JPH06211890A - 2'-deoxy-2'@(3754/24)s)-substituted alkylcytidine derivative - Google Patents

Abstract

(57) [Summary] [Structure] Formula (I) (In the formula, R ¹ represents a hydroxyl group or amino, R ² represents a hydroxyl group, acyloxy or halogen atom, and R ³ represents a hydrogen atom or a phosphoric acid residue.) 2′-deoxy-2 ′
(S) -Substituted alkyl cytidine derivative or a salt thereof, a method for producing the same, and an antitumor agent containing the same as an active ingredient. [Effect] The compound of the present invention has excellent antitumor activity.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel compound, a 2'-deoxy-2 '(S) -substituted alkylcytidine derivative, a method for producing the same, and an antitumor agent containing the same as an active ingredient. is there.

[0002]

2. Description of the Related Art Conventionally, various nucleic acid-related compounds having antitumor activity have been synthesized, and some of them have been put to actual clinical use. However, these compounds have various problems in terms of spectrum of antitumor activity and side effects such as toxicity, and are not always satisfactory. Recently,
2'-deoxy-2'-methylidene cytidine
3-230699, JP-A-2-256698), 2'-deoxy-2'-fluoromethylidene cytidine (JP-A-2-178272), 2'-deoxy-2 '(S) -C-. Methylcytidine (JP-A-63-2
15694, J. Med. Chem., 1991, 34 , 234) and 2'-C-cyano-2'-deoxy-1-β-D-arabinofuranosylcytosine (JP-A-4-235182, J. Med. Chem., 1991, 34 , 2917) and other 2'-substituted pyrimidine nucleosides having a cell growth inhibitory activity have been attracting attention and are being developed as pharmaceuticals.

[0003]

[Problems to be Solved by the Invention] In Japan today,
Cancer is one of the three major adult diseases, and a great deal of time and money is being spent on its eradication. However, it is far from achieving that goal, and lung cancer, for example, is rapidly increasing the number of patients. Thus, cancer diversity,
Considering various problems in cancer treatment such as resistance to conventional anti-cancer agents, it is the current situation that the development of many effective medicines even with one kind is desired. Then, the objective of this invention is to provide a novel compound useful as an antitumor agent. Another object of the present invention is to provide a method for producing the compound. Yet another object of the invention is to provide anti-tumor uses of the compounds.

[0004]

Means for Solving the Problems The present inventors have conducted research to achieve the above object with reference to a 2′-substituted pyrimidine nucleoside, which has recently been attracting attention, and as a result, are represented by the following formula (I). It was found that the 2′-deoxy-2 ′ (S) -substituted alkylcytidine derivative obtained has excellent antitumor activity. The present invention has been completed based on this finding.

That is, the present invention has the formula (I)

[0006]

[Chemical 6]

(Wherein R ¹ is a hydroxyl group or amino, R ^{2 is}
Is a hydroxyl group, an acyloxy or a halogen atom, and R ³ is a hydrogen atom or a phosphoric acid residue. 2'-deoxy-2 '(S) -substituted alkylcytidine derivative (hereinafter sometimes referred to as compound (I)) or a salt thereof.

The present invention also comprises 2'-deoxy-2 'represented by the above formula (I), which comprises the following first to fourth steps.
The present invention relates to a method for producing an (S) -substituted alkylcytidine derivative. Step 1: Epoxidizing the sugar moiety 2'position of the compound represented by the following formula (II) (hereinafter referred to as compound (II)) with iowilide to form the compound represented by the following formula (III) III))).

[0009]

[Chemical 7]

(In the formula, R ¹ represents a hydroxyl group or amino, and Z represents a protecting group.) Step 2: The epoxy ring at the 2′-position of the sugar moiety of the compound (III) is opened with a nucleophile to give the following formula: A step of obtaining a compound represented by (IV) (hereinafter referred to as compound (IV)).

[0011]

[Chemical 8]

(In the formula, R ² represents a hydroxyl group, an acyloxy or a halogen atom, and R ¹ and Z have the same meanings as described above.) Step 3: Acylation of the 2'-hydroxyl group of compound (IV) followed by reduction A step of reducing with an agent to obtain a compound represented by the following formula (V) (hereinafter referred to as compound (V)).

[0013]

[Chemical 9]

(In the formula, R ¹ , R ² and Z have the same meanings as described above.) Fourth step: Amination of the 4-position of the base moiety of the compound (V) as necessary, followed by deprotection of the sugar moiety protecting group. And further, if necessary, phosphorylating the 5′-position of the sugar moiety to obtain the compound (I).

[0015]

[Chemical 10]

(In the formula, R ³ represents a hydrogen atom or a phosphoric acid residue, and R ¹ , R ² and Z have the same meanings as described above.)

