JPH02196789A - Stabilization of rhizoxin compounds - Google Patents

Abstract

PURPOSE:To store the subject compounds having an antineoplastic effect in a stabilized state for a long period by addition of an antioxidant having phenolic hydroxyl groups. CONSTITUTION:For example, to a rhizoxin of the formula, etc., 10-50wt.% antioxidant having phenolic hydroxyl groups, preferably such as quercetin ellagate of 0.01-5wt.% concentration is added and the mixture is dissolved into diethyl ether, N,N-dimethylacetamide, etc. The resultant solution is subdivided into amples and stored as a water-soluble liquid preparation. In addition, a nonionic surfactant such as a polyoxyethylene castor oil ester may be added thereto in case of use as a dilution (before using) type preparation or a freeze- dried preparation.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for stabilizing rhizoxin compounds having antitumor activity and preserving them for a long period of time by adding an antioxidant having a phenolic hydroxyl group.

Conventional rhizoxin compounds are extremely unstable in aqueous solutions, non-aqueous solutions, or lyophilized formulations, and it is difficult to formulate them while retaining their activity.

We have been intensively researching formulations that can maintain stability over long periods of time, even in non-aqueous solutions or freeze-dried formulations.

As a result, the inventors discovered that these compounds can be stored stably for a long period of time by adding an antioxidant having a phenolic hydroxyl group to these compounds, thereby completing the present invention.

The present invention relates to a method for stabilizing rhizoxin compounds by adding one or more kinds of antioxidants having phenolic hydroxyl groups.

The rhizoxin compounds of the present invention include, for example, the following compounds.

CH3 The present inventors have developed a rhizoxin compound in which the rhizoxin compound has an aqueous solution.

Rhizoxin having formula (1) was published in the 43rd (1981)
This is a known compound described in the article of the Japanese Cancer Society general meeting, page 283, title 1,005.

2) Formula (IQ 3) Formula (2) (In the formula, R' represents a linear or branched alkyl group. However, a methyl group is excluded. A represents a single bond or an oxygen atom.) A rhizoxin derivative comprising a carboxylic acid having the following, a pharmacologically acceptable salt thereof, or a closed ring lactone thereof.

Rhizoxin derivatives having the formula (If) are disclosed in JP-A-62-8
It is a known compound described in No. 7, and its production method is described in the publication.

(In the formula, R" is a C1 to C25 straight chain or branched alkyl group having a substituent, a C3 to C25 which may have a substituent
8 Schiff Award alkyl group, C2-C which may have a substituent
25 straight or branched alkenyl group, C2 to C25 straight or branched alkynyl group which may have a substituent, C1 to C18 straight or branched chain which may have a substituent It represents an alkoxy group, a phenyl group which may have a substituent, or an aromatic heterocyclic group which may have a substituent. A represents a single bond or an oxygen atom. ), a rhizoxin derivative consisting of carganic acid, a pharmacologically acceptable salt thereof, or a closed ring lactone thereof.

Rhizoxin derivatives having formula (2) are disclosed in Japanese Patent Application Laid-Open No. 1983-24.
It is a known compound described in No. 6380, and its production method is described in that publication.

4) Formula (Goods) (In the formula, n represents an integer of 1 to 25, X represents oxygen, sulfur or nitrogen atom, or carbonyl group, and when X is oxygen atom, R# represents hydrogen atom, aliphatic acyl group, alkoxycarbonyl group, phosphono group, alkylphosphono group or dialkylphosphono group, and when X is a sulfur atom, R1 is a hydrogen atom, an aliphatic acyl group, an alkoxycarbonyl group, an alkylthio group, an aralkylthio group or represents a terocyclylthio group, when X is a nitrogen atom, R″ represents R, R, and R1 and R2 are the same or different and are a hydrogen atom, an aliphatic acyl group, an alkyl group, an alkoxycarbonyl group, a phosphono group, an alkyl It represents a phosphono group or a dialkylphosphono group, and when X is a nyl group, R'' represents an alkyloxy group.

The alkyl group in the alkyl moiety of the above alkyl group, aliphatic acyl group, alkoxycarbonyl group, alkylphosphono group, dialkylphosphono group, alkylthio group, and alkoxy group has a C1 to C25 straight chain or branched chain substituent. Indicates an optional alkyl group. ) or a salt thereof.

Rhizoxin derivatives having the formula
It is a novel compound having antitumor activity as described in No. 8152.

The acyl group in the above formula (transformation) is, for example, acetyl,
n-propionyl, n-butyryl, 1so-butyryl, 2-methylzolopionyl, n-pentanoyl, 2-
Methylbutyryl, pivaloyl, n-hexanoyl, 2-
Methylintanoyl, 3-methylpentanoyl, 4-methylpentanoyl, n-heptanoyl, 2-methylhexanoyl, 3-ethylhexanoyl, n-octanoyl, 2-methylheptanoyl, 3-ethylheptanoyl,
2-ethyl-3-methylpentanoyl, 3-ethyl-2
-Methyl-intanoyl, n-nonanoyl, 2-methyloctanoyl, 7-methyloctanoyl, 4-ethylheptanoyl, 3-ethyl-2-methylhexanoyl, 2
-Ethyl-1-methylhexyl, n-decyl, 2-methylnonanoyl, 8-methylnonanoyl, 5-ethyloctanoyl, 3-ethyl-2-methylheptanoyl, 3.
3-diethylhexanoyl, n-undecenoyl, 2-
Methyldecenoyl, 9-methyldecenoyl, 4-ethylnonanoyl, 3,5-dimethylnonanoyl, 3-propyloctanoyl, 5-ethyl-4-methyloctanoyl, n-dodecanoyl, 1-methylundecanoyl, 10
-methylundecanoyl, 3-ethyldecenoyl, 5
-7' lobyrnonyl, 3,5-diethyl octanoyl,
n-tridecanoyl, 11-methyldodecanoyl, 7-
Ethylundecenoyl, 4-propyldecenoyl, 5-
Ethyl-3-methyldecenoyl, 3-pentyloctanoyl, n-tetradecyl, 12-methyltridecanoyl, 8-ethyldodecyl, 6-propylundecyl, 4-
Butyldecenoyl, 2-pentylnonanoyl, n-pentadecanoyl, 13-methyltetradecenoyl, 10-
Ethyltridecenoyl, ↓-7-propyldecanoyl, 5-ethyl-3-methyl-dodecanoyl, 4-pentyldecanoyl, n-hexadecanoyl, 14-methyl-pentadecanoyl, 6-ethyltetradecanoyl, 4-propyltridecanoyl, 2-
Butyldecenoyl, n-heptadecanoyl, 15-methylhexadecanoyl, 7-ethylintertadecanoyl, 3
-Propyltetradecanoyl, 5-pentyldodecanoyl, n-octadecanoyl, 16-ethylheptadecanoyl, 5-f propylbentadecanoyl, n-nonadecanoyl, 17-methyloctadecanoyl, 4-ethylheptadecanoyl Noyl, icosanoyl, 18-methylnonadecanoyl, 3-ethyl octadecanoyl, hexanoyl,
Docosanoyl, tricosanoyl, tetracosanoyl, intacosanoyl, pehenoyl, are crushed.

