JPS636080B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides 1-(β-D-arabinofuranosyl)
Cytosine derivatives, specifically 1-(β-D-arabinofuranosyl) having a side chain containing a reactive amino group
This invention relates to cytosine 5'-phosphoric acid diester and its production method.

1-(β-D-arabinofuranosyl)cytosine (hereinafter abbreviated as AraC) is associated with acute myeloid leukemia,
Focusing on chemotherapy for blood cancers such as acute lymphocytic leukemia, when used in combination with other drugs such as DCMP (daunomycin, AraC, mitomycin, prednisolone) therapy and MFC (mitomycin, 5-fluorouracil, AraC) therapy, gastrointestinal It is also a drug widely used in chemotherapy for solid cancers such as cancer and breast cancer. However, conventionally, the method of administration of AraC has been limited to intravenous or intramuscular injection, and oral administration has been difficult. Recently, a phosphate ester of araC, namely 1-(β-D-arabinofuranosyl)cytosine 5'-phosphate (hereinafter referred to as
It was discovered that phosphoric acid diesters obtained by reacting long-chain alcohol with Ara-CMP (abbreviated as Ara-CMP) exhibit antitumor activity similar to that of Ara-C, and can be administered orally. The use of its derivatives as antitumor agents is being expanded (for example, JP-A-55-2601, JP-A-55-2601;
(See Publication No. 2602).

The present inventors have arrived at the present invention as a result of intensive research to develop a more effective araC derivative as an antitumor agent based on the knowledge in the prior art.

That is, the present invention is based on the general formula [1] [In formula [1], R ₁ represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. R ₂ is carbon number 2~
Represents 20 divalent aliphatic hydrocarbon groups. R ₃ represents a hydrogen atom or an alkali cation. ] 1-(β-D-arabinofuranosyl) represented by
It is a cytosine derivative (hereinafter abbreviated as araC derivative).

The araC derivative of the present invention exhibits antitumor activity equal to or higher than that of araC, and therefore, when the side chain fatty hydrocarbon group (R ₂ ) is long, it has excellent orally administrable properties. Can be used as an antitumor agent. Furthermore, since the araC derivative of the present invention has a reactive amino group in its side chain, it can form an amide bond with a carboxyl group-containing compound and an imino bond with an aldehyde-containing compound, for example. It can be used as a pharmaceutical synthesis intermediate, making it possible to produce complexed or polymerized antitumor drugs.

In the general formula [1], R ₁ is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, and a hydrogen atom is particularly preferred. R ₂ is a divalent aliphatic hydrocarbon group having 2 to 20 carbon atoms, examples of which include ethylene (C ₂ ), propylene (C ₃ ), butene (C ₄ ), and pentene (C ₅ ). , hexene (C ₆ ), heptene (C ₇ ), octene (C ₈ ), nonene (C 9 ), dekene (C ₁₀ ), undekene (C ₁₁ ), dodekene (C ₁₂ ), tridekene ( _{C 13 )} , tetragekene (C ₁₄ ), Pentadekene (C ₁₅ ), Hexadekene (C ₁₆ ), Heptadekene (C ₁₇ ), Octadekene (C ₁₈ ), Nonadekene (C ₁₉ ),
Examples include eicosene (C ₂₀ ).

The aliphatic hydrocarbon group may have an unsaturated bond or a branch. R ₃ is a hydrogen atom or an alkali cation. Examples of the alkali cation include alkali metal ions such as sodium and potassium, alkaline earth metal ions such as calcium and magnesium, and ammonium ions. When utilizing the araC derivative of the present invention, it can also be used in the form of a salt such as a hydrochloride, and these are also included within the scope of the present invention.

The araC derivative represented by the general formula [1] is a protected 1-
(β-D-arabinofuranosyl)cytosine 5'-phosphate, [In formula [2], R ₄ represents a protecting group for the amino group.
R ₅ and R ₆ represent the same or different hydroxyl protecting groups. ] Amino alcohol with a protected amino group represented by the following general formula [3] [In formula [3], R ₇ represents a protecting group for an amino group.

The definitions of R ₁ and R ₂ are the same as in formula [1]. ] to form a phosphodiester bond, and then the protecting groups R ₄ , R ₅ , R ₆ and R ₇ are removed from the product.
It can be produced by removing and optionally converting into an alkali salt.

