CS208178B2 - Method of making the compounds from the antibiotic group - Google Patents

Description

The invention relates to a process for the preparation of novel compounds, namely a new class of antibiotics, in particular a new series of important antibacterial agents derived from reductive alkylation of the antibiotic BM123.

In particular, the invention relates to a process for the preparation of compounds of formula I, trans O p A-CH-CH-C-CH, (t) wherein:

R denotes residues of formulas II or III

and where further denotes

R 2 C 1 -C 5 -alkyl,

R 5 is C 1 -C 6 -alkyl, N, N-di, propylaminomethyl,

2-chloroethyl, benzyl, styryl, cyclohexyl, 4-meithylpenten-3-yl, 1,3-dimethyl, tert -hexyl, 0-imdolylmethyl, cyclopenten-2-ylimethyl, n-heptyl, n-ctytyl, (o-tolylcarbamoyl) ) methyl, propenyl, methoxymethyl, cyclohexylmethyl, 2- (4-hydroxyphenyl) ethyl, n-ethyl, n-dodecyl, meithoxyivinyl, 1-meithyl-2-hydroxyethyl, 3,4-dimethoxybenzyl, N, N-diethylaminominoethyl, carbethoxychlairinethyl, 1-methyl-1-hydroxyethyl, 4-chlorobenzyl, (t-butylbutylcaribamoyl) methyl, dimethoxymethyl, ethyl, 3-methoxybenzyl, phenoxymethyl, ethinyl, N, N-dimethythylaminomethyl, N, N-dimethylaminopropyl , cyclopropyl, 2,2-dimethylethyl, 2-methoxyibenzyl, acetonyl, 3-phepylethyl, 3-chloropropyl, 2-chlorobenzyl, 3-chlorobenzyl, buten-3-yl, 1-acetamidoethyl, 3-pyridyl, carbethoxymethyl, 5-noirbornen-2-yl, cyclopentyl, 1-kanbethoxyeithyl, 1-hydroxyethyl and 3-hydroxypropyl, wherein:

R 2 and R 3 together with the attached meithylidine group may be cycloibutyl, cyclopentyl, mionomethylcyclopentyl, dimethylcyclopentyl, trimethylcyclopentyl, cyclohexyl, monomelthylcyclohexyl, dimethylcyclohexyl, trimethylcyclohexyl, cylcloheptyl, cycloocityl, cyclopentyl, cyclopentyl, cyclopentyl, cyclopentyl, cyclopentyl, cyclopentyl,

3-tert-pentylcyclopemtyl, 2- (1-cyanohexene) cyclohexyl, 2-cyclohexylcyclohexyl, 2-ethylcyclohexyl, 2-propylcyclohexyl, 4-tetrahydrothlopyranyl, 2-ethylcyclopentyl, 3-methyl-6- propylcyclohexyl, 3,3-dimethylcyclohexen-3-yl, 2-phenylcyclohexyl, 2-cyclohexenyl,

4- tert -butylcyclohexyl, 2-ethylidencyiklohexyl, defcahydro-1-methyl-2-naphthyl, deicahydro-3-methyl-2-naphthyl-1-benzyl-4-piperidyl, 1-methyl-4-pi, peridyl, bicyclo [ 3.2.1] octan-2-yl, 3-quinuclidinyl, 5-methoxy-2-tetralyl, adamantyl or 2-nonboirnyl, as well as their physiologically acceptable acid addition salts.

The object of the invention is to alkylate an amine of the formula IV, trans O '-Ti, 2 ^ (IV) in which:

R is as defined above, with the ketone of formula V,

O

Rz — c_ r ₃ (V), in which

R2 and R3 are as defined above, in the presence of a reducing agent in a solvent inert to the reactants; wherein the reaction mixture is maintained at a pH of 6.0 to 8.0 for the duration of the reaction and at a temperature of +10 to -35 ° C.

