CH626895A5 - Process for the preparation of alkyl derivatives of the antibiotics BM 123 - Google Patents

Description

The invention relates to a process for the preparation of a new group of antibiotics with a strong antibacterial effect, which is obtained by the antibiotic BM 123 gamma 1, gamma 2 or mixtures thereof with an aldehyde or a ketone of the formula:

Ri-CHO or R2-C-R3 wherein

Ri for hydrogen, lower alkyl, halogen-substituted lower alkyl, lower alkenyl, phenyl, monosubstituted phenyl, phenyl-lower alkyl, styryl, 2-furyl, methyl-substituted 2-furyl, 2-thienyl, methyl-substituted 2-thienyl, 2-pyrryl, methyl-substituted 2-pyrryl, 2 -Pyridyl or 2-quinolyl, R2 is lower alkyl and

R3 for alkyl with up to 6 carbon atoms, N, N-dipropylaminomethyl, 2-chloroethyl, benzyl, styryl, cyclohexyl, 4-methylpenten-3-yl, 1,3-dimethylhexyl, ß-indolylmethyl, cyclopenten-2-ylmethyl , n-heptyl, n-octyl, (o-tolylcarbamoyl) methyl, propenyl, methoxymethyl, cyclohexylmethyl, 2- (4-hydroxyphenyl) ethyl, n-nonyl, n-dodecyl, 2-methoxyvynyl, l- Methyl 2-hydroxyethyl, 3,4-dimethoxybenzyI, N, N-diethylaminoethyl, carbäthoxychlormethyl, 1-methyl-l-hydroxyethyl, 4-chlorobenzyl, (t-butylcarbamoyl) methyl, dimethoxymethyl, 2-, 3- or 4-methoxybenzyl , Phenoxymethyl, ethynyl, N, N-dimethylaminomethyl, N, N-diethylaminopropyl, cyclopropyl, 2,2-dimethoxyethyl, acetonyl, ß-phenylethyl, 3-chloropropyl, 2-chlorobenzyl, 3-chlorobenzyl , Buten-3-yl, 1-acetamido-ethyl, 3-pyridyl, carbäthoxymethyl, 5-norbornen-2-yl, cyclopentyl, 1-carbäthoxyethyl, 1-hydro-xyethyl, 3-hydroxypropyl, chloromethyl, isobut-2-enyl , Phenyl, m-chlorophenyl, p-bromophenyl, p-trifluoromethylphenyl, m-nitrophenyl or p-dimethylaminoph enyl, or R2 and R3 together with the methylidine group cyclobutyl, cyclopentyl, monomethylcyclopentyl, 2,4-dimethylcyclopentyl, 2,4,4- or 1,3,3-trimethylcyclopentyl, cyclohexyl, 3-methylcyclohexyl, 3,5-dimethylcyclohexyl , Cycloheptyl, cyclooctyl, cyclononyl, 2,2,4,4-tetramethylcyclopentyl, 2-cyclopentylcyclopentyl, 3-tert.pentylcyclopentyl, 2- (l-cyclo-hexene) cyclohexyl, 2-cyclohexylcyclohexyl, 2-ethyl-cyclohexyl, 2- Propylcyclohexyl, 4-tetrahydrothiopyranyl, 2-ethylcyclopentyl, 3-methyl-6-isopropylcyclohexyl, 3,3-dimethylcyclohexen-4-yl, 2-phenylcyclohexyl, 2-cyclohexenyl, 4-t-butyl -cyclohexyl, 2-ethylidene cyclohexyl, decahydro-l-methyl-2-naphthyl, decahydro-3-methyl-2-naphthyl, l-benzyl-4-piperidyl, l-methyl-4-piperidyl, bicyclo [3.2.1 ] octan-2-yl, 3-quinuclidinyl, 5-methoxy-2-tetralyl, 2-adamantyl or 2-norbornyl,

reductively alkylated, with aldehydes depending on the amount of the reagents, the terminal amino group single or double and the proximal secondary amino group is not or also alkylated and the terminal amino group is simply alkylated with ketones.

Suitable lower alkyl groups (R 1, R 2 and R 3) or halo-substituted lower alkyl groups (R 1) are those with up to 6 carbon atoms, in which halogen generally represents chlorine, bromine or iodine, such as methyl, ethyl, isopropyl, sec-butyl , n-amyl, dichloromethyl, 2-bromoethyl, 2,3-diiodopropyl or gamma-chloropropyl. The lower alkenyl groups (R 1) include, for example, those with up to four carbon atoms, such as vinyl, allyl, propenyl, isopropenyl, 1-butenyl, 2-butenyl, 3-butenyl or isobutenyl. Examples of suitable monosubstituted phenyl groups (R 1) are p-acetamidophenyl, m-nitrophenyl, m-mercaptophenyl, o-anisyl, p-anisyl, 0-is tolyl or p-tolyl. Suitable phenyl-lower alkyl groups (Ri) are, for example, benzyl, a-phenylethyl or β-phenylethyl, suitable methyl-substituted 2-furyl, 2-thienyl or 2-pyrryl radicals are, for example, 5-methyl-2-furyl, 3,4- Dimethyl-2-furyl, 4-methyl-2-thienyl, 3,5-dimethyl-2-20 thienyl, 5-methyl-2-pyrryl or l, 3,4-trimethyl-2-pyrryl.

The reductive alkylation by which the new antibacterial compounds are produced is preferably carried out as follows. An antibiotic BM123gamma, BM123gammai or BM123gamma2 is dissolved in a suitable solvent such as water, methanol, methyl cellosolve or mixtures thereof, an excess amount of the desired aldehyde or ketone in excess of an equimolar amount is added to the solution obtained, followed by one Sufficient amount of sodium cyanoborohydride for reduction. The pH of the reaction mixture is kept at 6.0 to 8.0 during the reaction with dilute mineral acid. After 1 to 24 hours of reaction at room temperature (10 to 35 ° C.), the reaction mixture is evaporated to dryness in vacuo, the residue is treated with methanol and the whole is filtered. The filtrate is diluted with acetone and the solid which precipitates is removed by filtration and dried in vacuo.

Aldehydes that can be used in the above process. 40 are, for example, acetaldehyde, propionaldehyde, butyraldehyde, isobutyraldehyde, crotonaldehyde, valeraldehyde, benzaldehyde, p-cyanobenzaldehyde, salicylaldehyde, cinnamaldehyde or trichloroacetaldehyde. Examples of ketones that can be used in the above process include acetone. 45 2-butanone, chloroacetone, acetophenone, m-chloroacetophenone. p-bromoacetophehori, 'p-trifluoromethylacetophenone, m-nitro-acetophenone or p-dimethylaminoacetophenone.

The desired products can be obtained from the reaction mixtures obtained in the reductive alkylation by 50 customary processes, such as precipitation, concentration, solvent extraction or combinations thereof. After isolation, these products can be cleaned by any of the well known cleaning procedures. Such purification processes consist, for example, of recrystallization from various solvents and solvent mixtures, chromatographic techniques or countercurrent distribution, and these processes can usually all be used for this purpose.

The new antibacterial compounds are organic 60 bases and can therefore form acid addition salts with a number of organic or inorganic salt-forming compounds. Such salts are prepared by mixing the free base of the respective antibacterial agent with up to 3 equivalents of an acid, expediently in a neutral solvent. Acids suitable for salt formation are, for example, sulfuric acid, phosphoric acid, hydrochloric acid, hydrobromic acid, sulfamic acid, citric acid, maleic acid, fumaric acid, tartaric acid, acetic acid, Ben

5

626895

zoic acid, gluconic acid or ascorbic acid. The acid addition salts of the process products are generally crystalline solids which generally dissolve relatively well in water, methanol or ethanol, but are relatively insoluble in non-polar organic solvents such as diethyl ether, benzene or toluene. The free bases of the antibacterial agents are considered equivalent to their non-toxic acid addition salts.

The antibiotics called BM123ßi,

BM123ß2, BM123gammai and BM123gamma2 arise during the breeding of a new strain of an undetermined species of Nocardia under controlled conditions. This new antibiotic-producing strain was isolated from a sample of garden soil found at Oceola, Iowa, and is listed in the culture collection of the Lederle Laboratories Division, American Cyanamid Company, Pearl River, New York, as Culture No. BM123 kept. A living culture of the new microorganism was deposited with Northern Utilization Research and Development Division, United States Department of Agriculture, Peoria, Illinois, and included in the permanent culture collection. From there it is under the deposit no. NRRL 5646 freely available. The term BM123ß here refers to any mixture of BM123ßi and BM123ß2, and the term BM123gamma generally refers to any mixture of BM123gammai and BM123gamma2.

The microorganism Nocardia sp., *. NRRL 5646, generally described due to the characteristic features determined by him. The cultural characteristics, physiological characteristics and morphological characteristics of the organism were determined according to the methods used in the boarding school. Journ. of system. Bacteriol. 16: 213-240 (1966). The chemical composition of the culture was according to the in Advan. Appi. Microbiol. 14: 47-72 (1971). The underlined color specifications and the color names were based on Color Harmony Manual, 3rd edition (1948), Container Corp. of America, Chicago, Illinois. The details of the description can be found in the following tables I to V.

