MXPA99001306A - Antibacterial compounds for ralstonia solanacea - Google Patents

Abstract

The present invention relates to antibacterial compounds for Ralstonia solanacearum having substantially (S) -3- (3-indoryl) butanoiso acid of a specific structure or its salt as active component can selectively inhibit the growth of Ralstonia solanacearum and reliably suppress wilting by bacteria even when used at a relatively low concentration

Description

ANTIBACTERIAL COMPOUNDS FOR RALSTONIA SOLANACEARÜM BACKGROUND OF THE INVENTION This invention relates to antibacterial compounds for Ralstonia solanacearum. Wilting by bacteria that affects tomatoes, eggplants, green peppers, tobacco plants, Japanese radishes, strawberries and the like is a type of bacterial disease caused by Ralstonia solanacearum. Ralstonia solanacearum is known for its high velocity of propagation, and its second invasion is very strong. Therefore, the wilt by bacteria caused by it can be a source of severe damage particularly for greenhouse crops and repeated crops. This invention relates to antibacterial compounds capable of reliably suppressing such wilting by bacteria even if used at a low concentration. Conventional methods for suppressing wilting by bacteria include disinfection by solar heat, restriction of root growth by means of sheets and grafts on resistant crops such as root stocks. With these conventional methods, however, the manifestation of the effects is extremely insufficient. The use of a disinfectant has also been, but the use of a disinfectant has negative effects not only on plants and soil but also on workers, in addition to suppressing wilting by bacteria. In view of the foregoing, Japanese patent publication Tokkai 7-228502 describes the use of synthesized 3- (3-indoyl) butanoic acid and its salts as antibacterial compounds capable of suppressing Ralstonia solanacearum. These compounds have the desirable characteristic of being capable of selectively inhibiting the growth of Ralstonia solanacearum which is the cause of wilting by bacteria and therefore suppressing wilting by bacteria without having any harmful effects on other bodies. However, the 3- (3-indoyl) -butanoic acid synthesized from the prior art and its salts have the disadvantage that they must be used at a relatively high concentration in order to relatively inhibit the growth of Ralstonia solanacearum.

BRIEF DESCRIPTION OF THE INVENTION It is therefore an object of this invention to provide an antibacterial compound capable of selectively suppressing the growth of Ralstonia solanacearum even when used at a relatively low concentration. This invention is based on the present discovery of the inventors that 3- (3-indoyl) butanoic acid is a racemic body containing both optical isomers of (S) -3- (3-indoyl) butanoic acid (which is the S body of 3- (3-indoryl) butanoic acid) as (R) -3- (3-indoyl) butanoic acid (which is the R-body of 3- (3-indoyl) butanoic acid) and which is the last of these two which has a significant chemical effect on the selective inhibition of the growth of Ralstonia solanacearum and is therefore able to reliably suppress wilting by bacteria, even when used at a relatively low concentration, without having any negative effect on other bodies.

DETAILED DESCRIPTION OF THE INVENTION This invention relates to antibacterial compounds for Ralstonia solanacearum, of which the active component is substantially acidic (S) -3 (3-indoyl) -butanoic shown by equation (1) given below or its salts: Formula 1) The synthesized 3- (3-indoyl) butanoic acid is a racemic body which contains as optical isomers both the above-mentioned (S) -3- (3-indoyl) butanoic acid shown by equation (1) given above and the acid ( R) -3- (3-indoryl) butanoic shown by equation (1) shown by formula (2) given below: Formula (2) As will be shown in more detail below, the (S) -3- (3-indoryl) butanoic acid given by the formula (1) above can be obtained by the following three procedures: (1) first method according to which a product of condensation of Meldrum acid and acetoaldehyde is passed through an addition reaction with indole to obtain a reaction product; (2) the second procedure according to which the reaction product obtained in the first process is dissolved in a mixed liquid of pyridine and alcohol, the copper is added thereto and a decarboxylation reaction is caused by heating under reflux to obtain body esters racemic acid 3- (3-indoyl) butanoic acid; and (3) third method according to which the esters obtained in the second process are subjected to an enzymatic process using lipase which is capable of selectively dissociating by hydrolysis only (S) -3- (3-indoryl) butanoic acid esters contained in the esters before said obtained in the second process to obtain therefore (S) -3- (3-indoryl) butanoic acid.

