

Neutralize with acetic acid, remove the solvent under reduced pressure, collect the residual aroma using a silica gel column (φ2.4×17c weight), elute with ethanol/chloroform, crystallize from ethanol-ethyl acetate, and obtain 2゛-deoxy-2゛. -Methylidene-5-methylcytidine was obtained. Yield ff1137■, yield 89. θ%,
Melting point 198-204°C (decomposition).

Mass (+s/z) = 253 (M”).

Elemental analysis value C+ + H+ s N, as Oa:
Calculated values: Ci 52.17 Hi 5.97
N; 16.59 Actual value: Ci 52.02
H, 5,99N; 16.37 Example 3 (1) 1Og of 5-chlorouridine to 100-1 of pyridine
1,3-dichloro-1,1,3,3-
12.44N+ of tetraisopropyldisiloxane was added and stirred for 3 hours. After returning to room temperature and stirring for 2 hours, 5 ml of ethanol was added and the solvent was distilled off under reduced pressure. After separating the residual aroma with 200ml of ethyl acetate and 200ml of water, the organic layer was dried over sodium sulfate, distilled off under reduced pressure, and the residual aroma was transferred to a silica gel column (φ3
．． OX25cm) and eluted with 25% ethyl acetate/hexane, 3',5'-0-
Tetraisopropyldisiloxanyl-5-chlorouridine was obtained. Yield 14.63 g, yield 78.2%. M
ass (m/z): 47B (M'-isopropyl).

(2) Dissolve 5.21 g of the compound obtained in (1) in methylene chloride 15-I and mix with 4 equivalents of chromic acid complex (chromium oxide 4 g, pyridine 5.0 + 1. acetic anhydride 4 + al in methylene chloride 100 + Ill). Then, the 0 reaction solution stirred for 2 hours was added dropwise to ethyl acetate 4001, and φ
6. OX 1. Filtered through Ocm silica gel. The solvent was distilled off under reduced pressure, and the residue was transferred to a silica gel column (φ2.4×
30c+a), eluted with 25% ethyl acetate/hexane, and crystallized from ethyl acetate/hexane to yield 3".

5″-O-tetraisopropyldisiloxanyl-2″-
Oxo-5-chlorouridine was obtained. Yield 3.75g,
Yield 72%. Melting point 199~201″C, Mass (I
S/Z) F476 (M”-isopropyl).

Elemental analysis value CzrH2sNzOtCIS Two calculated values as il: Ci 4B, 49 H: 6.78 N
; 5.39 Actual value: Ci 48.24 Hi
7.02 N, 5.18(3) 5.72 g of methyltriphenylphosphonium bromide was suspended in 50*L of tetrahydrofuran under an argon stream, and 10.1 ml of n-butyllithium was added dropwise under water cooling. Returned to room temperature, stirred for 1 hour, cooled to 0°C, and obtained compound 2.08 obtained in (2).
A solution of 10 g in 10 ml of tetrahydrofuran was added dropwise thereto, and after 30 minutes the temperature was returned to room temperature.

After 5 hours, the mixture was neutralized with IN ammonium bromide, and 100 ml of ethyl acetate and 100 s+1 of water were added to separate the layers.

The organic layer was washed with water, dried over sodium sulfate, and evaporated under reduced pressure. Transfer the remaining amount to a silica gel column (φ5.0X14C+a)
When fractionated and eluted with 25% ethyl acetate/hexane,
3',5'-0-tetraisopropyldisiloxanyl-2'-deoxy-2'-methylidene-5-chlorouridine was obtained as a foamy substance. Yield 1.7g, Yield 81
．． 7%. Mass (m/z): 517 (M”).

(4) 3 ml of triethylamine and 1.5 ml of 1.2.4-triazole in 20 ml of acetonitrile under a stream of argon.
2g was dissolved and cooled on ice. Phosphorus oxychloride 6204 was added dropwise, and after 30 minutes, the mixture was returned to room temperature and stirred for 30 minutes. The precipitate was filtered off, and 1.04 g of the compound obtained in (3) was added to the filtrate.
Stirred at room temperature for 3 hours. Ammonia gas was passed through sodium hydroxide for 2 hours, and 100 ml of chloroform and 100 ml of water were added to separate the layers.