Strength of growth Moderate growth on yeast extract, asparagine dextrose, Benedict, Bennett, potato dextrose and tartar agar, slight growth on Hickey and Tresner, tomato paste oatmeal and pablo agar, trace growth on inorganic salts Starch, Kuster Oatmeal, Czapek Solution and Rice Agar.

Aerial mycelium

The aerial mycelium, if present, is whitish, is only formed on yeast extract, asparagine dextrose, Benedict, Bennet and potato dextrose agar.

Soluble pigments No soluble pigments are produced.

Bottom color Colorless to yellowish shades.

Different physiological reactions No gelatin liquefaction, nitrates are produced within

valley

Cultural characteristics vc

7 days reduced to nitrites, no melanoid pigments are formed on peptone-iron agar, no peptonization or coagulation in purple milk, sodium chloride tolerance in yeast extract agar,

s

> 4% but <7%,

optimal growth at a temperature of 32 ° C; Carbon utilization according to J. Bacteriol. 56: 107-114 (1948), good utilization of glycerol, salicin, d-trehalose and dextrose, moderate utilization of isoinosit and poor to no utilization of d-fructose, maltose, adonite, 1-arabinose, lactose, d-mannitol , d-melibiose, d-raffinose, 1-rhamnose, sucrose and d-xylose.

xs

Chemical composition The organism belongs to cell wall type IV, i.e. H. it contains meso-2,6-diaminopimelic acid and has a total cell sugar spectrum of type A, which means that it contains arabinose 20 and galactose. Methylated whole cell extracts show fatty acid spectra in the gas chromatogram which are similar to those formed by Nocardia asteroides ATCC 3308.

Micromorphology 25 The aerial mycelium grows in shape from the carrier mycelium. weakly branched out long tortuous elements that usually end in elongated simple spirals. The sinuous elements are irregularly segmented into short elliptical to cylindrical sections (spores?), Which are easily broken down. The spiral end areas are less clearly segmented. The segments generally have dimensions of 0.8 to 1.7 fxm X 0.3 to 0.5 (im, with an average of 0.4 (im X 1.2 (im.

3s identification of the strain

The morphological characteristics of culture No. BM123 are difficult to observe and interpret on most media due to the poor development of aerial mycelium. Chemical analysis to determine the general relationships of the organism is therefore of considerable importance. On the basis of the Lechevalier et al. proposed system contains the culture No. BM123 meso-2,6-diaminopimelic acid in their whole cells, and sugar analysis shows 45 the presence of arabinose and galactose. The culture therefore belongs to cell wall type IV. A comparison of the gas chromatograph spectrum of culture No. BM123 with that of Nocardia asteroides ATCC 3308 shows that both are remarkably similar. Other characteristics of the culture No. BM123, which are consistent with the Nocardia concept, are its fragmented air growth on some media and the total lack of air growth on most media. Because of the lack of adequate criteria for the characterization of Nocardia with regard to the species ss, no such determination was made. Culture No. BM123 is therefore considered an undetermined species of Nocardia until such identification can be made.

ellel i Nocardia sp. NRRL 5646

medium

Incubation: 14 days extent of aerial mycelium

Growth and / or spores

Temperature: 32 ° C soluble underside

Color pigment

Remarks

Medium yeast extract agar

Aerial mycelium whitish, light no mustard-colored dark zones in the

(3 ie) substrate mycelium; Coremie

education on the mycelium surface

626895 ö

Table I (continued) Culture characteristics of Nocardia sp. NRRL 5646

Incubation: 14 days

Temperature: 32 ° C

medium

Extent of

Air mycelium soluble

Subpages

Remarks

Growth and / or spores

Pigment color

Hickey and Tresner

low no aerial mycelium none colorless to the peripheral zones of the

Agar -.

yellowish green

Colonies turn olive green

Asparagine dextrose

mediocre

Trace of whitish no amber

Surface light

Agar

Aerial mycelium

(31c)

wrinkled

Benedict agar mediocre

Aerial mycelium whitish none transparent on the mycelium surface bright

tan (4gc)

abundant coreme formation

Bennett agar mediocre

Trace of a whitish no camel color

Surface light

Aerial mycelium

(3ie)

wrinkled

Inorganic salt

Trace no aerial mycelium no colorless

Starch agar

Kuster oatmeal

Trace no aerial mycelium no colorless

Agar

none colorless

Czapek solution agar

Trace no aerial mycelium

Potato dextrose agar medium

Aerial mycelium, whitish,

none camel

bright

(3ie)

Tomato paste oat

low no aerial mycelium none colorless

flour agar

Pablum agar low no aerial mycelium none colorless

Rice agar

Trace no aerial mycelium no colorless

Weinstein agar medium no aerial mycelium none colorless to yellowish

Tables

Micromorphology of Nocardia sp. NRRL 5646 medium aerial mycelium and / or sporiferous structures

Yeast Extract - The aerial mycelium emerges from the carrier agar in the form of slightly branched convoluted

Elements that usually end in elongated simple spirals. The tortuous elements are irregularly segmented into short sections (spores?) That easily disarticulate. The spiral end areas are less clearly segmented. The segments generally have dimensions of 0.8 to 1.7 p.m X 0.3 to 0.5 | un, with an average of 0.4 (in X 1.2 (in.

Table III

Different physiological reactions of Nocardia sp. NRRL 5646

medium

incubation period

Extent of growth

Physiological response

gelatin

7 days low no liquefaction

gelatin

14 days good no liquefaction

Organic nitrate broth

7 days good

Nitrates became nitrites

reduced

Organic nitrate broth

14 days good

Nitrates became nitriles

_ -

reduced

Peptone iron agar

24 to 48 hours well no reduction in melanin

-

pigments

Purple milk

7 days well no peptonization or

Coagulation _

Yeast extract agar

7 days medium

NaCl tolerance> 4%

plus (4,7,10 and 13%) NaCl

however <7%,

7

626 895

Table IV

Carbon utilization behavior of Nocardia sp. NRRL 5646

Incubation: 10 days

Temperature: 32 ° C

Carbon source

Utilization *

Adonit

0

1-arabinose

0

Glycerin

3rd

d-fructose

1

i-inositol

2nd

Lactose

0

d-mannitol

0

Salicin

2nd

d-melibiosis

0

d-raffinose

0

Rhamnose

0 ~

Maltose

1

Sucrose

0

d-trehalose

3rd

d-xylose

0

Dextrose

3rd

Negative control

0

+ 3-good utilization

2-Passionate utilization

1- Bad utilization

0-no utilization

Table V

Chemical composition of Nocardia sp. NRRL 5646

Cell wall type

Main ingredients

Type IV

meso-DAP

Arabinose

Solve galactose, but dissolve much more. The antibiotics BM123gammai and BM123gamma2 are structural isomers that can be represented by the following structural formulas:

x «

15

20th

25th

30th

35

(M

! a i

fN

£ C U

I.

K Z I

m

CM K U

The production of BM123ß and BM123gamma is not limited to this particular organism or the organisms that fully meet the growth behavior described above and the microscopic properties mentioned, which are given for illustration purposes only. Mutants are therefore also formed from this organism by various means, such as, for example, by X-ray radiation, ultraviolet radiation, nitrogen mustard, actinophage or the like. A living culture of a typical such mutant strain has been deposited with the Northern Utilization Research and Development Division, United States Department of Agriculture, Peoria, Illinois, and has been included in the permanent collection from where it can be obtained under number NRRL 8050. Although the cultural characteristics, the physiological characteristics and the morphological characteristics of NRRL 8050 are practically the same as those of NRRL 5646, the former produces larger amounts of BM123gamma during an aerobic fermentation. The organism NRRL 8050 also differs from the parent organism NRRL 5646 in the following ways:

(a) it reduces nitrates to nitrites and more slowly

(b) it forms a rosewood-tan-colored mycelium pigment on Bennett and yeast extract agar.

The new antibacterial compounds are generally crystalline solids which are relatively limited soluble in non-polar solvents, such as diethyl ether or hexane, and are soluble in solvents, such as water or lower alkali.

40

so

60

65

626895

8th

The reductive alkylation of BM123gamma and BM123gammai or BM123gamma2 with ketones takes place on the spermadin side chain to form derivatives of the formula:

(N ffi 2 I

CM

K U

«W»

1

ta

2 i

M

fM E U

O

SJ II ffi z— o — z f-

M H

S oa d Il / «2

R-V V-CH = CH-C-NH- (CH2) 3-NH- (CH2) 4-NH-CH

io where R for a remainder of the formulas

R2

^ • R,

or

25th

30th

35

h O H

H \

OH H /

\. V NH-

H NH 1

c = o

1

nh2

stands and the substituents R2 and R3 have the meanings given above.