The reductive alkylation process by which the novel aintibacterial compounds of the invention can be prepared is carried out as follows. Antibiotics ΒΜ123χ, BM123 / 1 or ΒΜ12,3χ2 are dissolved in a suitable solvent, such as water, methanol, methylcellosolv or a mixture thereof, then an excess of eiquimolecular or amount of the desired aldehyde or ketone is added, followed by addition of an amount of sodium cyanohorohydride sufficient for reduction. . The pH of the reaction mixture is maintained at 6.0 - 8.0 by adding dilute mineral acid during the course of the reaction. After 24 hours at normal temperature (10-35 ° C), the reaction mixture was evaporated to dryness in vacuo and the residue was triturated with methanol and filtered, the filtrate was diluted with acetone ¹ and the solid which precipitated was removed by filtration and dried under vacuum.

Aldehydes which may be used in the above process are, for example, acetaldehyde, propiolnaldehyde, butyraldehyde, iso-buityraldehyde, crotonaldehyde, valeraldehyde, benzaldehyde, p-cyanohenzaldehyde, salicylaldehyde, cinnamaldehyde, trichloraicetaldehyde, which may be mentioned above; of the method, for example, 198178 are acetone, 2-butanone, 1,3-dibromoacetone, chloroacetone, acetophenem, m-chloraeeltaphemon, p-toromacetophenone, p -trifluoroironiethylaccelophenone, nunitroacetophenone, p-di-diethylaminoacetophenone and the like.

The reductive alkylation products are obtained by conventional methods such as precipitation, concentration, solvent extraction or a combination of these methods. After isolation, the products can be purified by a method known per se. These include precrystallization from various solvents and mixed solvent systems, chromatography techniques and countercurrent shaking, all of the methods which are usually used for this purpose.

The new antibacterial agents of the present invention are organic bases and are capable of forming acid addition salts with various organic and inorganic salt forming agents. Acid addition salts formed by mixing the free antibacterial base, preferably in a neutral environment, with up to three equivalents of acid, are formed with acids such as sulfuric, phosphoric, hydrochloric, hydrobromic, sulfamic, citric, maleic, fumaric, tartaric, acetic, benzoic acids. , gluconic, ascorbic and related acids. The acid addition salts of the antibacterial agents of the invention are typically crystalline solids, soluble in water, methanol, ethanol, but relatively insoluble in non-polar organic solvents such as diethyl ether, benzene, toluene, and the like. For the purposes of the invention, antibacterial free bases are equivalent to the corresponding non-toxic addition salts.

Antibiotics designated BM123 (Si, BM123 / 32, BM123 / 1 and BM123p are produced by culturing a new strain of undetermined Nocardia species under controlled conditions. This new antibiotic-producing strain was isolated from garden soil collected in Oceole, Iowa and maintained in the Lederle Laboratories culture collection Dlvision, American Cyanamid Company, Pearl River, New York as a culture under BM123 The living culture of this new microorganism was deposited in a laboratory of culture collection of the Northern Utilization Research and Development Division, United States Department of Agrtculture, Pecria, Illinois and was added to its It is freely available to the public in the depository under No. NRRL 5646. The antibiotic BM1'23 | 3 discussed here applies to any mixture of BM123 /? and ΒΜ123 (3ζ and ΒΜ123χ refers to any mixture of BM123 _r ia ΒΜ123χ ₂₎ .

The following is a general description of Nocardia, NRRL 5646, based on the observed diagnostic traits. The organism was monitored for cultural, physiological and moirphological properties according to the methods described in detail by Sihirling and Goittlieb in In ternat. Journ. Syst. Bacteriol. 16: 213-240 (1966). The chemical composition of the culture was determined by the procedures described by Lechevalier, et al. in Advan. Appl. Microbiol. 14: 47-72, (1972). Color data are taken from Jacobson et al. Color Harmoiny Manua.1, 3rd edition (1948} Container Coirp. Chicago, Illinois. Details are given in Tables I through V below.

The amount of growth

Medium on yeast extract, asparaginose dextrose, Benedict's, Bennett's dextrose from potatoes and Weinetein agar; • weak on Hicfcey and Treaner paste of potatoes, oatmeal, pablum agars and trace growth on inorganic salt-starch mixtures, Kuster's oatmeal, Czajpek's solution and rice agar.

Aerial mycelium

If airborne mycelium is present, it is whitish; humiliation is formed on yeast extract, asparagine dextrose, on Benedict, Benneft and potato dextrose and potato dextrose agar.