Alkyl groups include, for example, ethyl, n-propyl, 1-methylethyl, n-butyl, 1-methylpropyl, 2-methylpropyl, 1,1-dimethylethyl, n-pentyl,
3-methylbutyl, 2,2-dimethylpropyl, 1,1
-dimethylpropyl, n-hexyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, n-heptyl, 1-methylhexyl, 2
-Methylhexyl, 5-methylhexyl, 3-ethylpentyl, n-octyl, 2-methylheptyl, 5-methylheptyl, 2-ethylhexyl, 2-ethyl-3-methylpentyl, 3-ethyl-2-methylpentyl, n-
Nonyl, 2-methyloctyl, 7-methyloctyl, 4
-ethylheptyl, 3-ethyl-2-methylhexyl,
2-ethyl-1-methylhexyl, n-decyl, 2-methylnonyl, 8-methylnonyl, 5-ethyloctyl,
3-ethyl-2-methylheptyl, 3,3-diethylhexyl, n-undecyl, 2-methyldecyl, 9-methyldecyl, 4-ethylnonyl, 3,5-dimethylnonyl, 3-nia'ropyloctyl, 5--1- thyl-4-methyloctyl, n-dodecyl, 1-methylundecyl,
10-methylundecyl, 3-ethyldecyl, 5-propylnonyl, 3,5-diethyloctyl, n-)lysocyl, 11-methyldodecyl, 7-ethylundecyl, 4
-propyldecyl, 5-ethyl-3-methyldecyl, 3
-pentyloctyl, n-tetradecyl, 12-methyltridecyl, 8-ethyldodecyl, 6-probylundecyl, 4-butyldecyl, 2-intylinonyl, n-interdecyl, 13-methyltetradecyl, lO-dityltridecyl, 7 -propyldodecyl, 5-ethyl-3-
Methyldodecyl, 4-inchildecyl, n-hexadecyl, 14-methylpentadecyl, 6-ethyltetradecyl, 4-propyltridecyl, 2-butyl-decyl, n
-heptadecyl, 15-methylhexadecyl, 7-ethylpentadecyl, 3-propyltetradecyl, 5-pentyldodecyl, n-octadecyl, 16-methylheptadecyl, 5-propylpentadecyl, n-nonadecyl,
17-methyloctadecyl, 4-methylheptadecyl,
Examples include eicosyl, 18-methylnonadecyl, 3-ethyloctadecyl, henicosyl, docosinyl, tricosinyl, tetracosinyl, intacosinyl, and the like.

Substituents on the alkyl moiety of the above alkyl groups and aliphatic acyl groups, alkoxycarbunyl groups, alkylphosphono groups, dialkylphosphono groups, alkylthio groups and alkoxy groups include fluorine, chlorine, bromine or iodine. Rodan atom; Cal is xyl: -CO2Rhz (
Rhz is a group obtained by removing the 13th-position OH group in formula (1), and will be described later. ): -CON(eye)CONH(eye
) (e7e represents a cyclohexyl group); Examples include a hydroxyl group or an aryl group such as phenyl, nitrophenyl or cyanophenyl.

Examples of the pharmacologically acceptable salts of the compound of the present invention having the above general formula (goods) include metal salts, amino acid salts, and amine salts. Examples of metal salts include alkali metal salts such as sodium and potassium, alkaline earth metal salts such as calcium and magnesium, and aluminum salts, iron salts, zinc salts, copper salts, nickel salts, and cobalt salts. Among them, alkali metal salts, alkaline earth metal salts and aluminum salts are preferred, and sodium salts, potassium salts, calcium salts and aluminum salts are most preferred. Preferred amino acid salts include basic amino acids such as arginine, lysine, histidine, α, γ-diaminobutyric acid, and ornithine. Suitable examples of the amine salt include t-octylamine, dibenzylamine, dicyclohexylamine, morpholine, D-phenylglycine alkyl ester, and D-glucosamine.

In the following, Rhz is shows.

The novel rhizoxin derivative having the above-mentioned general ω in the present invention can be produced by a reaction process as shown in the following formula. In the formula, n, R'R2 and Rhz have the same meanings as described above. m represents an integer of 1 to 25, and R represents an aliphatic alkyl group such as methyl, ethyl or propyl, or an aralkyl group such as benzyl phenethyl or phenylglobil.

Next, the reaction reagents and reaction conditions in each step will be described below.

1) Esterification step (1st, 6th, 10th, 15th and 16th steps) This step involves reacting an acid anhydride in the presence of a deoxidizing agent or reacting a substituted caldic acid such as DCC. In this step, an ester bond is formed between the hydroxyl group at the 13th position of rhizoxin using a condensing agent. The condensing agent used in this step is DCC (dicyclohexylcalgediimide),
CDI (N, N'-L is nyldiimidazo/L/
), DPPA (diphenylphosphoryl azide)
, HOBT (1-hydroxybenzotriazole),
HONB (N-hydroxy-5-nordelene-2,
3-dicardeximide) or EDAPC (1-ethyl-3-(3-dimethylaminepropyl)cardiimide), etc. can be used. Note that the reaction proceeds more quickly when pyridine, dimethylaminopyridine, or pyrrolidinopyridine is used. There are no particular restrictions on the reaction solvent used, but halogenated hydrocarbons such as methylene chloride, chloroform, and dichloroethane, benzene, and toluene are usually used. Aromatic hydrocarbons such as , xylene are preferred. The reaction temperature may be anywhere from 100°C to 0°C, but
Usually at room temperature. The reaction time varies from 10 minutes to 48 hours depending on the reaction temperature, but the reaction is completed in 3 hours at room temperature.

1i) Removal step of iJ chloroethyl group (2nd.5th, 11th.14th step) This step removes trichloroethyl group from trichloroethoxycar-nyloxy group, trichloroethoxycarbunylamino group, and trichloroethylphosphoryl group. In this step, free hydroxyl groups, phosphoryl groups, and amino groups are generated. The reagent used is not particularly limited as long as it can remove trichloroethyl groups, but zinc dust is preferably used. The reaction solvent used is not particularly limited, but alcohols such as methanol and ethanol, organic acids such as water-containing formic acid and acetic acid, aqueous solutions with - adjusted to 1 to 8, tetrahydrofuran, acetone, and dioxane. , a mixture with an organic solvent such as ethyl acetate can be used. The reaction temperature and reaction time are 10
The reaction time ranges from 0°C to 90°C for 10 minutes to 50 hours, although this varies depending on the reaction solvent. When a mixture of -4,2 phosphate buffer and tetrahydrofuran is used as the reaction solvent, stirring at room temperature for 3 hours is suitable.

111) Disulfide reduction step (7th step) This step reduces the disulfide α peak of acylated rhizoxin,
This is a step to obtain an acylated rhizoxin derivative (9) having a mercapto group at the end of the acyl group. The reagents and reaction solvent used are not particularly limited as long as they can reduce disulfide bonds to mercapto groups, but as a reaction solvent, a mixed solvent of a buffer solution with a pH of 1 to 7 and an organic solvent such as tetrahydrofuran or acetone is used. In such cases, good results can be obtained by using zinc dust as a reaction reagent.

The reaction temperature is preferably from 100°C to O'c, and the reaction time varies depending on the reaction temperature, but when the reaction is carried out at room temperature, the reaction is completed in 3 hours.

iv) Acylation step (3rd, 8th, 12th steps) In this step, an acylated rhizoxin derivative having a hydroxyl group, mercapto, or amine group at the end of the acyl group is acylated by reacting with an activated carboxylic acid. It is a process. As a reaction reagent, an acid halide such as a carboxylic acid chloride or a carboxylic acid bromide is used, or a carboxylic acid is used as a DC reaction agent as described in "Esterification process of the 13-position hydroxyl group of rhizoxin".
C and a condensing agent compatible with it can be used.

Furthermore, pyridine, dimethylaminopyridine, or pyrrolidinopyridine can be used as a catalyst to promote the reaction. The reaction solvent is not particularly limited as long as it does not hinder the reaction, but it usually includes halodanized hydrocarbons such as methylene chloride, chloroform, and dichloroethane;
Alternatively, aromatic hydrocarbons such as benzene, toluene, and xylene can be used. The reaction temperature is -3 from 80℃
Although it depends on the reagent used up to 0°C, the reaction is completed in 1 to 10 hours at 0°C when using an acid halide, and in 1 to 10 hours at room temperature when using a condensing agent such as DCC.