Further, the present invention relates to an antitumor agent containing a 2'-deoxy-2 '(S) -substituted alkylcytidine derivative represented by the above formula (I) or a salt thereof as an active ingredient. is there.

The present invention will be described below. (1) Compound of the present invention The 2'-deoxy-2 '(S) -substituted alkylcytidine derivative of the compound of the present invention is a novel compound, and is represented by the above formula (I).
It is represented by. In formula (I), the acyloxy represented by R ² is preferably an acyloxy having an acyl having 1 to 5 carbon atoms, and specific examples thereof include formyloxy, acetyloxy (acetoxy), propionyloxy, butyryloxy, pivaloyloxy and the like. The halogen atom of R ² means fluorine, chlorine, bromine and iodine.

The compound of the present invention also includes a salt form. For example, when R ¹ of the above formula (I) is amino, acid addition with an inorganic acid such as hydrochloric acid or sulfuric acid is mentioned. When R ³ is a phosphoric acid residue, a salt or an acid addition salt with an organic acid such as citric acid or succinic acid is used, such as an alkali metal salt such as sodium salt, potassium salt or lithium salt, calcium salt or the like. Examples are pharmaceutically acceptable salts such as alkaline earth metal salts and ammonium salts.

Specific examples of the compound of the present invention include 2'-deoxy-2 '(S) -fluoromethylcytidine, 2'-deoxy-2' (S) -chloromethylcytidine, 2'-deoxy-2 '. (S) -Bromomethylcytidine, 2'-deoxy-2 '(S) -iodomethylcytidine, 2'-deoxy-2' (S) -acetoxymethylcytidine, 2'-deoxy-2 '(S) -hydroxy Examples thereof include compounds such as methylcytidine, and their 5'-phosphate esters, or salts thereof. Among these compounds of the present invention, 2′- in which R ² is fluorine
Deoxy-2 '(S) -fluoromethylcytidine has a particularly strong antitumor activity.

(2) Method for producing the compound of the present invention The compound of the present invention can be produced by the following four reaction steps.

(First Step) In the first step, the starting compound (I
This is a reaction step of epoxidizing the sugar moiety 2'position of I) with iowilide.

The protecting group for Z in the formula (II) may be any of those usually used as a protecting group for a hydroxyl group of a nucleoside, and examples thereof include acyl such as acetyl, propionyl, butyryl, pivaloyl and benzoyl; benzylidene and the like. Alkylidene; benzyl; silyl protecting groups such as tetraisopropyldisiloxyl (TIPDS) and t-butyldimethylsilyl; trityl protecting groups such as triphenylmethyl (trityl) and dimethoxytrityl; ethers such as tetrahydropyranyl and methoxymethyl Examples include system protecting groups.

The iowilide used in this step is prepared by a known method in a mixed solvent of dimethyl sulfoxide or tetrahydrofuran with a commercially available trimethylsulfoxonium iodide and a base (for example, sodium hydride,
(For example, potassium hydride).

In the reaction, iowilide is used in an amount of 2 to 10 mol, preferably 2 to 4 mol, per 1 mol of compound (II),
It can be carried out by reacting at room temperature to 100 ° C. in an inert gas atmosphere such as argon or nitrogen in dimethyl sulfoxide or a mixed solvent with tetrahydrofuran.

The compound (III) thus prepared may be isolated by the usual means for separating and purifying nucleosides. For example, after partitioning with ethyl acetate and water, it is subjected to silica gel column chromatography to obtain n- It can be performed by a method of eluting with an organic solvent such as hexane-ethyl acetate.

(Second Step) The second step is a reaction step in which the 2'position epoxy ring of the compound (III) is opened with a nucleophile.

The reaction solvent and nucleophile used for the cleavage reaction of the epoxy ring depend on R ² of the target compound (IV). For example, when R ² is a halogen atom,
A glycol solvent such as methyl cellosolve is used as a reaction solvent, and potassium hydrogen halide such as potassium hydrogen fluoride or lithium halide such as lithium chloride is used as a nucleophile. When R ² is a hydroxyl group or acyloxy, an organic acid such as acetic acid or propionic acid can be used as the reaction solvent, and an organic acid salt such as sodium acetate or sodium propionate can be used as the nucleophile. The reaction can be carried out by using 2 to 50 mol of a nucleophile for 1 mol of compound (III) and reacting at room temperature to solvent reflux temperature.

Compound (IV) produced as described above
Isolation can be carried out by using the usual means for separating and purifying nucleosides. For example, the solvent can be distilled off, and the mixture can be partitioned with ethyl acetate and water and then isolated by silica gel column chromatography.