Soluble pigments

No soluble pigments are formed.

Variable pigments

Colorless to yellow.

General physiological reactions

There is no liquefaction of gelatin; nitrates are reduced to nitrites over a period of 7 days; melanoid pigments are not formed on peptone ferric agar; there is no peptonisation or curd formation in purple milk; tolerance of sodium chloride in agar with yeast extract § 4% but <7%; the optimal growth temperature was 32 ° C. Utilization of the carbon source by Pridham and Gottlieb [J. Bacteriol. 58: 107-114 (1948)] was as follows: good utilization was observed for glycerin, salicin, d-trelhalose and dextrose; sufficient utilization observed after invasion; insufficient or no utilization of d-fructose, maltose, adomite, 1-arabinose, laictose, d-mannitol, d-mielibioose, d-raffinose, 1-rhamnose, sucrose and xylose.

Chemical composition

The organism belongs to cell wall type IV, i.e. it contains acid meso-2, 6-diamine ^and, iinopimelovou type A with the cell wall sugar permeated whole, i.e. contains arabinose and galajktózu. When methyl cell wall extracts are subjected to gas ehromatography, they exhibit an ointment of ointments similar to those produced by Nocardia astetroides; ATC.C 3308.

Mikromorfologio

The aerial mycelium grows from the substrate mycelium in the form of spirally branched elongated elements which usually end with elongated basic spirals. The flexible elements are irregularly divided into short elliptical to cylindrical sections [spores?] That easily disintegrate. The spiral terminal parts are less likely to be broken down. The size of the segments typically ranges from 0.8 to 1 + 7 pans, x 0.30-0.5 pni, with an average of 0.4 µm-x 1.2 µm.

Diagnosa

Morphological characteristics of culture no. BM123 is difficult to follow and interpret, for; aerial mycelium in most; media is underdeveloped. For this reason, it is of great importance; attributed _f ohemické analysis, providing ;: generic relations mikrnonganismu. On.-. basis; system; designed; Lechevalier et al., Culture no. BM123 contains meso-2,6-diamine-diimelic acid permeated by whole cells, analgesia, and guikr ru demonstrates the presence of arablmase and galaxy. toses, Culture; therefore belongs; krtypu. IV cell walls. Gas-stunning anatomical reunion of kinship, culture. BM123 ·. Nosardia asteroides; ATGG 3308 showed that both cultures did; they are prominent; similar characteristics of culture No. BM12-3, which give rise to the concept of Nocardia, are fragme-n? tative air growth - on some media; and the complete absence of air growth in most media; Because it was missing '; adequate criteria for the characterization of Nocardia-na?, ύηο, νιπΐ dnu? hu, an attempt at this determination has not been tested. Culture No. BM123. it is, therefore, considered; as an unspecified Nocardia species, if such diagnosis is possible.

TABLE IV

Utilization of carbon sources Nocardia sp. NRRL 5646

Incubation: 10 days Temperature: 32 ° C

Carbon source Utilization * adonit 0 ’

L-arabinose 0 glycerine 3 d-fructose, 1-iodine 2 lactose 0 d-immannit 0 saliciin 2 d-melibiosis 0 d-raffinose 0 rhamnose 0 xnaltose 1 sucrose 0 d-trehalose 3 d-xylose 0 dextrose 3 O ¹ · * good utilization = 3 sufficient utilization = 2 unsuitable utilization = 1 no utilization = 0 '

TABLE V

Chemical composition Nocandia sp. NRRL 5646 Cell wall type Main components

Type IV meiso-DAP, arabinose, galactose

Generation of the antibiotics BM, 103j3 and BM123γ is not lilmifted - for this individual organism or for organisms fully corresponding to the above growth and microscopic characteristics, which are presented for illustrative purposes only. In fact, mutants generated from this organism can also be used in a variety of ways, such as exposure to X-rays, ultraviolet radiation, nitrogen mustard, actinophags, etc. A living culture of such a typical strain mutant has been deposited in the laboratory culture collection of the Nortlhenn Utilization Research and Developmeht Division , Department of Agriculture, Peoria, Illinois, and was added to her permanent collection of poid number NRRL 8050. Although the culture, physiological, morphological features of NRRL 8060 are essentially the same as NRRL 5646, they produce increased amounts of BM123 γ during aerobic fermentation. NRRL 8050 also differs from NRRL 5646 in the following:

(a) lower nitrate to nitrite reduction;

(b) formation of a rosewood tan of mycelial pigmeint on Bennett yeast extract agar.