40 The reductive alkylation of BM123gamma,

BM123gammai and BM123gamma2 with aldehydes take place on the spermadin side chain with formation of mono-, di- and tri-substituted derivatives of the formulas:

45

o trans ||

R-v ^ y-CH = CH-C-NH- (CH2) ^ - NH-.tCHj) 4 ~ NH-CH2-R ^

en // i \ trans ||

R-y y-CH = CH-C-NH- (CH,) 3-NH- (CH2) 4-N,

\ - / -i_n2 ~ 'vi

/ CH2-R! ^ CH, -R,

55

60

O

/ 7 ~~ \ trans |

i - ^ __ y-CH = CH-C-NH- (CH2) 3-N- (CH2) 4-NH-CH2-R1

CH2-Rj

CH2-RI

^ ch2-R1

/ ■; —trans 1) /

y-CH = CH-C-NH- (CH2) 3-N- (CHj) 4 ~ N>

'CH2-Ri

65 wherein the substituents R and Ri have the meanings given above.

Antibiotic compounds were already known (US Pat. Nos. 3,350,387,3,784,541 and 3,978,041 and British Pat.

9

626895

No. 1.033.394), which can be recognized by their origin or their chemical structure a certain relationship with the new derivatives.

The new compounds are valuable medicinal products and, as already mentioned, represent interesting antibiotics. This favorable property of the alkyl derivatives of BM 123gamma is demonstrated by their ability to fight systemic lethal infections in the mouse. The new substances show a strong antibacterial effect on the mouse against Escherichia coli US311 in vivo if they are administered to Carworth groups by subcutaneous single dose

Farms (CF-1 mice weighing approximately 20 g administered intraperitoneally infected with a lethal dose of these bacteria in a 10 "dilution of trypticase soy broth TSP from a 5 hour old TSP blood culture. In the s The following table VI shows the in vivo activity of typical products obtainable according to the invention, which have been produced from the specified carbonyl compounds, against Escherichia coli US311 on the mouse The activity is given in ED50 values or that dose in milligrams per kilogram of body weight required to protect 50% of the mice against E. coli.

10th

Table VI

Carbonyl compound used

Name of the derivative

EDJ0 in i body Gi l-DipropyIamino-2-propanone l-Methyl-2- (N, N-dipropylamino) ethyl-BM123gamma

0.3

l-chloro-3-pentanone l-ethyl-3-chloropropyl-BM123gamma

0.12

Cyclooctanone

Cyclooctyl-BM123gamma

0.18

4-methyl-2-pentanone 1,3-dimethylbutyl-BM123gamma

<0.12

Phenylacetone l-methyl-2-phenylethyl-BM123gamma

0.18

trans-4-phenyl-3-buten-2-one l-methyl-3-phenylpropene (-2-) yl-BM123gamma

0.25

1-cyclohexyl-l-propanone •

1-Cyclohexylpropyl-BMl 2 3 gamma

0.37

6-methyl-5-hepten-2-one 1,5-dimethylhexene (-4-) yl-BM123gamma

0.06

3-methyl-2-pentanone

... 1,2-dimethylbutyl-BM123gamma

0.12

5-methyl-2-hexanone 1,4-dimethylpentyl-BM123gamma

0.12

3-ethyl-2-pentanone l-methyl-2-ethylbutyl-BM123gamma

0.18

3,5-dimethyl-2-octanone 1,2,4-trimethylheptyl-BM123gamma

0.37

3-octanone l-ethylhexyl-BM123gamma

0.18

3-methyl-2-hexanone 1,2-dimethylpentyl-BM123gamma

0.18

3-indyl acetone l-methyl-2- (β-indolyl) ethyl-BM123gamma

0.12

2-pentanone

1-methylbutyl-BMl 2 3 gamma

<0.12

2-butanone l-methylpropyl-BM123gamma

<0.12

2-Cyclopentene (-l-) yl-acetone l-methyl-2-cyclopentene (-2 -) - yl-ethyl-BM123gamma

<0.12

acetone

Isopropyl BM123 gamma

<0.12

3-decanone l-ethyl octyl-BM123 gamma v

0.25

3-undecanone l-ethylnonyl-BM123gamma

0.38

o-acetoacetotoluidide l-methyl-2-o [tolylcarbamoylethyl] -BM123gamma

0.25

Mesityl oxide 1,3-dimethylbutene (-2-) yl-BM123gamma

0.3

Methoxyacetone l-methyl-2-methoxyethyl-BM123gamma

0.3

Cyclohexylacetone l-methyl-2-cyclohexylethyl-BM123gamma

0.18

4- (p-hydroxyphenyl) -2-butanone l-methyl-3- (4-hydroxyphenyl) propyl-BM123gamma

0.3

4-methyl-2-hexanone 1,3-dimethylpentyl-BM123gamma

<0.12

2,2,4,4-tetramethylcyclopentanone

2,2,4,4-tetramethylcyclopentyl-BM123gamma

0.75

2,4,4-trimethylcyclopentanone

2,4,4-trimethylcyclopentyl-BM123gamma

0.3

2-cyclopentylcyclopentanone

2-cyclopentylcyclopentyl-BM123gamma

0.37

2- (cyclo-l-hexenyl) cyclohexanone

2- (l-cyclohexene) cyclohexyl-BM123gamma

0.19

3-tert-pentylcyclopentanone

3-tert-pentylcyclopentyl-BM123gamma

0.5

2-cyclohexylcyclohexanone

2-cyclohexylcyclohexyl-BM123gamma

0.75

2-ethylcyclohexanone

2-ethylcyclohexyl-BM123gamma

0.19

3,3-dimethyl-2-butanone 1,2,2-trimethylpropyl-BM123gamma

<0.12

2-undecanon

1-Methyldecyl-BMl 23 gamma

> 2nd

Tetrahydrothiopyran-4-one

4-tetrahydrothiopyranyl-BM123gamma

0.38

3,5-dimethylcyclohexanone

3,5-dimethylcyclohexyl-BM123gamma

<0.12

2-tetradecanone

1-methyltridecyl-BM123gamma

2.0

l-methoxy-l-buten-3-one l-methyl-3-methoxypropen (-2-) yl-BM123gamma

> 2.0

4-hydroxy-3-methyl-2-butanone 1,2-dimethyl-3-hydroxypropyl-BM123gamma

0.12

Menthon

3-methyl-6-isopropylcyclohexyl-BM123gamma

0.38

Cyclononanone

Cyclononyl-BM123gamma

0.18

l-methyl-2-decalon

Decahydro-l-methyl-2-naphthyl-BM123gamma

0.25

3,3-dimethyl-4-cyclohexen-1-one

3,3-dimethylcyclohexene (-4-) yl-BM123gamma

0.37

3-methyl-2-decalon

Decahydro-3-methyl-2-naphthyl-BM123gamma

0.37

l- (3,4-Dimethoxyphenyl) -2-butanone l-ethyl-2- (3,4-dimethoxyphenyl) ethyl-BM123gamma

1.0

l-diethylamino-3-butanone l-methyl-3- (N, N-diethylamino) propyl-BM123gamma

0.18

Ethyl 2-chloroacetoacetate l-methyl-2-chloro-2-carbäthoxyäthyl-BM123gamma

1.5

3-hydroxy-3-methyl-2-butanone 1,2,2-dimethylhydroxypropyl-BM123gamma

0.18

3-pentanone l-ethyl propyl-BM123 gamma

0.12

3-methyl-2-butanone 1,2-dimethylpropyl-BM123gamma

0.19

626895

10th

Table VI (continued)

Carbonyl compound used Name of the derivative EDS0 in mg / kg

Body weight p-chlorophenylacetone l-methyl-2- (4-chlorophenyl) ethyl-BM123gamma 0.25

N- (tert-Butyl) acetoacetamide 2- (tert-Butylcarbamoyl) -l-methylethyl-BM123gamma 0.38

1,1-dimethoxyacetone l-methyl-2,2-dimethoxyethyl-BM123gamma 0.5

4-heptanone l-propylbutyl-BM123gamma 0.25 3-methoxyphenylacetone l-methyl-2- (3-methoxyphenyl) ethyl-BM123gamma 0.30

2-phenylcyclohexanone 2-phenylcyclohexyl-BM123gamma 0.38 phenoxy-2-propanone l-methyl-2-phenoxyethyl-B123gamma 0.3

3-butyn-2-one l-methylpropine (-2-) yl-BM123gamma <2.0 dimethylaminoacetone l-methyl-2- (N, N-dimethylamino) ethyl-BM123gamma 1.5

