DE2327639C3 - L-trans-2-amino-4- (aminoethoxy) but-3-en-1-acid and acid addition salts thereof and processes for their preparation and herbicidal compositions containing them - Google Patents

Description

NH,

and their acid addition salts.

2. Herbicidal agent containing a compound according to claim 1 in addition to the usual ones Carriers.

3. A method for producing a compound according to claim 1, characterized in that a microorganism of the species Streptomyces X-11085 (NRRL 5331) in an aqueous nutrient solution, which is an assimilable source of carbon and nitrogen and inorganic salts, cultivated submerged under aerobic conditions, contain the fermentation solution filtered, the compound of formula 1 present in the filtrate on a cation exchanger or through Partition chromatography binds to silica, then from the cation exchanger or from Recovered silica gel and, if necessary, ίγ, transferred to an acid addition salt.

The present invention relates to L-trans-2-amino-4- (2-aminoäiho) cy) -but-3-en-1 -acid of the formula

11, N-CH, -CH, -O

C -C

CH COOH

I.
NH,

(I) Department of Agriculture deposited in Peoria, Illinois and registered under No. NRRL 5331.

The Streptomyces strain X-Il 085 can be characterized by the following criteria:

Growth behavior

The culture forms a gut in many media

developed and branched substrate mycelium as well as a characteristic powdery aerial mycelium. The colonies are convex and have wavy edges. on

Bennett agar generally develop fine to coarse-grained, sometimes smooth colonies in the center,

: partly - media dependent - with Newtonian

Wrestling.

Color of vegetative mycelium and spores

After 2 days of growth on ISP-2, asparagine and Amidex medium, the spore mass shows a Sand, leather and ivory colors. The leather color is stable for about 10 days. The back of the colonies d. H. the vegetative part of the mycelium is yellow, yellow-orange or light brown in color.

The color coding and the \ "irbfächerzuordnung comply with by the International Streptomyces

myces Project (ISP): E. B. Shirling and D. G & 111 i e b, "Methods for Characterization of Streptomyces Species, "International Journal of Systematic Bacteriol. Vol. 16, (1966) 313-340 Methods.

45

Acid addition salts of this compound, a microbiological process for the preparation of this compound and herbicidal compositions containing this compound.

The compound of formula 1 is from a new Streptomycete, namely from Streptomyces X-Il 085 educated. The process for their preparation is thereby characterized in that a microorganism of the species Streptomyces is obtained in a manner known per se X-H 085 in an aqueous nutrient solution which is an assimilable carbon and nitrogen source as well contains inorganic salts, under acrobic conditions Cultivated submerged, the compound of formula I isolated from the aqueous nutrient solution and, if necessary in an acid addition salt, leads to exercise.

The new Streptomyces strain was found in a soil sample in Arlington, California. One a virulent culture of this strain, labeled in the laboratory with Streptomyces X-Il 085, was found in the Northern Utilization Research and Development Division, Agriculture Research. Service, United States morphology

Long-thread mycelium. Long and short sporophores with up to 50 spores per chain, some stretched to the Part curved [RF Group Pridham]. No spirals.

Sporophores are irregularly branched, often tangled and split at the ends, or they form multifarious tufts. Under the electron microscope at a magnification of 50,000 these appear Spores elongated, cylindrical, smooth to slightly wrinkled, covered with a skin. Their dimension is 0.6 χ 0.4 up to 1.0 χ 0.5 μ.

Physiology Soluble pigment

Low to moderate pigmentation after 4 days in some media e.g. B. in asparagine, Bennett, Emerson, yeast or malt extract as well as in ISP-2, milk and gelatine media but not in ISP-3, 4 and 5 or in Amidex, Pablum, Tomato / agar tomato / oatmeal, tomato / soy flour or in Sp. 5 media. The abbreviations ISP 2, 3, 4 and 5 relate to media which are described in International J. of Systematic Bacteriol., 16, 313 to 340 (1966). The abbreviation Sp. ^C denotes a medium which is described as medium 5 in the ATCC catalog, 1 HI edition, 1974.

After 11 days, Sp. 5-, Amidex-, aul Tomato / oatmeal and pablum / agar nutrient bait excreted a brown-black pigment.

Color of the back of the colonies

After an incubation period of 11 days at 28 ° C, this varies from white to buff to black, depending on the medium used for growth; (e.g. on Pablum, ISP-J, ISP 5 and on tomato agar) and from smoky gray to black (e.g. on Sp. 5 ^: Amidex, asparagine, Bennett, tomato / oatmeal and Emerson agar).

