JPH0215555B2 - - Google Patents

Description

[Detailed description of the invention]

(Field of Industrial Application) The present invention relates to a novel halogenated derivative of SF-2370 substance having antibacterial, antifungal, antihypertensive, and diuretic effects, and also to a method for producing this novel derivative. (Conventional technology and problems to be solved) The antibiotic SF-2370 is an actinomycete belonging to the genus Actinomadura, SF-2370.
This is a new antibiotic isolated from the culture solution of the 2370 strain (Feikoken Bibori No. 7760), which exhibits antibacterial and antifungal effects (Patent application No. 59-210524 filed by the present applicant).
(see specification). The SF-2370 substance has a strong control effect on rice and leaf blight as an agricultural fungicide, for example (Japanese Patent Application No. 60-93755).
-2370 substances have insufficient antibacterial activity. Therefore, in order to utilize SF-2370 substance more effectively, it is desired to enhance its activity through formulation technology, chemical conversion, etc. The present inventors investigated the chemical structure of SF-2370 substance, and found that the following formula () It was revealed that it has the chemical structure shown in (J.
Antibiotics, Vol. 38, pp. 1437-1439 (1985)).
Next, we inferred the chemical reactivity of the SF-2370 substance from the chemical structural formula () and conducted various studies for the purpose of enhancing antibacterial activity, and found that the aromatic ring hydrogen of the SF-2370 substance was replaced with a halogen atom. Find out that you can use the formula below () We succeeded in newly synthesizing a halogenated derivative of SF-2370 substance represented by the following formula: [wherein X represents a chlorine, bromine or iodine atom, and Y represents a hydrogen, chlorine or bromine atom]. When we investigated the antibacterial, antifungal and other physiological activities of this novel SF-2370 substance halogenated derivative of formula (), we found that it not only enhanced its antibacterial and antifungal effects, but also exhibited various physiological activities, such as It has now been discovered that it also exhibits blood pressure-lowering and diuretic effects. That is, the present invention was completed by not only discovering a novel active substance, SF-2370 derivative, but also converting it into a halogen derivative, expanding the use of SF-2370 substance to the pharmaceutical field. (Means for solving the problems) Therefore, the gist of the first invention is as follows:
The formula below () In the formula, X represents a chlorine, bromine or iodine atom, and Y represents a hydrogen, chlorine or bromine atom. In the halogenated derivative of formula (), when X is chlorine and Y is a hydrogen atom, when X and Y are both chlorine atoms, when X is bromine and Y is a hydrogen atom, when X and Y are both bromine If it is an atom, X
When is iodine and Y is hydrogen atom, etc.,
It can be a monohalogenated product or a dihalogenated product. The second aspect of the present invention is a method for producing a halogenated derivative of the SF-2370 substance of the formula (), which is obtained by halogenating the SF-2370 substance of the formula (). The compound of the present invention can be produced by reacting SF-2370 substance () as a raw material with a halogenating agent, preferably in an inert solvent, to perform a halogenation reaction, and The derivative can be obtained by purification and isolation using conventional methods. Examples of halogenating agents used include known ones, such as elemental chlorine, bromine, iodine,
Examples include sulfuryl halides and N-halogenosuccinimides. Among these, N-halosuccinimide is preferred because it is readily available in pure form and is easy to handle. Examples of inert solvents that can be used include alcohols such as tetrahydrofuran, dioxane, lower alkanols, and halogenated alkanes. of starting material
Tetrahydrofuran and dioxane are preferred because they dissolve SF-2370 materials well. The halogenation reaction proceeds at room temperature, for example, 20 to 25°C, but it can be heated to, for example, 60 to 80°C for the purpose of promoting the reaction.
The progress of the reaction can be easily observed by analytical methods such as silica gel thin layer chromatography. Isolation of the new derivative () from the reaction solution is carried out by commonly used purification methods, such as solvent extraction, washing, recrystallization, or column chromatography. derivative () is obtained. When the physiological activity of the novel SF-2370 derivative () of the present invention was investigated, the SF-2370 substance ()
It was found to have enhanced antibacterial and antifungal activities, as well as strong antihypertensive and diuretic effects. Examples of experiments illustrating the hypotensive and diuretic effects of the compounds of the present invention are shown below. Example 1 Antihypertensive effect of DOCA/saline in hypertensive rats. DOCA/saline hypertensive rats were prepared by the method described below, and the antihypertensive effect of the halogenated derivative of the SF-2370 substance of the present invention () was examined using a non-invasive method. Specifically, one kidney of an SD rat weighing 180 to 200 g was removed under ether anesthesia, and then the rat was raised with 1% saline as drinking water.
During the breeding period, DOCA (deoxycorticosterone)
acetate) 4% gum arabic suspension once a week,
Administered subcutaneously at 10 mg/Kg. Among the rats reared in this manner, those whose blood pressure was consistently 170 mmHg or higher after 4 weeks or more after surgery were selected and used as test animals. SF-2370 substance derivative () was used as a test drug.
% aqueous gum arabic solution and administered orally.
Blood pressure was measured at a certain time after administration using the following method. That is, the test rat was kept warm at 37°C for about 5 minutes in advance to allow the tail artery to dilate well, and a non-invasive blood pressure measuring device (manufactured by Ueda Seisakusho, UM101) was used.
It was measured by the tail cuff method. The results are shown in Table 1.