Examples of (S) -3- (3-indoryl) butanoic acid salt shown by equation (1) include those of an alkali metal such as potassium and sodium, those of an alkaline earth metal such as calcium and magnesium and ammonium salt . As will be described in more detail below, the acid (S) -3- (3-Indoryl) butanoic shown by equation (1) and its salts is more effective to selectively prevent the growth of Ralstonia solanacearum than (R) -3- (3-indoyl) -butanoic acid or its salts, and also more effective than the racemic body of 3- (3-indoyl) butanoic acid which is a mixture of these two, as well as its salts, and therefore can reliably suppress wilting by bacteria even at low concentration without bring a negative effect to other bodies. The embodiments of this invention can be considered to include the following: (1) antibacterial compounds for Ralstonia solanacearum having (S) -3- (3-indoyl) butanoic acid shown by equation (1) as an active component; (2) Antibacterial compounds for Ralstonia solanacearum having sodium salt of (S) -3- (3-indoryl) utanoic acid shown by equation (1) as an active component; and (3) antibacterial compounds for Ralstonia solanacearum "having ammonium salt of (S) -3- (3-indoryl) butanoic acid shown by equation (1) as an active component.

The invention will be explained immediately by means of the results of the experiments carried out.

EXPERIMENT PART 1 3 g of indole and 4.43 g of Meldrum's acid were dissolved in 30 ml of acetonitrile, and 3 ml each of refined acetaldehyde immediately before by distillation were added both to the start time of the reaction and two hours later to cause a reaction at 30 ° C for 6 hours with stirring. After the liquid reaction was condensed at a reduced pressure, it was dissolved in a mixed solvent of 30 ml of pyridine and 3 ml of ethanol, and 150 mg of copper particles were added for a reaction by heating under reflux for 6 hours. After the reaction liquid was filtered and the filtrate was made acidic (according to its pH) by adding 2N-hydrochloric acid, ether was used for extraction. The extracted liquid was washed with water and then dried with hydrated magnesium sulfate, the solvent was distilled under a reduced pressure. The residue was refined by silica gel column chromatography (gel: Wakogel C-200 (product trade name by Wako Puré Chemical Industries, Lts.); Solvent: hexane / ethyl acetate - 5/1) to obtain 2.25 g of racemic ethyl ester of 3- (3-indoryl) butanoic acid. A mixture l / l of 20 ml of ethanol and 10% of aqueous solution of potassium hydroxide were added to this racemic ethyl ester of 2.25 g of 3- (3-indoyl) butanoic acid, and was dried and hardened by means of of an evaporator after 6 hours of reflux. The water was added to this hardened dry substance, and was washed with ether. After activated carbon was added for heat decolorization, it was filtered to remove the activated carbon. 1.85 g of racemic body crystal of 3- (3-indoryl) butanoic acid were abstinated by adding 2N-hydrochloric acid to the bleached liquid to make the acidic solution.

EXPERIMENT PART 2 53. 6 mg of racemic ethyl ester of 3- (3-indoyl) butanoic acid, produced during the course of the part 1 experiment were dissolved in 0.1M acetic acid buffer (pH = 5) containing t-butanol by 10%, and Lipase AK (commercial name of product by Amano Pharmaceutical Co., Ltd.). The reaction liquid was filtered with celite and, after the filtered liquid was made acidic (according to its pH) by adding IN-hydrochloric acid, it was extracted using ethyl acetate. After the extracted liquid was dried with hydrated sodium sulfate, the solvent was distilled under reduced pressure. The recurrence was analyzed through a column of silica gel (gel: akogel).

First, 13.4 mg of (R) -3- (3-Indoryl) butanoic acid ethyl ester not decomposed by Lipase AK were levigated out using a dichloromethane / ethyl acetate = 9/1 mixture as a solvent. Then, 13.1 mg of (S) -3- (3-indoryl) butanoic acid were obtained by levigation using a dichloromethane / ethyl acetate = 1/1 mixture. The optical purity of (S) -3- (3-indoyl) butanoic acid by a high performance liquid chromatography method was 99%.