The amino group protecting group R ₄ and the hydroxyl group protecting groups R ₅ and R ₆ in the general formula [2] may be any group as long as it is stable under the condensation reaction conditions and can be easily removed after the reaction, such as an acetyl group, Acyl groups such as butyryl group and benzoyl group are preferably used.

The amino group protecting group R ₇ in general formula [3] is
Any group may be used as long as it is stable under the condensation reaction conditions and can be easily removed after the reaction, but t-butyloxycarbonyl group and benzyloxycarbonyl group are particularly preferred.

The reaction for forming a phosphodiester bond between the compound of general formula [2] and the compound of general formula [3] is carried out in an organic solvent using a condensing agent. As the condensing agent, carbodiimides such as dicyclohexylcarbodiimide, triisopropylbenzenesulfonyl chloride, benzenesulfonyl chloride, O-tosyl chloride, P-
Arylsulfonyl chlorides such as tosyl chloride are preferably used. The reaction solvent is preferably an aprotic organic solvent that has sufficient dissolving power and does not hinder the progress of the reaction. In order to obtain the best results, the solvent should be selected depending on the type of reaction substrate and the condensing agent used, but generally preferred solvents include pyridine, N/N-dimethylformamide, and N/N-dimethylacetamide. , ethyl acetate, tetrahydrofuran, dioxane, chloroform, etc. can be used alone or as a mixed solvent. When using arylsulfonyl chlorides as a condensing agent, the protected ara-CMP of general formula [2] is reacted with at least the same molar amount of tertiary amines, such as tri-n-butylamine, tri-n-octylamine, and triethylamine. By adding and mixing with a solvent, the solubility of the phosphoric acid ester can be increased and favorable results can be obtained. When using carbodiimides as condensing agents, the use of the above-mentioned tertiary amines does not give good results, and therefore their use is not preferred.

The reaction time for the condensation reaction to form a phosphodiester bond varies depending on the reaction substrate, type of condensing agent, and solvent, but is generally 1 hour to 1 week.
The reaction temperature is 0°C to 100°C, preferably at room temperature, but if the reactivity is poor, heating may be used.

After the condensation reaction is complete, the method for removing protecting groups R ₄ , R ₅ and R ₆ depends on the type of protecting group, but in the case of acyl groups such as acetyl and butyryl, it is recommended to remove aqueous ammonia, ammonia/methanol, etc. By doing so, it can be easily removed.

After this operation, the obtained 1-(β-D-arabinofuranosyl)cytosine 5'-phosphoric acid diester having a protected amino group in the side chain was subjected to liquid-liquid extraction, ion exchange chromatography, It can be isolated by normal operations such as recrystallization.

The method for removing the protecting group _R7 of the side chain amino group is as follows:
Depends on the type of _R7 . When R ₇ is a t-butyloxycarbonyl group, it can be easily removed by the action of an acid. Examples of acids that can be used include acetic acid, formic acid, oxalic acid, dilute trifluoroacetic acid, dilute methanesulfonic acid, dilute hydrochloric acid, and dilute sulfuric acid. In order to proceed with the reaction, it is necessary to appropriately select the concentration of the acid used. The reaction temperature is -10°C to 100°C, preferably 0°C to room temperature. Reaction time is 1 minute to 8 hours, preferably 10 minutes to
It is 5 hours.

After the reaction is completed, the desired product, the araC derivative of the present invention represented by the general formula [1], can be obtained by performing conventional methods such as extraction, crystallization, ion exchange chromatography, and partition chromatography. Can be done.

When R ₇ is a benzyloxycarbonyl group,
The protecting group is reductively removed by hydrogenolysis using a palladium catalyst or the like. As for the reaction conditions, it is preferable to complete the reaction in 30 minutes to 10 hours under a hydrogen atmosphere at a normal pressure of usually 1 atm to a medium pressure of several atm at a temperature of room temperature to 60°C. Any solvent may be used as long as it does not impede the reaction, but alcohols such as methanol and ethanol, and ethers such as THF and dioxane are preferably used. In order to improve solubility, a tertiary amine in the same molar amount as the substrate, preferably tri-n-butylamine, tri-n-octylamine, or triethylamine, may be added. After the reaction is complete, by performing normal operations such as filtration, extraction, ion exchange chromatography, partition chromatography, and recrystallization,
The araC derivative of the present invention can be obtained.