5-diethylamino-2-pentanone l-methyl-4- (N, N-diethylamino) butyl-BM123gamma 0.5 2-cyclohexen-l-one 2-cyclohexenyl-BM123gamma 0.25 cyclopropylmethyl ketone l-cyclopropylethyl-B123gamma 0.25 4, 4-dimethoxy-2-butanone l-methyl-3,3-dimethoxypropyl-BM123ganuna 0.75 2-methoxyphenylacetone l-methyl-2- (2-methoxyphenyl) ethyl-BM123gamma 0.7 acetylacetone l-methyl-2- acetylethyl-BM123gamma 1.5 cyclobutanone - • Cyclobutyl-BM123gamma 0.38 p-chlorophenylacetone l-methyl-2- (4-chlorophenyl) ethyl-BM123gamma 0.25 2-octanone l-methylheptyl-BM123gamma 0.38

4-phenyl-2-butanone l-methyl-3-phenylpropyl-BM123 gamma 0.38

5-chloro-2-pentanone l-methyl-4-chlorobutyl-BM123gamma 0.37

0-chlorophenylacetone - l-methyl-2- (2-chlorophenyl) ethyl-BM123gamma 0.37 m-chlorophenylacetone l-methyl-2- (3-chlorophenyl) ethyl-BM123gamma 0.38 5-hexen-2-one „l -Methylpentene (-4-) yl-BM123gamma 0.38 cyclohexanone cycloliexyl-BM123gamma 0.75 2-hexanone l-methylpentyl-BM123gamma 0.38 2-heptanone "l-methylhexyl-BM123gamma 0.38 cycloheptanone cycloheptyl-BM123gamma 0.3 Cyclopentanone Cyclopentyl-BM123gamma 0.25 4,4-Dimethyl-2-pentanone 1,3,3-Trimethylbutyl-BM123gamma 0.18

2-acetamido-3-butanone 2-acetamido-l-methylpropyI-BM123gamraa 0.50 2,6-dimethyl-3-heptanone l-isopropyl-4-methylpentyl-BM123gamma 0.39

4-octanone l-propylpentyl-BM123 gamma 0.39

3-Acetylpyridine _ 1- (3-pyridyl) ethyl-BM123gamma 0.75

3-heptanone l-ethylpentyl-BM123gamma 0.25 ethylbutyl acetate l- (carbätboxymethyl) butyl-BM123gamma 0.75

1-benzyl-4-piperidone l-benzyl-4-piperidyl-BM123gamma 0.18

1-methyl-4-piperidone l-methyl-4-piperidyl-BM123gamma 0.75 3-methylcyclopentanone 3-methylcyclopentyl-BM123gamma 0.25

3,3-dimethyl-2-butanone l-methyl-2,2-dimethylpropyl-BM123gamma 0.18

2-acetyl-5-norbornene '1- [5-norbornene (2)] ethyl-BM123gamma <0.12 bicyclo [3.2. l] octan-2-one bicyclo [3.2.1] octanyl-2-BM123gamma <0.25 3-quinuclidinone 3-quinuclidinyl-BM123gamma 0.39

5-methoxyl-2-tetralone 5-methoxyI-2-tetralyl-BM123gamma 0.18

4-methyl-2-heptanone 1,3-dimethylhexyl-BM123 gamma 0.5

3,4-Dimethyl-2-hexanone l, 2,3-trimethylpentyl-BM123gamma 0.39 1,3,3-trimethylcyclopentanone l, 3,3-trimethylcyclopentyl-BM123gamma 0.37 acetylcyclopentanone l-cyclopentylethyl-BM123gamma 0.18

5-Hexen-2-one l-methylpentene-4-yl-BM123gamma 0.18 2-methylcyclopentanoa 2-methylcyclopentyl-BM123gamma 0.25 2,4-dimethylcyclopentanone 2,4-dimethylcyclopentyl-BM123gamma 0.18

2-ethylcyclopentanone 2-ethylcyclopentyl-BM123gamma 0.12 "2-adamantone adamantyl-2-BM123gamma 0.25

3-hexanone l-ethylbutyl-BM123gamma 0.39 ethyl-2-methylacetoacetate l-methyl-2-carboethoxypropyl-BM123gamma 0.18 norbornanone norbomyl-BM123gamma 0.18 5-hexen-2-one l-methyl-4-pentenyl- BM123 gamma 0.18

3-hydroxy-2-butanone l-methyl-2-hydroxypropyl-BM123gamma 0.37

4-hydroxy-3-methyl-2-butanone l-methyl-2- (3-hydroxy-2-methylpropyl) -BM123gamma 0.18 2-nonanone l-methyloctyl-BM123gamma 0.37

5-Hydroxy-2-pentanone l-methyl-4-hydroxybutyl-BM123gamma 0.18 2-decanone l-methylnonyl-BM123gamma 0.18 4-tert-butylcyclohexano 4-tert.-butylcyclohexyl-BM123gamina 0.12 2-ethylidene cyclohexanone 2-ethylidene cyclohexyl BM123 gamma 0.12

11

Table VI (continued)

626 895

Carbonyl compound used Name of the derivative EDJ0 in mg / kg

body weight

Phenylacetaldehyde

2-phenylethyl-BM123gamma

0.18

p-methoxyphenylacetaldehyde

2- (p-methoxyphenyl) ethyl-BM123gamma

0.18

2,2-dimethylbutanal

2,2-dimethylbutyl-BM123gamma

0.12

2,2-dimethylpropanol

2,2-dimethylpropyl-BM123gamma

0.18

2-ethyl-2-butenal

2-ethyl-2-butenyl-BM123gamma

0.18

trans-2-methyl-2-butenal trans-2-methyl-2-butenyl-BM123gamma

0.18

trans-2-methyl-2-pentenal trans-2-methyl-2-pentenyl-BM123gamma

0.18

formaldehyde

Methyl BM123 gamma

0.12

acetaldehyde

ÄthyI-BM123gamma

0.38

Fermentation process selected for the production of predominantly BM123ß and BM123gamma. The cultivation of Nocardia sp. NRRL 8050 can be done in a number of different liquid culture media. Media suitable for forming the antibiotics contain assimilable carbon sources, such as starch, sugar, molasses or glycerol, assimilable nitrogen sources, such as protein, protein hydrolyzate, polypeptides, amino acids or corn steep liquor, and inorganic anions and cations, such as potassium, magnesium, calcium, ammonium, sulfate, Carbonate, phosphate or chloride. Trace elements, such as boron, molybdenum or copper, are supplied as impurities in other components of the media. Aeration in tanks or flasks is carried out by passing sterile air through the fermentation medium or onto the surface of the fermentation medium under pressure. Another mixing in tanks is carried out by a mechanical stirrer. If necessary, an anti-foaming agent such as Hodag (R) FD 82 can also be added.

Preparation of the inoculum for BM123ß and BM123gamma

A primary shaker bottle inoculum from Nocardia sp. NRRL 8050 is made by mixing 100 ml of sterile liquid

35

Meat extract

15 g

Ammonium sulfate

3g

Potassium dihydrogen phosphate

3g

Calcium carbonate lg

Magnesium sulfate heptahydrate

1.5 g

glucose

10 g

Water on

1000 ml

The glucose is sterilized separately.

25th

The inoculum obtained in the third stage is aerated at a rate of 0.4 to 0.8 liters of sterile air per liter of broth per minute, the fermentation mixture being mixed by a stirrer at a rate of 150 to 300 revolutions per minute. The temperature is maintained at 25-29 ° C, usually 28 ° C. The whole is left to grow for 48 to 72 hours, after which the inoculum obtained in this way inoculates a 3000 liter fermentation tank.

Tank fermentation for the production of BM123 ß and BM123gamma For the production of BM123ß and BM123gamma in a medium by scraping or washing off spores from 40 tank fermenters, the following one of the culture agar slants inoculated in 500 ml flasks is preferably used. The following medium is usually used for this:

Fermentation medium:

Bactotrypton yeast extract bovine extract glucose water

5g 5g 3g

10 g 1000 ml

The flasks are incubated at a temperature of 25 to 29 ° C., preferably 28 ° C., and kept vigorously in motion on a rotary shaker over a period of 30 to 48 hours. The inoculum is then transferred to sterile screw cap culture bottles and stored at below -17.8 ° C (0 ° F). This stock of vegetative inoculum is then used instead of inclined agar preparations for inoculating additional shake flasks for the production of this first-stage inoculum.

The piston inocula obtained from this first stage are used to inoculate 12 liter batches of the same medium in 20 liter glass fermenters. The inokulum pulp is aerated with sterile air while being allowed to grow for 30 to 48 hours.

The 12 liter batches of the inocula obtained in the second stage are used to inoculate tank fermenters which contain 300 liters of the following sterile liquid medium in order to form the third and last inoculum:

Meat extract

30 g

Ammonium sulfate

6g

Potassium dihydrogen phosphate

6g

Calcium carbonate

2g

Magnesium sulfate heptahydrate

3g

glucose

20 g

Water on

1000 ml

The glucose is sterilized separately.