The novel antibacterial agents of the invention are mostly crystalline solids with relatively limited solubility in non-polar solvents such as diethyl ether and hexane, but substantially more soluble in solvents such as water and lower alkanol. Antibiotics BMT23 y, BM123 γϊ are structural isomers and can be represented by the following structural formulas:

trans $

CH = CH-C-NH- (CH 2 -NH-CH 2 NH 4)

trance

O

II

CH * CH-NH-C- fCHjfcNH these ^ _NH-t

Reductive alkylation of BM123 γ tons takes place in the spermadine side and the BM12'3 / i or BM123 p ts chain to form derivatives of the general formula:

trance

R-γ-CH = CH-C-NH- (CR _S wherein

R represents the structure section:

and R 2 and R 5 are as defined above. Re-speradiadine side chain for the highly predictive alkylation of BM123 χ, BM- Ku mono-, di- and tri-substituted derivatives

1,23 / 1 or BM123 / 2 with aldehydes proceeds in the formula:

.—. tra os O trans O / CH2 / Ri / T ~, trans J?

KV __) -CR = CH-C-NH- (CH 2 -N- (CH 2 RH-CRCH ₂ R _t , trans)

RCH-Cf-C-NH-ICH ^ H- (CR CR _{and Z-R 1}

CR ^ R ₁ β

O

CU a

oj>

Q

-M 'CO £

j§ £

y í>

* + 73

WITH

Q \ φ>

$

S £

λ o> y ú>

g ° Φ ř »

CO ° £ w 'm s m S 8 g Φ what y> ú' what <p 0> ° kú>

g ú

E>

O á

B 'cO

Si— <

C '

3 £

O-i O-i

AT

O £ o> údd &> fo

-H ^three £

O 'CO £

CD y

n y □ 33 y £ y

CO p_,>

ČD O ŕ ý a '5 0 £ y-f

O > 30 '

> y £

V) CO 3 ->

N ú

>. ů> Pm y

£ £ y '>>

>

CD

what '£ y (1 * 1 'WHAT > 'CD Sp — 1 IT '>> > '> p > '>> > t3. '>> > >> y > N O hive O uu > O at Oo £ Q y at CD AT CD AT CD £ ό y at CD Ú Ú cd no WHAT 1 > y > what - £ what cn ÝrU 3 what pQ sa pQ pQ sa § what ía JO-30 sa n '-' sa CD £ > t4 \_at y y y y y y y y y pQ 4- ^ £ 1— < CD 4— · > 30 30 30 > 30 30 30

what

Three cp to § ^y z

and

T3 ú

CM

WHAT

PU y

H

< CD N5 43 WITH ΰ O OF 0 CQ £ < t-l £ £ 43 L-H y y and >> 'y íg £ 4-1 y £ • ρ — 1 t-l y 4- » I.e M >

WHAT

AT

I · §

Til y

o cd £

Pi

S .3 'Φ í>

JU

CO y

'S ^ s ^ \ '> S '> p '>> £ £ £ £ £ £ £ e e £ e e c I.e I.e rJ I.e I.e I.e TJ TÍ tJ T3 ie 'CD 'WHAT '£ 'CD 'CD 'CD 'CD '3' honors 'CD '3 13 MŮ > N > N > N > N > tsi > sa > N> N > N > N> N> N

cp y

CJ

O g

> § £

sa> cO a

£ «I — I y

cj> s a

O 'Φ £

> y £

Ie>

Sf

3 <0 'CD gg>

'CD'>

c0 'CD £

> y £

That is CD

Tj 'CO> 65 _rt _ '05 '05 * S 3J d Λ 2 5 o ij) to -ø '05 5 ^ ph' Φ § 63 «>

> y

From o

Ό>

£> y> y cd a

O'

