JP2782597B2 - Anticancer agent ester compound and method for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an ester cancer drug which is hydrolyzed in cancer cells but hardly decomposed in blood, and a method for producing the same.

[0002]

2. Description of the Related Art p-Hydroxyaniline mustard (formula IV) and 5-
In the case of fluorouridine (formula V), in order to reduce its side effects, its hydroxyl group is esterified to make it temporarily inactive, and administered to a living body, and then the cancer cell is hydrolyzed by esterase in cancer cells. Attempts have been made to convert it to the active form only within T.
J. Bardos et al., Ann. NYAcad. Sci., 163, 1006 (196
9) and JP-A-57-91998).

[0003]

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[0004]

However, in the ester compounds which have been used in the past, a considerable portion of the acyl groups are hydrolyzed by the action of esterases present in the blood before they reach cancer tissues. I couldn't help. An object of the present invention is to provide an ester compound which is hydrolyzed in cancer cells but is hardly hydrolyzed in blood, and a method for producing the same.

[0005]

Means for Solving the Problems The present inventors have studied various structures of esters that are hydrolyzed in cancer cells but hardly decomposed in blood. It has been found that the three-dimensional structure greatly influences this hydrolyzability. That is, an ester obtained by acylating p-hydroxyaniline mustard with a specific acyl group (but racemic) is treated with various cancer cells and whole blood of rat or human, and the absolute configuration and optical purity of the carboxylic acid produced are determined. Examined. As a result, the absolute configuration is generally high with stereoselectivity in hydrolysis by cancer cells.
R carboxylic acid is formed, while ( R ) in blood
-The carboxylic acid tends to be preferentially produced, but its stereoselectivity is low, and more surprisingly, depending on the acyl group, the ( R ) form is also preferentially hydrolyzed in human leukemia cell U937. In normal human whole blood, however, it was found that the ( S ) -form was preferentially hydrolyzed, and that stereoselectivity could be reversed between cancer and normal blood.
Furthermore, even in the case of a 5-fluorouridine ester in which the 5'-position is acylated with a specific acyl group (but a racemate), the ester of the ( R ) -acyl group is stereoselectively hydrolyzed in cancer cells. On the other hand, it was found that the ( S ) -form is preferentially hydrolyzed in blood. The present invention has been made based on this finding. In the present invention, a carcinostatic agent having a hydroxyl group due to an asymmetric acyl group having a steric structure that conforms to the stereoselectivity of esterase in cancer cells, while contradicting the stereoselectivity of esterase in blood or normal cells. Is acylated to temporarily inactivate the anticancer agent. When this is administered to a living body, the acyl group is preferentially hydrolyzed and removed in the cancer cells based on the stereoselectivity of the enzymatic reaction, and the activity of the anticancer agent is expressed.

That is, the present invention provides (1) an ester compound having a structure represented by the formula (I):

[0007]

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(Wherein R ₁ represents a methyl group, a methoxy group or a trifluoroacetamide group, R ₂ represents a phenyl group or a phenylmethyl group, and the absolute configuration of the asymmetric center marked with an asterisk is R And (2) a 5-fluorouridine compound represented by the following formula (II) or the corresponding 5-fluorouridine compound:
A protected 2 ', 3'-isopropylidene compound of a fluorodeoxyuridine compound and an acid chloride of a carboxylic acid represented by the formula (III) or a compound obtained by activating the carboxylic acid with dicyclohexylcarbodiimide (DCC) or carbonyldiimidazole; Wherein the 5'-position hydroxyl group is acylated to deprotect the 2 ', 3'-hydroxyl group, or the 5-fluorouridine ester compound or its corresponding 5-fluoro group according to (1). A method for producing an ester compound of deoxyuridine,

[0009]

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[0010] is provided.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION The anticancer ester according to the present invention is a novel compound. Further, it was found for the first time in the course of the present invention that esterases of cancer cells and blood / other normal tissues have different stereoselectivities.

Preferred embodiments of the ester compound of the present invention and the method for producing the same will be described. In the formula (I) or (III), when R ₁ is a methyl group and R ₂ is a phenyl group, R ₁
Is a methoxy group and R ₂ is a phenyl group, R ₁ is a trifluoroacetamide group and R ₂ is a phenylmethyl group. Preferred acyl groups in the acylation with the carboxylic acid compound of the formula (III) are represented by the following formulas (IIIa), (IIIb) or (IIIc).