Wash the aqueous layer with chloroform, collect the chloroform layer,
After drying with sodium sulfate, the residue was distilled off under reduced pressure.

The residue was collected using a silica gel column (φ3.5 x 17 cm), eluted with 75% ethyl acetate/hexane, and crystallized from ethyl acetate to yield 3", 5°-O-tetraisopro and 2°- Deoxy-2°-methylidene-5-chlorocytidine was obtained. Yield 944■, yield 9
0.7%, melting point 108-111'C, Mass (w+
/z): 517 (M”).

(5) 900 μl of the compound obtained in (4) was dissolved in 10 ml of tetrahydrofuran, 4+sl of tetra-n-butylammonium fluoride was added, and the mixture was stirred at room temperature for 30 minutes.

Add 5 ml of methanol, evaporate the solvent under reduced pressure, and collect the residue using a silica gel column (φ3.5 x 12 cm).
Elution with 20% ethanol/chloroform and crystallization from ethanol-ethyl acetate gave 2'-deoxy-2-methylidene-5-chlorocytidine.

Yield fi441■, yield 93.4%, melting point 168-170
°C.

Mass (s/z): 273 (M”)
.

Example 4 (1) 3.32 g of 5-bromouridine was mixed with 30 g of pyridine.
1,3-dichloro-1,1,3 dissolved in O″C
, 3-tetraisopropyldisiloxane were added thereto, and the mixture was stirred for 2 hours. The mixture was further returned to room temperature and stirred for 1 hour and 40 minutes. 51 g of water was added and the solvent was distilled off under reduced pressure.

After ethanol azeotropy, the remaining residue was transferred to a silica gel column (φ2.4
25 cm) and eluted with 25% ethyl acetate/hexane to obtain 3', 5', 0-tetraisoprobyl disiloxanyl-5-bromouridine as a candy-like substance. Yield, If 5.05 g, Yield 89.2%.

Maaa (m/z): 522 (M''-isopropyl).

(2) Add 3.57 g of the compound obtained in (1) to 4 equivalents of a chromic acid complex (2.4 g of chromium oxide, 4 Il of pyridine, 2.4 + acetic anhydride in 701 methylene chloride)
1) and stirred at room temperature for 40 minutes. The reaction solution was added dropwise to ethyl acetate 4001, and the solution was added to φ6. OX 1. Oc
The mixture was filtered through silica gel. The filtrate was concentrated, 150 ml of ethyl acetate and 150 ml of water were added, and the layers were separated. The organic layer was washed with water, dried over sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was collected using a silica gel column (φ5.0×14C1), eluted with 20% ethyl acetate/hexane, and crystallized from ethyl acetate/hexane to give 3°, 5′-0-tetraisopropyldisiloxanyl-2′. -Oxo-5-bromouridine was obtained. Yield 13.01g, yield 84.8%,
Melting point 204-205°C, Mass (g+/z): 5
20 (M'-isopropyl).

Elemental analysis value C*+H3sNzOtS As 1tBr: Calculated value: C; 44.75 H; 6.26 N
i 4.97 Actual value: C; 44.57 Hi
6.32 N? 4.92(3) 5.72 g of methyltriphenylphosphonium bromide are suspended in 50 ml of tetrahydrofuran under a stream of argon, and 10.1+*l of n-butyllithium are added dropwise at 0°C. Return to room temperature, 1
After an hour, a solution of 2.25 g of the compound obtained in (2) in 15 mol of tetrahydrofuran was added dropwise at 0"C. After 3 hours, the temperature was returned to room temperature, stirred overnight, neutralized with IN ammonium bromide, and acetic acid. 150ml of ethyl.150ml of water was added and the layers were separated.The ethyl acetate layer was washed with water, dried over sodium sulfate, and the solvent was distilled off under reduced pressure.The residue was collected using a silica gel column (φ5.0
X14C■) and eluted with 25% ethyl acetate/hexane, 3',5'-0-tetraisopropyldisiloxanyl-2'-deoxy-2'-
Methylidene-5-bromouridine was obtained. Yield 1.72
g1 yield 76.4%, Mass (s/z): 51
B (M”-isopropyl).

