JPS60258196A - Novel dactimicin derivative and its preparation - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION This invention relates to a derivative or a salt thereof. In addition, the present invention provides a novel novel method by microbially converting the chemical structure of hothymidine B using a strain of the genus Streptomyces that has the ability to produce hothymidine B but also has the ability to convert hothymidine B into a dactymicin derivative. The present invention relates to a method for producing dactymicin derivatives.

The novel dactymicin derivatives provided by the present invention exhibit rapid antibacterial activity and are extremely useful as antibacterial agents.

Hotimidine B, represented by the following formula, is one of the known antibiotics (*Kaisho Guter/No. 261P2, "Gynal.
"Op Antibiotic" Vol. 30, pp. 133-70 (
However, their antibacterial activity is not always satisfactory. The present inventors found that f
'L7'5 During research to produce antibiotics, when a certain type of microorganism was used, hothymidine B was converted to the following formula [I]
All findings indicate that dactymicin is converted into a derivative of dactymicin. The Dakuchi sewing machine is made by Tokukai Sho! ! It is an f'L'fc compound described in the T4TR Publication No. 7 under the name of SF-, 20j substance.

Accordingly, the seventh aspect of the present invention provides a novel dactymicin derivative represented by the following formula % and an acid addition salt thereof.

The compound of the present invention of formula [1] has a chemical structure corresponding to nfc, in which the amine group at the 7-position of hothymidine B is converted to shrimp, and the methylamino group at the G-position is formed to formimidoyl glycylation.

Examples of stereoisomers of the compound of the present invention represented by formula (1) include n/-evidactymicin, i.e., /-evidactymicin, represented by the following three-dimensional structural formula, i.e., /-shrimp-2''
- Formimidoylformimidoylformimidine k.

The compound of formula (1-/) (lyophilized product) exhibits the following physical and chemical properties.

(1) Basic sulfate of this substance: white powder (2) Solubility: extremely soluble in water, methanol.

Ethanol, acetone, chloroform, benzene, ethyl acetate, butyl acetate, ether.

It is insoluble in organic solvents such as n-hexane.

(3) Elemental analysis value (OlsHssNsOs・2H snow SO
4.g H4o): OHN Theoretical value (%) 30. ♂j tJO //, Tata experimental value (
@ 30.71 7,0/ /, 2.00 (→ Melting point: 2
Gradual heat content M (browning) at temperatures above 0j°C.

(accompanied by foaming and melting) (ri optical rotation: [α]
7) Infrared absorption spectrum (KBr tablet): As shown in FIG. 1:: Maximum absorption (am-”): 31100, 2po
, ,2o<to.

/7/! , /130. /100. /1100. /3tO
.

/32. f, /, 2tO, // 10, 10170 (♂)
Nuclear magnetic resonance spectrum (in heavy water): As shown in Figure 2.

Characteristic signal: /, J, jppm (d, JH, J=
Aj Hz )---・A' -C-methyl, /, A
p, pm (m/H)...g'-methylene, , 2
．． 0 j ppm ~ λ, /jppm (ml J H)
・,, 3/, 4t/-methylene, J, /j
ppm(s,J)()・4-N-methyl, 3. II
/ppm(m, /HJ6', □H3=1.
JHz)-A'-mefy.

3J/ppm (s, JH) J-0-methyl.

3, tOppm (m,/H)-co'-methine, 3. r♂ppm (/'H) ・r'-methine, 3
．． ri J ppm (t.

/ H, J! ;2=J,7H2,JB6=JjHz)
−/−Methine, Il, Ot ppm (d, d
,/H,J3;2==J,JHz, ,J3;4
= / / Hz) 3-methine, 440.23
ppm (t, 1H)-A-methine, 3.31 ppm (
d.

/ H, J = / 7. j Hz) and llj!
7 ppm (d.