[0013]

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The anticancer ester according to the present invention has the formula (III)
a) the ( R ) -carboxylic acid corresponding to the acyl group represented by (IIIb) or (IIIc) is obtained by acid chloride method, DCC method,
It is obtained by activating with a known method such as the carbonyldiimidazole method and coupling with 2 ′, 3′-isopropylidene-5-fluorouridine. In this case, the isopropylidene protecting group is promptly removed by acid treatment. Among the raw materials, ( R ) -N -trifluoroacetylphenylalanine can be prepared by a known method (MH Benn et al., J. Chem. S.
oc., 2365 (1961); MW Holladay et al., J. Org. Che.
m., 3900 (1991)), and 2 ', 3'-isopropylidene-5-fluorouridine can be prepared from commercially available 5-fluorouridine by a known method (KA Watanabe et al., J. Am.
Med. Chem., 24, 893 (1981)). ( R )
-2-phenylpropionic acid and ( R ) -2-methoxy-
2-Phenylacetic acid is commercially available. The amount of ( R ) -carboxylic acid compound used for 2 ′, 3′-isopropylidene-5-fluorouridine may be a stoichiometric amount. The ester compound of the present invention represented by the formula (I) is an anticancer agent ester which is easily hydrolyzed in cancer cells but relatively resistant to degradation in blood. In the case of rats, homogenates of normal liver, pancreas, and muscle were also tested for the hydrolysis of the above-mentioned esters. As a result, similar to blood, these tissues sometimes showed stereoselectivity different from that of cancer. Therefore, if the steric structure of the acyl group is properly selected, it is useful as an ester-type anticancer drug that can avoid damage to normal cells as much as possible even if cancer cells attack.

[0015]

Next, the present invention will be described in more detail with reference to Reference Examples, Examples and Test Examples.

Reference Example 1 (Synthesis of (±) -4-bis (2-chloroethyl) aminophenyl 2-methoxy-2-phenylacetate) 52 mg of (±) -2-methoxy-2-phenylacetic acid was added to 0% of tetrahydrofuran. Then, 60 mg of carbonyldiimidazole was added thereto while stirring under ice-cooling. After stirring for 10 minutes, 200 μl of a tetrahydrofuran solution containing 20 mg of p-hydroxyaniline mustard was added, and the mixture was stirred for 1 hour under ice-cooling and then for 1 day at room temperature. The product was separated by preparative thin layer chromatography (silica gel F ₂₅₄ , developed with 5% EtOAc / benzene) to give 8 mg of Ib as an oil. Yield 28%. M
S m / z: 381.0740 (M ⁺ ); calculated value C ₁₉
H ₂₁ Cl ₂ NO ₃ = 381.0898. ^{The 1} H-NMR spectrum was consistent with the expected structure.

Reference Example 2 (4-bis (2-chloroethyl)
Synthesis of optically active form of aminophenyl 2-methoxy-2-phenylacetate) ( R )-(-)-2-methoxy-2-manufactured by Aldrich
Using phenylacetic acid, the reaction was carried out in the same manner as in Synthesis Example 2 to obtain levorotatory Ib as an oil in a yield of 44%.
[Α] _D ²⁸ -60.8 ° (c = 1.9, ethanol).
The reaction and treatment were carried out in the same manner as above using ( S )-(+)-2-methoxy-2-phenylacetic acid manufactured by Aldrich to obtain dextrorotatory Ib in 51% yield as an oil.
[Α] _D ²⁸ + 82.7 ° (c = 1.7, ethanol).
The Rf values of these optically active compounds in thin layer chromatography were consistent with those of the above-mentioned racemic Ib.

Example 1 (Synthesis of 5 ′-( RS )-(2-phenylpropionyl) -5-fluorouridine (Ia)) 25 mg of 2 ′, 3′-isopropylidene-5-fluorouridine and (±) 80 of 2-phenylpropionic acid
mg was coupled in 1.8 ml of tetrahydrofuran using 75 mg of carbonyldiimidazole in the same manner as in Reference Example 1. The product (39 mg, crystallized from methanol, mp 169-170 ° C.) was purified by preparative thin-layer chromatography (silica gel F ₂₅₄ , 50% E).
(Developed with tOAc / benzene)), dissolved in a mixture of 0.2 ml of methanol and 0.8 ml of trifluoroacetic acid, and left at room temperature for 30 minutes to decompose the isopropylidene group. The reaction solution was concentrated under reduced pressure, and the residue was subjected to preparative thin-layer chromatography (silica gel F ₂₅₄ , 2% MeOH / Et).
(Developed with OAc) to give 26 mg of Ia as fine needles. Yield 80%, melting point 133-134 ° C, MS
m / z: 394.1048 (M ⁺ ); calculated C ₁₈ H
₁₉ FN ₂ O ₇ = 394.1176. ^{The 1} H-NMR spectrum was consistent with the expected structure.

Example 2 (Synthesis of 5 '-( RS )-(2-methoxy-2-phenylacetyl) -5-fluorouridine (Ib)) 25 mg of 2', 3'-isopropylidene-5-fluorouridine And 80 mg of (±) -2-methoxy-2-phenylacetic acid were reacted and treated in the same manner as in the above example to give 33 mg
mg of IIb was obtained in the form of a syrup. 97% yield. MS m
/ Z ^: 410.1154 (M ⁺ ); calculated value C ₁₈ H ₁₉ F
N ₂ O ₈ = 410.1125. ^{The 1} H-NMR spectrum was consistent with the expected structure.