Different physiological responses

The culture is strongly chromogenic; it produces melanin on a peptone-iron agar medium, but not on tyrosine agar. Starch is hydrolyzed and gelatine is partially liquefied after just 4 days. Good growth in milk with low peptone formation. The culture grows well at 28 ° C. After 11 days it forms a colorless exudate on some agar media, e.g. B. on Sp. 5, Amidex, asparagine, Bennett, Emerson, Pablum, tomato and tomato / soy agar. However, the culture does not grow well between 28 and 35 ° C at 45,47 and 50 ⁰ C.

From the ornaments formed by the spores, the general morphology of the spores, the branching the sporophores, the colors of many media and from certain biochemical and physiological reactions it can be concluded that the microorganism Streptomyces Sp. X-Il 085 is different from all different cultures of the RF group described in the literature.

The cultivation of the Streptomyces strain X-Il 085 various fermentation methods can be used to produce the desired compound of the formula I. In general, can Standard methods of both bottle and tank fermentation are used.

Streptomyces X-Il 085 can be of the most varied liquid media are cultivated. Media that have an assimilable Carbon source, e.g. B. starch, glucose, molasses, an assimilable nitrogen source e.g. B. protein, protein hydrolyzate, Polypeptides, amino acids, corn steep liquor, ammonium salts, and generally the cations and Anions of inorganic salts, ζ Β. the sodium, potassium, calcium, magnesium ions and the sulfate and Chloride ion. Trace elements like cobalt, copper, iron, molybdenum, boron etc. are inevitable as impurities present.

The compound of the formula I obtained by microbiological means has antibiotic properties. In particular, it has an antimicrobial and herbicidal effect.

The size of the zone of inhibition against Streptomyces cellulosae is a measure of the antimicrobial effectiveness 097. The quantitative and qualitative determination of the inhibitory effect of the compound of the formula I can be based on a filter paper disc can be determined with the agar diffusion method. Proceed as follows: An inoculum of Streptomyces cellulosae 097 is placed in a 500 ml Erlenmeyer flask containing 60 to 80 ml a nutrient solution of the following composition:

2.0 g / l glucose
7.0g / lK2HPO4
3.0 g / l KH2PO4
0.5 g / l sodium citrate 2 HO
0.1 g / l MgSO ₄ · 7 HO
1.0g / l (NH4): SO4 and
15.0 g / l agar

precultured for about 3 days at 28 ° C.

The cell mass formed is then washed 3 times with water to remove excess nutrients, then adjusted to a density of 1.2 (wavelength 500 nm, layer thickness 15.5 mm). From the washed 30 ml of cell mass suspension are poured into 1 liter of liquid minimal agar () immediately before the Überimofen.

Bacteriology, 16 [19501 17-28) transferred. 5 ml each of this inoculum are transferred into Petri dishes (diameter 100 mm, layer thickness 15 mm). As soon as the agar has solidified, the dishes ^{are stored at 4 °} C. They can be used for one week. The filter papers soaked with the Streptomyces cellulosae 097 culture described above are placed on the agar surfaces. The Petri dishes are incubated for 12 hours at 28 ° C. The size of the inhibition zones is then measured. The diameter of the zone of inhibition is proportional to the logarithm of the concentration of the compound of the formula I, which is between 10 and 100 μg / ml. Doubling the concentration of the compound of the formula 1 increases the diameter of the zone of inhibition to 3.5 mm.

Preferred fermentation media and the antibiotic yields that can be achieved with them in the glass shake bottle are compiled in Table 1. The data presented in this table show that complex, of Nitrogen-containing substances from various sources promote the formation of amibiotics, in particular z. B. plant materials such as soybean meal, animal substances such as meat meal extracts and microbiological Cell material such as yeast.

The compound of the formula I can be isolated in various ways after fermentation has ended getting cleaned. Suitable isolation and purification methods are ion exchange chromatography, partition chromatography and adsorption chromatography.

According to a preferred process, the compound of the formula I is obtained from the culture medium by the fact that the mycelium and the insoluble solids in the usual manner, for. B. Filtration or Centrifugation separates the fermentation solution and the compound of formula 1 from the clear fermentation solution with Isolated using ion exchange or adsorption chromatography. By adsorption chromatography all impurities are advantageously removed on an activated carbon. I ur ion exchange chromatography acidic and basic ion exchangers, especially those containing sodium ions, be used. Partition chromatography is advantageously carried out on silica gel (eluent:

Alcohol, water, ammonia). The methods described above for isolation and Purification of the compound of formula I can also be combined.