/ H, J = / 7.1 Hz) 2”-methylene II, lI2ppm (/H) ・-j methylene, l
lj A ppm (d, d, /H)・g-methylene, 4t, 7ppm (t, /H)・=x-methine, ! , 11 A ppm (d*/H, JI
', 2' ==J, JHz)...//-Anomeric proton, 7.7 gppm (S, /H)...
Molecular ion peak in the proton mass spectrum (SIM8) of 3# (formimidoyl): 4tJJ (M+/) C10) Retention time by high performance liquid diurography:
As shown in Table 1.

Table l/) Column...Dipero Seal 0D8-7 (0-20
%, jμ), 6mφ×cooo.

h) Solvent liquid...θ, 2 moles of liquid, I and 0,
0.0 J mol containing sodium l-pentanesulfonate.
/ (1) Acetonitrile solution of acetic acid aqueous solution 3) Detection method: UV detection of 3g 4'mμ by orthophthalaldehyde reaction method (//) Rf value by thin layer chromatography: As shown in Table 2 Table 2: nf value by thin layer chromatography (shown in comparison with known aminoglycoside antibiotics)
) Silica gel thin layer plate: Kiesel gel OF2B4 ((:', -2, tw
m, 200wm×λθ, etc.) developing solvent; n-pronognol: t OV/W sodium acetate:
Acetic acid (,!':≠2/) Antibacterial spectrum of the compound of formula CI-/) according to the invention [)
・Hochimidine B (abbreviated as FM-B), hochimidine xjpM-person), $cutimicin (D
(abbreviated as AO) is shown in Table 3 below.

As is clear from the above table, the compound of the present invention of formula (1) exhibits a wide range of and strong antibacterial activity against various Gram-positive and Gram-negative bacteria, and in particular, it exhibits several times as much antibacterial activity as the starting material hothymidine B. The antibacterial activity is enhanced several tens of times, and it has a stronger antibacterial activity that is comparable to Hotimidine A and Dactymicin.

Examples of acid addition salts of the compounds of the invention include pharmaceutically acceptable inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, or organic acids such as acetic acid.

There are salts with propionic acid, citric acid, methanesulfonic acid, etc.

According to the first aspect of the present invention, a bacterium belonging to the genus Streptomyces and having the ability to convert the amino group at the 7-position of hotimidine B and introduce a formimidoylglycyl group into the methylamino group at the hi-position is used to act on hotimidine B. Provided is a method for producing a dactymicin derivative represented by the following formula or an acid addition salt thereof, which is characterized by:

In the method of the present invention, compound [I] is hothymidine B
is produced by contacting with a strain belonging to the genus Streptomyces and having the above-mentioned conversion ability, or a mutant strain thereof, and the
The amino group at the position is converted to a shrimp, and the methylagno group at the g position is converted to formimidoyl 1).

There are no particular restrictions on the strain used in the method of the present invention, as long as it is capable of converting glycine.

An example of a strain that can be used in the method of the present invention is Streptomyces tenzimariensis, which is known as an istamycin-producing bacterium.
mariensis) S S-ta 3 strain (FFiRM
-P! 2) (refer to Japanese Patent Application Laid-Open Publication No. 2003-111004), and Streptomyces tenzimariensis 88/!07.The present 88-1107 strain It has been deposited with the National Institute of Microbial Technology, and its accession number is FERM-P.

In addition, mutant strains can be obtained from istamycin-producing strains belonging to the genus Streptomyces, for example, by using ultraviolet irradiation, cobalt zO irradiation, X-ray irradiation, or mutagenic agents such as nitrone compounds, acridine dye compounds, and nucleobase analogues. There are many things that can be obtained through artificial mutation. Suitable mutant strains to be used in this method include those that do not have the ability to produce -f stamycin or have extremely reduced production ability;
In addition, it has the property of converting the amine group at the 7-position of hothymidine B and converting the methylamino group at the μ-position to formimidoyl glycylation. A representative example of a mutant strain that can be used in the method of the present invention is Streptomyces tenjimari: 1-7, which the present inventors derived from Streptomyces tenjimariensis 5s-itO7.
8treptomyces tenjlmar
iensia) SS-/, t07U-11/f. This strain has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, and its accession number is FERM-'P77λo. Streptomyces tenzimariensis (Streptomyces tenzimariensis) by Rin et al.
cestenjimariensis) S S -/
707 Streptomyces 'tenj1mari
ensis) S S -/! 07 U-4t
The mycological properties of both are exactly the same and are as shown below.