(Third Step) The third step is a reaction step in which the 2'-hydroxyl group of compound (IV) is acylated and then this is reduced with a reducing agent.

As the acylating agent, an ordinary acylating agent such as an acid anhydride such as acetic acid, propionic acid, butyric acid, benzoic acid, substituted benzoic acid, oxalic acid or an acid chloride thereof can be used. Particularly, considering the subsequent reduction reaction, it is preferable to use methyl oxalyl chloride as the acylating agent. The acylation reaction at the 2'-position may be carried out by a conventional method. For example, in a reaction solvent (a single solvent or a mixed solvent of pyridine, picoline, dimethylaminopyridine, dimethylformamide, acetonitrile, methylene chloride, triethylamine, etc.), compound (IV) It can be carried out by reacting 3 to 10 times the molar amount of an acylating agent at 0 to 50 ° C.

As the reducing agent in the reduction reaction, an organic tin hydride is preferable, and for example, tri-n-butyltin hydride, triphenyltin hydride or the like is used. The amount of the reducing agent used may be appropriately selected within the range of 1 to 5 times the molar amount of the compound (IV). The reduction reaction can be carried out by reacting the reducing agent at 50 to 150 ° C. in an organic solvent such as toluene in the presence of a radical initiator such as azobisisobutyronitrile or di-t-butylperoxide. Compound (V) thus synthesized
Can be isolated by ordinary silica gel column chromatography and the like.

(Step 4) When R ¹ is amino as the desired product, if necessary, the compound (V) is subjected to an amination reaction and then deprotected. When R ¹ is a hydroxyl group as the target product, the compound (V) is directly deprotected.

The amination reaction may be carried out according to a conventional method,
For example, by reacting a compound (V) with a reagent prepared from phosphorus oxychloride and triazole in acetonitrile in the presence of triethylamine to temporarily triazole the 4-position of the base, and then reacting with ammonia water or ammonia gas. You can The reaction temperatures are both 0 to 50 ° C. Deprotection may be carried out by appropriately selecting an ordinary treatment such as acidic hydrolysis, alkaline hydrolysis, tetrabutylammonium fluoride treatment, catalytic reduction or the like depending on the protective group used.

When R ³ is a phosphoric acid residue for the purpose of producing a compound, a phosphorylating agent used for the usual 5′-selective phosphorylation of nucleosides after completion of the above-mentioned deprotection. By reacting with (phosphorus oxychloride, tetrachloropyrophosphate, etc.), the free acid type or salt type target compound can be obtained by a conventional method.

The compound of the present invention thus synthesized can be separated and purified by appropriately combining the methods used for the isolation and purification of general nucleosides and nucleotides. For example, a nucleoside (R ³ is a hydrogen atom)
In this case, after distilling off the solvent, it may be crystallized from an appropriate solvent such as ethanol, and if necessary, it may be obtained in a salt form. In the case of a nucleotide body (R ³ is a phosphoric acid residue), it can be purified by ion exchange column chromatography, adsorption column chromatography such as activated carbon and the like, and the free acid form can be obtained by lyophilization or crystallization,
If necessary, it can be obtained in a salt form.

(3) Uses of the compound of the present invention The compound of the present invention has antitumor activity,
It is useful for treating malignant tumors in mammals such as dogs, mice and rats. Oral for the treatment of tumors,
It can be administered by any route such as enteral, parenteral and topical administration. The dosage depends on the patient's age, disease state,
It is appropriately determined depending on the body weight, etc., but usually 0.1 to 1000 mg / kg body weight per day, preferably 1 to 10
The dose is selected from the range of 0 mg / kg body weight and is administered once or in multiple doses.

In formulating the compound of the present invention, it is usually used as a composition containing a carrier, an excipient and other additives which are usually used. As the carrier, lactose, kaolin, sucrose, crystalline cellulose, corn starch, talc, agar, pectin, stearic acid, magnesium stearate, lecithin, solid carriers such as sodium chloride, glycerin, peanut oil, polyvinylpyrrolidone, olive oil, ethanol Examples of liquid carriers include benzyl alcohol, propylene glycol, and water.

The dosage form may be any form, for example, when a solid carrier is used, tablets, powders,
Granules, encapsulating agents, suppositories, lozenges and the like can be exemplified, and when a liquid carrier is used, syrup, emulsion, soft gelatin capsule, cream, gel, paste, spray, injection and the like can be exemplified.

[0040]

EXAMPLES The present invention will be described in detail below with reference to examples.