■) Phosphorylation step (4th and 13th steps) In this step, acylated rhizoxin (4) and (to) having a hydroxyl group and an amino group with an acyl chain at the end are reacted with an activated phosphoric acid derivative. This is a phosphorylation step. As for the reagents used, a phosphoric acid derivative such as phosphorochloridate may be reacted in the presence of a deoxidizing agent, or a phosphoric acid derivative may be reacted using a condensing agent such as DCC. The reaction solvent is not particularly limited, but includes aromatic hydrocarbons such as benzene and toluene, ionahalogenated hydrocarbons such as methylene chloride, chloroform, and dichloroethane, amides such as dimethylacetamide and dimethylformamide, and hexamethylphosphorocarbons. It is also possible to use phosphoric acid ami-Ps such as amidate, trimethyl phosphate, and triethyl phosphoric acid triesters such as triethyl phosphate. Further, as the deoxidizing agent used, in addition to pyridines such as pyridine, dimethylaminopyridine, and 4-pyrrolidinopyridine, tertiary amines such as triethylamine, triethylamine, and tributylamine can also be used. Although the reaction temperature and reaction time vary depending on the reagents and deoxidizers used, it is usually preferable to carry out the reaction at a temperature of 80°C to 0°C, particularly preferably around room temperature, and in this case, the reaction time is 3 to 15 hours. It is.

■1) Disulfide formation step (9th step) This step consists of acylated rhizoxin having a sulfhydryl group at the end (
This is a step of reacting 9) with another compound having a sulfhydryl group to obtain acylated rhizoxin α■ having an asymmetric disulfy Y bond.

The reagent for forming an asymmetric disulfide bond used in this step is not particularly limited, but 2,4 dinitrobenzenesulfenyl chloride is preferably used. Further, the reaction solvent is not particularly limited, but acetonitrile, acetone, dimethylformamide, etc. are usually used. The reaction temperature may be anywhere from 100°C to -20°C,
It is usually carried out at a temperature of 0°C to room temperature. The reaction time varies depending on the reaction temperature, but when carried out at 0°C to room temperature, the reaction time is 1 to 2
It is 4 hours.

Next, among the target compounds of the present invention, the carboxylic acid having the general formula (1) is a carboxylic acid having the general formula (1) among the target compounds.
It is obtained by contacting a pharmacologically acceptable salt of carbic acid having 1) with an acid. The method is a conventional method, for example, the following table method. That is, the obtained pharmacologically acceptable salt of the carboxylic acid having the general formula (1) is dissolved in a solvent such as methanol, acetone, dimethylformamide, or dimethyl sulfoxide, and an equivalent to a slight excess amount of the acid is dissolved. By adding and K, we obtain. The acid used is not limited to organic acids or mineral acids as long as it does not affect the target compound, and trifluoroacetic acid, hydrochloric acid, sulfuric acid, etc. are preferably used.

The target compound thus obtained can be collected, separated, and purified by appropriately combining various methods. For example, pour the reaction solution into water, use a solvent that is immiscible with water,
For example, extract with benzene, ether, ethyl acetate, etc.
It is obtained by distilling off the solvent from the extract. If necessary, the target compound thus obtained may be subjected to adsorption or ion exchange chromatography using various carriers such as activated carbon or silica dal, or gel filtration using a Sephadex column, or organic solvents such as ether, ethyl acetate, or chloroform. This is done by recrystallization using

The rhizoxin derivative of the present invention is derived from p388 transplanted into mice.
It showed a stronger antitumor effect on cells than the rhizoxin derivative described in JP-A-62-87.

Reference example 1 Rhz-ooC(Cu2)110COOCH2CCt.

Rhizoxin 3.1211 was dissolved in methylene chloride, 12
- (2,2,2-) +)chloroethoxycarboxyloxy)dodecanoic acid 4.30 g, DC'C(N,N-
4.12 g of dicyclohexylcarbodiimide) and a catalytic amount of 4-pyrrolidinopyridine were added and stirred for about 3 hours. Methylene chloride was distilled off under reduced pressure, acetic ester was added, and the mixture was washed successively with 0.2N hydrochloric acid, a saturated aqueous sodium bicarbonate solution, and water, dried over anhydrous magnesium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel chromatography (cyclohexane:ethyl acetate=1:1) to obtain the target compound 4.62.9.

NMR (270MH2lnCDC23) δppm:
4.79 (2H, s.

cz, ccH2o), 4.2s (iH, da,, r=1
o, 3.4. sHz, 13-a).

4.24 (21(,t, J=6.5Hz, C
23CCH20COOCH,), 1.24~1.4
s (18H, bs, oca2 (Ca2), cH
2co) Reference Example 2 Rh E-00C (CH2), 10H Rhizoxin 1
2-(2,2,2-)lichloroethoxycarponyloxy)dodecanoic acid ester 4,261 was dissolved in acetone, an aqueous IM-sodium phosphate solution was added, and the mixture was stirred vigorously. 10 g of zinc powder was added thereto and stirred for about 2 hours. Insoluble matter was filtered off, and the solvent was distilled off under reduced pressure. Ethyl acetate and water were added to the residue to separate the layers, and the ethyl acetate layer was washed with a saturated aqueous sodium bicarbonate solution and water, then dried over anhydrous magnesium sulfate, and the solvent was distilled off. The residue was purified by silica gel chromatography (methylene chloride: methanol =
99.5:0.5) to obtain the target compound 73.
I got 0■.

NMR (270M) (z i n CDCt3 ) δp
prn: 4.25 (IH, q.

J=10.3, 4.6Hz, 13-H), 3.64(2
H, t, J = 6.5Hz.

HOCI(2(CH2)1oCoo), 2.30~2
．． 41 (2H, m, CH3COO).

1.50~1.85 (2H1m, CH2CH2Co
o), 1.24-1.42 (16H.

b color, HOCH2(CH2)8CH2CI(2Coo
) Reference Example 3 Roughy (methylene chloride:methanol=99.5:0.
5) to obtain the target compound 761n9.

NPa (270'WfHz 1 n CDCt, )
δppm: 4.25 (IH.

dd, J=4.3Hz, 13-H), 4.
06 (2H, t, J=6.6Hz.

(CH2), CH2C00CH□), 1.14 (
3) I, t, J=7.7H1.

0 (3CH2COOCH2) Reference example 4 Rhz-00C (CH2), 10COCH2CH.

Rhizoxin 12-hydroxydodecanoic acid ester 288
rn9 was dissolved in toluene, and propionyl chloride 112119, pyridine 1931V, and a catalytic amount of dimethylaminopyridine were added under water cooling. The mixture was returned to room temperature and stirred for 3 hours. Ethyl acetate was added, and the mixture was washed successively with 0.1N hydrochloric acid, saturated sodium hydrogen carbonate, and water, dried over anhydrous magnesium sulfate, and the solvent was distilled off. The residue was converted to silica chromatograph RhZ-00C(CH2) 1,0CO(CH2) 3
CH3 rhizoxin 12-hydroxydodecanoic acid ester #
Using 2881n9 and 198 rng of U-valeryl bromide, the reaction, treatment, and purification were performed in the same manner as in Reference Example 3 to obtain target compounds 209-.

NMR (270MIH21nCDCt3) δppm:
4.25 (IH, dd.

J = 4.3Hz, 13-H), 4.05(2
H,t, J=6.8Hz.

CH2C00CH□"), 2.34~2.42 (
2H, m, CM, C00CH2), 1.25~
1.42 (16H, m), 0.91 (3H, t,
J=4.6Hz.