A) Streptomyces tenjimariensis that grew well on the morphological agar medium.
ariansis) S S -/! 07 strain and Stresotomyces tenzimariensis (Strep
tomyces tenjimariensis) S
The S −/ and t07U-μ/ strains simply branch, elongating straight or slightly curved aerial hyphae from well-elongated basal hyphae, and when mature, have io and t at the tips.

Forms a chain of long cylindrical spores (width 1 micron, length μ~! micron). Spirals and axle branches are not shown.

Under an electron microscope, the spore surface was smooth and no spine-like or hair-like structures were observed.

It is a typical Streptomyces without flagella or sporangia.

B) Characteristics on each weighing medium ■ Sucrose/nitrate agar medium (cultured at 27°C);
White aerial hyphae appear on weak colorless growth, which gradually turns blue-greenish gray (/7ec, aqua blue,
Color) --- According to Mony Manual, the same applies hereafter)
increase. It does not produce significant diffusible pigments in the medium.

■ Glycerin/Asunoguragin agar medium (cultured at 27°C): Almost the same as that, but aerial mycelia are difficult to attach to.

(2) Starch agar (cultured at -27°C): Almost the same as that, but aerial mycelium grows on it, and the starch around the bacterial growth becomes transparent.

(2) Tyrosine agar medium (cultured at 27°C); almost the same findings as (2); melanin-like pigment is produced.

■ Nutrient agar medium (cultured at 27°C); White aerial mycelium grows on the colorless growth, and the medium becomes brownish.

(2) Yeast/malt agar medium (cultured at 27°C); white aerial mycelium grows on colorless growth, gradually turning blue-greenish gray (/9 dc aquagray). The medium appears brownish in color.

■ Oatmeal agar medium (cultured at 27°C); white to blue-gray color on colorless growth (/7ec.

Aerial mycelia with aqua blue color are attached.

0) Physiological properties. -7,. A growing Yuno Ao Ao. )■ Hydrolyze starch on starch agar.

■ Hardly any coagulation or peptonization of skim milk is observed.

■ Produces melanin-like pigments on tyrosine agar and peptone-yeast-iron agar.

D) Assimilability of carbon source (Bridham-Bott) IJ −
(On agar medium) Only glucose and inositol are assimilated, and arabinose, D-xylose, sucrose, rhamnose, raffinose, and D-mannitol are not assimilated. Assimilation of D-7 lactose is complicated.

The above properties are similar to those of the istamycin-producing strain Streptomyces tenzimariensis 5S-23 (see Japanese Patent Application Laid-Open No. 2003-111027), which the present inventors have already reported, and the ability to form aerial hyphae and melanin. Streptomyces tenzimariensis 8B-/! has exactly the same pigment productivity, color tone of aerial mycelia, and assimilation of carbon sources. 07U-
The 4AI strain is unique in that it has almost no ability to produce istamycin, and is convenient for use in the method of the present invention.

To carry out the method of the invention, Ho Chi Minh B is combined with compound C
For conversion to I), the above-mentioned strains of bacteria, particularly Streptomyces tenzimariensis, which can be used in the method of the present invention, are usually cultured in a medium containing hotimidine B. That is, hothymidine B is present in the culture solution during both of the above-mentioned whole cultures and is allowed to act. In culturing bacteria according to the method of the present invention, conventional culture methods for producing antibiotics are not used. Various sources of nutrients can be used for culture. Glucose as a carbon source? Powdered dextrin, cane sugar, molasses, etc. are used alone or in combination, and depending on the assimilation ability of C, hydrocarbons, alcohols,
Organic acids, animal oils, vegetable oils, etc. may also be used. Inorganic nitrogen sources, and ammonium chloride and ammonium nitrate as organic nitrogen sources.