The compound of formula I can after filtering or centrifuging the fermentation solution with the help of Thin layer or paper chromatography can be analyzed. The characteristic spots of this connection can be visualized on the plates by spraying with ninhydrin. Also the bioautography can also be used to advantage for identification. The chromatography can be done with Filter papers are carried out. However, it is advantageous to use silica gel or cellulose coated glass plates. The following solvent mixtures are used for developing the thin-layer chromatogram in question: ethanol / water / ammonia 49: 49: 2 for silica gel plates, and phenol / 80:20 water for cellulose panels. The compound of the formula I precipitates in the form of a upon final crystallization Zwitterions or as a mono- or di-acid addition salt, z. Bi. As mono- or di-hydrochloride.

The new compound of formula I forms pharmaceutically with inorganic and organic acids

usable acid addition salts. Among the inorganic acids that are suitable are Hydrohalic acids, e.g. B. hydrochloric and hydrobromic acid, also mineral acids such as sulfuric acid and phosphoric acid. The organic acids include the ants, vinegar, Citric, tartaric, succinic and maleic acids are in question. The sake formed with the acids mentioned can be used in Forßi of the zwitterion as bioeid. In In dissolved form, however, the monohydrochlorides of the ι ο compound of the formula I are more stable than the zwitterions. It is therefore advisable to work under weakly acidic conditions when cleaning.

The compound of formula I has antibiotic properties. It is antimicrobial, antiprotozoic and anthelmintically effective. It also has herbicidal properties.

The antimicrobial spectrum can be found in Table 2. As a test, the one above became closer The agar diffusion test described is used.

The antiprotozoal properties of the compound of formula 1 can inter alia in the effect against Trichromonas vaginalis can be illustrated. Groups of 8 mice each are used in this test. When Control serves one infected or one untreated: s te group of animals. The animals become subcutaneously in the abdominal area with about 500,000 pathogens Inoculated the test animals are immediately afterwards at the same place subcutaneously with 1 ml of a solution the compound of formula 1 inoculated in various concentrations. The next day the Animals checked for lesions in the area of infection. The number of animals with and without lesions is determined. The dose curativa CDso is according to Reed and M u e η c h (Am. J. of Hyg. VoI 27 [1938], 493). It was found that the dose curativa CDw of the compound of formula I as an antiprotozoicum 1 μ / ml.

The anthelmintic properties of the compound of the formula I can be _{measured by the activity against Ascaris 4} c suum in the mouse. In this test, 4 mice per group are used. The same number is infected, but remains untreated as a control group. In this case, the compound of formula 1 is administered 24 hours before infection, as an admixture to the feed in an amount of 100 to 200 mg test substance per 100 g feed. The mice are infected by the oral administration of about 2000 to 4000 Ascaris eggs in the embryonic state per mouse. The treatment with the test substance is continued for 7 days as indicated. The animals are then killed. The lungs of the animals are pressed between two microscope slides and examined under the microscope. The number of larvae in the lungs of the infected and treated animals is compared with the number of larvae in the lungs of the infected but untreated animals. It was found that the lungs of the infected mice treated with the compound of the formula I contained 71% fewer larvae than the lungs of the control animals. The compound of the formula I is accordingly clearly active against Ascaris suum.

The toxicity of the compound of formula I is low when administered orally or parenterally. the DL50 is 500 mg in mice (per os or subcutaneously).

The compounds according to the invention are particularly suitable for the preparation of post-emergence herbicides, which the acid of the formula I or an addition salt of this acid in connection with usually in herbicidal preparations contain existing carriers or additives. Examples of the above Carriers and additives are: Extenders, thinners or conditioning agents, which the Production of solutions, emulsions, dispersions, as well as dusts and wettable powders facilitate.

The amount of active ingredient contained in the herbicidal use form is normally less than 50 percent by weight

It is advisable to achieve the greatest possible herbicidal post-emergence effect, always sufficient To use amounts of the use form according to the invention, i.e. about 0.1 kg to about 1.8 kg, preferred 0.45 kg to about 0.9 kg of active ingredient per 0.4 ha.

From Plant Physiology, Volume 40, pp. 931-933 (1965) a "rhizobitoxin" is already known, the production of which is extraordinarily complex. From Plant Physiology, 1971, Volume 48, pp. 1 to 4 it is known that this rhizobitoxin described above, the compound L-2-Ami no-4- (2'amino-3'-hydroxypropoxy) -trans-3-butenoic acid and this acid inhibits ethylene production.