Example 1: 2'-deoxy-2 '(S)-
Fluoromethyl cytidine [Formula (I), R ¹ = NH ₂ , R
² = F, R ³ = H] (1) 1- [3,5-di-O-trityl-2,2 '
Synthesis of (S) -spiroepoxy-β-D-erythro-pentofuranosyl] uracil [formula (III), R ¹ = OH, Z = trityl (Tr)] trimethylsulfoxonium iodide 7.75 g in DM
Suspend in 100 ml of SO-THF (1: 1) and add 6
1.28 g of 0% sodium hydride was added, and the mixture was kept at 90 ° C. for 45 minutes under an argon stream. After cooling to room temperature,
50 ml of a THF solution in which 9.33 g of 3 ′, 5′-di-O-trityl-2′-ketouridine [formula (II), R ¹ ═OH, Z = Tr] was dissolved was added, and the mixture was stirred at room temperature for 30 minutes under an argon stream. It was stirred. After quenching the reaction with 1N aqueous ammonium chloride, the mixture was extracted with ethyl acetate and the organic layer was dried.
After distilling off the solvent, the residue was purified by silica gel column chromatography, and the portion eluted with 50% ethyl acetate-n-hexane was collected and concentrated to give 6.16 g of the desired product (yield 65%).
Got

¹ H-NMR (CDCl ₃ ) δ: 8.04 (1H,
brs), 7.37-7.20 (31H, m), 6.70 (1H, s), 5.26 (1H,
dd, J = 2.0, 8.3Hz), 4.25-4.22 (1H, m), 4.07 (1H,
d, J = 2.0Hz), 3.04 (1H, dd, J = 3.2, 10.5Hz), 2.92 (1
H, dd, J = 5.6, 10.5Hz), 2.52 (1H, dd, J = 4.4Hz), 2.23
(1H, d, J = 4.4Hz)

(2) 1- [3,5-di-O-trityl-
2-Fluoromethyl-β-D-arabinofuranosyl] uracil [Formula (IV), R ¹ = OH, R ² = F, Z = Tr]
The above-mentioned spiro epoxy compound (3.09 g) was dissolved in methyl cellosolve (80 ml), potassium hydrogen fluoride (3.26 g) was added thereto, and the mixture was refluxed for 1 day under an argon stream. After the solvent was distilled off, the residue was dissolved in ethyl acetate, washed with water and dried. After filtration, the filtrate was concentrated to dryness, the residue was purified by silica gel column chromatography, and the portion eluted with 50% ethyl acetate-n-hexane was concentrated to obtain 1.33 g of the desired product.
(Yield 42%) was obtained.

¹ H-NMR (CDCl ₃ ) δ: 8.17 (1H,
brs), 7.47 (1H, d, J = 8.3Hz), 7.44-7.22 (30H, m),
6.39 (1H, s), 5.50 (1H, d, J = 2.0, 8.3Hz), 4.64 (1
H, dd, J = 10.3, 20.0Hz), 4.53 (1H, dd, J = 9.8, 20.5H
z), 4.02 (1H, d, J = 1.5Hz), 3.98 (1H, t, J = 2.0Hz),
3.54 (1H, brs), 3.10 (1H, dd, J = 2.4, 10.7Hz), 2.96
(1H, dd, J = 4.4, 10.7Hz)

(3) 1- [2-deoxy-3,5-di-
O-TIPDS-2-fluoromethyl-β-D-arabinofuranosyl] uracil [formula (V), R ¹ = OH, R ²
= F, Z = TIPDS] 1.02 g of the fluoromethyl compound was suspended in 80% acetic acid and kept at 80 ° C. for 2 hours. After the solvent was distilled off, the residue was azeotropically distilled with toluene and pyridine and dissolved in 15 ml of pyridine. 1,3-dichloro-1,1,3,3-
Add 0.63 ml of tetraisopropyldisiloxane,
The mixture was stirred overnight at room temperature under a stream of argon. After distilling off the solvent, the residue was dissolved in ethyl acetate, washed with water, dried and further concentrated, and the obtained residue was subjected to silica gel column chromatography and eluted with 33% ethyl acetate-n-hexane. Is concentrated and 3 ', 5'-di-O-TIPDS-
0.41 g (yield 59%) of 2'-fluoromethyl body was obtained. 360 mg of this TIPDS body was added to 10 m of methylene chloride.
It was dissolved in 1, and 4-dimethylaminopyridine 254
mg and 0.19 ml of methyl oxalyl chloride were added, and the mixture was stirred overnight at room temperature under an argon stream. After adding water to stop the reaction, the mixture was extracted with chloroform, dried, and the solvent was distilled off. The residue was dissolved in 15 ml of toluene, 0.56 ml of hydrogenated tributyltin and 30 mg of azoisobisbutyronitrile were added, and the mixture was refluxed for 2 hours under an argon stream. After evaporating the solvent, the residue was purified by a silica gel column, and the portion eluted with 20% ethyl acetate-n-hexane was concentrated to obtain 353 mg (100%) of the desired product.

