JP4565236B2 - Cytochalasin derivative and method for producing the same - Google Patents

Description

  The present invention relates to a cytochalasin derivative and a method for producing the same.

  There are soil microorganisms that weaken and die plants due to toxins that secrete themselves in the roots of plants. In recent years, there are many cases in which Rosellinia necatrix Prillieux infects fruit trees and causes damage in recent years, and the economic damage caused by this has become more serious every year. When one tree is infected in a farm, etc., there is a high possibility that the infection will spread to other trees through the soil, and it is necessary to detect the infection early and take measures to prevent such a situation from occurring. . If the appearance of white-white feather-infected trees is found abnormal, the infection has already progressed considerably and is considered too late.

Therefore, for early detection of infection, it is strongly desired to establish a method for detecting the toxin (Cytochalasin E) produced by the white fungus and absorbed in trees at an early stage of infection.
Immunoassay has high sensitivity and specificity, and if a specific antibody is obtained, multiple specimens can be examined quickly and at low cost. However, the specific antibody cannot usually be obtained by immunizing a low molecular weight compound such as Cytochalasin E. For this reason, it is necessary to synthesize a complex by covalently binding a low molecular compound (hapten) to a carrier protein molecule, and using this complex to obtain a specific antigen as an immunity. Accordingly, an object of the present invention is to provide a cytochalasin derivative capable of synthesizing complexes of Cytochalasin E and various carrier proteins for the purpose of developing a rapid and simple detection technique for Cytochalasin E.
Ed Harlow et al, Antibodies-A LABORATORY MANUAL, Cold-Harbor-Laboratory, pp 75-85 (l988). Satoko Kanematsu et al, Ann Phytopathol. Soc. Jpn., 63, 425-431 (1997).

（上記式中のＲ₁は、水素原子、炭化水素基またはスクシノイル基を表わし、Ｒ₂は水素原子またはメトキシ基を表わす。） The above-mentioned subject is achieved by the following present invention.
1. A cytochalasin derivative represented by the following general formula (1) or (2):

(R ₁ in the above formula represents a hydrogen atom, a hydrocarbon group or a succinoyl group, and R ₂ represents a hydrogen atom or a methoxy group.)

（上記式中のＲ₂は水素原子またはメトキシ基を表わす。） 2. 2. A method for producing a cytochalasin derivative represented by the general formula (1) or (2), wherein the cytochalasin represented by the following general formula (3) is reacted with a compound that reacts with the lactam. .

(R ₂ in the above formula represents a hydrogen atom or a methoxy group.)

  Since the cytochalasin derivative of the present invention can form complexes with various carrier proteins, monoclonal and polyclonal antibodies can be prepared using this complex, and enzyme immunoassay necessary for antibody activity evaluation can be performed. .

Next, the present invention will be described in more detail with reference to the best mode for carrying out the invention.
Cytochalasin E and Phenochalasin B represented by the general formula (3) are known substances per se and are available as reagents, and the derivatives of the present invention were developed using the reagents. Cytochalasin E and Phenochalasin B are compounds with low photostability, so it is preferable to use them while being shielded from time to time. The compound represented by the general formula (1) or (2) can be produced by the following production method.
[Production Method 1]

Examples of the acid anhydride used in the above reaction include malonic anhydride, succinic anhydride, glutaric anhydride, adipic anhydride, and the like, and a particularly preferred acid anhydride is succinic anhydride. The acid anhydride used in the reaction is preferably used in an amount of about 0.3 to 5 mol per mol of Cytochalasin E. Examples of the base used in the above reaction include n-BuLi (normal butyllithium), LDA (lithium diisopropylamide), Et ₃ N (triethylamine), pyridine, DBU (1,8-diazabicyclo [5.4.0]. And inorganic bases include potassium t-butoxide, sodium hydride, potassium carbonate, potassium bicarbonate, sodium carbonate, sodium bicarbonate, lithium hydroxide, sodium hydroxide, potassium hydroxide. Etc.
Solvents used for the reaction include ether solvents such as THF (tetrahydrofuran), dioxane, Et ₂ O (diethyl ether), IPE (isopropyl ether), DME (1,2-dimethoxyethane), dichloromethane, chloroform, carbon tetrachloride. And halogen solvents such as dichloroethane, benzene solvents such as benzene, toluene, xylene and chlorobenzene, and hydrocarbon solvents such as pentane, hexane and heptane. The reaction temperature is -78 to 100 ° C, and the reaction time is 0.5 to 24 hours.