Φ ω

what and »» - <

r-H \ φ s y> w α z 0 TJ 65 Ě>

'CD £

Tj '£> N y y y y> >>

'CD £

> w £

Tj>

'Φ £

Jj cd> N Φ £

> y £

I.e

N>

CD g

'cd> N y

CJ> s a

a .a y

o a

BBS

3 3

05 05 O U O * r * s! >>

a and a

'CD '> p 'y 'Φ 'Φ 'Φ > 33 £ £ £ £ CD > y CD y > y £ > y £ > w £ pQ I.e I.e I.e I.e sa 65 65 sa 'Φ —4 > > > C SQ> \ φ sQ) s <D £ £ £ £ £ I.e I.e I.e I.e I.e 65 'CD 'CD *CD 'CD > > 64 > 65 > N5 > N

'fcs > >> '> S £ s s £ £ O and n c 30 sg % I at Sr — 1 UU at & © 4U Φ 3 + j 3 £ w B with 0 4- » y y co g y

sne 3 Č5- £}

3ΐ5 and y

+ u

4ti cO β

X y

g '> s £

> u • pU three c / 5>

co aj bo (9

Ú cd cd o§ fH y

£ y

y

JU

H cO>

ȣ> y

and ffi £

O sa

Ό · &

X y

Tj £

LU 00'd

JU cd

P-l y

MS

Ju cd co>

° £ • w • pu

Tj y

£ y

JU

CO $ 0 co> ° £ 4— 'y

£ £

y '>

Š pQ

O

JU> y three cd <£ o

y

Ό y

£ <0

Ú o

í-l oš

SP s

05 cli bo b 3 bO '>' š

Ň

Ό ú X y

CO> á, - ri y _ a

í> x

2? N ω y '£ o y

£ &lt; Q &amp; CO &lt; 5P &gt;

o íu

O £

Pi Cd t-1 w

> 3 ό £!

S 23 Š3

Í φ / 3> N a a g 3 a and co y O-f í £ - £

TABLE II

Mifcromorphology of Noca.rdia NRRL 5646

Medium Air mycelium, m or / and spore-like structures

The airborne mycelium agar grows from a destructive substrate mycelium in the forexitractic me of spirally branched, flexible elements that usually culminate in elongated underlying spirals. The flexible elements are irregularly divided into short sections (spores? Which easily disintegrate. The spiral terminal portions are less likely to be segmented. The segments usually have a size of 0.8-1.7 μην x 0.3 to 0.5 mn, average 0 4 μην xx 1.2 am

TABLE III

General physiological reactions Nocardia sp. NRRL 5646

Medium Time Quantity Physiological reactions of growth incubation

gelatine 7 days faintly no liquification gelatine 14 days good no liquification organic-nitric acid culture medium 7 days good nitrates reduced to nitrites organic nitrate broth ,14 days good nitrates reduced to nitrites peptone-iron agar. 24—28 hours good melanin pigments reduced milk ("purpie piilk") 7 days good no pejptonization or clots yeast extract agar + 4, 7, 10 and 13% sodium chloride 7 days moderate NaCl tolerance ≥ 4% but š 7%

wherein R and R1 have the above-mentioned meanings.

The usable un-provided derivatives of the BM123 χ antibiotic are demonstrated by their ability to control systemic fatal infections in mice. These new compounds exhibit high antibacterial activity in vivo against Escherichia coli US3.11 in mice following a single subcutaneous administration to a group of carw0 · mice. CF farms, weighing about

The carbenynyl reagent 1-ditpropylamino-2-pircpanone used

1-Chloro-3-pentanone cycloocitanitanone

4-Methyl-2-pentanone phenylphenyl-4-phenyl-3-buten-2-one 1-cyclohexyl-1-piperidone

6-Methyl-5-heipten-2-one

3-methyl-2-ipentanone

5-Methyl-2-hexanone 3-Ethyl-2-pentanone 3,5-Dimethyl-2-octanone 3-Octanone 3-methyl-2-hexanone 3-Indolylacetone

2- pentanone

2-buitanone

2-cyclopentene (-1-) yl-acetone acetone

3-decanone

3-undecanone o-acetoacetotoluidide mesyloxto methoxy methoxy acetone cytolohexylacetone