The araC derivative obtained as described above can be converted into a phosphate (R ₈ in the general formula [1] is an alkali cation) by a known alkali treatment.

Hereinafter, the present invention will be explained in detail with reference to Examples.

Example 1 In this example, acetylated ara-CMP and 2-t-
An example of producing the araC derivative of the present invention from butyloxycarbonylaminoethanol by the following reaction will be shown.

(1) ^N4・^O2 ′・^O3′ -triacetyl-1-β-D
- Dissolve 348 mg of arabinofuranocytocytosine 5'-monophosphate in 10 ml of pyridine, add a solution of 125 mg of 2-t-butyloxycarbonylaminoethanol dissolved in 5 ml of pyridine, and add condensing agent dicyclohexylcarbodiimide (DCC). 797 mg was added, the mixture was sealed, and the mixture was stirred at room temperature for 3 days. Next, 5 ml of ice water was added, and after stirring overnight, the solvent was distilled off under reduced pressure, and the residue was extracted with water-ethanol (1:1). The extract was distilled off under reduced pressure, the residue was dissolved in 30 ml of methanol, 6 ml of saturated ammonia/methanol was added, and the mixture was left overnight at room temperature under a nitrogen atmosphere. The solvent was then distilled off under reduced pressure, and the residue was subjected to DE-52 (acetate type) ion exchange column chromatography in 50% ethanol. The eluate was obtained by increasing the acetic acid concentration linearly from 0 to 2N in 50% ethanol.

Fractions belonging to the main peak detected by absorption at 273 nm (absorption of the ara C base moiety) were collected, the solvent was distilled off under reduced pressure, and the resulting residue was solidified by adding acetone and collected. By drying under reduced pressure, 1-[5'-(2-N-t-butyloxycarbonylaminoethylphosphoryl)-
β-D-arabinofuranosyl cytosine powder
Obtained 184 mg. The yield was 51%. The melting point is
199.0~202.0℃ (decomposition), NMR, IR,
The UV data was as follows.

NMR (expressed as δ value from TMS in DMSOd ₆ ): 1.36 (9H, S), 3.10 (2H, m), 3.70 (2H,
m), 3.94 (5H, br, m), 5.97 (1H, d, 7.5
Hz), 6.00 (1H, d, 3.8Hz), 6.20 (3H,
hump), 6.80 (2H, hump), 7.92 (1H, d,
7.5Hz), 9.2 (1H, hump).

IR: ν (KBr) 3425 (br, s), 2995 (m), 2950 (m), 1720
(s), 1684 (s), 1652 (sh), 1538 (m),
1450, 1410, 1281 (m), 1208 (m), 1061 (s)
cm ^-1 .

UV: λmax 272.5nm (in EtoH-H ₂ O, 1:1) (2) 1-[5'-(2-N-t-butyloxycarbonylaminoethylphosphoryl)-β-D-arabinofuranosyl] Cytosine powder 100mg 98%
After dissolving in 4 ml of formic acid and stirring at room temperature for 3 hours,
The solvent was removed under reduced pressure. The residue was subjected to DE-52 (acetate type) column chromatography, developed and eluted with ethanol-water (1:1). 273n
Corresponding fractions detected by absorption at 72 mg of 1-[5'-(2-aminoethylphosphoryl)-β-D-arabinofuranosyl]cytosine powder was obtained. Yield is 94
%, and the melting point was 205.0-212.0°C (decomposition).

In addition, the NMR, IR, and UV data were as follows.

NMR (Sodium3−(trimethylsilyl)− in D ₂ O
(expressed as δ value from propansulfonate): 3.33 (2H, t, 5.0Hz), 4.04 (2H, t, 5.0
Hz), 4.20 (4H, m), 4.52 (1H, m), 6.17
(1H, d, 8.0Hz), 6.33 (1H, d, 5.0Hz),
7.97 (1H, d, 8.0Hz).