[Table]
Experimental Example 2 Antihypertensive effect in spontaneously hypertensive rats (SHR). SHR purchased from Charles River Japan
Among them, those with blood pressure of 170 mmHg or higher were adopted as test animals. The rest was the same as in Experiment 1.
SF-2370 substance derivative () was administered, and blood pressure drop values were measured. The results are shown in Table 2.

[Table] Experimental Example 3 Effect on urine volume and electrolyte excretion. Using SD rats (5 rats per group) that had been fasted overnight,
The test compound was suspended in 5% gum arabic solution and administered orally. 30 minutes after administration of test compound, physiological saline solution
The rats were orally loaded at a rate of 2.5 ml/100 g, placed in individual metabolic cages, and the urine excreted within 5 hours was collected.
The urine volume and the amount of Na ⁺ in the urine were measured. Urinary Na ⁺ amount is measured using ion analyzer Mo−
Measured by del407A (ORION Res.). The general properties of urine were qualitatively examined using urine test strips (Maluteistakes). The results are shown in Table 3.

[Table] Experimental Example 4 The acute toxicity of the compound of the present invention was tested by oral administration using mice. The results are shown in Table 4.

[Table] In addition, the present invention compound 1, present invention compound 2, present invention compound 3, present invention compound 4, and present invention compound 5 referred to in Experimental Examples 1 to 4 above refer to Examples 1 to 5 described later, respectively. Dibrominated SF-2370 substance, monobrominated SF-2370 substance, monoiodination obtained in 4.
This refers to SF-2370 substances, dichlorinated SF-2370 substances, and monochlorinated SF-2370 substances. From the above animal experiment examples, it is clear that the halogenated derivative of SF-2370 of the present invention has antihypertensive and diuretic effects, and its effects were equivalent to or better than that of the known drug Hydroflumethiazide. In addition, its acute toxicity in mice is low, so it is highly expected to be used as a medicine. Examples of the production of the compound () of the present invention are shown below as examples. Example 1 Dibrominated SF-2370 substance (Compound 1 of the present invention)
Manufacturing of. 1.0g of SF-2370 substance in 30ml of tetrahydrofuran
600 mg of N-bromosuccinimide was added thereto, and the mixture was reacted at 20 to 25°C for 7.5 hours. Concentrate the reaction solution under reduced pressure, and add 20 ml of methanol to the solid residue.
was added, heated, stirred, cooled, and filtered through a crystalline substance to obtain a dibrominated SF-2370 substance. Yield 1.07g Yield 79%, decomposition point 280℃ or higher. Rf value (Merck silica gel TLC; solvent system, ethyl acetate): 0.64. ¹ H-NMR spectrum (CDCl ₃ , ppm); 8.60s,
7.98s, 7.85d, 7.61dd, 7.23d, 7.13d, 6.77dd,
5.63brs, 5.26s, 4.60d, 4.37s, 4.11s, 3.69dd,
3.04d, 2.15s, Mass spectrum (FD); 623, 625, 627 (M ⁺ ) Example 2 Monobrominated SF-2370 substance (invention compound 2)
Manufacturing of. 1.0g of SF-2370 substance in 30ml of tetrahydrofuran
360 mg of N-bromosuccinimide was added thereto, and the mixture was reacted at 20 to 25°C for 7.5 hours. The reaction solution was concentrated under reduced pressure, and the residue was washed with 20 ml of methanol.
958 mg of crude crystals were obtained (crude yield 82%). The crude crystals were purified by column chromatography using 140 g of silica gel (solvent system, toluene-ethyl acetate (1:1 to 0:1)), and monobrominated SF-
Obtained 488 mg of 2370 substances. Yield 42%, decomposition point 275℃. Rf value (measurement conditions are the same as Example 1): 0.54 ¹ H-NMR spectrum (CDCl ₃ , ppm); 8.59m,
8.05d, 7.90d, 7.57m, 7.46m, 7.23m, 7.09d,
6.75dd, 6.04brs, 4.71brs, 4.50d, 4.37m, 4.10s,
3.78dd, 3.29dd, 2.16s, Mass spectrum (FD); 545, 547 (M ⁺ ) Example 3 Monoiodinated SF-2370 substance (invention compound 3)