4- (p-hydroxyphenyl) -2-hutanone diimethyl (2-oxoheptyl) phoate

4-Methyl-2-hexane

2.2.4.4- tetramethylcyclopentanone

2.4.4-Trimethylcyclopentanone 2-cyclopentylcycloipentanone

2- (cyclo-1-hexenyl) cyclchexanone

3-tert-pentylcyclohexanone 2-cylcyclohexylilcyclohexanone 2-ethylcyclohexanone

3,3 -d ime th y 1-2 -b u t a n he

2-undecanone tetrahydirothloipyran-4-one

3,5-dimethylcyclohexanone

2-Tetradecanone 1-methoxy-1-buten-3-one

4-Hydroxy-3-methyl-2-butanene menthone cyclononan-1-methyl-2-decalon isophorone g, infected with a lethal dose of this bacterium at a dilution of 10 ^-3 in tryptic soybean. TSP medium for 5 hours in TSP blood culture. Table VI below shows the in vivo activity of typical products from the shown carboinin agents against Escherichia coli US311 in mice. Activity is expressed by NMR.

TABLE VI

Derivative name [5] primary NMR values of 1-methyl-2- (Ν, Ν-dipropylamino-ethyl-BM123 · / 1-ethyl-3-chloirpiropyl-BMH23yclooctyl - Β M123)

1,3-dimethylibutyl-BM123y 1-methyl-2-denylethyl-BMT23y

1-Methyl-3-phenyl-open (-2-) yl-BM123y 1-Cyclohexyl, projyl-BM123y

1,5-Dimethylhexen (-4-) -yl-BM123y

1,2-dimethylbutyl-BM123

1,4-dimethylpentyl-BMT2,33

1-methyl-2-ethylbutyl-BM123v

1,2.4-tri, methylheptyl-BM123 / 1-ethylhexyl-BM1'23y

1,2-dimethylpentyl-BM123y

- 1-methyl-1- (phenylamino) ethyl-BM 123v 1-methylbutyl-BM123y 1-methylpiropropyl-BM1237 '

1-Methyl-2'-cyclopentene (-2-) yl-ethyl-BM123 / i with qpropyl-Μ123χ 1-ethyl, ktyl-BM123y

T-ethylmethyl-BM123 / · methyl 1-2-0- (tolylcarbamoylethyl) -ΒΜ123χ

1.3- dimetihylbuten '(- j 2-yl-L-im BM12Sý ^and ethyl 2-mrethoxyethyl BM123ý-l-methyl-2-cyclohexylethyl-B'M123y l-Methyl-3- (4-hydroxyphenyl) etlryl-BM123y

1 - [(dimetethoxyoxyphosphinyl) methyl] hexyl-BM1-23]

1,3-thylpentyl-BMlZSy

2.2.4.4- tetramethylcyclopentyl-BM123)

2.4.4-trimethylcyclopentyl-BMT23y

2-cyclopentylcyclopentyl-BM123y

2- (1-Cyclotreatin) cyclohexyl-BM123 /

3- tert -pentylcyclopentyl-BM123j '

2-Cyclohexylcyclotoexyl-BM123 /

2-ethylcyclohexyl-BM123y

1.2.2-trimethylpiropyl-BMT23 /

-met h yldeey 1 - Β Μ12 3 γ

4-Tetrahydrothiopyranyl-B'M123 /

3.5-d ^: methylcytkilohexyl-BM123y 1-methyltridecyl-BMT23y 1-methyl-1S-melthoxypropipen (-2-) yl-BM123y

1,2-dimethyl-3-hydroxy; protyl-BM12 [beta] /

3-methyl-6-isopropylcyclohexyl-BM123 / cycloonyl-BM12.3y decahydro-1-methyl-4-naphthyl-BM1 / 1Sy

3.3-Dimethylcyclohexane (-4-) yl-BM123y

0.3

0.12 <0.18.