50

Each tank is inoculated with 5 to 10% of the inoculum obtained in the third stage 55 manufacture of the inoculum described above. The fermentation slurry is kept at a temperature of 25 to 28 ° C, usually 26 ° C. The slurry is aerated with sterile air at a rate of 0.3 to 0.5 liters of sterile air per liter of slurry per minute and kept in motion by a stirrer with a stirring speed of 70 to 100 revolutions per minute. The fermentation is allowed to run for 65 to 90 hours, after which the mash is harvested.

The invention is further illustrated by the following examples.

65

example 1

Preparation of the inoculum for BM123ß and BM123gamma using the following typical nutrient medium

626 895

12th

let the first and second stages of an inoculum grow:

Bactotrypton 5 g

Yeast extract 5 g

Beef extract 3 g

Glucose 10 g

Water to 1000 ml

Two 500 ml flasks, each containing 100 ml of the above sterile medium, are each treated with 5 ml of a frozen vegetative inoculum from Nocardia sp. Inoculated with NRRL 8050. The flasks are then placed on a rotary shaker and shaken vigorously over a period of 48 hours at a temperature of 28 ° C. The flask inoculum obtained is then transferred to a 22.7 liter glass fermenter containing 12 liters of the above sterile medium. The porridge is aerated with sterile air during the approximately 48 hours of growth, after which the fermenter contents are inoculated into a 455 liter tank fermenter which contains 300 liters of the following sterile liquid medium:

Meat extract 15 g

Ammonium sulfate 3 g

Potassium dihydrogen phosphate 3 g

Calcium carbonate 1 g

Magnesium sulfate heptahydrate 1.5 g

Glucose 10 g

Water to 1000 ml

The glucose is sterilized separately.

The glucose is sterilized separately.

The above inoculum porridge of the third stage is aerated with sterile air, which is blown into the fermenter at a rate of 0.4 liters per liter of porridge per minute. The contents of the fermenter are mixed with a stirrer at a stirring speed of 240 revolutions per minute. The slurry is kept at a temperature of 28cC, using Hodag (R) FD82 as defoamer. After a 48 hour growth period, the inoculum mash obtained is used to inoculate a 3000 liter fermentation.

Example 2

Fermentation using Nocardia sp.

NRRL 8050 and a medium that promotes the formation of BM123ß and BM123gamma

A fermentation medium is made from the following ingredients:

Meat extract 30 g

Ammonium sulfate 6 g

Potassium dihydrogen phosphate 6 g

Calcium carbonate 2 g

Magnesium sulfate heptahydrate 3 g

Glucose 20 g

Water to 1000 ml

The glucose is sterilized separately.

The glucose is sterilized separately.

The fermentation medium is sterilized at 120 ° C with steam of 1.41 kg / cm2 (20 pounds) for 60 minutes. The pH of the medium after sterilization is 6.9. In a 4000 liter tank fermenter, 3000 liters of sterile medium are inoculated with 300 liters of the inoculum described in Example 1, whereupon the whole is fermented at a temperature of 26 ° C. using Hodag (R) FD82 as a defoamer. The tank is aerated with sterile air at a flow rate of 0.35 liters per liter of mash per minute. The mash is kept in motion by an agitator that is driven at a speed of 70 to 72 revolutions per minute. After a total of 67 hours of fermentation, the mash is harvested.

10th

Example 3

Isolation of BM123ß and BM123gamma 3000 liters of a fermentation mash with a pH of 4.3 produced as described in Example 2 are adjusted to pH 7.0 with sodium hydroxide and filtered using 5% diatomaceous earth as a filter aid. The filter cake is washed with about 100 liters of water and then discarded. The combined filtrates and wash liquors are passed from bottom to top through three parallel 20 stainless steel columns with dimensions 21X122 cm, each of which is 15 liters of CM Sepha-dex® C-25 [Na +] resin (a cross-linked dextran-epichlorohydrin Cation exchange gel from Pharmacia Fine Chemicals, Inc.) contains. The loaded columns are washed with a total of about 390 2s liters of water, whereupon they are first developed with 200 liters of 1 percent aqueous sodium chloride and then with 560 liters of 5 percent aqueous sodium chloride. The 5% aqueous sodium chloride eluate is clarified by filtering it through diatomaceous earth, and the clarified filtrate obtained is then passed through a glass column measuring 20.3 × 152.5 cm, the 25 liters of granular Darco® G-60 (0.37 to 0.83 mm mesh size) (a granular activated carbon from Atlas Chemical Industries, Inc.) contains. The loaded column is washed m: 3s 120 liters of water and then first developed with 120 liters of 15 percent aqueous methanol, then with 340 liters of 50 percent aqueous methanol and finally with 12 liters of 50 percent aqueous acetone. The 15 percent aqueous methanol eluate is concentrated in vacuo to about 7-40 liters of an aqueous phase, after which its pH value is determined with Amberlite® IR-45 (OH ~) resin (a weakly basic polystyrene-polyamine anion exchange resin) pH 4.5 adjusted to pH 6.0. The resin is then separated by filtration, and by concentrating the 45 filtrate in vacuo to about 1 liter and then lyophilizing, 38 g of material consisting mainly of BMl23ß are obtained, together with a small amount of BM123gamma (predominantly BM123gamma2). The 50% aqueous methanol eluate is adjusted with Amberlite® IR-so 45 (OH ~) resin from pH 4.65 to pH 6.0. The resin is then separated off by filtration, and by concentrating the filtrate in vacuo to about 6.3 liters and then lyophilizing, 213 g of material are obtained, which mainly consists of BM123gamma. The 50 percent aqueous ss acetone eluate is adjusted with Amberlite® IR-45 (OH ~) resin from pH 4.0 to pH 6.0. By filtering off the resin, then concentrating the filtrate obtained in vacuo to about 1.5 liters and subsequent lyophilization, 56 g of impure BM123gamma are obtained.

60

Example 4

Further purification of BM123gamma A slurry of CM Sephadex® C-25 [NHÎ] in 2 percent aqueous ammonium chloride is poured into a glass 65 column with a diameter of 2.6 cm until a resin height of approximately 62 cm is obtained. The excess of 2 percent aqueous ammonium chloride is drained off. Then dissolve 5.0 g of that prepared according to Example 3

13

626 895

BM123gamma in about 10 ml of 2 percent aqueous ammonium chloride and put the whole thing on the column. The column is then eluted with a gradient between 6 liters of 2 percent and 4 percent aqueous ammonium chloride. Fractions of approx. 75 ml each are collected automatically every 15 minutes. The position of the antibiotic BM123gamma is monitored by constant examination of the column effluent in ultraviolet light and by bioautography of soaked paper disks on large agar plates inoculated with Klebsiella pneumoniae, strain AD. The majority of BM123gamma comes between fractions 71 to 107.

130 ml of granular Darco® G-60 (0.37 to 0.83 mm mesh size) are suspended in water and poured into a glass column. There you let the material settle and the excess water runs off. Fractions 84 to 96 of the above CM Sephadex chromatogram are combined and passed through the column filled with carbon granules. The loaded column is washed with 600 ml of water and then developed with 1 liter of 50 percent aqueous acetone. The eluates, which both contain BM123gamma, are concentrated in vacuo to aqueous phases and lyophilized, whereby a total of 886 mg of BM123gamma is obtained as the hydrochloride salt. To prepare a sample for microanalysis, the above material is again subjected to the above procedure.

The antibiotic BM123gamma has no pronounced melting point, but it gradually begins to decompose at around 200 ° C. The microanalysis of a sample, which is allowed to balance for 24 hours at a temperature of 22.2 ° C and a relative humidity of 23%, leads to the following values: C = 39.44%; H = 6.10%; N = 16.19%; Cl (ionic) = 11.54%. The weight loss on drying is 8.19%. In water, BM123gamma shows a UV absorption maximum at 286 nm with a value EJot = 250. The position of this maximum does not change with the pH value. The BM123gamma shows a specific rotation of [alpha] ^? ° = + 71 ° (C = 0.97 in water).

The antibiotic BM123gamma shows a characteristic absorption in the infrared region of the spectrum at the following wavelengths: 770, 830, 870, 930, 980,1035, 1105,1175, 1225,1300,1340,1370,1460, 1510,1555, 1605, 1660,1740 , 2950 and 3350 cm "1. A conventional infrared absorption spectrum of BM123gamma produced with a KBr pellet is shown in the attached FIG. 1.

Example 5 Isolation of BM123gammai

A slurry of CM Sephadex (R) C-25 [Na +] in 2 percent aqueous sodium chloride is poured into a 2.6 cm diameter glass column to give a resin height of about 70 cm. The excess 2 percent aqueous sodium chloride is drained off. Then 4.11 g of a sample, which mainly contains BM123gammai together with some BM123gamma2 and other impurities and has been prepared according to Example 3, is dissolved in about 10 ml of 2% aqueous sodium chloride and the whole is brought onto the column. The column is then eluted at a gradient between 4 liters with 2 percent and 4 percent aqueous sodium chloride. Fractions of approximately 75 ml each are automatically collected every 15 minutes. The position of the antibiotic BM123gamma is monitored and determined by constant monitoring of the column flow in ultraviolet light and by bioautography of soaked paper discs on large agar plates inoculated with Klebsiella pneumoniae, strain AD. The main part of BM123gamma lies between the fractions 64 and 90. The initial fractions (64-80)

contain a mixture of BM123gammai and BM123gamma2, and the last fractions (81-90) contain practically pure BM123gammai.