IR: ν (KBr) 3425 (br, s), 2950 (m), 1646 (s), 1607
(sh), 1528 (m), 1492 (m), 1285 (m), 1217
(s), 1074 (s), 1020 (s) cm ^-1 .

UV: λmax 272.5nm (in EtOH-H ₂ O, 1:1) (3) 1-[5'-(2-aminoethylphosphoryl)-
Biological activity of β-D-arabinofuranosyl]cytosine.

DNA synthesis of cultured tumor cells L1210 and MM48
The 50% inhibitory concentration was measured and was as follows (determined by measuring the amount of 3H-thymidine incorporated).

L1210: 0.1 μM MM48: 1.0 mM When the biological activity of AraC was measured for comparison, it was 0.1 μM in the case of L1210.

Example 2 In this example, the 2-t-
An example is shown in which 6-Nt-butyloxycarbonylaminohexanol is used instead of butyloxycarbonylaminoethanol.

(1) ^N4・^O2 ′・^O3′ -triacetyl-1-β-D
- 2.50 g of arabinofuranosylcytosine 5'-monophosphate and 1.21 g of 6-Nt-butyloxycarbonylaminohexanol in pyridine.
The solution was dissolved in 120 ml, 5.75 g of dicyclohexylcarbodiimide (DCC) was added thereto, the mixture was sealed, and the mixture was stirred at room temperature for 3 days. Then add 20ml of ice water,
After stirring overnight, the solvent was distilled off under reduced pressure, and the residue was extracted with water-ethanol (1:1). The extract was distilled off under reduced pressure, and the residue was dissolved in 130 ml of methanol.
To this was added 30 ml of saturated ammonia/methanol, and the mixture was left overnight at room temperature under a nitrogen atmosphere. The solvent was then distilled off under reduced pressure, and the residue was taken up in 50% ethanol and subjected to DE-52 (acetate type) ion exchange column chromatography. Eluate is 50%
Acetic acid concentration was increased linearly from 0 to 2N in ethanol. Corresponding fractions detected by absorption at 273 nm were collected, the solvent was distilled off under reduced pressure, and the resulting residue was solidified with acetone-methanol (1:1) and collected. By drying under reduced pressure, 810 mg of 1-[5'-(6-Nt-butyloxycarbonylaminohexylphosphoryl)-β-D-arabinofuranosyl]cytosine powder was obtained.
The yield was 28%, and the melting point was 205.0-208.0°C (foam decomposition).

In addition, the NMR, IR, and UV data were as follows.

NMR (shown as δ value from TMS in DMSOd ₆ ): 1.36 (9H, s), 1.15-1.80 (8H, hump),
2.90 (2H, m), 3.80 (2H, m), 3.95 (5H,
m), 5.20 (4H, hump), 5.98 (1H, d, 8.0
Hz), 6.05 (1H, d, 3.8Hz), 6.71 (1H,
hump), 7.98 (1H, d, 8.0Hz), 9.30 (1H,
hump).

IR: ν (KBr) 3425 (br, s), 2952 (m), 1721 (s), 1680
(s), 1656 (sh), 1542 (m), 1283 (m), 1252
(w), 1181 (m), 1063 (s) cm ^-1 .

UV: λmax 272.5nm (EtOH- _H2O , 1:1).

(2) 70 mg of 1-[5'-(6-N-t-butyloxycarbonylaminohexylphosphoryl)-β-D-arabinofuranosyl]cytosine powder
The solution was dissolved in 20 ml of % acetic acid, heated and stirred at 80°C for 3 hours under a nitrogen atmosphere, and then the solvent was distilled off under reduced pressure. The residue was subjected to DE-52 (acetate type) column chromatography, developed and eluted with ethanol-water (1:1). Corresponding fractions detected by absorption at 273 nm were collected, the solvent was distilled off under reduced pressure, and a mixture of acetone and ethanol (1:1) was added to the resulting residue to solidify, collected, and dried under reduced pressure. −
[5'-(6-aminohexylphosphoryl)-β-
D-Arabinofuranosyl Cytosine Powder 119
I got mg. Yield is 74%, melting point is 183-191℃
It was (decomposition). In addition, the NMR, IR, and UV data were as follows.