A comparison of the known compound and the compound according to claim 1 shows that the invention Compound inhibits the formation of ethylene in apple tissue slices more than the previously known one Compound rhizobitoxin.

connection

Percentage inhibition of ethylene formation
2h 6h

Rhizobitoxin 73

(State of the art)
Connection according to the invention 75

76

81

The activity of the compounds which can be prepared according to the invention as post-emergence herbicides can be illustrated using the following tests on a number of different weeds. The plants used for the individual tests are cultivated in plastic ^{dishes measuring approximately 1.2 × 1.2 m 2.} The height of growth varied between 3.8 and 10 cm depending on the plant. Each species of plant was grown in its own container. The active ingredient was the hydrochloride of the acid of the formula I in the form of an aqueous solution containing 0.5 percent by weight of polyoxyethylene sorbitan monostearate. For this purpose, the active ingredient was dissolved in the carrier in concentrations of 0.226, 0.45, 0.9 and 1.8 kg of active ingredient per 0.4 ha. The plants were each sprayed once with the respective active ingredient solution and observed for 14 days. The test was then terminated and the results evaluated. The test results are summarized in the table below. The degree of damage is expressed by numbers that indicate the following:

0 = no visible effect,

1,2,3 = slight damage (the plants recover

usually low or none

Reduction in growth),
4,5,6 = moderate damage (the plants recover

normally, the growth is

but reduced),
7,8,9 = severe damage (the plants recover

usually not),
10 = all plants have died.

Herbicidal effect of L-trans-2-amino-4- (aminoethoxy) -but-3-en-l-acid
14 day Tcsi

Active ingredient concentration
kg / 0.4 ha

1) xanthium
pensy! -
vanicum

2) Cyperus exculentus

3) Sorghum halepense

4) Datura 5) Cirsium

stramonium arvense

6) Cyperus rotondus

1.8 9 7th 9 8th 5 4th 0.9 8th 5 8th 8th 4th 2 0.45 8th 4th 8th 6th 1 0 0.226 6th 2 7th 4th 1 0

Active ingredient concentration
kg / 0.4 ha

7) Sida acuta

8) Daubentonia texana

9) Chenopodium album

10) Agropyron
repens

11) Bromus
secalinus

12) Abutilon theophrastl

1.8 8th 6th 5 2 6th 7th 0.9 7th 3 3 1 4th 4th 0.45 5 4th 1 0 2 2 0.226 4th 2 0 0 2 2

The invention is explained in more detail with reference to the following examples, the percentages being based on each other Refer to weight.

example 1
Fermentation of Strepto.nyces X-11 085

A spore suspension of Streptomyces X-II 085 of the eluate, which increases the pH to over 9.0

is from a nutrient agar slant in a 6-1 alder leaves, contain almost all of the active material,

Meyer's flask swept away, the 21 of a trypticase- You are under reduced pressure to about 2 1,

Contains soy nutrient solution. The inoculated nutrient solution containing 64 g of solid, concentrated. The concentrate

on a rotary shaker 72 hours after adjusting the pH by adding

incubated at 28 ° C. Thereafter, 340 g of activated charcoal are added to 41 of the inoculum in 5 η hydrochloric acid to 3.3. the

a 227 1 nutrient solution of the following composition is then initially through a sintered glass

Composition filter and then through one with 70 g of activated carbon

coated silica gel pad filtered. The filter

Glucose - 10.0 g / l 40 is washed off with water. That with the

Bacto-Pepton 5.0 g / l wash water combined filtrate (approx. 51) contains the

Bacto yeast extract 3.0 g / L total active material (approximately 42 g solids). The

Iron ammonium sulfate · 6 H2O 0.03 g / l filtrate is then under reduced pressure

evaporated. From the concentrate obtained are

transfer. The pH of the fermentation batch is dissolved by 45 to 31 ml containing 18 g of solid in 65 ml of ethanol / water addition of sodium hydroxide solution to 6.8 ser / ammonia 80: 15: 5 before sterilization. The solution is adjusted by. The fermentation batch is in a 380-1 Fermen a column (height 90 cm, diameter 8 cm), filled with ter at 28 ° C with stirring (number of revolutions of the 4.7 l silica gel (grain size 0.05 to 0, 2 mm), which is slurried in agitator 200 rpm) and aeration (amount of air 1 Lpm) ethanol / water / ammonia 80: 15: 5 slurried The foam formation is filtered by adding a 50, the column is with the same silicone-based antifoam under control Solvent mixture eluted The active substance is retained. After 41 hours, the fermentation solution is mixed with a total of 121 solution infusor earth after fractional elution and enriched in 4.81 by centrifugation. This active fraction is filtered and concentrated to 200 ml. The concentrate is filtered through