100 ml of granular Darco® G-60 (0.37 to 0.83 mm s internal mesh size) are suspended in water and poured into a glass column. The suspension is allowed to settle in this and the excess water is drained off. Fractions 81-90 of the above CM Sephadex chromatogram are pooled and passed through the column filled with carbon granules. The loaded column is washed with 500 ml of water and then developed first with 500 ml of 10 percent aqueous methanol and then with 1 liter of 50 percent aqueous methanol. The 50 percent aqueous methanol eluate, which contains the bulk of BM123gammai, is adjusted to pH 5.9 to 6.0 with Amberlite® IR-45 (OH_1) resin. The resin is then separated off by filtration, the filtrate is concentrated in vacuo onto an aqueous phase and this filtrate is lyophilized, giving 294 mg of white amorphous BM123gammai in the form of the hydrochloride 20 salt.

The antibiotic BM123gammai has no defined melting point, but it gradually decomposes at a temperature around 200 ° C. The microanalysis of a sample which is allowed to balance 24 hours beforehand at a temperature of 21.1 ° C. and a relative humidity of 60% gives the following values: C = 37.84%; H = 5.73%; N = 15.58; Cl (ionic) = 10.01%. The weight loss on drying is 10.45%. In methanol, BM123gammai shows a UV absorption maximum at 286 nm with a value of 30 E IOT = 225. The position of this maximum does not change with the pH value. The BM123gammai shows a specific rotation of + 55 ° (c = 0.803 in water).

The antibiotic BM123gammai shows a characteristic absorption in the infrared region of the spectrum at the following wavelengths: 770, 830, 870, 930, 980, 1045, 1080, 1110, 1125, 1175, 1225, 1305, 1345, 1380, 1465, 1515, 1560, 1605, 1660, 1730, 2950 and 3350 cm-1. A conventional infrared absorption spectrum of BM123gammai in a KBr pellet is shown in the attached FIG. A conventional magnetic proton resonance spectrum from BM123gammai can be found in FIG. 4, which was determined on a D2O solution in a spectrometer with 100 megacycles.

Example 6

45 Isolation of BM123gamma2

. 25 g of a sample prepared according to Example 3, which predominantly contains BM123gamma2 and BM123ß, are dissolved in about 120 ml of 2% aqueous sodium chloride and then placed in the form of this solution on a column which is filled with 1800 ml of CM Sephadex® C-25 [ Na +] is filled in 2 percent aqueous sodium chloride. The column is then eluted under a gradient between 20 liters of 2 percent and 4 percent aqueous sodium chloride. The first 12 liters of eluate are collected in a large flask and discarded. Subsequently, fractions of about 800 ml each are automatically collected every 40 minutes. The position of the antibiotic BM123gamma is determined by constant examination of the fractions coming from the column in ultraviolet light. The majority of 60 BM123gamma are between fractions 7-18. The initial fractions (7-15) contain practically pure BM123gamma2. The final fractions (16-18) contain a mixture of BM123gammai and BM123gamma2.

600 ml of granular Darco® G-60 (0.37 to 0.83 mm clear mesh size) are suspended in water and then poured into a glass column. The suspension is allowed to settle here and excess water is drained off. Fractions 7-15 from the CM Sephadex chromatogram above

626895

14

united and sent through the column filled with coal granules. The loaded column is washed with 3 liters of water, whereupon it is developed first with 3 liters of 10% aqueous methanol and then with 6 liters of 50% aqueous methanol. The 10% aqueous methanol eluate contained in this process is adjusted to pH 5.8 to 6.0 with Amberlite® IR-45 (OH ~) resin. The resin is then separated off by filtration. By concentrating the filtrate in vacuo to an aqueous phase and lyophilizing, 595 mg of white amorphous BM123gamma2 are obtained in the form of the hydrochloride salt. The 50 percent aqueous methanol eluate is adjusted with Amberlite® IR-45 (OH_) resin from pH 4.6 to 6.1. The resin is then removed. Concentrating the filtrate in vacuo to an aqueous phase and then lyophilizing gives 3.645 g of slightly less pure white amorphous BM123gamma2 in the form of the hydrochloride salt.

The antibiotic BM123gamma2 has no pronounced melting point, but gradually decomposes at around 200 ° C. The microanalysis of a sample, which is allowed to balance for 24 hours at a temperature of 21.1 ° C and a relative humidity of 60%, gives the following values: C = 36.14%; H = 5.67%; N = 15.1%; Cl (ionic) = 11.11%. The weight loss on drying is 10.87%. In methanol, BM123gamma2 shows a UV absorption maximum at 286 nm with a value E iSi • = 220. The position of this maximum does not change with the pH value. The BM123gamma2 shows a specific rotation of + 60 ° (c = 0.851 in water).

The antibiotic BM123gamma2 has a characteristic absorption in the infrared region of the spectrum at the following wavelengths: 770, 830, 870, 950, 980,1035, 1110, 1175,1225,1285,1345,1380,1470,1515,1560,1605,1660, 1755, 2950 and 3350 cm-1. A conventional infrared absorption spectrum of BM123gamma2 in a KBr pellet is shown in the attached FIG. The attached FIG. 5 shows a conventional magnetic proton resonance spectrum of BM123gamma2, determined on a D20 solution in a spectrometer with 100 megacycles.

Example 7

Paper distribution and thin layer chromatography of BM123ß and BM123gamma The BM123 antibiotics can be differentiated from one another by paper chromatography. For this purpose, Whatman No. 1 paper strip swab water or methanol solutions of the respective substances and equilibrate the whole for 1 to 2 hours in the presence of both the upper and lower phases. The strips are then developed overnight with the lower (organic) phase, which is a mixture of 90 percent phenol: m-cresol: acetic acid: pyridine: water (100: 25: 4: 4: 75 by volume). The developed strips are removed from the chromatograph chamber, air-dried for 1 to 2 hours, washed with ether to remove residual phenol and bioautographed on large agar plates inoculated with Klebsiella pneumoniae, strain AD. The typical Rf values obtained are shown in Table VII below:

Table VII

component

RF value

BM123gamma

0.85

BM123ß

0.50, 0.70

The β component is a mixture of the two antibiotics using this system. The BM123ß consists of a larger amount of an antibiotic (Rf = 0.50) called BM123ßi and a smaller amount of an antibiotic (Rf = 0.70) called BM123ß2.

The antibiotics BM123 can also be distinguished from one another by thin-layer chromatography. For this purpose, appropriate swabs are applied with a water solution of the substance to be chromatographed (approximately 20 to 40 micrograms per swab) on pre-coated Cellulose F® plates (0.10 millimeters thick), namely a form of a strong cellulose layer, as described by EM Laboratories, Inc., Elmsford, New York. The plates are then developed overnight with a solvent obtained by mixing 1-butanol: water: pyridine: acetic acid (15: 12: 10: 1 by volume). The developed plates are then removed from the chromatograph chamber and air-dried for about an hour. The antibiotics are detected either using conventional ninhydrin reagents or using a Sakaguchi spray reagent. The typical Rf values obtained are shown in Table VIII below:

Table VIII

component

RF value

BM123gamma

0.17, 0.23

BM123ß

0.08, 0.14

Both antibiotics BM123ß and BM123gamma are mixtures of two components using this system. The BM123ß consists of a larger amount of a component (Rf = 0.08), which is called BM123ßi, and a smaller amount of a component (Rf = 0.14), which is called BM123ß2. The less polar component of BM123gamma (Rf = 0.23) is BM123gammai, and the more polar component (Rf = 0.17) is the BM123gamma2.

Example 8

General procedure for the reductive alkylation of the antibiotic BM123gamma A suspension of 100 mg of antibiotic BM123gamma in 20 ml of methanol is mixed with 5 ml (or 5 g) of the respective aldehyde or ketone and 100 mg of sodium cyanoborohydride. The pH of the solution obtained is kept at about 7.0 with 0.1N methanolic hydrogen chloride over a period of 3 to 24 hours. The reaction is monitored by thin layer chromatography until BM123gamma disappears. The reaction mixture is then filtered, whereupon the filtrate is evaporated to dryness. The residue is treated with 3 ml of methanol and the whole is filtered. The filtrate is diluted with 50 ml of acetone, and the precipitate which forms is separated off by filtration and then dried. The methanol used as the solvent can also be replaced by 20 ml of water, provided that the aldehydes or ketones used as starting materials are soluble in water.