Sodium nitrate, soy flour, defatted soy flour, cottonseed meal.

Gluten meal, cornmeal, wheat embryo? ', Hesoton, meat extract, yeast extract, dry yeast, corn steep liquor, etc. may be used alone or in combination. Other amino acids as required.

Nucleic acids, vitamins, and inorganic salts such as sodium chloride, calcium carbonate, phosphates, magnesium sulfate, and conolt chloride can also be added.

As a culture method, a liquid culture method, particularly a method using a deep stirring method, is suitable. The culture temperature should be 20°C to 1°C, preferably 2t°C to 3.2°C, and the pH should be around neutral. Also, the culture medium composition, liquid properties of the medium, amount of additives, temperature, number of stirring 9
It goes without saying that culture conditions such as the amount of aeration are appropriately selected depending on the bacterial strain used. Ibi compound [1)'fr
In order to obtain this, hotimidine B, which is a raw material, may be added at the start of the culture, or after the bacteria have grown, but it is preferable to add it within about 22 hours after the start of the culture.

The amount of raw materials added should be medium/! l) 0. / r to 10f
It may be added at once in small amounts, or it may be added in portions. Further, the fotimidine BFj to be added may be in the form of a free base, but may also be in the form of a salt such as a sulfate or a hydrochloride. The action time of the bacteria and hothymidine B is selected so that the maximum amount of compound [I] accumulates after the addition of the raw materials.

For example, Bacillus subtilis
The amount of compound (1) in the culture solution can be monitored by the Benosedesk method using P CI 2 /li as a test strain, but it usually takes 3 to 2 days.

The method for collecting compound (I), including its isolation and purification from the culture solution, is the method normally used for collecting aminoglycoside antibiotics. That is, adsorption and desorption methods using cation and anion exchange resins, adsorption and desorption methods using porous polymer resins using counter ions, and adsorption and desorption methods using activated carbon.

Methods such as silica gel column chromatography can be used in appropriate combination.

Specifically, compound CI)? In order to collect, for example, after adjusting the pH of the culture solution to 3 to 3,
The bacterial cells are temporarily removed, the mixture is again adjusted to pie j or t, and the substance having this chemical structure is absorbed. □, □ka22. □
,7. Amberly as a cation exchange resin with off □, y)
:), DOWEX, row (trade name) [Na''1, etc., and elute with 0.0N sulfuric acid to obtain a crude solution containing compound [T].

To further purify the material, use a counterionic agent and 17/-sodium pentanesulfonate.

Suitable from /-sodium hexanesulfonate, /-sodium hebutanesulfonate, /-sodium octanesulfonate, /-sodium dodecanesulfonate, /-sodium dedecylsulfonate, sodium nogradluenesulfonate, etc. Choose something, knit it, dissolve it, and gradually O0! ~/After adding a specified amount of sodium hydroxide and adjusting the pH to OK, annoglite XAD-2 (trade name), which is a porous polymer resin,
Amberlight X A, D-4t (product name), Diaion) IP-, 20 (product name), Diaion O
Adsorb and mix with PH-2'OP (trade name) etc. After absorbing the temperature of the resin and thoroughly washing the resin with water, increase the metal content of the elution solvent gradually from O or by using the concentration gradient method to elute the substance. Raise it until it reaches the top. In this way, 'f1. Collect the fractions of each substance and collect O8! After neutralizing the pH to 0 with a specified amount of sodium hydroxide, the cation exchange tree (Amberley) IRQ-jO (trade name) (Nm"), 0
Adsorb to G-jO (trade name) [Na'':], wash with hx < water, and elute with O1! specified sulfuric acid.

This measures the separation from the counter ion agent used. This eluate contains a large amount of sodium sulfate in addition to the substance of formula (1), so it is adsorbed on activated carbon and washed with water to remove sodium sulfate. 1 containing specified sulfuric acid
Elute with 0.0 g of methanol.