0.12

0.18

0.25

0.37

0,03 '

0.12

0.12

0.18

0.37

0.18

0.18

0.12 <0.12 '<0.12 <0.12 <0.12 0.25 0.38 9.25

3 0 3 0.18 0.3

0.3 <0.12 0.73

0.3

0.37

0.19

0.5

0.75

0.19

0.12

0.38

0.12

2.0> 2.0 0.12 0.38 0.18 0.25 0.37

The carbonyl reagent used

3-iinomethyl-2-decalone

1- (3,4-dimethoxyphenyl) -2-buitanone

2-chloroacetacetic acid, 1-diethylamino-3-butainone ethyl ester

3-hydroxy-3-methyl-2-butanone

3-pentanone

3-methyl-2-butanone p-chlorophenylacetone

N- (tert-butyl) acetoacetamide

1,1-dimethoxyacetone

4- hep'tanoin

3-Methoxy-phenyl-acetone.

and 1,3-acetone and rhoxylic acid

2-Phenylcyclohexanone phenoxy-2-propanone

3-Butyn-2-one dimethylaminoacetone

5-diethylamino-2-pentanoin 2-cyclohexen-1-one cyclohexylpentylmethyl-4,4-dimethoxy-2-butanone

1,3-dimethylacetondicairboxylate

2-methoxyphenylacetoin acetylaceitone cyclobutanone p-chlorophenylacetate 2-oiktanone

4-phenyl-2-butanone

5-chloro-2-pentanone o-chlorophenyl aceto m-chlorophenylacetone 5-hexein-2-one cyclohexanone 2-hexanoin 2-heptanone cyclolheptanone cyclopentanone

4,4-dimethyl-2-pentanone

2-acetamido-3-butanone 2,6-di-methyl-3-heptane

4-oiktanone

3-Aceitylpyridine

3-heptainone ethylbutyrylacetate 1-benzyl-4-piperidone

1-methyl-4-ipiperidone 3-methylcyclopentanone

3,3-dimethyl-2-butanone

2-Acetyl-5-norbornene bicyclo [3.2.1] octan-2-one

3-quinuclidinone

5-Methoxy-2-tetralone

4-Methyl-2-heptanone

3,4-diimetyl-2-hexanone 1,3,3-trimethylcyclopentainone acetylcyclopentanone

5-hexen-2-one

Derivative Name [E] Primary NMR Value of Dicahydro-3-methyl-2-naphthyl-BMli23y 0.37

2-ethyl-2- (3,4-dimethoxyphenyl) ethyl-BM123 / 1.0 1-methyl-3- (N, N-diethylamino) propyl-0,18

-BM123 / 1-methyl-2-chloir-2-carbethoxyethyl-BM123 / 1.5

1,2.2-dimethylhydroxy oxypropyl-BM 123 / 0,18 1-ethylpropyl-BM123 / 0,12

1,2-Dimethylpropyl-BM123 / 0.19

1-methyl-2- (4-chlorophenyl) ethyl-BM 123 / 0,26 ^:

2- (tert-butylcarbamoyl) -1-imethylethyl-0.38

-BM123 / 1-methyl-2,2-diimethoxyethyl-BM123 / 0.5 1-propylbutyl-BM, 123y 0.25 1-methyl-2- (3-methoxyphenyl) ethyl-BM123 / 0.30 (2- (carboxy-1-carboxymethyl) ethyl-BM123 / 0/5

2-Phenyicyclohexyl-BM123y 1-methyl-2-phenoxyethyl-BM123 /

1-methyl-bultyn- (2-] yl-BM12,33y1-methyl-2- (N, N-dimethylamino) propyl-BM123 /

1-methyl-4- (Ν, Ν-diethylamino) hutyl-BM1, 23 /

2- cyclohexenyl BM123 / l-cyclopropylethyl-BMI 23Y ^l l-methyl-3,3-dimethoxypropyl-BM123 / l kairbomethoxy-2-carbomethoxyethyl-BM123 / l-methyl-2- (2-methoxyphenyl) ethyl-BM123 (1-methyl-2-acetylethyl-BM123) cyblobityl-BM123 / 1-methyl-2- (4-chlorophenyl) ethyl-BM123 / 1-methylhexyl-BM123 / 1-methyl-3-phenynyl-propyl- BM123 / 1-methyl-4-chlorobutyl-BM123 / 1-methyl-2- (2-chlorophenyl) ethyl-BM12,3 / 1-methyl-2- (3-chlorophenyl) ethyl-BM123 / 1-methyl-pentene , (- 4-) y-BM 12 3 / cyclohexyl-BM123 (1-methylpeptide, 1-BM123)