(4) Triethylamine 31 and 1,2,4-triazole in 20w1 acetonitrile under a stream of argon],
52g was dissolved and cooled on ice. Phosphorus oxychloride 620P4
was added dropwise, and after 30 minutes, the temperature was returned to room temperature, and the precipitate was removed by filtration. 1.12 g of the compound obtained in (3) was added to the filtrate, and the mixture was stirred at room temperature for 3 hours. Passing ammonia gas through sodium hydroxide for 3 hours, chloroform 1001, water 1001
was added and separated. The aqueous layer was washed with chloroform, the chloroform layer was collected, dried over sodium sulfate, and the solvent was distilled off under reduced pressure. Transfer the remaining amount to a silica gel column (φ3.5×17
．． ) and eluted with 75% ethyl acetate/hexane.
Crystallization from ethyl acetate hexane gives 3°, 5'-0-
Tetraisoprobyl disiloxanyl-2°-deoxy-
2°-methylidene-5-bromocytidine was obtained. Yield 9
26■, yield 82.7%, melting point 169-17F (, Ma
ss(II/Z): 560 (M'').

(5) 900 ml of the compound obtained in (4) was dissolved in 10 ml of tetrahydrofuran, 41 ml of tetra-n-butylammonium fluoride was added, and the mixture was stirred at room temperature for 30 minutes.

51 ml of methanol was added and evaporated under reduced pressure. The residue was collected using a silica gel column (φ3.5×11 cm), eluted with 20% ethanol/chloroform, and crystallized from ethanol-ethyl acetate to give 2°-deoxy-2°-methylidene-
5-bromocytidine was obtained.

518■, yield 102%, melting point 172-180°C (decomposition), Mass (II/Z): 318 (M).

Example 5 (1) 10 g of 5-iodouridine to 100 pyridine 1
8.91 of 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane was added at 0°C, and 1.
Stirred for 5 hours. The temperature was returned to room temperature, 10 ml of water was added for 3 hours, and the solvent was distilled off under reduced pressure. After ethanol azeotropy, the residue was fractionated using a silica gel column (φ3.OX30cm) and 25%
Elution with ethyl acetate/hexane yields 3°, 5″-0-tetraisoprobyl disiloxanyl- as a syrupy substance.
5-iodouridine was obtained. Yield 14.7 g,
Yield 89% 0Mass (m/z): 612 (M”
).

(2) 5.62 g of the compound obtained in (1) was mixed with 4 equivalents of chromic acid complex (3.55 g of chromium oxide, 5.91 g of pyridine, 3.1 g of acetic anhydride in 100 l of methylene chloride).
551) and stirred at room temperature for 40 minutes. The reaction solution was added dropwise to 500 s+l of ethyl acetate, and φ6.0×15C1
The filtrate filtered through silica gel was distilled off under reduced pressure. The remaining amount was collected using a silica gel column (φ5.OX21cm), and 25
Elute with 3% ethyl acetate/hexane.

5'-0-tetraisopropyldisiloxanyl-21-
Oxo-5-iodouridine was obtained. Yield 1.72g,
Yield 76.4%, Mass (++/z): 610
(M').

(3) Methyltriphenylphosphonium bromide 7.94g
suspended in tetrahydrofuran 801 under a stream of argon,
At 0°C, n-butyllithium 141 was added dropwise. After returning to room temperature for 1 hour, cooling to O'C, compound 3. obtained in (2).
39 g of tetrahydrofuran 151 solution was added dropwise.

After 1 hour, the mixture was returned to room temperature and stirred overnight. Neutralize with IN ammonium bromide and add 200ml of ethyl acetate, 2Q of water
Oml was added and the liquid was separated. The organic layer was dried over sodium sulfate and evaporated under reduced pressure. The residual fragrance was removed through a silica gel column (φ5.
0X22c+*) and eluted with 20% ethyl acetate/hexane to give 3',5'-0-tetraisoprobyldisiloxanyl-2'-deoxy-2°-methylidene-
5-iodouridine was obtained as a candy-like substance. Collection 11.
6g, yield 47.2%, Mass (It/Z):
608 (M').