[Production Method 2]

Examples of the dicarboxylic acid halide used in the above reaction include oxalic acid dichloride, malonic acid dichloride, succinic acid dichloride, glutaric acid dichloride, adipic acid dichloride, oxalic acid dibromide, malonic acid dibromide, succinic acid dibromide, glutaric acid dibromide, Adipic acid dibromide and the like can be mentioned, and a particularly preferred dicarboxylic acid halide is succinic acid dichloride.
The dicarboxylic acid halide used in the reaction is preferably used in an amount of about 0.3 to 5 mol per mol of Cytochalasin E. The base, solvent, reaction temperature and reaction time used in the above reaction are the same as in Production Method 1. Hydrolysis (removal of halogen) after the reaction is extremely easy and occurs easily in the presence of water, acid or alkaline water.

[Production Method 3]

  Examples of the halogenated fatty acid ester used in the above reaction include chloroacetic acid ethyl ester, chloropropionic acid ethyl ester, chlorobutyric acid ethyl ester, bromoacetic acid ethyl ester, bromopropionic acid ethyl ester, bromobutyric acid ethyl ester, chloroacetic acid methyl ester. Chloropropionic acid methyl ester, chlorobutyric acid methyl ester, and the like, and particularly preferred halogenated fatty acid ester is bromoacetic acid ethyl ester. The halogenated fatty acid ester used in the reaction is preferably used in an amount of about 0.3 to 5 mol per mol of Cytochalasin E. The base, solvent, reaction temperature and reaction time used in the above reaction are the same as in Production Method 1. The hydrolysis of the ester bond after the reaction is preferably hydrolysis with thiolate or hydrolysis with iodotrimethylsilane.

[Production Method 4]

The N-hydroxysuccinimide used in the above reaction is preferably used in an amount of about 0.3 to 5 mol per 1 mol of the compound of the general formula (1) (R ₁ = H). Examples of the dehydrating agent used in the above reaction include DCC (dicyclohexylcarbodiimide), DIPC (diisopropylcarbodiimide), EDC (N-ethyl-N′-3-dimethylaminopropylcarbodiimide) and its hydrochloride, BOP (benzotriazole- 1-yl-tris (dimethylamino) phosphonium hexafluorophosphide salt), DPPA (diphenylphosphoryl azide) and the like, and particularly preferred dehydrating agent is EDC (N-ethyl-N′-3-dimethylaminopropylcarbodiimide) hydrochloric acid. Salt. The dehydrating agent used in the reaction is preferably used in an amount of about 0.3 to 5 mol per mol of Cytochalasin E. As the solvent used in the above reaction, DMF (dimethylformamide), DMSO (dimethylsulfoxide), DMAC (dimethylacetamide), AcOEt (ethyl acetate), CH ₃ CN (acetonitrile), THF (tetrahydrofuran), dioxane, Et ₂ O (diethyl ether), IPE (isopropyl ether), ether solvents such as DME (1,2-dimethoxyethane), halogen solvents such as dichloromethane, chloroform, carbon tetrachloride, dichloroethane, benzene, toluene, xylene, chlorobenzene, etc. Examples thereof include hydrocarbon solvents such as benzene solvent, pentane, hexane and heptane. The reaction temperature is −20 to 50 ° C., and the reaction time is 0.5 to 24 hours.

  Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited thereto. NMR was measured using JNM-AL300 (manufactured by JEOL Datum).