Example 9

Preparation of Methyl-BM123gamma A solution of 1.0 g of BM123gamma and 2.5 ml of a 37 percent aqueous formaldehyde solution in 50 ml of water

io

15

20th

25th

30th

35

40

45

50

55

60

65

15

626 895

400 mg of sodium cyanoborohydride are added in portions. During this addition, the pH of the reaction mixture is kept at 7.0 with in hydrochloric acid. The reaction mixture is then stirred for a further 10 minutes at room temperature and then evaporated to dryness in vacuo. The residue is treated with 20 ml of methanol, the whole is filtered off and the filtrate is diluted with 250 ml of acetone. The product which precipitates is separated off by filtration and dried. The yield is 667 mg.

Example 10 Preparation of Isopropyl-BM123gamma A solution of 200 mg of BM123gamma in 30 ml of methanol is mixed with 5 ml of acetone. This solution is then mixed with 139 mg of sodium cyanoborohydride and the reaction mixture is stirred for 30 minutes at room temperature. During this time, the pH of the reaction mixture is kept at 7.4 to 7.8 by adding 0.1 in methanolic hydrogen chloride. The resulting small amount of precipitate is separated off by filtration, and the filtrate is then evaporated to dryness in vacuo. The residue is treated with 2 ml of methanol and the whole is filtered. The filtrate is diluted with 100 ml of acetone and the solid obtained is separated off by filtration and dried. The yield is 184 mg.

Example 11

Preparation of ß-phenylethyl-BM123gamma A solution of 200 mg of BM123gamma in 15 ml of water and 25 ml of acetonitrile is mixed with a solution of 2 ml of phenyl-acetaldehyde in 4 ml of ethanol. 103 mg of sodium cyanoborohydride are then added. The reaction mixture is then stirred for 30 minutes at room temperature, the pH of the mixture being kept at 7 with 0.2N hydrochloric acid. The reaction mixture is then filtered and the filtrate is evaporated to dryness in vacuo. The residue obtained is treated with 2 ml of methanol and the whole is filtered. The filtrate is diluted with 100 ml of acetone, and the product obtained is separated off by filtration and dried. The yield is 180 mg.

Example 12

Preparation of 1,3,3-trimethylbutyl-BM123gamma A solution of 200 mg of BM123gamma-hydrochloride in 50 ml of methanol is mixed with 3 ml of 4,4-dimethyl-2-pentanone and 106 mg of sodium cyanoborohydride. The reaction solution is kept at pH 7 by dropwise addition of methanolic hydrogen chloride. The reaction mixture is stirred at room temperature for 18 hours and then filtered. The filtrate is evaporated to dryness in vacuo. The residue is dissolved in 3 ml of methanol, whereupon the solution is diluted with 50 ml of acetone and filtered. The yield is 125 mg.

Example 13

Preparation of l-methylphenylethyl-BM123gamma A solution of 200 mg of BM123gamma in 50 ml of methanol is mixed with 5 ml of phenylacetone. The solution is then mixed with 170 mg of sodium cyanoborohydride and the reaction mixture is stirred for 3.5 hours at room temperature. During this time, the pH of the reaction mixture is kept at 7.0 by adding methanol saturated with hydrogen chloride gas. The reaction mixture is then concentrated to a volume of about 5 ml, diluted with 2 ml of methanol and filtered. The filtrate is then poured into 100 ml of acetone, whereupon the solid obtained is diluted with 50 ml of acetone and filtered. The yield is 233 mg.

Example 14

Preparation of l-methylnonyl-BM123gamma Sodium cyanoborohydride (100 mg) is added to a solution of BM123gamma (200 mg) and 2-decanone (1 ml) in 40 ml of methanol. The pH of the solution is adjusted to 7.0 by adding 0.1 in methanolic hydrogen chloride gas and kept at a value of 7.0 ± 0.2 if necessary by further adding this acid. After reaction for 19.5 hours, the reaction mixture is filtered and the filtrate is concentrated in vacuo at 30 ° C. The residue thus obtained is slurried in 5 ml of methanol and the whole is filtered. The filtrate is added to 50 ml of acetone. The resulting white precipitate is separated off by filtration, washed with acetone and dried in vacuo. The yield of crude 1-methylnonyl-BM 123gamma is 167 mg.

Example 15

Preparation of 1,3-dimethylbutyl-BM123gamma A solution of 2.10 mg of BM123gamma in 50 ml of methanol is mixed with 5 ml of methyl isobutyl ketone. This solution is then mixed with 166 mg of sodium cyanoborohydride and the reaction mixture is stirred for 5 hours at room temperature. During this time, the pH of the reaction mixture is kept at 7.0 with methanol saturated with hydrogen chloride gas. The reaction mixture is then evaporated to dryness in vacuo. The residue is treated with 2 ml of methanol and the whole is filtered. The filtrate is diluted with 100 ml of acetone, whereupon the separated solid is filtered off and dried. The yield is 210 mg.

Example 16

Preparation of 2-norbornyl-BM123gamma Sodium cyanoborohydride (100 mg) is added to a solution of BM123gamma (200 mg) and 2-norboraanone (400 mg) in 40 ml of methanol. The pH of the solution is adjusted to 7.0 with 0.1 methanolic hydrogen chloride.

By adding 0.1 in hydrogen chloride as required, the pH is kept at 7.0 ± 0.2. After 21.5 hours of reaction, the reaction mixture is filtered and the filtrate is concentrated in vacuo at 35 ° C. The residue is slurried in 5 ml of methanol and the whole is filtered. The filtrate is added to 50 ml of acetone. The whitish solid which precipitates is collected by filtration, washed with acetone and dried in vacuo. The yield of crude 2-norbornyl-BM123gamma is 175 mg.

Example 17 Preparation of Isopropyl-BM123gammai Fin Mixture of BM123gammai, 5 ml acetone and 60 mg sodium cyanoborohydride in 35 ml methanol is stirred for 40 minutes at room temperature. The pH of the solution is kept at 7 by dropwise addition of methanolic hydrogen chloride solution. The mixture is then evaporated to dryness in vacuo. The residue is treated with 5 ml of methanol and the solution obtained is diluted with 50 ml of acetone. The precipitate obtained is filtered, washed and dried in vacuo. The yield is 49 mg.

Example 18 Preparation of isopropyl-BM123gamma2 A mixture of 41 mg of BM123gamma2, 5 ml of acetone and s

10th

IS

20th

25th

30th

35

40

45 ■

SO

SS

60

65

626895

16

50 mg sodium cyanoborohydride in 35 ml methanol is stirred for 40 minutes at room temperature. The pH of the solution is kept at 7 by dropwise addition of a methanolic hydrogen chloride solution (saturated). The resulting mixture is filtered and then evaporated to dryness in vacuo. The residue is treated with 5 ml of methanol and the solution obtained is diluted with 50 ml of acetone. This gives 46 mg of product.

Example 19

Preparation of l-methyl-2-phenylethyl-BM123gamma2 A mixture of 200 mg of BM123gamma2, 5 ml of phenylacetone and 170 mg of sodium cyanoborohydride in 50 ml of methanol is stirred for 3.75 hours at room temperature. During this time, the pH of the reaction mixture is kept at a value of 7 by dropwise addition of a methanolic hydrogen chloride solution (saturated). The mixture is then evaporated to dryness in vacuo. The residue is treated with 5 ml of methanol and the resulting methanol solution is then diluted with about 50 ml of acetone. In this way, 233 mg of product are obtained.

Example 20 ■

Preparation of 2-ethylcyclopentyl-BM123gamma A solution of 200 mg of BM123gamma, 3 ml of 2-ethylcyclopentanone and 101 mg of sodium cyanoborohydride in 50 ml of methyl alcohol are left at room temperature for 18 hours. During this time, the pH of the solution is maintained at 7 by adding a saturated solution of sodium chloride in methanol. The reaction mixture is then evaporated to dryness. The residue is treated with 3 ml of methanol, the whole is filtered and the filtrate is diluted with 40 ml of acetone. This gives 157 mg of product.

Example 21

Preparation of 3,5-dimethylcyclohexyl-BM123gamma A solution of 200 mg of BM123gamma, 5 ml of 3,5-dimethylcyclohexanone and 200 mg of sodium cyanoborohydride in 50 ml of methanol is left for one hour at room temperature. During this time, the pH of the solution is maintained at 7 by adding a saturated solution of sodium chloride in methanol. The reaction mixture is then treated with 3 ml of methanol and filtered off, after which the filtrate is diluted with 40 ml of acetone. This gives 200 mg of product.

Example 22

Preparation of 2,4-dimethylcyclopentyl-BM123gamma A solution of 206 mg of BM123gamma, 3 ml of 2,4-dimethylcyclopentanone and 104 mg of sodium cyanoborohydride in 50 ml of methanol is left at room temperature for 6 hours. During this time, the pH of the solution is maintained at 7 by adding a saturated solution of sodium chloride in methanol. The reaction mixture is then treated with 3 ml of methanol and filtered off, after which the filtrate is diluted with 40 ml of acetone. The yield of the desired product is 101 mg.