Example 3 (5 '-( RS )-(2-trifluoroacetamido-3-phenylpropionyl) -5
Synthesis of Fluorouridine (Ic)) A reaction treatment was performed in the same manner as in Reference Example 2 except that 70 mg of (±) -N-trifluoroacetylphenylalanine was used, to obtain 51 mg of IIc in the form of a syrup. Yield 61
%. MS m / z: 505.1002 (M +); calcd _{C 20 H 19 F 4 N 3} O 8 = 505.1108. ¹ H-N
The MR spectrum was consistent with the expected structure.

Test Example 1 The anticancer drug ester synthesized as described above was incubated with cancer cells, rat whole blood, normal tissue homogenate, or human whole blood, and the hydrolysis rate and the absolute configuration and optical purity of the generated carboxylic acid were examined. Was. In each reaction solution, 1 mg of the ester was dissolved in 10 μl of ethanol, and 90 μl of 0.1 M phosphate buffer (pH 7.5) was mixed with 100 μl of cell suspension, 20 to 30 μl of whole blood, or 50 μl of tissue homogenate. Prepared. Cancer cells are described in the literature (T. Okada et al., Inorg. Chem. Acta. 178, 13).
(1990) and the catalog of American Type Culture Collection), and washed thoroughly with the above ice-cooled phosphate buffer. Cell suspension 1 used for reaction
The number of cells in 00 μl is as follows: rat hepatoma cell Anr4, 7 × 10 ⁶ ; rat pancreatic cancer cell ARI
P, 8 × 10 ⁶ ; rat sarcoma cells XC, 5 × 10 ⁶ ; human histiocytic leukemia cells U937, 1 × 10 ⁷ ; human pancreatic cancer cells MIA PaCa-2, 8 × 10 ⁶ : and human colon cancer cells Colo320 1 × 10 ⁷ pieces. Also,
50 μl of tissue homogenate corresponded to 20 mg of wet tissue. After stirring these reaction solutions at 30 ° C. for 12 hours with a magnetic stirrer, the pH was adjusted to about 2.5 by adding 1.5 μl of 2N hydrochloric acid, and the mixture was diluted with 1 ml of ethyl acetate in the presence of saturated sodium chloride. Extracted times.
Combine the ethyl acetate layers for each sample,
After adjusting to 1, the mixture was divided into 1.7 ml and 0.3 ml. The former was concentrated under reduced pressure, and the contained carboxylic acid was isolated by preparative thin-layer chromatography. An ether solution of diazomethane was added thereto, and methylation was performed. Then, absolute configuration and optical purity were determined by high performance liquid chromatography using a chiral column. The latter was methylated as it was,
After concentrating and preparing the resulting carboxylic acid, the methyl ester of the carboxylic acid produced was quantified by gas chromatography, and the hydrolysis rate was determined from the value. The column used for high performance liquid chromatography was Daicel's Chiral Cell OJ or OD.
Column (4.6 x 250 mm), elution 10% 2
-Performed with a propanol / hexane mixed solution at a flow rate of 1 or 0.75 ml / min. The column used for gas chromatography was an OV-1 capillary column (0.
25 mm × 25 m) and the analysis temperature is 120 to 180
° C. The methyl ester used as a standard sample in these chromatography was prepared from commercially available optically active and racemic carboxylic acids. The results are summarized in Table 1.

[0022]

[Table 1]

[0023]

The compound of the present invention is used as an ester-type anticancer agent which is hardly decomposed in blood, but is rapidly hydrolyzed and activated in cancer cells. According to the present invention, an anticancer agent is esterified so as to be more easily activated by a hydrolysis reaction in cancer cells than in blood or normal cells, thereby increasing stability in blood and reducing side effects on normal cells. It can reduce and enhance selective attack on cancer cells.

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int. Cl. ⁶ , DB name) C07H 19/067-19/073 A61K 31/70 CA (STN) REGISTRY (STN)

Claims (2)

(57) [Claims] 1. An ester compound having a structure represented by the formula (I). Embedded image (In the formula, R ₁ represents a methyl group, a methoxy group or a trifluoroacetamide group, R ₂ represents a phenyl group or a phenylmethyl group, and the absolute configuration of the asymmetric center marked with an asterisk is R. ) 2. A protected 2 ′, 3′-isopropylidene compound of a 5-fluorouridine compound represented by the following formula (II) or a corresponding 5-fluorodeoxyuridine compound, and a carboxylic acid represented by the formula (III): Acid chloride or the carboxylic acid is converted to dicyclohexylcarbodiimide (DC
C) or by reacting with a compound activated with carbonyldiimidazole to acylate the hydroxyl group at the 5′-position,
3. The deprotection of 3'-hydroxyl group is performed.
The method for producing the ester compound of 5-fluorouridine or the corresponding ester compound of 5-fluorodeoxyuridine according to the above. Embedded image