¹ H-NMR (CDCl ₃ ) δ: 8.34 (1H,
brs), 7.75 (1H, d, J = 8.3Hz), 6.30 (1H, d, J = 7.3H)
z), 5.67 (1H, dd, J = 2.0, 8.3Hz), 4.83-4.51 (3H,
m), 4.19 (1H, dd, J = 1.0, 13.7Hz), 4.06 (1H, dd, J =
2.9, 13.7Hz), 3.84-3.79 (1H, m), 2.76-2.64 (1H,
m), 1.11-0.98 (28H, m)

(4) 2'-deoxy-2 '(S) -fluoromethylcytidine [Formula (I), R ¹ = NH ₂ , R ² =
Synthesis of F, R ³ = H] 0.216 ml of phosphorus oxychloride and 534 m of triazole
1.0 g of triethylamine in an acetonitrile solution (10 ml) in which g was dissolved at 0 ° C. under an argon stream.
1 was added, and the mixture was stirred at room temperature for 1 hour. The deposited precipitate was filtered off under an argon stream, and then 353 mg of the above-mentioned reductant was added to the filtrate.
Was added and stirred at room temperature overnight. 10 ml of concentrated aqueous ammonia was added to this solution, and the mixture was stirred for 1 hour. Then, acetonitrile was distilled off and the mixture was extracted with chloroform. After drying the organic layer, the solvent was concentrated, the residue was purified by silica gel column chromatography, and the portion eluted with 4% methanol-chloroform was concentrated to give 208 mg of cytidine (yield 5
9%) was obtained. 200 mg of this cytidine compound was added to 7 m of THF.
It was dissolved in 1 and 0.8 ml of a tetrabutylammonium fluoride THF solution was added thereto, followed by stirring at room temperature for 30 minutes. After evaporating the solvent under reduced pressure, the residue was purified by silica gel column chromatography, and the portion eluted with 16% methanol-chloroform was concentrated. Further, the residue was applied to an ion exchange chromatograph (Dowex 50Wx8) and eluted with a linear concentration gradient of 0-0.1N ammonia water, and the obtained fraction was concentrated to obtain 63 mg of the desired product.
(Yield 61%) was obtained. In addition, an analytically pure compound was obtained by crystallization from ethanol.

Melting point:> 180 ° C. (decomposition) Elemental analysis value: Calculated value as C ₁₀ H ₁₄ N ₃ O ₄ F C: 46.33, H: 5.44, N: 16.2
1 Measured value C: 46.13, H: 5.49, N: 15.9
3 ¹ H-NMR (D ₂ O) δ: 7.85 (1H, d, J = 7.8Hz), 6.
37 (1H, d, J = 7.3Hz), 6.04 (1H, d, J = 7.8Hz), 4.63-4.
64 (2H, m, J _{H, F} = 46.9Hz), 4.26 (1H, t, J = 8.3Hz),
4.01 (1H, dd, J = 2.4, 12.5Hz), 3.98-3.94 (1H, m),
3.87 (1H, dd, J = 4.4, 12.5Hz), 3.00-2.86 (1H, m)

Example 2: 2'-deoxy-2 '(S)-
Hydroxymethyl cytidine [Formula (I), R ¹ = NH ₂ ,
R ² = OH, R ³ = H] For the compounds described in (1) and (2) below, Ch
It is a known compound reported in em. Pharm. Bull., 33, 3617 (1985). (1) 1- [3,5-di-O-TIPDS-2,2 '
(S) -Spiroepoxy-β-D-erythro-pentofuranosyl] uracil [Formula (III), R ¹ = OH, Z = TI
PDS] Synthesis of 6.10 g of trimethylsulfoxonium iodide in DM
Suspend in 80 ml of SO-THF (1: 1) and add 60
% Sodium hydride 1.10 g was added to the mixture under an argon stream,
Hold at 90 ° C. for 45 minutes. After cooling to room temperature,
3 ', 5'-di -O--2'Ketourijin [Formula ^{(II), R 1 = OH} , Z = TIPDS ] 4.90g
40 ml of a THF solution in which was dissolved was added, and the mixture was stirred under an argon stream at room temperature for 1 hour. After the reaction was stopped with 1N aqueous ammonium chloride, extraction was performed with ethyl acetate, and the organic layer was dried. After evaporating the solvent, the residue was purified by silica gel column chromatography, and the portion eluted with 33% ethyl acetate-n-hexane was collected and concentrated to obtain 4.00 g of the desired product (yield 79%).