Manufacturing of. 1.0g of SF-2370 substance in 30ml of tetrahydrofuran
900 mg of N-iodosuccinimide was added thereto, and the mixture was reacted at 20 to 25°C for 2 days. The reaction solution was extracted with 100 ml of ethyl acetate and 50 ml of water, and the ethyl acetate layer was separated, washed with water, and dried over anhydrous sodium sulfate. The solid residue obtained by concentrating the ethyl acetate solution was purified by column chromatography using 100 g of silica gel (solvent system: toluene-dioxane (5:1)). Fractionate the eluate into 7ml portions, examine each fraction by thin layer chromatography (developing system: ethyl acetate), collect the fractions with an Rf value of 0.57, and concentrate under reduced pressure to obtain a crystalline substance. Ta. 5ml hot methanol
After washing and drying, 240 mg of monoiodized SF-2370 material was obtained. Yield 18.9%, decomposition point 257-259℃ ¹ H-NMR spectrum (CDCl ₃ , ppm); 8.80d,
8.04d, 7.91d, 7.56m, 7.46t, 7.43dd, 7.09d,
6.78dd, 5.90brs, 4.53d, 4.42d, 4.10s, 3.84dd,
3.25dd, 2.16s. The ¹ NMR spectrum curve diagram is shown in Figure 1 of the attached drawings. Mass spectrum (SIMS); 594 (M ⁺ ) Example 4 Dichlorinated SF-2370 substance (compound of the present invention 4)
and production of monochlorinated SF-2370 substance (invention compound 5). SF-2370 substance 467mg in tetrahydrofuran 15ml
199 mg of N-chlorosuccinimide was added thereto, and the mixture was reacted at 20 to 25°C for 12 days. The reaction solution was concentrated under reduced pressure, and the resulting residue was subjected to column chromatography using 70 g of silica gel (solvent system: toluene-
ethyl acetate (1:1)). 4ml of eluate
Each fraction was analyzed by thin layer chromatography in the same manner as in Example 3. Fractions of a substance exhibiting an Rf value of 0.59 were collected, concentrated under reduced pressure, and 7 ml of methanol was added to filter out the precipitated crystals to obtain 167 mg of dichlorinated SF-2370 substance. Yield 31%, decomposition point 298-300°C Mass spectrum (FD): 537 (M ⁺ ). Fractions in which a substance exhibiting an Rf value of 0.52 was eluted were collected and concentrated under reduced pressure. When methanol was added, crystals of monochlorinated SF-2370 substance precipitated and were collected. Yield 166 mg (34% yield), decomposition point 293-294
°C Mass spectrum (FD); 501 (M ⁺ ). Note that the SF-2370 substance used as a raw material compound in the method of the present invention is itself a new substance, so
A manufacturing example thereof is shown below as a reference example. Reference Example 1 (Manufacture of SF-2370 substance) Glucose 2.0g, wheat germ 1.0%, peptone 0.5
%, yeast extract 0.5%, and calcium carbonate 0.1% (PH 7.0) was dispensed into a 100 ml Erlenmeyer flask and sterilized at 120° C. for 15 minutes. This was inoculated with the microorganism Actinomadilla sp. SF-2370 (Feikoken Bacteria No. 7760), and cultured at 28°C for 7 days at 220 revolutions per minute. 20 ml of this culture was mixed into 600 ml of production medium (PH 7.0) consisting of 1.5% glucose, 1.0% wheat germ, 1.0% cornstarch liquor, 0.5% firma media, and 0.3% calcium carbonate.
Inoculate a 1-volume jar containing 28
Culture with aeration at ℃ for 5 days (aeration rate: 600 ml per minute,
The rotation speed was 500 revolutions per minute). After the culture was completed, diatomaceous earth was used as an auxiliary agent to obtain cultured bacterial cells. 500 ml of 70% acetone water was added to the bacterial cells to extract the active ingredient, and the bacterial cells were separated. The bacterial cell extract was then concentrated under reduced pressure to remove acetone, and 250 ml of ethyl acetate was added to 250 ml of the resulting concentrated solution and shaken to extract the active ingredients. This extraction operation was repeated twice, and 500 ml of the resulting ethyl acetate extract was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain an oily substance. N-hexane was added to this oily substance, and the resulting precipitate was collected to obtain 286 mg of a crude substance. This crude substance was dissolved in a mixture of chloroform and ethyl acetate (10:1), and silica gel C-200 (manufactured by Wako Pure Chemical Industries, Ltd.) was used.