EXPERIMENT PART 3 13 mg of the ethyl ester of (R) -3- (3-indoryl) -butanoic acid obtained from the experiment part 2 above were dissolved in 0.1 M phosphoric acid buffer (pH = 7.3) containing t-butanol by 10%. 62 mg of porcine liver esterase (commercial product name by Sigma-Aldrich Co.) were added to this solution for a reaction at 30 ° C for 24 hours with stirring. The reaction liquid was filtered and after 1N hydrochloric acid was added to the filtrate to make it acidic (according to its pH), it was extracted using ethyl acetate. The extracted liquid was washed with water and then dried using hydrated sodium sulfate, the solvent was distilled under reduced pressure. The residue was refined by silica gel column chromatography (gel: Wakogel C-200, solvent: dichloromethane / ethyl acetate = 1/1) to obtain 7 mg of (R) -3- (3-indoryl) butanoic acid. Its optical purity by the HPLC method was 99 ' EXPERIMENT PART 4 By the method of minimum inhibitory concentration (MIC) which will be described in detail immediately, the antibacterial activities against the race 1 of Ralstonia solanacearum of the racemic body, the body S and the body R of the 3- (3-indoryl) butanoic acid obtained in the experimental parts 1, 2 and 3 described above were measured. The results are shown in table 1 below. According to this MIC method, the racemic body, body S and body R of the 3- (3-indoyl) butanoic acid obtained in experiment parts 1, 2 and 3 were each dissolved in 4% methyl alcohol and these solutions were each mixed with an equal amount of a dual resistance PCG liquid medium (10 g of bacto-peptone + 1 g of casamino acid + 10 g of glucose per liter) such that the concentrations (in μg / ml ) of the racemic body, body S and body R would be as given in table 1. The liquid medium thus prepared was filtered through a 0.22 μm pore size membrane to reduce the bacteria count to provide a medium test. Separately, race 1 of Ralstonia solanacearum bacteria was preliminarily cultured using a liquid PCG medium and after they were pooled, a bacterial suspension of 5 x 10 5 cells / ml was obtained in physiological saline. These 60 μl of bacterial suspension were injected into 3 ml of the above test medium and shaken for cultivation at 30 ° C for 24 hours. The cultivation of strain 1 of Ralstonia solanacearum was measured using the turbidity of this culture liquid at 600 nm as an indicator. Because the turbidity of the culture liquid would be the same as that of a non-inoculated culture liquid if the cultivation of racemic bacteria of Ralstonia solanacearum was completely inhibited by the racemic body, S or R, the concentration of the racemic body, S or R at this time would be defined as the MIC.

TABLE 1 In the box: +: Cultivation observed -: Inhibited culture Table 1 shows that the MIC of 3- (3-indoryl) butanoic acid against race 1 of Ralstonia solanacearum is almost 10 μg / ml in the case of the R body and almost 2.5 μg / ml in the case of the racemic body but this is only 1.0 μg / ml. This means that the use of (S) -3- (3-indoryl) butanoic acid or its salt as an antibacterial compound is much more effective in inhibiting the growth of Ralstonia solanacearum and therefore in reliably and selectively suppressing bacterial wilt yet if used at a relatively low concentration without causing any harmful effect on other bodies.

Claims (2)

NOVELTY OF THE INVENTION CLAIMS

1. An antibacterial compound for Ralstonia solanacearum, the active component of said compound consisting of (S) -3- (3-indoyl) butanoic acid shown by formula 1 given below or a salt thereof: Formula 1

2. The antibacterial compound according to claim 1, further characterized in that said (S) -3- (3-indoryl) butanoic acid shown by formula 1 is obtained by a first method according to which a condensation product of Meldrum acid and acetoaldehyde is passed through an addition reaction with indole to obtain a reaction product, a second process according to which said reaction product is dissolved in a mixed liquid of pyridine and alcohol, the copper is added thereto and a reaction of decarboxylation is caused by heating under reflux to obtain racemic body esters of 3- (3-indoryl) butanoic acid, and a third method according to which the esters obtained in said second process are subjected to an enzymatic procedure using lipase which is capable of disassociating selectively by hydrolysis only esters of (S) -3- (3-indoyl) butanoic acid contained in said esters obte nests in said second process to thereby obtain (S) -3- (3-indoryl) butanoic acid.