[0050] Elemental _{analysis: C 22 H 38 N 2 O} 7 Si 2 · 1
Calculated as / 2H ₂ O C: 45.11, H: 6.06, N: 15.7
8 Measured value C: 44.95, H: 6.06, N: 15.5
4 ¹ H-NMR (CDCl ₃ ) δ: 8.29 (1H, brs), 7.44
(1H, d, J = 8.3Hz), 6.20 (1H, s), 5.74 (1H, dd, J = 2.
0, 8.3Hz), 4.48 (1H, d, J = 9.3Hz), 4.14 (1H, dd, J =
2.4, 13.2Hz), 4.08 (1H, d, J = 2.9, 13.2Hz), 3.91-3.
87 (1H, m), 3.24 (1H, d, J = 5.4Hz), 3.00 (1H, d, J =
5.4Hz), 1.12-0.92 (28H, m)

(2) 1- [3,5-di-O-TIPDS
2-Acetoxymethyl-β-D-arabinofuranosyl] uracil [Formula (IV), R ¹ = OH, R ² = OCOC
Synthesis of H ₃ , Z = TIPDS] 2.59 g of the above-mentioned spiro epoxy compound was dissolved in 60 ml of acetic acid, 4.92 g of sodium acetate was added thereto, and the mixture was kept at 100 ° C. for 2 hours under an argon stream. After cooling to room temperature, the solvent was evaporated, the residue was dissolved in ethyl acetate, washed with water, saturated sodium hydrogen carbonate solution and saturated brine, and the organic layer was dried. The solvent was evaporated, the residue was purified by silica gel column chromatography, and the portion eluted with 33% ethyl acetate-n-hexane was concentrated to obtain 1.91 g of the desired product (yield 58%).

¹ H-NMR (CDCl ₃ ) δ: 8.59 (1H,
brs), 7.76 (1H, d, J = 8,3Hz), 5.99 (1H, s), 5.70
(1H, dd, J = 2.0, 8.3Hz), 4.52 (1H, d, J = 12.2Hz), 4.
42 (1H, d, J = 12.2Hz), 4.30 (1H, d, J = 9.3Hz), 4.15
(1H, dd, J = 2.0, 13.2Hz), 3.99 (1H, dd, J = 2.9, 13.2
Hz), 3.84-3.81 (1H, m), 3.48 (1H, brs), 2.18 (3H,
s), 1.11-0.97 (28H, m)

(3) 1- [2-deoxy-3,5-di-
O-TIPDS-2-acetoxymethyl-β-D-arabinofuranosyl] uracil [Formula (V), R ¹ = OH, R
² = OCOCH ₃ , Z = TIPDS] 1.91 g of the above acetoxymethyl derivative was added to methylene chloride 5
After dissolving in 0 ml, 1.26 g of 4-dimethylaminopyridine and 0.947 ml of methyl oxalyl chloride were added, and the mixture was stirred overnight at room temperature under an argon stream. After water was added to stop the reaction, the mixture was extracted with chloroform, dried and the solvent was distilled off. The residue was dissolved in 70 ml of toluene, 2.77 ml of hydrogenated tributyltin and 20 mg of azoisobisbutyronitrile were added thereto, and the mixture was refluxed for 1 hour under an argon stream. 1 hour later, 20 m of azobisisobutyronitrile
g was added, the mixture was refluxed again for 1 hour, and after 1 hour, the same operation was repeated. After 1 hour, the mixture was cooled to room temperature, the solvent was distilled off, and the residue was purified by a silica gel column.
The portion eluted with 3% ethyl acetate-n-hexane was concentrated to obtain 1.55 g of the desired product (yield 83%).

Melting point: 140.0-142.0 ° C. Elemental analysis value: C ₂₄ H ₄₂ N ₂ O ₈ Si ₂ calculated value C: 53.11, H: 7.80, N: 5.16 measured value C : 53.30, H: 7.87, N: 5.41 ¹ H-NMR (CDCl ₃ ) δ: 8.32 (1H, brs), 7.79
(1H, d, J = 8.3Hz), 6.30 (1H, d, J = 7.3Hz), 5.70 (1H,
dd, J = 2.4, 8.3Hz), 4.45 (1H, dd, J = 8.8, 10.8Hz),
4.43 (1H, dd, J = 2.0, 12.5Hz), 4.20 (2H, m), 4.05
(1H, dd, J = 2.9,13.7Hz), 3.81 (1H, dd, J = 2.4, 8.3H
z), 2.88-2.82 (1H, m), 1.89 (3H, s), 1.12-1.00 (28
H, m)