CH3(CH2), Co) Reference example 5 00CCH2CH2S CH2CH2Coo) Reference example 6 StellRhz-00C(CH2)288(CH2)Coo-R
hz Rhizoxin 900 Iv was dissolved in benzene, dithiodipropionic acid 605 rv, DCC 1,1911 and a catalytic amount of 4-pyrrolidinopyrrolidine were added, and the mixture was stirred at room temperature for about 2 hours. (Add ethyl acetate and remove saturated hydrogen carbonate) IJum aqueous solution, 0.1N acid resistant solution, saturated sodium hydrogen carbonate aqueous solution, wash with water, dry over anhydrous magnesium sulfate, distill off the solvent, A residue containing various products was obtained. The mixture was subjected to silica gel chromatography, eluted with cyclohexane:ethyl acetate=1:1, and the fractions eluted later were collected, concentrated, and freeze-dried in benzene to obtain 400 Da of the target compound.

NMR (270MHz in cnc, )δpp
m: 4.29 (2H, dd.

J=3.8, 10.8Hz, 13-H,13'-
H), 2.99-3.05 (4H, m.

00CCH2CH2SSCH2CH2COO), 2
．． 83-2.88 (4H, rn.

Rhz-00C (CHz) 2SS (CH2) 2
CONCONHC6H1゜6H11 Reference Example 50 When the target compound is purified by silica gel chromatography, both the first eluted fractions are collected, concentrated,
The product was washed with benzene and freeze-dried to obtain 350 ml of the target compound.

NMR (270MH2lnCDCt3) δppm:
4.29 (IH, dd.

J=3.8, 11.1Hz, 13-H), 3
．． 95 (IH, m, NH), 3.70 (IH, 0
11, NH), 2.80-2.90 (4HIms00
CCH2CH2SSCH2CH2Coo ), 2.
90-3.08 (4H, m.

00CCH2CI! 2SSC'! ! 2C Dan, coo) Reference Example 7 Rhz-00C(CH2) 28H Dirizoxin 3,3'-dithiodipropionic acid diester 330# was reacted, treated and purified in the same manner as in Reference Example 2 to obtain the target compound 220In9. Ta.

NMR (270MFfz in CDCL, )δp
pm: 4.31 (IH, dd.

J=10.8, 3.8Hz, 13-H), 2
．． 70-2.90 (8H, m.

H8(CH2)2CO) Reference Example 8 Ester Rbz-00C(CH2)2SCO(CH2)8CH3
Rhizoxin 3-mercaptopropionic acid diester 12
0da was reacted, treated, and purified in the same manner as in Reference Example 3,
97 da of the target compound was obtained.

NR11'R(270MHz in cDcz3)δ
ppm: 4.27 (IH, dd.

J=10.5.3.8Hz, 13-H), 2.
71 (2H,t, J=6.9Hz.

SC! ! 2CH2Co), 2.57 (2H,t
, J=6.9H2, 5ca2ca2co).

1.20-1.40 (12H, bl, CH, (C
H2)8CO8), 0.88 (3H.

t, J=6.5Hz, CH, (CH2) 8)
Reference Example 9 Rhizoxin 3-(lauroylthio)f ropionic acid ester Rhz-00C(CHz) 2SCO(CH2),
. CH3 rhizoxin 3-mercaptopropionic acid ester 120 ml was reacted, treated and purified in the same manner as in Reference Example 8 to obtain the target compound 85rn9.

NMR (270MHz in CDC23) δppm
: 4.27 (IH, dd.

J=10.8, 3.8Hz, 13-H), 2
．． 70 (2H, t, J=7.0Hz.

5CH2C! (2Co), 2.57 (2H, t
, J=7.0Hz, 5CH2CH2Co).

1.45 (4H, bs, CH, (C) (2),.C
o), 1.26 (12H, bs.

CH3(C!!2)10CO), 0.88 (3H,
t, J=6.8Hz mCdan! 5 (CH2) 1゜c
o) Reference example 10 Rhz-00C (CHz) 2SCO (CH2) 12
CH3 rhizoxin 3-mercaptopropionic acid ester 90rn9 was reacted, treated and purified in the same manner as in Reference Example 8 to obtain the target compound 57da.

NMR (270MHz in CDC1,) δppr
n: 4.27 (IH, dd.

J=11.1, 4.6Hz, 13-H), 2
．． 71 (IH, t, J=6.8Hz.

5CH2CH2Co), 2.57 (IH,t,
J=6.8Hz, SC! ! 2CH2Co).

1.61 (16H, bs, CH3(CH2)12CO)
, 1.26 (4H+bs.

C13 (CH2), 2Co), 0.88 (3H,
t, J=6.5Hz.

C! ! 3(CH2)12CO) Reference Example 11 2.66 (2H, t, J=6.2H1, NHCH
2CH2C0) Reference example 12 Rh! -00C(CH2) 2NHCOOCH2C
C1゜Rizoxin 1.25# and 3- (2,2,2-
) Lichloroethoxylic? nylamino)teropionic acid 1
．． 59. F was dissolved in toluene, DcC1,031 was added thereto, a catalytic amount of 4-pyrrolidinopyridine was added, and the mixture was stirred at room temperature for about 2 hours. Add ethyl acetate to 0.1
After washing with normal hydrochloric acid, then with a saturated aqueous sodium bicarbonate solution, and with water, it was dried over anhydrous magnesium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel chromatography (cyclohexane:ethyl acetate=1:1) to obtain the target compound 1.251.

NMR (270MHz in CDCt3) δP P
”: 5-68 (I H* b s + zu), (2
H, l, CCt, CH2), 4.32 (IH, dd, J
=11.1°3.2 Hz, 13-H), 3.5
7 (2H, q, J=6.2 Hz, NHCH2C
H2Co).

Rhz-000(CH2) 2NHCOOCH2C6H
Using 315m9 of 5-lisoxin and 560g of 3-(penzoloxycargenylamino)f-ropionic acid, the reaction, treatment, and purification were carried out in the same manner as in Reference Example 11 to obtain the target compound 415r.
I got v.

NMR (270MH2lnCDC23) δppm:
7.28-7.40 (5H1m r C6H5), 5
．． 37 (IHlb t, J=6.2Hz -N!ri + 5.11 (2H,t, J=12.5Hz, C6
H3CH2), 4.29 (IH, dd, J=11.1
, 3.2Hz, 13-H), 3.53(2H, q, J=
6.2Hz.

NHcH2ca2co ), 2.63 (2H, t
, J=6.2H1, +cH2cH2co) Example 1
3 Rhz-00C(CH2) 2NHcooc(co3)
3Rizoxin 315■ and 3-(t-butoxycar?
Reference Example 1 using 284 Da (nylamino)propionic acid
React, treat, and purify in the same manner as in 1 to obtain the target compound 383~
I got it.

NMR (270MH7lnCDCt3) δppm:
5.08 (IH, bs.

N'H) s 4.32 (IH, dd 1.r==1
i, o -3,2Hz, 13-H), 3.45(2
H, (1, J=6.2, 6.2H1, xca2cH2
co), 2.60 (2H,t.

J”” 6.2 Hz,? 'J) (CH2Cdan, c
o), 1.44 (9H,m, (CH3)3C)
Reference Example 14 Rhizoxin 3-aminozolopionic acid ester Rhz-
00C(CH2) 2NH2 Lysoxin 3- (2,2,2-) IJ dichlorotoxycarbonylamino)furopionic acid ester 1.046
1 was dissolved in 30 g of tetrahydrofuran, and IM-
30ml of sodium phosphate aqueous solution was added and stirred vigorously. Zinc dust 5. OII was added in three portions every 30 minutes. After the reaction was completed, the zinc powder was removed by filtration, and ethyl acetate was added to separate the layers. The ethyl acetate layer was washed with a saturated aqueous sodium bicarbonate solution, then water, dried over anhydrous magnesium sulfate, and the solvent was distilled off under reduced pressure. . The residue was subjected to silica gel chromatography (methylene chloride:ethanol = 90:1
0) to obtain the target compound 635.