(4) In 10 ml of acetonitrile under an argon atmosphere, triethylamine 1.51 and 1.2.4-) Riazole 7
60 μm was dissolved and cooled on ice. Phosphorus oxychloride 310#&
was added dropwise, the temperature was returned to room temperature after 30 minutes, the precipitate was filtered off, the compound 321■ obtained in (3) was added to the filtrate, and the mixture was stirred at room temperature for 30 minutes.
Stir for hours. Ammonia gas was passed through sodium hydroxide for 3 hours, and 50a1 of chloroform and 5011 of water were added to separate the layers. The aqueous layer was washed with chloroform, and the organic layers were collected, dried over sodium sulfate, and evaporated under reduced pressure. The residual fragrance was collected using a silica gel column (φ2.4 x 20 cm), and 10
Elution with % ethanol/chloroform and crystallization from ethyl acetate yielded 3',5°-0-tetraisoprobyl disiloxanyl-2'-deoxy-2'-methylidene-5-
Iodocytidine was obtained. Yield 169■, yield 52.7%
.

Melting point 185-187℃, Mass (poor/z): 6
07 (M”).

(5) Compound 160 .mu. obtained in (4) was dissolved in 51 ml of tetrahydrofuran, 1-1 of tetra-n-butylammonium fluoride was added, and the mixture was stirred at room temperature for 30 minutes.

Two parts and one part of methanol were added, and the solvent was distilled off under reduced pressure. The residual fragrance was collected using a silica gel column (φ3.5 x 7.0 cm) and eluted with 20% ethanol/chloroform.
2'-deoxy-2'-methylidene-5-iodocytidine was obtained as a candy-like substance. Yield 192■, yield 97.
3%, melting point 159-I61'C, Mass (m/z
):365 (M”).

Example 6 (1) 2.1 g of 5-ethynyluridine was mixed with 50-
Dissolved in 1,3-dichloro-1,1,3,3 under water cooling.
- 2.61 g of tetraisopropyldisiloxane was added and stirred for 3 hours. Furthermore, after returning to room temperature and stirring overnight, methanol 5
ml was added, and the solvent was distilled off under reduced pressure. 11 ethyl acetate for residual fragrance
001, 100 m+ of water was added, the layers were separated, and the organic layer was washed twice with water, dried over sodium sulfate, and the solvent was distilled off under reduced pressure. The residual aroma was collected using a silica gel column (φ5.OX17cm) and eluted with 25% ethyl acetate/hexane.
'-0-Tetraisopropyldisiloxanyl-5-ethynyluridine was obtained as a foam. Yield 3.26
g, yield 82%, Mass (m/z): 510
(M”).

(2) Mix 2.04 g of the compound obtained in (1) with 4 equivalents of chromic acid complex (1.55 g of chromium oxide, 2.57+*l of pyridine, 1.55 ml of acetic anhydride in 40-1 methylene chloride). and stirred at room temperature for 40 minutes.
The reaction solution was added dropwise to ethyl acetate 2001, and the φ6. OX
1. Filtered through Ocm silica gel. The filtrate was distilled off under reduced pressure, and the residual aroma was separated between 100 Ill of ethyl acetate and 100+ g of water.

The organic layer was washed twice, dried over sodium sulfate, and the solvent was distilled off under reduced pressure. Remove the residual fragrance using a silica gel column (φ3.5X1
5c carbon dioxide) and eluted with 20% ethyl acetate/hexane to obtain 3°, 5'-0-tetraisoprobyl disiloxanyl-2'-oxo-5-ethynyl uridine as a candy-like substance. Yield 11.62g, yield 81%, Mass
(m/z): 508 (M”).

(3) Methyltriphenylphosphonium I 4.2
9 g was suspended in 40 ml of tetrahydrofuran under an argon stream, and 7.58 ml of n-butyllithium was added dropwise under water cooling. After returning to room temperature and stirring for 1 hour, the mixture was cooled with water, and a solution of 1.52 g of the compound obtained in (2) in 1 mL of tetrahydrofuran was added dropwise, and after 30 minutes, the mixture was returned to room temperature. After 6 hours, neutralize with IN ammonium bromide, add 100 parts of ethyl acetate and 10 parts of water.
01 was added and the liquid was separated. The organic layer was washed twice with water, dried over sodium sulfate, and evaporated under reduced pressure. The residual aroma was collected using a silica gel column (φ3.5 x 20c carbon dioxide), and 20% ethyl acetate/
Elution with hexane gave 3',5'-0-tetraisopropyldisiloxanyl-2'-deoxy-2'-methylidene-5-ethynyl uridine as a candy-like material.