(4) 2'-deoxy-2 '(S) -hydr
Roxymethyl cytidine [Formula (I), R ¹= NH₂, R²
= OH, R³= H] 0.308 ml of phosphorus oxychloride and 543 m of triazole
g in acetonitrile solution (10 ml)
1.53 ml of triethylamine at 0 ° C under gon stream
Was added, and the mixture was stirred at room temperature for 1 hour. The deposited precipitate is
After filtering off under a stream of air, 543 mg of the reductant described above was added to the filtrate.
In addition, the mixture was stirred at room temperature overnight. Concentrated aqueous ammonia in this solution
After adding 5 ml and stirring for 1 hour, distill off acetonitrile.
After that, it was extracted with chloroform. Dried organic layer
After that, the solvent is concentrated and the residue is subjected to silica gel column chromatography.
Purify more and elute with 4% methanol-chloroform
The concentrated portion was concentrated to give 241 mg of cytidine (yield 49
%) Was obtained. 214 mg of this cytidine compound is added to 7 ml of THF.
Dissolved in tetrabutylammonium fluoride
Add 0.79 ml of THF solution and stir at room temperature for 15 minutes
did. After evaporating the solvent under reduced pressure, the residue was washed with silica gel.
Purified by Rum chromatography, 16% methanol-chloro
The portion eluted with form was concentrated. The obtained residue
Was dissolved in 5 ml of methanol and concentrated ammonia water was added to it.
5 ml was added, and the mixture was stirred at room temperature for 5 hours. Distill off the solvent and leave
Dissolve the residue in water and use an ion-exchange chromatograph (Dove
x 50Wx8) and applied 0-0.1N ammonia
A) Elute with a linear concentration gradient of water and concentrate the resulting fraction
As a result, 73 mg (yield 72%) of the desired product was obtained.
It was Also, by crystallizing from isopropanol,
An analytically pure compound was obtained.

Melting point: 178.0-179.0 ° C. Elemental analysis value: C ₁₀ H ₁₅ N ₃ O ₅ 1 / 2H ₂ O calculated value C: 52.04, H: 7.74, N: 5. 52 Found C: 51.93, H: 7.43, N: 5.56 1 H-NMR (DMSO-d 6) δ: 7.90 (1H, d, J = 7.
3Hz), 7.17, 7.10 (2H, brs), 6.09 (1H, d, J = 6.8Hz),
5.69 (1H, d, J = 7.3Hz), 5.22 (1H, brd, J = 5.4Hz), 5.
05 (1H, brt, J = 5.1Hz), 4.42 (1H, brdd, J = 3.7, 7.6H
z), 3.88-3.85 (1H, m), 3.76-3.58 (3H, m), 3.43-3.38
(1H, m), 3.18-3.12 (1H, m), 2.52-2.45 (1H, m)

Example 3: 2'-deoxy-2 '(S)-
Acetoxymethyl cytidine [Formula (I), R ¹ = NH ₂ ,
R ² = OCOCH ₃ , R ³ = H] 0.308 ml of phosphorus oxychloride and 543 m of triazole
1.5 g of triethylamine in an acetonitrile solution (10 ml) in which g was dissolved at 0 ° C. under an argon stream.
Was added, and the mixture was stirred at room temperature for 1 hour. The deposited precipitate was filtered off under an argon stream, and the filtrate was obtained with 1- [2-deoxy-3,5-di-O-TIPDS-obtained in (3) of Example 2.
2-acetoxymethyl-β-D-arabinofuranosyl]
Uracil (543 mg) was added, and the mixture was stirred at room temperature overnight. 5 ml of concentrated aqueous ammonia was added to this solution, and the mixture was stirred for 1 hour, then acetonitrile was distilled off and the mixture was extracted with chloroform. After drying the organic layer, the solvent was concentrated, the residue was purified by silica gel column chromatography, and the portion eluted with 4% methanol-chloroform was concentrated to obtain 241 mg of cytidine (yield 49%). This cytidine body 0.
68 g was dissolved in 10 ml of THF, 2.5 ml of a solution of tetrabutylammonium fluoride in THF was added, and the mixture was stirred at room temperature for 1 hour. After evaporating the solvent under reduced pressure, the residue was purified by silica gel column chromatography (eluted with 16% methanol-chloroform) to obtain 0.13 g of the desired product (yield 34%).