NMR (270MHz 1 n CDCt, )δpp
m: 3.15-3.30 (2H, m, NHCH2C
H2C0), 2.75 (2H,t, J=5.4
H2.

NHCH2CH2CO) Reference Example 15 Acid ester Rhz-000(CH2)2NHCO(CH2)8CH
3Rizoxin 3-aminopropionic acid ester 210m
9 and 155 μm of capric acid were dissolved in toluene and DCC
155Pn9 and a catalytic amount of 4-pyrrolidinopyridine were added and stirred for about 2 hours. The product was treated and purified in the same manner as in Reference Example 1 to obtain 160 mg of the target compound.

NMR (270MHz in CDCt, )δpp
m: 6.14 (IH, bt.

J=8.0Hz, N) (), 4.31 (IH,
dd, J=11.1, 3.2Hz, 13-H
), 3.59 (2H, (1, J=5.9Hz,
NI (cx+, cH2co), 2.60 (2H=
t, J = 5-9 Hz, NHCH2CH2CO)
, 1.51-1.73 (4H.

m, CH2CH2C0NH), 1.15-1.
39 (12H, bs.

CH3(CH2) 6 CH2CH2C0NT(),
0.88 (3H, t, J" 4.6 HE.

Reference example 16 Rhz-00C(CH2) 2NHCO(CH2),
. CH3 rhizoxin 3-aminopropionic acid ester 2
70 Da and lauric acid 800III9 were dissolved in toluene, DC0618In9 was added thereto, a catalytic amount of 4-pyrrolidinopyridine was added, and the mixture was stirred at room temperature for about 2 hours.

Treated and purified in the same manner as in Reference Example 15 to obtain the target compound 192I.
I got n9.

NMR (270MHz 1 n CDCl2) δppm
: 6.17 (IH, bt.

J=8.0Hz, NU), 4.32(IH, dd
, J=11.1, 3.2Hz, 13-H)
= 3.58 (2H, q 1J=5.9 Hz, N
HcH2CH2Co), 2.60 (2H2tlJ=
7.6H2, CH2C0■), 1.52-1.70 (2
1m.

CH2CH2C0NH), 1.17~1.40 (
16H, bs.

CH, (C')! 2)8CH2CH2CONH),
0.88 (3H,t, J=4.6H1.

CH3 (CH2),. C0NH) Reference Example 17 Onic acid ester Rhz-ooc(CH2) 2NHco(C1(2)
, 2CH.

Rhizoxin 3-aminopropionic acid varnish f, Ay275
Dissolve da in toluene and add 0.39 ml of myristoyl chloride.
ml was added and cooled on ice. There, triethylamine 0
．． 231d and a catalytic amount of 4-dimethylaminopyridine (
DMAP) was added, the temperature was returned to room temperature, and the mixture was stirred for 2 hours. Ethyl acetate was added to hide the 0.1N salt, washed with water, dried over anhydrous magnesium sulfate, and the solvent was distilled off. The residue was subjected to silica dal chromatography (cyclohexane:
Purified with ethyl acetate = 1:1) to obtain target compound 80I.
I got it.

NMR (270MHz l n CDCtx ) δpp
m: 6.17 (IH, bt.

J=8.1Hz, NH), 4.31 (IH, d
d, J=11.1, 3.2Hz, 13-H)
, 3.58 (2H,q, J=5.9Hz,
NHCH2CH2C0), 2.60 (2H.

t r J=5-9 Hz -NHCH2CH2C0)
+ 2.34 (2H, t, J=8.9 Hz −
CH2C0■H), 1.53~1.70 (2H,
m, CM2CH2C0NH), 1.20-1.
40 (20H, b8, CHs (C!!2)
1 o CH2CH2Co ) + 0.88 (3H
.

t, J=6.8Hz, CH, (CH2), 2C
o) Reference Example 18 Rhizoxin-13-0-(3-(n-decyloxy-trichloroethoxyphosphonoamidite)) propionic acid ester 0CH2CC23 15 ml of dry methylene chloride to 1 ml of n-decyl alcohol
．． 1 of triethylamine was added and dissolved, and 1.4 of triethylamine was added. While cooling with water, 1.33N of trichloroethyl horsphorodichloridate was added with stirring, and stirring was continued at room temperature for 3 hours. After cooling on ice, 690 da of the compound of Reference Example 14 was added, moisture was removed, and stirring was continued for 5 hours. Methylene chloride and 30 #L of sphagnum moss were added to the reaction solution, and after stirring for 30 minutes, the organic layer was separated. The organic layer was diluted with 0.1N hydrochloric acid, saturated saline, and 5% hydrogen carbonate). The mixture was washed with water, dried over magnesium sulfate, and the solvent was distilled off to obtain a caramel-like substance. This was dissolved in 20 g of a 1:1 mixture of cyclohexane and ethyl acetate, subjected to silica column chromatography packed with the same solvent, and purified using a 2:1 mixture of cyclohexane and ethyl acetate as the effluent. The target compound was obtained as a yellow powder of 380 rv by lyophilization with benzene.

NMR (270MHz ln CDC23) δppm
: 4.55 (IH, d.

J=1.0Hz, CCt, CH2), 4.53
(IH, d, J=1.0Hz.

CH2CH2), 4.31 (IH, d, J=
11.1, 3.2Hz, 13-H).

4.09 (2H-q, J=5.9 Hz-NI(
CH2CH2Co) -2,65(2H,t.

J=5.9Hz, NHCH2CH2Co), 1.
15-1.43 (12H, bs.

CH, (CH2), O) Reference Example 19 Rhizoxin-13-0-(3-(n-decyloxyphosphonoamidite)]] Soropionate Sodium Na Compound 272m9 of Reference Example 18 was dissolved in 27 m of THF, 1 molar sodium phosphate buffer with pH 4.2 27
While stirring vigorously, 2.79 g of zinc powder was added. After stirring at room temperature for 1 hour, the solvent was distilled off under reduced pressure, 30 ml each of ethyl acetate and 30 ml of sodium bicarbonate were added to the residue to dissolve it, and after filtering off insoluble matter, the organic layer was separated. The organic layer was washed with 0.1N hydrochloric acid, saturated brine, and 5% sodium bicarbonate for 20 hours each, and dried over magnesium sulfate.

After removing the desiccant by filtration, the solvent was distilled off under reduced pressure, and the residue was freeze-dried from benzene to obtain the desired product 202 as a yellow powdery substance.

NMR (270MH2lnCDC23) δppm: 4.
22 (IH, dd.

J=11.1, 3.2H2, 13-H), 3.7
3 (2H, bd, J=5.9Hz.

NHCH2CH2Co), 2.56 (2H, b
s, NHCH2CH2Co'), 1.12~1
．． 48 (IOH, bs, CH3 (CH2), 0) 10.
82 (3H, t, J=6.4Hz, CH3(CH2
), O) Reference Example 20 Rhizoxin 6-(2,2,2-)lichloroethoxycarponylamino)caproic acid ester Rh! -00
C(CH2) 5NHCOOCH2CC13 Rhizoxin 2.51. ! ? and 3.68i of 6-(2,2,2-trichloroethoxycarnylamino)caproic acid were dissolved in toluene, 2,06N of DCC was added, and 4 of the solvent amount was added.
- Pyrrolizonoviridine was added and stirred at room temperature for about 2 hours. Treatment and purification were performed in the same manner as in Reference Example 11 to obtain the target compound.