Yield: 11.12g/yield: 74%. Mass (s/z):
506 (M") a(4) 500 μ of the compound obtained in (3) was dissolved in tetrahydrofuran LOsl, 2.2111 tetra-1-butylammonium fluoride was added, and the mixture was stirred at room temperature for 20 minutes. Neutralized with acetic acid. The solvent was distilled off under reduced pressure.The residual aroma was removed using a silica gel column (φ2.4
+a), eluted with 10% ethanol/chloroform, and crystallized from ethanol-hexane to obtain 2°-deoxy-2′-methylidene-5~ethynyluridine.

Yield 230■, yield 87%, melting point 172-174"C,
Mass (m/z): 264 (M”).

Example 7 (1) In 10 ml of acetonitrile under an argon stream, 1.5 ml of triethylamine and 760 ml of 1.2.4-) lyazole were dissolved and cooled on ice. Phosphorus oxychloride 31OP
After 30 minutes, the mixture was returned to room temperature and stirred for an additional 30 minutes. The precipitate was removed by filtration, and the 3" obtained in Example 8 (3) was added to the filtrate.
5°-0-tetraisopropyldisiloxanyl 2′-deoxy-2°-methylidene-5-ethynyluridine 50
0 g was added and stirred at room temperature for 3 hours.

Ammonia gas was passed through sodium hydroxide for 2 hours, and chloroform 80-1 and water 80a+1 were added to separate the layers. The aqueous layer was washed with chloroform, the chloroform layers were collected, dried over sodium sulfate, and then evaporated under reduced pressure. The residual fragrance was collected using a silica gel column (φ3.5×14C+a), and 75
Elution with % ethyl acetate/hexane gave 3'',5'-0-tetraisoprobyldisiloxanyl-2'-deoxy-2'-methylidene-5-ethynylcytidine as a candy.

Yield 131 (l■, yield 62%, Mass (m/z
): 505 (M”).

(2) 300 μl of the compound obtained in (1) was dissolved in 10 ml of tetrahydrofuran, 1.2@l of tetra-n-butylammonium fluoride was added, and the mixture was stirred at room temperature for 30 minutes. 2+wl of methanol was added and the solvent was distilled off under reduced pressure.

The residual aroma was collected using a silica gel column (φ2.OX12cm) and eluted with 20% ethanol/chloroform to produce 2'-deoxy-2'-methylidene- as a candy-like substance.
5-ethynylcytidine was obtained. Yield 9.7■, Yield 62
%.

Example 8 (1) 3', 5'-0-tetraisoprobyl disiloxanyl-5-ethynyl uridine 3 obtained in Example 6 (1)
．． 8 g was dissolved in 50 ml of methanol, 100 μl of 10% palladium on carbon was suspended, and medium pressure catalytic reduction was carried out at 20 psi for 4 hours. The reaction solution was filtered through Celite and the solvent was distilled off under reduced pressure. Remove residual fragrance using silica gel column (φ3, OX20cm)
When fractionated and eluted with 33% ethyl acetate/hexane,
3゛,5′-〇-tetraisoprobyl disiloxanyl-
5-Ethyluridine was obtained as a candy-like substance. Collection 13.
22■, yield 84%.

Mass (m/z): 514 (M.

(2) 3.15 g of the compound obtained in (1) was mixed with 4 caps of chromic acid complex (in 60+ml of methylene chloride,
Chromium oxide 2.37g2 Pyridine 3.93ml1. The reaction solution, which was mixed with acetic anhydride (2.371) and stirred at room temperature for 50 minutes, was added dropwise to 300 ml of ethyl acetate, and the
Filtered through c+m silica gel. The filtrate was concentrated, and 100 s+1 ethyl acetate and 100 ml water were added to separate the layers. The organic layer was washed twice with water, dried over sodium sulfate, and evaporated under reduced pressure. Remove the residual fragrance using a silica gel column (φ3.5X15C1
m), eluted with 20% ethyl acetate/- xane, crystallized from ethyl acetate-hexane,
0-tetraisopropyldisiloxanyl-2°-oxo-5-ethyluridine was obtained.

Yield: 2.54 g, yield: 80.8%. Melting point 176-177
"C, Mass (II/Z): 512 (M").