It was applied to a 60 ml column and developed with 600 ml of a chloroform-ethyl acetate (10:1) mixture. The developing solution is silica gel thin layer chromatography (Merck, Kieselgel, 60F254, 5714; developing solvent: ethyl acetate)
The spot showed an Rf value of 0.58 when irradiated with ultraviolet light (254 nm), and Sarcina.
Fractions showing antibacterial activity were collected in a paper disc bioassay using Sarcina lutea as the test bacterium. This active fraction was concentrated to dryness under reduced pressure to obtain 216 mg of pale yellow powder. This crude powder was dissolved in ethyl acetate and subjected to preparative silica gel thin layer chromatography (Merck Kieselgel 60F254,
5744, developing solvent: ethyl acetate), the active portion (Rf value 0.58) was scraped off, and extracted with ethyl acetate.
After concentrating the extract under reduced pressure, methanol was added and left overnight to obtain 79 mg of pale yellow crystals of SF-2370 substance. Reference Example 2 (Manufacture of SF-2370 substance) Seed culture 1 of Actinomadilla sp. SF-2370 obtained in the same manner as described in Reference Example 1 was placed in a 50-volume microplate containing 35 of the production medium. The cells were inoculated into two Yafa Armenters, and cultured with aeration at 28° C. for 5 days (aeration rate: 35 per minute, rotation speed: 200 revolutions per minute). After the cultivation was completed, 25% of 70% acetone water was added to the microbial cells obtained by straining the culture to extract the active ingredients twice, and the microbial cells were separated to obtain 50% of an extract. This extract was concentrated under reduced pressure to remove acetone, and the resulting concentrate was added with 30% ethyl acetate.
was added and stirred for 15 minutes to extract the active ingredient.
The ethyl acetate extract was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain 33 g of pale yellow crude crystals. The crude crystals were recrystallized twice from a chloroform-methanol mixture to obtain 15 g of pale yellow crystals of SF-2370 substance. For more detailed physical properties and manufacturing method of SF-2370 substance, please refer to the patent application filed by the applicant in 1982-
Reference is made to the description in specification No. 210524.

[Brief explanation of drawings]

FIG. 1 is a ¹ H-NMR spectrum (measured at 400 MHz in deuterated chloroform) of the monoiodized SF-2370 substance obtained in Example 3 of the present invention.

Claims (1)

[Claims] 1. The following formula () A compound which is a halogenated derivative of SF-2370 substance represented by the following formula: [wherein X represents a chlorine, bromine or iodine atom, and Y represents a hydrogen, chlorine or bromine atom]. 2. The compound according to claim 1, wherein in formula (), X is a chlorine atom and Y is a hydrogen atom. 3. The compound according to claim 1, wherein in formula (), X and Y are chlorine atoms. 4. The compound according to claim 1, wherein in formula (), X is a bromine atom and Y is a hydrogen atom. 5. The compound according to claim 1, wherein in formula (), X and Y are bromine atoms. 6. The compound according to claim 1, wherein in formula (), X is an iodine atom and Y is a hydrogen atom. 7 The following formula () The following formula () is characterized by halogenating the antibiotic SF-2370 substance shown by A method for producing a compound which is a halogenated derivative of SF-2370 substance represented by the following formula: [wherein X represents a chlorine, bromine or iodine atom, and Y represents a hydrogen, chlorine or bromine atom].