NMR (270MHz in CDCl2) δppm
: 5.67 (IH,b@.

NH), 4.71 (2H, l, ccz, cH2
o), 4.24(11(, dd, J=11.1.
3.2Hz, 13-H), 3.15 (2H2m
, NT(CH2) -2,30~2.45 (2H2m
-CH2C) Reference Example 21 Rhizoxin 6-aminocaproic acid ester Rbz-00
C(CH2)5NH.

2.95JF was dissolved in 60 d of tetrahydrofuran, and 60 ntl of IM-sodium phosphate aqueous solution was added thereto, followed by vigorous stirring. Zinc dust 10. OII was added in three portions every hour. After 3 hours, treatment and purification were carried out in the same manner as in Reference Example 14 to obtain 1.65.9 of the target compound.

NMR (270MHz 5n CDC13) δppm
: 4-25 (I H-dd pJ=11.1,
3.2Hz, 13-H), 2.96(2H,t
, J=5.4Hz.

NH2cH2), 2.30-2.4 s (2H,
m, CH2Co) Reference Example 22 Rhizoxin 6-(hectanoylamine) caproic acid ester Rhz-OOC(CH2)5NHCO(CH2)5CH
3 Rhizoxin 6-aminocaproic acid ester 246~ and heptanoic acid 130η were dissolved in toluene, and DCC17
1/V and a catalytic amount of 4-pyrrolidinopyridine,
The mixture was stirred at room temperature for about 2 hours. Treated in the same manner as Reference Example 1,
The target compound 187rn9 was obtained by purification.

NMR (270MH2lnCDC63) δppm:
5.83 (IH, bt.

J = 6.8Hz, NH), 4.28 (IH, d
d, J = 10.8, 3.8Hz, 13-H
), 1.18-1.35 (8H, bs, CH,
(CH2)40H2CO), 0.89(3H,t
, J=6.8Hz, CH, (CH2) 4CH2
Co ) Reference Example 23 Rhizoxin 6-(nonanoylamino)caproic acid ester Rh z -00C(CH2) 5N)(Co (CH
2) 246 to 7 CH5 rhizoxin 6-aminocaproic acid ester and 158 da of nonanoic acid were dissolved in toluene, DCC171rn9 and a catalytic amount of 4-pyrrolidinopyridine were added, and the mixture was stirred at room temperature for about 2 hours. Reference example 22
The product was treated and purified in the same manner as above to obtain the target compound 193Iv.

NMR (270MHz in CDC13) δppm
: 5.83 (IH, bt.

J = 6.8Hz, NH), 4.27 (IH, J
=10.8, 3.8Hz, 13-H).

1.23-1.36(12H, ba, CH3(CH2
)6CH2), 0.88(3H.

t, J=6.7H1, CH, (OH2>6CH, C
o) Reference Example 24 Rhizoxin 6-(undecanoylamino)caproic acid ester Rhz-00C(CH2)5NHCO(CH2) 9C
H3 lysoxin 6-aminocaproic acid Nisful 246■
and undecanoic acid 186 ~ dissolved in toluene, DCC
I71 and a catalytic amount of 4-pyrrolidinopyridine were added and stirred at room temperature for about 2 hours. The product was treated and purified in the same manner as in Reference Example 22 to obtain the target compound 175.

NMR (2701 z i CDCl2 ) δpp
m: 5.82 (IHlbt.

J = 6.8Hz, NH), 4.27 (IH, d
d, J=11.1, 3.7Hz, 13-H)
, 1.20~1.36 (16H, bs, CH
, (CH2)80M2Co) , 0.89(3H, t
, J==6.7Hz, CH3(CH2)8C1
(,Co) Reference Example 25 Rhizoxin 12- (2,2,2-)lichloroethoxylic nylamino)dodecanoic acid ester Rh z-00
C(0M2), , NHCOOCH2CC1゜Rizoxin 2.51g and 12-(2,2,2-)lichloroethoxylic? nylamino)dodecanoic acid 4.69.9
was dissolved in toluene, DCC2,0619 was added thereto, a catalytic amount of 4-pyrrolidinopyridine was added, and the mixture was stirred at room temperature for about 2 hours. Treated and purified in the same manner as in Reference Example 1,
The target compound 2.551 was obtained.

NMR (270MHz 1 n CDC1,) δppm
: 5.01 (IH, bv.

NH), 4.72(2H,l, CCA3CH2
0), 4.26 (IH, dd, J=10.0
, 4.1)(z, 13-H), 3.30(2H
, q, J=8.1Hz.

0CONHCH2), 2.38(2H,t, J
=8.1Hz, CH2Co), 1.23~x, 4
z(1sa, b Kasumi, NHC'H2(CH2), CH
2Co) Reference Example 26 Rhizoxin 12-aminododecanoic acid ester Rhz-0
0C (CHz) 11NH2 Rhizoxin 12- (2,
1.70 l of 2,2-)lichloroethoxylic nylamino) dodecanoic acid ester was dissolved in 40 m of tetrahydrofuran, and 4 m of an aqueous solution of IM-sodium phosphate was added thereto.
9m was added and stirred vigorously. Zinc dust 60Ii 3
It was divided into portions and added every hour. After 3 hours, reference example 14
Process and purify in the same manner as above to obtain the target compound 1.0! I got i.

NMR (270MHz in CDCt3) δP
pm: 4.26 (I Hs dd *J = 4.
3.10.3Hz, 13-H), 2.88(2H,
bt, J = 8.1Hz.

NI(2CH2), 2.35(2H,t, J=
10.8Hz, CH2Co), 1.66-1.7
8 (2H= m -CH2CH2C0) -1,25
~1.42 (16H2m -■2(CI(2), CH
2CH2C0) Reference Example 27 Rhizoxin 12-(propionylamino)dodecanoic acid ester Rh z-000(CH2) 1 , NHcoCH2C
Hs rhizoxin 12-aminododecanoic acid ester 411
Dissolve da and propionic acid 111 ~ in toluene, DC
C257In9 and a catalytic amount of 4-pyrrolidinopyridine were added and stirred at room temperature. The product was treated and purified in the same manner as in Reference Example 1 to obtain the target compound 267In9.

NMR (270MHz in CDCt, )δpp
m: 5.43 (IH, bs.

NH), 4.25 (IH, dd, J=4.3
, 10.3Hz, 13-H), 2.18(2H
= q, J=7.6 Hz -CB5 CH2C0)
-1,15(3H, t-J=7.6Hz, CB
, CH2Co) Reference Example 28 Rhizoxin 12-(valerylamine)dodecanoic acid ester Rhz-00C(CH2) 1. NHCO(CH2)
3CM3 Rhizoxin 12-aminododecanoic acid ester 4
111R9 and valeric acid 153 are dissolved in toluene, D
257 mg of CC and a catalytic amount of 4-pyrrolidinopyridine were added and stirred at room temperature. Processed in the same manner as Reference Example 27,
Purification yielded the target compound 319 da.

NMR (270MHz l n CDCt3) δpPr
n: 5.40 (IH, bs.

NH), 4.25 (IH, dd, J=10.0, 4.3
Hz, 13-H), 1.24-1.42 (15H, rn
), 0.91 (3H,t, J=7.0Hz,
CH, (CH2)Co) Reference Example 29 Rhizoxin-13-0-(3-n-nonyloxycarmonyl)propionic acid ester Rhizoxin i, ooIi was dissolved in 160 ml of anhydrous toluene, 3.2 g of succinic anhydride, and triethylamine. 3.2.
f. 961v of pyrrolidinopyridine was added, and the mixture was stirred at 60°C for 22 hours without moisture. The reaction solution was mixed with 0.2N hydrochloric acid, saturated saline, and 5% hydrogen carbonate) IJum aqueous solution x each
The organic layer was dried over anhydrous magnesium sulfate, and the solvent was distilled off under reduced pressure to obtain a caramel-like residue.