Elemental analysis value: calculated as C ₁₂ H ₁₇ N ₃ O ₆ .H ₂ O C: 45.75, H: 5.70, N: 12.7
4 Measured value C: 45.42, H: 5.40, N: 13.2
4 ¹ H-NMR (CDCl ₃ ) δ: 7.83 (1H, d, J = 7.3H
z), 7.08 (2H, brd, NH ₂ ), 6.16 (1H, brd, J = 7.3Hz),
5.68 (1H, d, J = 7.3Hz), 5.40 (1H, d, J = 5.4Hz), 5.06
(1H, brt, J = 4.9Hz), 4.01-3.59 (4H, m), 2.69-2.66
(1H, m), 1.84 (3H, s)

Example 4: 2'-deoxy-2 '(S)-
Fluoromethyl cytidine-5'-phosphoric acid [formula (I), R
¹ = NH ₂ , R ² = F, R ³ = phosphoric acid residue] The 2'-deoxy-2 '(S) -fluoromethylcytidine 2.5 g obtained in Example 1 was added to 60 ml of trimethyl phosphoric acid. It was cooled by cooling, 1.6 g of phosphorus oxychloride was added dropwise thereto, and the mixture was further stirred for 1 hour. The reaction solution was poured into ice water containing sodium hydrogen carbonate and stirred for 1 hour,
Ether was added for partitioning. The aqueous phase is concentrated, the target compound is purified using an anion exchange resin, and lyophilized to 2 '.
-Deoxy-2 '(S) -fluoromethylcytidine-
5'-phosphoric acid was obtained.

Test Example 1: Cell proliferation inhibitory effect The cell proliferation inhibitory effect of the compound of the present invention is tested by the following method.
did. (1) Human acute lymphoblastic leukemia cell (T-cell) C
RPMI1 with 10% fetal bovine serum added to CRF-HSB-2
Cultured in 640 medium and 1.1 × 10 in logarithmic growth phase ^FiveIndividual/
Dilute with medium to make up to ml. (2) Take 0.9 ml of the above cell diluent into a culture test tube,
0.51 og using medium or PBS_TenSerially diluted
Add 0.1 ml of sample solution and incubate at 37 ° C for 4 days.
Culture in a incubator. (3) After culturing, count the number of cells in each tube with a cell counter.
-For (Sysmex micro cell counter, F-300 type)
The growth inhibition rate is calculated by the following formula.

[0061]

[Equation 1]

(4) The growth inhibition rate was plotted against the sample concentration on a dose-effect graph paper, and the sample concentration (ID ₅₀ ) showing the 50% inhibition rate was determined from the dose-growth curve. As a result, Example 1 for CCRF-HSB-2
The ID ₅₀ of the 2′-deoxy-2 ′ (S) -fluoromethylcytidine synthesized in 1. was 0.030 μg / ml.

[0063]

INDUSTRIAL APPLICABILITY The compound of the present invention has excellent antitumor activity.

 ─────────────────────────────────────────────────── ─── Continued Front Page (72) Inventor Akira Matsuda 12-1-7-501 Kita-ku Kita-ku Kita-ku, Sapporo, Hokkaido

Claims (3)

[Claims] 1. Formula (I): (In the formula, R ¹ represents a hydroxyl group or amino, R ² represents a hydroxyl group, acyloxy or halogen atom, and R ³ represents a hydrogen atom or a phosphoric acid residue.) 2′-deoxy-2 ′
(S) -Substituted alkyl cytidine derivative or a salt thereof. 2. The method according to claim 1, comprising the following first to fourth steps.
A method for producing the described 2'-deoxy-2 '(S) -substituted alkylcytidine derivative. First step: a step of epoxidizing the sugar moiety 2'position of a compound represented by the following formula (II) with iowilide to obtain a compound represented by the following formula (III). [Chemical 2] (In the formula, R ¹ represents a hydroxyl group or amino, and Z represents a protecting group.) Second step: The epoxy ring at the 2′-position of the sugar moiety of the compound represented by the following formula (III) is opened with a nucleophile. And a step of obtaining a compound represented by the following formula (IV). [Chemical 3] (In the formula, R ² represents a hydroxyl group, an acyloxy or a halogen atom, and R ¹ and Z have the same meanings as described above.) Third step: the 2′-hydroxyl group of the compound represented by the following formula (IV) was acylated Then, a step of reducing with a reducing agent to obtain a compound represented by the following formula (V). [Chemical 4] (In the formula, R ¹ , R ² and Z have the same meanings as above.) Fourth step: after optionally aminating the 4-position of the base moiety of the compound represented by the following formula (V), a sugar moiety protecting group And then phosphorylating the 5'position of the sugar moiety, if necessary, to obtain a compound represented by the following formula (I). [Chemical 5] (In the formula, R ³ represents a hydrogen atom or a phosphoric acid residue,
R ¹ , R ² and Z are as defined above. ) 3. The 2'-deoxy-2 'according to claim 1.
An antitumor agent comprising a (S) -substituted alkylcytidine derivative or a salt thereof as an active ingredient.