Since the eluate is sulfuric acid, after condensing it to an appropriate concentration, it is mixed with Amberlite IRA-41, an anion exchange resin.
,j (product name) (OH-) After neutralizing T and sending it from door to door,
IRA-4, an anion exchange resin! o.

(Product name) (804-:] ', r, after passing through and concentrating the aqueous solution that has passed (by bundling and drying)
Obtain the target substance of formula [1].

This basic compound [■] is an inorganic or organic acid.

For example, when reacted with hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, stearic acid, tartaric acid, maleic acid, etc. in a conventional manner, non-toxic acid addition salts are easily formed.

Next, the present invention will be further explained with reference to Examples.

Example/ Streptomyces tenzimariensis (Streptomyces ten) grown well by culturing on a soluble starch inorganic salt (ISP Nog) agar medium for λ weeks
jlmarien-sis) SS/jO71U-171
Strain (FERM-P'711t) was treated with cornmeal T, 0%, wheat germ J, (1)%, magnesium sulfate 0. A loopful of a liquid medium (pH 7, O) containing Oj%, calcium carbonate Ot%, 00- was sterilized in a zoo77 flask and inoculated at 1.27°C for 4]g~
7. A seed culture solution was obtained by shaking culture for 2 hours.

Separately, prepare 1 ootrt of main culture medium in j00m17 Lasco, and inoculate 1 ml of the above seed mother culture into 1].

The composition of this medium is wheat germ x, tl, sodium palmitate 0. j%, magnesium sulfate o, or %,
Charcoal WR calcify 0g%, soybean oil 3. t%(
pH 7,0) 'C 11. Sterilize at 20°C for 7 minutes before use. 7-2 hours after inoculation - Thymidine B (base) was added to the medium in an amount of J 00 mc? After addition of the raw materials, shaking culture was carried out at 27°C for 141 hours.
When the antibacterial activity of the culture solution was measured by the paper dispersion method using the same strain, an inhibition circle of 28 strips in diameter was obtained.

After adjusting the pH of 22 flasks of the obtained culture solution to 2.0 with tN sulfuric acid, the bacterial cells were separated in a furnace, and the bacterial cells were further washed with water and the washing liquid was filtered. Combined with culture solution. 4j, j/t of the filtered culture solution obtained in this way
Return the pH to 6.0 with Hironori's sodium hydroxide, Amberly) IRQ-, tO (trade name) [Na":]!P
The target compound [1) t-
It was adsorbed. After washing the column thoroughly with water, The mixture was eluted with a specified amount of sulfuric acid/l to obtain a solution containing the crude target compound [D]. LOof of sodium paratoluenesulfonate was added to this solution, and 1 normal sodium hydroxide was gradually added to adjust the pH to 0. Add this solution to Diaion 0HP-CoOP, 8□, 7) 7, ka24□. 1
ゎIa81゛□It absorbed things. The column was washed with λoo, 1% hydrochloric acid solution (pH, 2,0), and then subjected to concentration gradient column chromatography with 0.1% methanol water using hydrochloric acid solution (pH -2,0). went. Each fraction (each/□m/)'e was searched by high performance liquid chromatography and the compound [:I)k was confirmed. By the way, the two operating conditions for high performance liquid chromatography used at this time were Diveroseal 0DS-! ((: J-, 20th section,!
μ) [Product name] (6I11++Iφ×, 200 order), the eluent was O, 2 mol of sodium sulfate and l-pentanesulfonic acid as a counterion agent) IJ
o, o, containing 2 moles of um. /% acetic acid solution and 4]% acetonitrile are added and the liquid is fed at a rate of //min. Further, in order to detect this substance, a solution prepared by dissolving an orthofucuraldehyde reagent (hereinafter abbreviated as OPA) in a boric acid buffer solution of pH 10 at a ratio of o, t~/g is used. After the sample was injected, it was taken out from the column, and the L eluent and OPA# solution were reacted in a mixing joint, and the target compound CI)k was detected using a UV detector at a wavelength of 3jμmμ. According to this operating method, the retention time of the target compound [I] was 7 minutes 2♂ seconds (Table 7 above). Compound (1), which was searched in this way, was fractionated from 2μ to 1μ2, so after collecting T'L and concentrating it to about 0.2μ, the compound (1) was fractionated from 2μ to 1μ2. The pH was adjusted to <1N using sodium chloride solution. Both parts of this target compound [I] were converted into Amberlite IRQ-jO (trade name) CNa+]
The target compound [1] was adsorbed through a 3 tnl column. Then, after washing thoroughly with water, 0. j Elute with normal sulfuric acid water! - were fractionated. The target compound [■] is Bacillus .
Plate containing S. subtilis PCI212 as a test bacterium.
- Tested using the Ri-disk method. Paper discs were neutralized with ammonia gas before assay. In addition, after confirming that fractions 1 to 2 have been fractionated by testing positive for ninhydrin, collect these fractions,
o, a with normal sodium hydroxide solution pH'fr: t,
It was adjusted to 0.2.