This residue was purified by silica dual column chromatography using methylene chloride containing 5% methanol as the effluent, and lyophilized with benzene to give a yellow powder of 1.2
21! Norizoxin-13-0-(3-calgexy)propionic acid ester was obtained. Dissolve 363 da of this yellow powder in 30 ml of anhydrous methylene chloride and dissolve n-no2#7A/:2.
216 Ing and 37 m9 of DCC154J19.4-pyrrolidinopyridine were added to the mixture, and the mixture was stirred at room temperature without moisture. After 2 hours, insoluble materials were removed by filtration, and the filtrate was mixed with 3Q each of 0.1N hydrochloric acid, saturated saline, and 5% sodium bicarbonate aqueous solution.
After washing with m/ and drying the organic layer with anhydrous magnesium sulfate,
The solvent was distilled off to obtain a caramel-like residue. This residue was purified by silica dull column chromatography using a 3:2 mixed solvent of cyclohexane and ethyl acetate as the effluent, the first peak was collected, and the solvent was distilled off. The residue was freeze-dried from benzene to obtain the desired product 214 as a yellow-white powdery substance.

NMR (270MHz ln CDC't3) δpp
m: 4.27 (IH, dd.

J=10.3, 4.0Hz, 13-H), 4
．． 10 (2H, t, J=6.8Hz.

C00CH2(CH2)7), 2.60-2.75(4
H1m, 00C(CH2)C00).

1.15-1.40 (14H, bs, C00CH2(
CH2), CH3), 0.87 (3H.

t, J=7, OHz, Coo(CH2)8CH
,)Reference Example 30 Rhizoxin-13-0-(3-n-(undecyloxycarbonyl))propionic acid ester O0 Instead of using n-nonyl alcohol in the reaction procedure to obtain the compound of Reference Example 29, n-undecyl alcohol was used. Prepare the target product as a yellowish-white powder in exactly the same manner except for using 1.
I got 77 da.

NMR (270MHz in CDCl2) δPpi
n: 4.27 (I H+ dd.

J=10.8, 3.8Hz, 13-H), 4
．． 10 (2H, t, J=6.8Hz.

C00CH2(CH2), ), 2.57-2.8
0 (4H, m, oc(ca2)2co).

1.15~1.40 (18H, bs, C00CH
2(CH2), CH3), 0.88(3H,t,
J=6.8H1), Coo(CH2), oC
H5) Reference Example 31 Rhizoxin-13-0-(3-n-()lysosyloxycarbonyl)]furopionate ester In the reaction procedure to obtain the compound of Reference Example 29, n-) The target product was obtained as a yellowish-white powder by the same procedure except that lysosyl alcohol 258~ was used.
2rn9 was obtained.

NMR (270MHz ln CDCl2) δppm
: 4.27 (IH, dd.

J = 10.3.3-2 Hz + 13-H), 4
10 (2H, t, J = 6-8 Hz + COOC
H2 (CH2) 4. CH3) -2,6o~2. s
o (4H, m.

00C(CH2)2Coo), 1.15~1.40
(22H, bs.

C00CH2(CH2)11CH3), 0.88(
3H,t, J==6.2Hz.

C00(CH2)1□CH3) Reference Example 32 3-(2,2,2-)IJdichlorotoxycarbonylamino)propionic acid 15 β-alanine in 170 ml of monoN sodium hydroxide
．． 06.9 was dissolved. 2.2.2-) Lichloroethoxycarponyl chloride 75F and -N hydroxide) IJium 340d were added dropwise thereto over 5 hours under ice-cooling. After the dropwise addition was completed, the mixture was returned to room temperature and stirred overnight.

Add 300 g of water and 200 rnl of ethyl ether to dissolve insoluble matter and separate the layers.
Washed twice. The aqueous layer was cooled with water and treated with concentrated hydrochloric acid to pH=1.
5 and extracted with 300M ethyl acetate. After washing with saturated brine and drying over anhydrous magnesium sulfate, the solvent was distilled off. Add 10% of cyclohexane and ethyl acetate to the residue.
0rlLl was added and left overnight at room temperature. The precipitate was collected by filtration, washed with 50 ml of cyclohexane, and dried to obtain the target compound 33.81.

Melting point m, p, 89.4-91.0℃6- (2,2
,2-) IJchloroethoxycardenylamino)caproic acid (melting point 87.3-89.0°C) and H12-(2
,2,2-)dichloroethoxycarnylamino)dodecanoic acid, melting point 69.8-72.6°C, was also synthesized using the same method as above.

Reference example 33 12-trichloroethoxylic? Nyloxydodecanoic acid 12-Hydroxydodecanoic acid 4.3211 was added to 200 ml of ethyl acetate. The mixture was dissolved in 30 WLt of water, added with diphenyldiazomethane, and stirred overnight at room temperature. After about 10 d of acetic acid was added to decompose excess diphenyldiazomethane, the organic layer was separated by washing with 100M water. This organic layer was mixed with 5 ml of sodium bicarbonate water, and 1001 nl of saturated salt solution.
The residue was washed twice with water, dried over anhydrous magnesium sulfate, and the solvent was distilled off to obtain 9.2 g of a yellow viscous crude residue. This residue was purified by silica gel column chromatography using cyclohexane containing 10% ethyl acetate as the eluent; 5.
12-Human tetraxidodecanoic acid penthydrylyl ester was obtained as a pale yellow viscous crude material of 21. Dissolve this ester in anhydrous ethyl acetate 400R1, add 13.7jlj of pyridine under ice cooling, and add trichloroethoxycarboxylic acid dissolved in 10ml of ethyl acetate. Nyl chloride 3.4
8.9 was added dropwise over about 15 minutes. Although insoluble salt precipitated, the mixture was returned to room temperature and stirred for an additional 2 hours. After washing the reaction solution with 0.5N hydrochloric acid 200M, saturated saline solution, 5
After washing the organic layer with magnesium sulfate and distilling off the solvent under reduced pressure, the resulting residue was poured into silica gel using cyclohexane containing 20 ethyl thiacetate as the eluate. Purification by column chromatography yielded a colorless and transparent viscous oil. This oily substance was dissolved in 50ml of anisole and 20ml of trifluoroacetic acid was cooled with water.
was added and left for 1 hour. The solvent was distilled off under reduced pressure, and the operation of adding and distilling off 30M toluene was repeated three times.
The residue was dissolved in ethyl acetate and 100 d of water. When saturated sodium bicarbonate water IQQm/ was added to the organic layer, it became emulsified, so the - was thickened to about 1 using concentrated hydrochloric acid. After separating the organic layer, it was washed with saturated brine Zoom/, and anhydrous magnesium sulfate was added. The solvent was distilled off under reduced pressure to obtain an oily residue. This was purified by silica gel column chromatography using a 2:l mixed solvent of cyclohexane and ethyl acetate as the effluent, and crystallized from n-hexane.3.96. The title compound was obtained as white disk-like crystals of F.