NMR (Sodium3−(trimethylsilyl)− in D ₂ O
(expressed as δ value from propansulfonate): 1.33 (8H, m), 2.87 (2H, m), 3.82 (2H,
m), 4.07 (4H, m), 4.40 (1H, m), 6.03
(1H, 8Hz), 6.22 (1H, 5Hz), 7.89 (1H,
d).

IR: ν (KBr) 3420 (br, s), 2950 (m), 1645 (s), 1608
(m), 1530 (w), 1492 (m), 1286 (w), 1204
(m), 1047 (s) cm ^-1 .

UV: λmax 272.5 nm (EtOH-H ₂ O, 1:1) Example 3 In this example, 2,4,6-triisopropylbenzenesulfonyl chloride was used as a condensing agent instead of DCC used in Example 2. An example is also shown in which a butyryl group is used instead of an acetyl group as a protecting group.

N ⁴・O ² ′・O ³ ′-tributyryl-1-β-D-
533 mg of arabinofuranosylcytosine 5'-monophosphate, 326 mg of 6-N-t-butyloxycarbonylaminohexanol, and 185 mg of tri-n-butylamine were dissolved in 30 ml of pyridine, and 2,4,6-triisopropyl 910 mg of benzenesulfonyl chloride was added and stirred at room temperature for 13 hours.
After that, 10 ml of ice water was added, and after further stirring for 30 minutes, the solvent was distilled off under reduced pressure, and the residue was extracted with a water-ethanol mixture (4:1). The solvent was distilled off from the extract under reduced pressure, and the residue was dissolved in 30 ml of 4N ammoniacal methanol and reacted for 4 hours at room temperature. The solvent was then removed under reduced pressure and the residue was taken up in 50% ethanol and DE
-52 (acetate type) ion exchange column chromatography. The eluate was obtained by increasing the acetic acid concentration in a linear gradient from 0 to 2N in 50% ethanol.
The corresponding fractions detected by absorption at 273 nm were collected and the solvent was distilled off under reduced pressure. The obtained residue was solidified with acetone-methanol (1:1) and dried under reduced pressure to give 1-[5'-(6-Nt-butyloxycarbonylaminohexylphosphoryl)-
β-D-arabinofuranosyl cytosine powder
395mg was obtained. The yield was 75.7%. This product showed the same physical properties as the intermediate product obtained in Example 2.

Next, the obtained 1-[5'-(6-N-t-butyloxycarbonylaminohexylphosphoryl)-β
-D-arabinofuranosylcytosine powder 164
mg was dissolved in 4 ml of formic acid and stirred at room temperature for 3 hours.
The solvent was distilled off under reduced pressure, and the resulting residue was subjected to DE-52 (acetate type) column chromatography, developed and eluted with ethanol-water (1:1). 273n
The corresponding fractions detected by the absorption of
The product was dried under reduced pressure to obtain 102 mg of powdered 1-[5'-(6-aminohexylphosphoryl)-β-D-arabinofuranosyl]cytosine. Yield is 77%
It was hot.

This product showed the same physical properties as the target product obtained in Example 2.

Claims (1)

[Scope of Claims] 1. A 1-(β-D-arabinofuranosyl)cytosine derivative represented by the general formula [1]. [In formula [1], R ₁ represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. R ₂ is carbon number 2~
Represents 20 divalent aliphatic hydrocarbon groups. R ₃ represents a hydrogen atom or an alkali cation. ] 2 Protected 1- represented by general formula [2]
(β-D-arabinofuranosyl)cytosine 5'-phosphate ester [Formula [2], R ₄ is a protecting group for an amino group,
R ₅ and R ₆ represent the same or different hydroxyl protecting groups. ] Amino alcohol with protected amino group represented by general formula [3] [In formula [3], R ₇ represents a protecting group for an amino group. The definitions of R ₁ and R ₂ are the same as in formula [1]. ] to form a phosphodiester bond, and then the protecting groups R ₄ , R ₅ , R ₆ and R ₇ are removed from the product.
1- represented by the following general formula [1], which is characterized by removing and optionally converting into an alkali salt.
A method for producing a (β-D-arabinofuranosyl)cytosine derivative. [In formula [1], the definitions of R ₁ , R ₂ and R ₃ are the same as in the above formula [1]. ]