[Example 1] (4- (6,7-epoxy-10-phenyl-5,6,16,18-tetramethyl-21,23-dioxa- [13] cytochalas-13,19-diene-17,22- synthesis of dione-2-yl) -4-oxo-butyric acid)

  Under a nitrogen atmosphere, sodium hydride (7 mg, 0.3 mmol: used after washing with dehydrated hexane) was suspended in dehydrated tetrahydrofuran (3 ml) and cooled to about −78 ° C. or lower. Cytochalasin E (65.6 mg, 0.13 mmol) dissolved in dehydrated tetrahydrofuran (3 ml) was added dropwise to the reaction solution, and the reaction was performed at −78 ° C. or lower for about 20 minutes and at 0 to 5 ° C. for about 15 minutes. . After cooling again to −78 ° C. or lower, succinic anhydride (25 mg, 0.24 mmol) dissolved in dehydrated tetrahydrofuran (3 ml) was added dropwise to the reaction solution, and the temperature was gradually raised to room temperature. To this reaction solution were added a saturated ammonium chloride solution (2 ml) and water (2 ml), and the mixture was extracted with ethyl acetate (3 ml × 5 times), then dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The obtained oil was purified by silica gel column chromatography (hexane: acetone = 2: 1) to give the title compound (10.1 mg) as white crystals in a yield of 15%. The results of identification of the obtained compound are shown below.

¹ H-NMR spectrum (300 MHz, CDCl ₃ ) δ ppm: 7.30 to 7.00 (5H, m), 6.26 (1H, d, J = 11.6 Hz), 5.70 to 5.60 ( 1H, m), 5.58 (1H, d, J = 11.6 Hz), 5.20 (1H, ddd, J = 14.9, 11.3, 3.8 Hz), 4.53 (1H, m ), 4.38 (1H, bs), 3.23 (2H, t, J = 6.0 Hz), 3.04 (1H, dd, J = 5.2, 1.9 Hz), 3.00-2 .80 (4H, m), 2.75 to 2.55 (3H, m), 2.50 (1H, d, J = 5.1 Hz), 2.10 to 2.00 (2H, m), 1 .49 (3H, s), 1.16 (3H, d, J = 6.8 Hz), 1.11 (3H, s), 0.90 (3H, d, J = 7.0 Hz)

¹³ C-NMR spectrum (75.5 MHz, CDCl ₃ ) δ ppm: 211.6, 172.3 × 2, 168.9, 148.8, 141.9, 135.0, 132.1, 130.4 × 2, 128.7 × 2, 127.9, 127.2, 120.7, 86.0, 76.7, 60.2, 57.3, 55.6, 45.7, 42.6, 40. 8, 40.7, 39.2, 35.8, 32.3, 27.6, 24.4, 20.0, 18.8, 12.6

[Example 2] (6,7-epoxy-10-phenyl-5,6,16,18-tetramethyl-21,23-dioxa- [13] cytochalas-13,19-diene-17,22-dione-2 -yl) -acetic acid ethyl ester)

  Under a nitrogen atmosphere, sodium hydride (0.6 mg, 0.02 mmol: used after washing with dehydrated hexane) was suspended in dehydrated tetrahydrofuran (0.5 ml) and cooled to about −78 ° C. or lower. Cytochalasin E (10 mg, 0.02 mmol) dissolved in dehydrated tetrahydrofuran (0.5 ml) was added dropwise to the reaction solution, and the reaction was performed at −78 ° C. or lower for about 20 minutes and at 0 to 5 ° C. for about 15 minutes. . After cooling again to −78 ° C. or lower, ethyl bromoacetate (3 μl, 0.02 mmol) was added dropwise to the reaction solution, and the temperature was gradually raised to room temperature. Saturated ammonium chloride solution (1 ml) and water (1 ml) were added to the reaction solution, and the mixture was extracted with ethyl acetate (3 ml × 5 times), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The obtained oil was purified by silica gel column chromatography (hexane: ethyl acetate = 1: 1) to give the title compound (5.2 mg) as white crystals in a yield of 45%. The results of identification of the obtained compound are shown below.