Elemental analysis value Ct x Ha. Two calculated values as NzOISig: C, 53,88) 1 i 7.86 N,
5.46 Actual value: C, 53,59H; 8.01
N, 5.56 (3) 5.72 g of methyltriphenylphosphonium bromide in 50 m of tetrahydrofuran
1 under an argon atmosphere, 10.1 ml of n-butyllithium was added dropwise under water cooling, the temperature was returned to room temperature, and the mixture was stirred for 1 hour. A solution of 2.05 g of the compound obtained in (2) in 15 ml of tetrahydrofuran was added dropwise under water cooling, and after 3 hours, the mixture was returned to room temperature and stirred for 5 hours. The mixture was neutralized with IN ammonium bromide, and 150 ml of ethyl acetate and 150 ml of water were added to separate the layers. The organic layer was washed twice with water, dried over sodium sulfate, and evaporated under reduced pressure. Remove the residual scent through a silica gel column (φ3,5 x 13C1l)
eluted with 20% ethyl acetate/hexane and crystallized from ethyl acetate/hexane to give 3',5'-0-tetraisopropyldisiloxanyl 2'-deoxy-2'.
-Methylidene-5-ethyluridine was obtained. Collection 111.
70g, yield 82.7%, melting point 153-155°C,
Mass (II/2): 510 (M”)
.

Elemental analysis value as CxaHazNtOhS iz: Calculated value: Ci 56.44 H; 8.29 N
; 5.48 Actual value: C, 56,67Hi 8.
41 N? 5.36(4) Dissolve 510 ml of the compound obtained in (3) in 101 ml of tetrahydrofuran, add 2.2 ml of tetra-n-butylammonium fluoride,
Stirred at room temperature for 30 minutes. The mixture was neutralized with acetic acid, and the solvent was distilled off under reduced pressure. Remove the residual fragrance using a silica gel column (φ3.OX12cm)
), eluted with 6.25% ethanol/chloroform, and crystallized from ethanol-hexane to obtain 2°-deoxy-2°-methylidene-5-ethyluridine.

Yield N234■, yield 87.2%, melting point 137-139'
C, Mass (II/2): 268 (M.

Elemental analysis value as C+ z HIh N z Os:
Calculated value: Ci 53.73 Hi 6.01
N? 10.44 Actual value: C; 53.79
H, 5,89N i 10.67 Example 9 (1) 3 ml of triethylamine and 1.5 ml of 1,2,4-triazole under a stream of argon in 20-1 acetonitrile
2g was dissolved and cooled on ice. 620 Pg of phosphorus oxychloride was added dropwise, and after 30 minutes, the mixture was returned to room temperature and stirred for an additional 30 minutes.

The precipitate was filtered off, and the 3',5 obtained in Example 8 (3) was added to the filtrate.
'-0-tetraisopropyldisiloxanyl 2'-deoxy-2°-methylidene-5-ethyluridine 1.2 g
The mixture was added and stirred at room temperature for 4 hours. Ammonia gas was passed through sodium hydroxide, and chloroform 10 (1+1) and water 100 ml were added to separate the layers.

The aqueous layer was washed with chloroform, the chloroform layers were collected, dried over sodium sulfate, and then evaporated under reduced pressure. The residual aroma was collected using a silica gel column (φ3.5×20c11), eluted with 9% ethanol/chloroform, and crystallized from ethyl acetate-hexane to give 3°, 5”-〇-tetraisopropyldisiloxanyl-2゛- Deoxy-2°-methylidene-
5-ethylcytidine was obtained. Yield 1.0g, yield 83.
3%, melting point 197-200"C, Mass (s/z
): 509 (M") *(2) 900 .mu. of the compound obtained in (1) was dissolved in 10 ml of tetrahydrofuran, 41 of tetra-n-butylammonium fluoride was added, and the mixture was stirred at room temperature for 30 minutes.

5 ml of methanol was added and the solvent was distilled off under reduced pressure. The residual fragrance was collected using a silica gel column (φ3.5 x 10 cm), and 20
Elution with % ethanol/chloroform and crystallization from ethanol-ethyl acetate gave 2'-deoxy-2'-methylidene-5-ethylcytidine.

Yield N402■, yield 85.4%, melting point 194-196℃
.

Mass (+a/z): 267 (M').