Melting point: 44.6-46.4°C Among the rhizoxin compounds of the present invention, preferably the formula (1
) having the rhizoxin formula (1) rhizoxin decanoate 2, butanoate 3, -3-methylbutanoate 4, rhizoxin octanoate 5, dodecanoate 6, tetradecanoate 7,
Hexadecanoate 8, Octadecanoate 9, Decanoate 5b
-Rikiru? In the compound having the formula (2), 1, rhizoxin benzoate 2, furocardate 3, l
nicotinic acid ester 4, z oleic acid ester 5, trans-2,2-dimethyl-3-(β-dichlorovinyl)cyclopropane caldic acid ester 6, 2-hexenecarboxylic acid ester 7, z methoxyacetic acid ester 8,
Phenylthioacetate 9, Rhizoxin α-
phenoxypropionic acid ester 10,
α-chloropropionic acid ester 11,
Cycloptan caldate ester 12, 2-methoxyethoxycarboxylic acid ester 13, orthofluorobenzoate ester 14, /subfluorobenzoate ester 15, cyclohexane caldate ester 16, 3-benzyloxypropionate ester 17, β - In compounds having the hydroxymyristate formula (transformation), 1, lysoxin 12-(2,2,2-)lichloroethoxylic? nyloxy)dodecanoic acid ester 2, #12-hydroxydodecanoic acid ester 3, 12-(propionyloxy)dodecanoic acid ester 4, 12-(valeryloxy)dodecanoic acid ester 5, l 3,3'-dithiodizolopionic acid diester 6゜7゜8゜9゜10゜11゜12゜13゜14゜15゜16゜17゜Rizoxin-13-0-7 Cyclohexylurea 1-
N(3,3'-dithiopropionate) 3-mercaptopropionate 3-(caprylthio)propionate 3-(lauroylthio)propionate 3-(myristoylthio)propionate 3- (2,2 , 2-trichloroethoxycarbonylamino)propionic acid ester 3-(penzyloxycarbonylamino)propionic acid ester 3-(t-butoxycarbonylamino)propionic acid ester 3-aminopropionic acid ester 3-(caprylamino)propionic acid Ester 3-(lauroylamino)propionic acid ester 3-(myristoylamino)propionic acid ester 18, Rhizoxin-13-0-(3-(n-decyloxy-trichloroethoxyphosphonoamidite))]
Propionate ester-13-0-[3-(n-decyloxyphosphonoamidite)]]Propionate ester-sodium-(2,2,2-)lichloroethoxycardenylamino)caproate ester 6-aminocaproic acid Ester 6-(heptanoylamino)caproic acid ester 6-(nonanoylamino)caproic acid ester 6-(undecanoylamino)caproic acid ester 12- (2,2,2-trichloroethoxycarbonylamino)dodecanoic acid varnish 77+z12 -aminododecanoic acid ester 12-(propionylamino)dodecanoic acid 21°26°19°23°25°20°24°22°27° ester 28, rhizoxin 12-(valerylamino)dodecanoic acid ester 29, -13-0- (3-n-nonyloxycarbunyl)propionic acid ester 30, -13-0- (3-n -(
undecyloxycarbonyl)]propionic acid ester 31, -13-0-(3-n-()ributyloxycarbonyl)]propionic acid ester.

Examples of the antioxidant having a phenolic hydroxyl group used in the present invention include salicylic acid, 2,6-di-t-butyl-cresol (BHT), 2(3)-butyl-4-
-hydric phenols and their salts such as oxyanisole (B)IA); resorcinic acid, gallic acid, ellagic acid,
Examples include low molecular weight polyphenols and their salts such as caffeic acid, pyrocatechol, and pyrogallol; tannins and their salts such as tannic acid, r-ranine, and capric acid; and flavonoids and their salts such as quercetin. Particularly preferred are ellagic acid, quercetin,
BHT and BHA.

Forms of formulations of rhizoxins and these antioxidants having phenolic hydroxyl groups include water, aromatic hydrocarbons such as benzene, ethers such as diethyl ether, organic acid esters such as ethyl acetate, N,N - Amides such as dimethylacetamide, pyrrolidones such as N-methyl-2-pyrrolidone, alcohols such as ethanol, soybean oil, sesame oil, peanut oil, corn oil, cottonseed oil, safflower oil, tung oil, ODO (dioctyldecyl triglyceride) ), organic solvents such as animal oils such as squalane; in particular, water-soluble solution preparations using N,N-dimethylacetamide, N-methyl-2-pyrrolidone, ethanol, etc., or water-soluble solution preparations and water. Mixed solution formulations, diluted preparations, and lyophilized formulations are preferred.

Furthermore, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene castor oil ester,
Contains a conventional amount of nonionic surfactant such as polyoxyethylene hydrogenated castor oil ester, polyoxyethylene fatty acid ester, polyoxyethylene alkyl ether, or polyoxyethylene/polyoxypropylene copolymer, and is diluted before use. It can also be a formulation or a lyophilized formulation.

The concentration of the antioxidant having a phenolic hydroxyl group in solution formulations, pre-diluted formulations, freeze-dried formulations, etc. is not particularly limited, but is preferably 0.01 to 51% KO, 01 to 51%.
1.0% is desirable.

The ratio of the antioxidant having a phenolic hydroxyl group to the rhizoxins is not particularly limited, but is 1 to 100%,
In particular, 10 to 50% is desirable.

Effects of the Invention The present invention will be explained in more detail below with reference to Examples.

Example 1゜Rizoxin 200II9 and antioxidant 20Da were added to N,N-
Brown ampoule K dissolved in dimethylacetamide 20a/
It was divided into 1aJ portions and kept at 60°C in a constant temperature bath, and the stability of rhizoxin over time was examined after 1 week, 2 weeks, and 4 weeks.

Quantification of rhizoxin was performed by high performance liquid chromatography using a reverse phase partition chromatography method.

That is, Ine was diluted to a concentration of acetonitrile KO.
The mixture was injected into rtsil 0DS-2 (manufactured by Gas Kuro Kogyo Co., Ltd.) and eluted with the above mobile phase at a flow rate of 2.0 ttl/min. Using Anthrone diluted in acetonitrile to a concentration of 0.05ψl as an internal standard, the absorption at 310 nm was measured and the retention time was 5.4.
The amount of silizoxin was determined by the area of the peak in minutes.

Note that this chromatography was performed at a temperature of 40°C. The results are shown in Table 1.

The numerical values in the table represent the residual rate (chi) of rhizoxin.

Table 1 The samples were kept at 60° C. in a warm bath, and their stability over time was examined after 1 week, 2 weeks, and 4 weeks. The quantification of rhizoxin was carried out in accordance with the method described in Example 1. The results are shown in Table 2. The numbers in the table represent the residual rate (chi) of zoxin.

Table 2 Example 2 Rhizoxin 600In9, polyoxyethylene hydrogenated castor oil ester (nonionic surfactant) 4Ii and antioxidant 60ml were dissolved in ethanol 4g and made into 10011LI with 10timannitol aqueous solution. 4d of this solution was divided into vials and freeze-dried, and 100 # of rhizoxin derivatives having the constant Example 3 formula (in IO, RL=c H, A=O) and the antioxidant were mixed with N,N-dimethylacetamide 2021/ The stability of the rhizoxin derivative over time was investigated by dissolving it in a thermostatic bath at 60°C.The rhizoxin derivative was quantified according to the method described in Example 1.The results are shown in Table 3. Values listed with antioxidants (
h) represents the concentration of the antioxidant, and the numerical value in the table represents the residual rate (1) of rhizoxin.

Table Example 4゜ 600 Ing of the rhizoxin derivative described in Example 3, 419 polyoxyethylene hydrogenated castor oil ester (nonionic surfactant) and 601 V of antioxidant were added in 4 ml of ethanol.
This was mixed with a 10 thimannitol solution to make 100 thimannitol. This solution was divided into vials of 4 d each and kept at 60° C. in a constant temperature bath to examine the stability of the rhizoxin derivative over time. Quantification of rhizoxin derivatives is
The method described in Example 1 was followed. The results are shown in Table 4.

The values in the table indicate the residual rate (%) of rhizoxin derivatives, and the rhizoxin compounds were well stabilized by adding an antioxidant having a phenolic hydroxyl group.

Claims (1)

[Claims] 1. A method for stabilizing rhizoxin compounds by adding one or more kinds of antioxidants having a phenolic hydroxyl group.