¹ H-NMR spectrum (300 MHz, CDCl ₃ ) δ ppm: 7.30 to 7.00 (5H, m), 6.29 (1H, d, J = 11.7 Hz), 5.88 (1H, dd, J = 14.9, 10.5 Hz), 5.54 (1H, d, J = 11.7 Hz), 5.16 (1H, ddd, J = 14.9, 10.5, 3.7 Hz), 4 .63 (1H, d, J = 17.5 Hz), 4.41 (1H, bs), 4.18 (2H, q, J = 7.2 Hz), 4.01 (1H, m), 3.69 (1H, d, J = 17.5 Hz), 3.02 (1H, m), 2.91 (2H, m), 2.70-2.50 (4H, m), 2.28 (1H, m ), 2.13 (1H, m), 1.47 (3H, s), 1.33 (3H, s), 1.28 (3H, t, J = 7.2 Hz), 1.15 (3H) d, J = 6.6Hz), 1.02 (3H, d, J = 7.3Hz), also observed NOE to 4.01ppm and 3.69pm

¹³ C-NMR spectrum (75.5 MHz, CDCl ₃ ) δ ppm: 211.5, 168.4, 167.6, 149.0, 141.9, 134.9, 131.1, 130.0 × 2, 128.8, 128.7 × 2, 127.3, 120.3, 86.8, 77.3, 61.6, 60.7, 57.8, 57.2, 46.1, 44.7, 42.8, 40.8, 40.1, 39.1, 35.7, 24.4, 20.0, 19.4, 14.2, 13.2

[Example 3] (4- [6,7-epoxy-10- (4-methoxyphenyl) -5,6,16,18-tetramethyl-21,23-dioxa- [13] cytochalas-13,19-diene- Synthesis of 17,22-dione-2-yl] -4-oxo-butyric acid)

  Under a nitrogen atmosphere, sodium hydride (2.4 mg, 0.1 mmol: used after washing with dehydrated hexane) was suspended in dehydrated tetrahydrofuran (3 ml) and cooled to about −78 ° C. or lower. Phenochalasin B (20.0 mg, 0.04 mmol) dissolved in dehydrated tetrahydrofuran (3 ml) was added dropwise to this reaction solution, and the reaction was performed at −78 ° C. or less for about 20 minutes and at 0 to 5 ° C. for about 15 minutes. . After cooling again to −78 ° C. or lower, succinic anhydride (10 mg, 0.1 mmol) dissolved in dehydrated tetrahydrofuran (3 ml) was added dropwise to the reaction solution, and the temperature was gradually raised to room temperature. To this reaction solution were added a saturated ammonium chloride solution (2 ml) and water (2 ml), and the mixture was extracted with ethyl acetate (3 ml × 5 times), then dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The obtained oil was purified by silica gel column chromatography (hexane: acetone = 2: 1) to give the title compound (4.5 mg) as white crystals in a yield of 19%. The results of identification of the obtained compound are shown below.

¹ H-NMR spectrum (300 MHz, CDCl ₃ ) δ ppm: 7.10 to 7.00 (2H, m), 6.70 to 6.60 (2H, m), 6.25 (1H, d, J = 11.6 Hz), 5.70-5.60 (1 H, m), 5.57 (1 H, d, J = 11.6 Hz), 5.19 (1 H, ddd, J = 14.9, 11.3) , 3.8 Hz), 4.52 (1 H, m), 4.37 (1 H, bs), 3.73 (3 H, s), 3.22 (2 H, t, J = 6.0 Hz), 05 (1H, dd, J = 5.2, 1.9 Hz), 3.00 to 2.80 (4H, m), 2.75 to 2.55 (3H, m), 2.49 (1H, d , J = 5.1 Hz), 2.10 to 2.00 (2H, m), 1.49 (3H, s), 1.16 (3H, d, J = 6.8 Hz), 1.09 (3H) , S) 0.91 (3H, d, J = 7.0Hz)

¹³ C-NMR spectrum (75.5 MHz, CDCl ₃ ) δ ppm: 211.1, 172.3 × 2, 168.9, 159.2, 148.8, 141.9, 132.1, 131.7, 129.3 × 2, 128.9, 120.7, 113.9 × 2, 86.2, 77.7, 60.1, 57.3, 56.0, 55.5, 45.7, 42. 5, 40.8, 40.7, 39.1, 35.8, 32.3, 27.7, 24.4, 20.1, 18.8, 12.5