Example 23

Preparation of 2-ethylcyclohexyl-BM123gamma A solution of 200 mg of BM123gamma, 5 ml of 2-ethylcyclohexanone and 213 mg of sodium cyanoborohydride in 50 ml of methanol is left at room temperature for 3 hours. During this time, the pH of the solution is kept at 7 by adding a saturated solution of sodium chloride in methanol. The reaction mixture is then treated with 3 ml of methanol and filtered, after which the filtrate is diluted with 40 ml of acetone. The title compound is obtained in a yield of 200 mg.

Example 24

Preparation of 3-methylcyclohexyl-BM123gamma A solution of 200 mg of BM123gamma, 1.5 ml of 3-methyl-cyclohexanone and 200 mg of sodium cyanoborohydride in 50 ml of methanol is left for 2 hours at room temperature. During this time, the pH of the solution is kept at 7 by adding a saturated solution of hydrogen chloride in methanol. The reaction mixture is then treated with 3 ml of methanol and filtered, after which the filtrate is diluted with 40 ml of acetone. The title compound is obtained in a yield of 200 mg.

Example 25

Preparation of 2,4,4-trimethylcyclopentyl-BM123gamma A solution of 200 mg of BM123gamma, 5 ml of 2,4,4-trimethylcyclopentanone and 179 mg of sodium cyanoborohydride in 50 ml of methanol is left at room temperature for 24 hours. During this time, the pH of the solution is kept at 7 by adding a saturated solution of sodium chloride in methanol. The reaction mixture is then treated with 3 ml of methanol and filtered, after which the filtrate is diluted with 40 ml of acetone. The desired compound is obtained in a yield of 176 mg.

Example 26

Preparation of 2-propylcyclohexyl-BM123gamma A solution of 200 mg of BM123gamma, 3 ml of 2-propylcyclo-clohexanone and 157 mg of sodium cyanoborohydride in 50 ml of methanol is left for 4 hours at room temperature. During this time, the pH of the solution is kept at 7 by adding a saturated solution of sodium chloride in methanol. The reaction mixture is then treated with 3 ml of methano and filtered, after which the filtrate is diluted with 40 ml of acetone. The yield of the desired product is 75 mg.

Example 27

Preparation of 2-methylcyclopentyl-BM123gamma A solution of 211 mg of BM123gamma, 3 ml of 2-methylcyclopentanone and 98 mg of sodium cyanoborohydride in 50 ml of methanol is left at room temperature for 3.5 hours. During this time, the pH of the solution is kept at 7 by adding a saturated solution of sodium chloride in methanol. The reaction mixture is then treated with 3 ml of methanol and filtered, after which the filtrate is diluted with 40 ml of acetone. The title compound is obtained in a yield of 157 mg.

s

10th

IS

20th

25th

30th

35

40

45

50

55

60

B

3 sheets of drawings

Claims (4)

626895 2 PATENT CLAIMS 1. A process for the preparation of new alkyl and substituted alkyl derivatives of the antibiotics BM 123 gamma 1, gamma 2 and their mixtures, and their acid addition salts, characterized in that said antibiotics or said mixtures with an aldehyde or a ketone of the formula: O II Ri-CHO or R2-C-R3, 10 wherein Ri for hydrogen, lower alkyl, halogen-substituted lower alkyl, lower alkenyl, phenyl, monosubstituted phenyl, phenyl-lower alkyl, styryl, 2-furyl, methyl-substituted 2-furyl, 15 2-thienyl, methyl-substituted 2-thienyl, 2-pyrryl, methyl-substituted 2- Pyrryl, 2-pyridyl or 2-quinolyl. R2 means lower alkyl and R3 for alkyl with up to 6 carbon atoms, NN-dipropylaminomethyl, 2-chloroethyl, benzyl, styryl, cyclohexyl, 4-20 methylpenten-3-yl, 1,3-dimethylhexyl, ß-indolylmethyl, cyclopenten-2-ylmethyl, n-heptyl, n-octyl, (o-tolylcarbamoyl) methyl, propenyl, methoxymethyl, cyclohexylmethyl, 2- (4-hydroxyphenyl) ethyl, n-nonyl, n-dodecyl, 2-methoxyvi-. nyl, l-methyl-2-hydroxyethyl, 3,4-dimethoxybenzyl, N, N- 2S diethylaminoethyl, carbethoxychloromethyl, 1-methyl-l-hydroxyethyl, 4-chlorobenzyl, (t-butylcarbamoyl) methyl, dimethoxymethyl, 2-, 3rd - or 4-methoxybenzyl, phenoxymethyl, ethynyl, N, N-dimethylaminomethyl, N, N-diethyIamino- propyl, cyclopropyl, 2,2-dimethoxyethyl, acetonyl, ß-phenyl-ethyl, 3-chloropropyl, 2-chlorobenzyl, 3-chlorobenzyl, buten-3-yl, 1-acetamidoethyl, 3-pyridyl, carbäthoxymethyl, 5-norbor- nen-2-yl, cyclopentyl, 1-carboethoxyethyl, 1-hydroxyethyl, 3-hydroxypropyl, chloromethyl, isobut-2-enyl, phenyl, m-chlorophenyl, p-bromophenyl, p-trifluoromethylphenyl, m-nitrophenyl or p-Dimethylaminophenyl, or R2 and R3 together with the methylidine group cyclobutyl, cyclopentyl, monomethylcyclopentyl, 2,4-dimethylcyclopentyl, 2,4,4- or 1,3,3-trimethylcyclopentyl, cyclohexyl, 3-methylcyclohexyl, 3,5 -Dimethylcyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, 2,2,4,4-tetramethylcyclopentyl, 2-cyclopentyl-cyclopentyl, 3-tert.pentylcyclopentyl, 2- (l-cyclo-hexene) cyclohexyl, 2-cyclohexylcyclohexyl, 2-ethylcyclohexy 2-propylcyclohexyl, 4-tetrahydrothiopyranyl, 2-ethylcyclo-pentyl, 3-methyl-6-isopropyl-cyclohexyl, 3,3-dimethylcyclo-hexen-4-yl, 2-phenyl-cyclohexyl, 2-cyclohexenyl, 4-t. -Butyl-cyclohexyl, 2-ethylidene ncyclohexyl, decahydro-l-methyl-2-naphthyl, decahydro-3-methyl-2-naphthyl, l-benzyl-4-piperidyl, l-methyl-4-piperidyl, bicyclo [3.2.1] octane-2 -yl, 3-quinu-clidinyl, 5-methoxy-2-tetralyl, 2-adamantyl or 2-norbomyl, reductively alkylated, with aldehydes depending on the amount of the reagents, the terminal amino group single or double and the proximal secondary amino group is not or also alkylated and the terminal amino group is simply alkylated with ketones. 2. The method according to claim 1 for the preparation of compounds of the formulas: 0 r. \ trans | 1 VCH = CH-C-NH- (CH2) 3-NH-CCHa) »-NH-SHa-Ri (I) 0 "XX trans II ^ CH2-RX R - // XVCH = CH-C-NH- (CH2) 3-NH- (CHz) ^ - Nn CH2-RI (II) • 0 CH2-RI f. \ trans |] I R-v ^> CH = CH-C-NH- (CH2) 3-N- (CH2) 4-NH-CH2- Rj (HD or 0 CH2-R1 r — n trans || | ^ .CHz-Ri R-C XVCÎ ^ CH-C-NH- (CH2) 3-N- (CH2) U-NX CH2-RI (IV) 626 895 where Ri has the meaning given above and R represents a radical of the formulas or H H / 0 \ / 0 H r »V, V» N CH- 0 II 0 N .OH NH. I. • C = 0 I. NH_ Vi H NH I. C = 0 I. NHo \ OH NH-C-Ì1N V II O 0 \ j i 0 - H NH I. C = NH I. NH- means, characterized in that an amine of the formula R 0 / .: i. \ trans || (f CH = CH-C-NH- (CH2) 3-NH- (CH2) 4 ~ NH2 # wherein R has the meaning given above, with an aldehyde of the formula Ri-CHO, where Ri has the above meaning, alkylated in the presence of a reducing period. tion agent in an inert to the reactants so 3. The method of claim 1 for the preparation of compound Use a solvent sufficient for the reductive alkylation of the formula: O R trans Jf ^ _J ^ -ch = ch-c-nh- (ch2) 3-nh- (ch2) 4-n-ch ^ r2 wherein R, R2 and R3 have the above meanings, characterized in that an amine of the formula: O trans R / 7 ~~ \ trans || y-CH = CHrC-NH- (CH2) 3-NH- (CH2) 4-NH2 where R has the above meaning, with a ketone of the formula: 626895 O II R2-C-R3 wherein R2 and R3 have the meanings given above, alkylated in the presence of a reducing agent in a solvent which is inert to the reactants for a period of time sufficient for the reductive alkylation. 4. The method according to any one of claims 1, 2 and 3, characterized in that compounds obtained are converted into the corresponding salt by reaction with an acid.