Example 10 When 2°-deoxy-2′-methylidene-5-fluorocytidine and phosphorus oxychloride are reacted and treated according to a conventional method, 2′-deoxy-2′-methylidene-5
-Fluorocytidine-5°-phosphoric acid is obtained.

Formulation Example 1 Compound of Example 1 50.0 ■ Finely powdered cellulose 25.0 mg Lactose
Tablet consisting of 49.5■ Starch 40.0■ Talc 5.0■ Magnesium stearate 0.5■.

Sugar-coated tablets or film-coated tablets can be obtained by sugar-coating or film-coating, if desired.

Formulation Example 2 Compound of Example 1 50.0■Lactose
50.0 ■ Starch 15.0 ■ Talc 5.0 ■ Capsules filled with the following composition.

Formulation Example 3 Compound of Example 1 10% Lactose
Fine granules consisting of 80% starch and 10%.

Formulation Example 4 Compound of Example 1 10% Lactose
55% finely powdered cellulose 20% starch
Granules consisting of 15%.

Formulation Example 5 50.0 μg of the compound of Example 1 and 100.0 μg of glucose are dissolved in purified water to prepare an injection solution with a total volume of 2+al.

Formulation Example 6 Compound of Example 1 1001g
7"Sole" 815 950mg Suppository consisting of Uitepsol @ E75 950■,
However, Uitepsol is a registered trademark owned by Wuitten (West Germany).

Formulation Example 7 Compound of Example 1 2 g Ethyl hydroxybenzoate 0.025 g Propyl hydroxybenzoate 0.015 g Sodium lauryl sulfate 1.5 g Propylene glycol
12.0g stearyl alcohol
22.0 g white petrolatum
Dissolve 25.0 g of the composition in purified water to obtain a total of IJ 1
00. Og's hydrophilic ointment.

〔Effect of the invention〕

The following pharmacological experiments demonstrate that the compounds of the present invention have excellent antitumor effects.

Pharmacological experiment: Effect on in vitro proliferation of various cultured tumor cells (96-well plate with culture solution containing various cultured tumor cells (104 cells) and test compound) (FALCON 3072)
Add to each person to make 100#j, 37°C, 5%
Cot, cultured under −95% air for 72 hours.

After incubation, 1 OPI of MTT (3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution (5 μ/ml) was added to each person, and the cells were incubated at 37° for another 4 hours. Cells were incubated under 15%C0X-95% air and 100μ of 10% sodium dodecyl sulfate-0.0IN hydrochloric acid solution was added to each person.After standing overnight, the absorbance at 570nm was measured using a microplate photometer. The inhibition rate was calculated from the following formula, and the concentration of the test compound that inhibited 50% (I
C. ) was sought.

As is clear from Table 1, the compounds of the present invention exhibit excellent growth-inhibiting effects on various cultured tumor cells and are useful as anticancer agents.

The test compound used was 2'-deoxy-2゜methylidene-5-fluorocytidine dissolved in distilled water. In addition, various tumor cell lines are RPM116 containing 1o% FC8.
40 medium (Hinaga Pharmaceutical Co., Ltd.).

The resulting IC. The values (pg/ml) are summarized in Table 1.

Table 1

Claims (5)

[Claims] (1) General formula ▲ There are mathematical formulas, chemical formulas, tables, etc. When R^1 is a hydroxyl group, R^2 is an alkyl having 2 to 4 carbon atoms, alkynyl or haloalkyl having 2 to 4 carbon atoms, and R^3 is hydrogen or a phosphoric acid residue. A 2'-methylidenepyrimidine nucleoside compound or a salt thereof represented by: (2) When R^1 is a hydroxyl group, its tautomer is the general formula ▲ Numerical formula, chemical formula, table, etc. ▼ (In the formula, R^2 is halogen, alkyl having 2 to 4 carbon atoms, Alkynyl or haloalkyl having 2 to 4 carbon atoms,
R^3 represents hydrogen or a phosphoric acid residue. ) or a salt thereof according to claim (1). (3) 2'-deoxy-2'-methylidene-5-fluorocytidine or a salt thereof. (4) An anticancer agent containing the compound according to claim (1) as an active ingredient. (5) An anticancer agent containing 2'-deoxy-2'-methylidene-5-fluorocytidine or a salt thereof as an active ingredient.