[Example 4] ([6,7-epoxy-10- (4-methoxyphenyl) -5,6,16,18-tetramethyl-21,23-dioxa- [13] cytochalas-13,19-diene-17, Synthesis of 22-dione-2-yl] -acetic acid ethyl ester

  Under a nitrogen atmosphere, sodium hydride (0.6 mg, 0.02 mmol: used after washing with dehydrated hexane) was suspended in dehydrated tetrahydrofuran (0.5 ml) and cooled to about −78 ° C. or lower. Phenochalasin B (10 mg, 0.02 mmol) dissolved in dehydrated tetrahydrofuran (0.5 ml) was added dropwise to the reaction solution, and the reaction was performed at −78 ° C. or less for about 20 minutes and at 0 to 5 ° C. for about 15 minutes. . After cooling again to −78 ° C. or lower, ethyl bromoacetate (3 μl, 0.02 mmol) was added dropwise to the reaction solution, and the temperature was gradually raised to room temperature. Saturated ammonium chloride solution (1 ml) and water (1 ml) were added to the reaction solution, and the mixture was extracted with ethyl acetate (3 ml × 5 times), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The obtained oil was purified by silica gel column chromatography (hexane: ethyl acetate = 1: 1) to give the title compound (4.5 mg) as white crystals in a yield of 39%. The results of identification of the obtained compound are shown below.

¹ H-NMR spectrum (300 MHz, CDCl ₃ ) δ ppm: 7.10 to 7.00 (2H, m), 6.80 to 6.70 (2H, m), 6.30 (1H, d, J = 11.7 Hz), 5.89 (1 H, dd, J = 14.9, 10.5 Hz), 5.55 (1 H, d, J = 11.7 Hz), 5.18 (1 H, ddd, J = 14) .9, 10.5, 3.7 Hz), 4.66 (1 H, d, J = 17.5 Hz), 4.39 (1 H, bs), 4.17 (2 H, q, J = 7.2 Hz) 4.01 (1H, m), 3.73 (3H, s), 3.69 (1H, d, J = 17.5 Hz), 3.00 (1H, m), 2.91 (2H, m) ) 2.70-2.50 (4H, m), 2.25 (1H, m), 2.13 (1H, m), 1.46 (3H, s), 1.31 (3H, s) 1.28 (3H, t, J = 7.2 Hz), 1.15 (3H, d, J = 6.6 Hz), 1.02 (3H, d, J = 7.3 Hz), and 4.01 ppm NOE observed at 3.69 pm

¹³ C-NMR spectrum (75.5 MHz, CDCl ₃ ) δ ppm: 211.3, 168.4, 167.6, 159.2, 149.1, 141.9, 131.7, 131.1, 129. 3 × 2, 127.3, 120.3, 113.9 × 2, 86.8, 77.7, 61.7, 60.7, 57.8, 57.2, 56.0, 46.1, 44.7, 42.5, 40.8, 40.5, 39.3, 35.7, 24.4, 20.1, 19.4, 14.2, 13.5

  Since the cytochalasin derivative of the present invention can form complexes with various carrier proteins, it is possible to produce monoclonal and polyclonal antibodies using this complex, enabling the enzyme immunoassay necessary for antibody activity evaluation. Become. Cytochalasin derivatives are known to have various pharmacological activities such as cell growth inhibitory action and cell death inducing action. The cytochalasin derivative of the present invention is also useful as a test drug for pharmaceutical use.

Claims (2)

A cytochalasin derivative represented by the following general formula (1) or (2):

(R ₁ in the above formula represents a hydrogen atom, a hydrocarbon group or a succinoyl group, and R ₂ represents a hydrogen atom or a methoxy group.) 2. The production of a cytochalasin derivative represented by the general formula (1) or (2) according to claim 1, wherein a compound that reacts with the lactam is reacted with the cytochalasin represented by the following general formula (3). Method.

(R ₂ in the above formula represents a hydrogen atom or a methoxy group.)