•. . ,, _. . , "... _v . 55 50 ml ion exchanger in H® form (passable through

Purification of the L-trans-2-amino-4- (2-aminoathoxy) -but- _{a Sjeb} ^ _{5Q to m meshes / cm2} ) _{filters the}

3-en-l-acid ion exchanger is successively mixed with water and

4261 of the clear fermentation solution obtained, which washed 3.1 kg of a 10% strength aqueous pyridine solution

Contains solids are replaced by an ion exchanger - active substance is then added with l, 5 molar

column (diameter: 30 cm. Contents: 501 exchangers, 60 ammonia eluted. The eluate is concentrated to 80 ml.

H®Form filtered The column is filtered one after the other. The concentrate is filtered after the

pH by adding 7.4 ml of 1 η-hydrochloric acid

501 Water 33 thickened to the syrup consistency

2001 containing 5% pyridine and 95% water and from 20 ml methanol / water 49: 1 crystallizing out

501 water 65 L-trans-2-amino-4- {2-aminoethoxy) -but-3-en-l-acid-

hydrochloride melts just like one made of methanol /

washed. The active material is then eluted with a second fraction which crystallizes out in 2001 water 49/1 in 185 1-molar aqueous ammonia. The first 801 to 186 ° C [ä] = +85.8 [c = 1 water].

Example 2

Purification of L-trans-2-amino-4- (2-aminoethoxy) -but-3-en-1 -acid

36 g of solid, partially purified by one of the customary methods, e.g. B. by adsorption chromatography, Partition chromatography or ion exchange chromatography are dissolved in 250 ml of water solved. The solution is, after lowering the pH value by adding 5 to 3.6, by a with 2.51 Cation exchanger, H® form (analogous to Example 1) filled column (height 60 cm, diameter 7 cm) filtered. The column is filled with a 0.2 molar sodium phosphate / citrate buffer solution (pH 5.5) elutes until the active substance appears in the eluate, which is after passing through about 6.2 to 9.1 1 is the case. The salts present in the active fraction are filtered through 1.2 l Cation exchanger, H® form (analogous to Example 1) removed. The active substance is made with 1.5 n ammonia eluted. The eluate is evaporated under reduced pressure. The remaining L-trans-2-amino-4- (2-aminoethoxy) -but-3-en-l acid hydrochloride crystallizes under those specified in Example 2 Conditions.

Table 1
Nutrient solutions

Components

g / l

Antibiotic effectiveness in y / ml

BB nutrient solution 2.0 glucose 7.0 K2H PO4 3.0 KH2PO4 0.3 Na3-citrai-2H2O O.i MgSr> 4-7H2O 1.0 (NH4) 2-SO4 CB nutrient solution 10.0 glucose 10.0 N-Z amines A 0.2 MgCb-6H ₂ O 0.025 CaCl2-2H2O 0, DOl FeCl3-6H2O 0.0005 ZnCl2 0.0005 CuCb-2 H2O 0.0005 MnCb-2H2O

1.0

0.1

IG

Components 10.0 Antibiotic! Κ 10.0 effectiveness 4.0 in "/ 'ml CC nutrient solution 2.0 Monosodium glutamate 0.2 0.1 glucose 0.025 K2HPO4 0.001 KH2PO4 0.000 ^r ) MgCb-6H2O 0.0005 CaCb-2 H2O 0.0005 FeCb-OH ₂ O ZnCb 10.0 CuCb-2H2O 10.0 MnCb-2 H ₂ O 0.5 CD nutrient solution 0.5 Soybean 0.5 10.0 Soluble starch 7.5 Corn steep water K2HPO4 10.0 CaCO ₃ 5.0 pH adjusted to 3.0 CE nutrient solution 0.031 glucose 10.0 Peptone Yeast extract Fe (NH4) 2 (SO4) 2-6H2O

Table 2

Antimicrobial spectrum of L-trans-2-amino-4- (2 aminoethyl) -but-3-en-1-acid
in vitro

Test organism

Inhibition zone diameter in mm

Active ingredient corporation !: ! mg / ml

Escherichia coli (17) Bacillus simplex (32) Staphylococcus aureus (24) Streptomyces cellulosae 097 51 Bacillus cereus 65 With fuzzy zones limit inhibition zones diameter in Brackets

Claims (1)

Patent claims: 1. L-trans -2-amino-4- {2-aminoethoxy) -but-3-en-1-acid the formula H, N-CH, -CH, -O (D CH COOH