Both parts'ii of the target compound (1) obtained in this way,
Filled with 2j- of activated carbon and adsorbed on a column of 100
-1 containing sulfuric acid of o, ozH constant after washing thoroughly with water.
It was eluted with 0% methanol water. 7 lacions each (each!-
) used the paper disk method to detect Bacillus, subtilis P.
The assay was performed using a CI plate culture medium. At this time, the paper disk was neutralized with ammonia gas before the assay. As a result, the target compound (1) was eluted from fraction 3 to ≠O, so f'Ly was collected and concentrated, and after methanol was removed, Amberlite IRA-μj (trade name) COH
After neutralizing with M and passing through, it was further concentrated to about 1-.

This concentrated solution was charged to an IO-column of Amberlite RA-4tOO (trade name) [5O4-), and eluted with water. It was fractionated as a fraction of d.

Each of these fractions is Ichirus subtilis PC1,
By assaying with the paper disk method using 2/ri and testing positive for ninhydrin, the target compound was found from 7-lactone I! After confirming that -4n was present in the sample, the fractions were collected, concentrated in O1j, and lyophilized to obtain a white powder of the target compound [1] of /r,i.

Based on the physical and chemical properties of this substance, especially the measurement results of mass spectra, nuclear magnetic resonance spectra, and infrared absorption spectra, and the fact that it was derived from fotimidine B, it was found that this substance has the above formula % formula % It was identified as A, l-Ebidactymicin.

[Brief explanation of the drawing]

FIG. 1 is an infrared absorption spectrum (KBr tablet) of /-evidactymicin according to the present invention, and FIG. 2 is a nuclear magnetic resonance spectrum of l-evidactymicin. 1:: FIG.

Claims (1)

[Claims] / A dactymicine derivative or an acid addition salt thereof represented by the following formula. 2 The dactymicin derivative is shown by the following three-dimensional structural formula.
- The compound according to claim 7, which is epidactymicin. 3. A patent characterized in that a bacterium belonging to the genus Streptomyces and having the ability to convert the amino group at the 1-position of hothymidine B and introduce a formimidoylglycyl group to the methylamine group at the μ-position is allowed to act on hothymidine B. A method for producing a dactemisin derivative represented by H3 1 1 or an acid addition salt thereof. Hiroshi Ho Chimidin B and Streptomyces tenjima
3. The method according to claim 3, wherein epidactymicin having the following three-dimensional structural formula (n/-epidactymicin) is produced by reacting Streptomyces tenjlma
rlensls)88-/! 07 Claim No. 3 in which U-4AI (Feikoken Bibori No. 7t, No. 21) acts
The method described in section. T, Streftmyces tenzimariensis (・
3. The method according to claim 3, wherein Hothymidine Bi is present in the culture solution during culturing and is caused to act.