1-methylhexy, 1-BM123 / cycloheptyl-BM123 / cyclo-pentyl-BM123 /

1.3 3-Trimethylbuty 1-B M123 /

2-Acaeta, mido-1-methylpropyl-BM123 (1-isopronyl-4-methylpyrtyl-BM123 (1-propyl) pentyl-BM123)

1- (3-pyridyl) ethyl-BM123 (1-ethyl, lpentyl-BM123)

1- (carbethoxymethyl) butyl-BM123 / 1-benzyl-4-piperidyl-BM123 / 1-methyl-4-piperidyl-BM123 /

3-methylcycloipentyl-BM123 (1-methyl-2,2-dimethyl) propyl-BM123)

1- [5-norboirneine (2)] ethyl-BM123 / hicyclophane 3.2.1] oiktanyl-2-BM123 /

3-quinuclidinyl-BM123 /

5-Methoxy-2-tetrayl-BM123y

1,3-dimethylhexyl-BM123

1,2.3-trimethylpentyl-BM123y

1.3.3-Trimethylcyclopentyl-1-M12 3/1-cycloipentylethyl-BM123 (1-methylhexen-4-yl-BM123)

0.38

0.3 <2.0 1.5

0.5

0.25

0.26

0.75

0.75

0.7

1.5

0.38

0,, 25

0.38

0.38

0.37

0.37

0.38

0.38

0.75

0.38

0.38

0.3

0.25

0.18

0.50

0.39

0.39

0.75

0.25

0.76

0.18

0.75

0.25

0.18 <0.12 <0.25

0.39

0.18

0.5

0.39

0.37

0.18

0.18

The carbonyl reagent used The name of the derivative [d] primary NMR values 2-methylcyclopentano ^: n 2-Methyl Lycycles 1- Β M123 / 0.25 2,4-dimethylethyl 1 op ent ano u 2,4-Dimethylcyclopentyl-B M12 3 / 0.18 2 - electronically bonding anon Z-ethylcyclopeintyl-BM (S) - 0.12 2-adamantine adam.antyl-2-BM123 0.25 3-hexanone 1-ethylbutyl-BM123y 0.39 ethyl 2-methylacetoacetate 1-Methyl-2-carboethoxypropyl-BM123y 0.18 norbornanone norbornyl-BM12.3y • 0.18 5-Hexen-2-one 1-methyl-5-pentenyl-BM1'23y 0.18 3-hydroxy-2-butenone 1-methyl-2-hydroxypropyl-BM123T ' 0.37 4-Hydroxy-3-methyl-2-butanone l-dimethyl-2- ^{(3-hydroxy-2-methylpropyl!} propyl) - -BM123 / 0.18 2-nomanon 1-methyloiktyl-BM123y 0.37 5-h y dr ox y-2-p nt an on 1-methyl-4-hydroxybutyl-BM123y 0.18 2-decanone 1-methylnonyl-BM123y 0.18 4-tert-butylcyclohexanone 4-t-butylcyclohexyl-BM123y 0.12 β-ethylidenecyclohexanone 2-ethylidency, kl'hexyl-BM123y 0.12 fenylacetaldebyd 2-phenylethyl-BM1.23y 0.18 p-methoxyphenylacetaldehyde 2- (p-methoxyphenyl) ethoxy-BM123y 0.18 2-ethylhexanal 2-ethylhexyl-BM123y 0.37 2,2-dimethylbutanal 2,2-dimethylbutyl-BM123y 0.12 2,2-dimethylproppanal 2,2-dimethylprojp1-BM123y 0.18 2-ethyl-2-butema 2-Ethyl-2-butenyl-BM123y 0.18 trans-2-methyl-2-butenal trans-2-methyl-1-2-butenyl-BMT23y 0.18 1-Methylcyclo-B-hexenyl- thanal (1-methylcyclo-3-hexenyl) methyl 1-BM123 / 0.18 trans-2-m -ethyl-2-penten.al Trans-2-methyl-2-pentenyl-BM123y 0.18 fownaldehyde methyl-BM123y • 0.12 acetaldehyde ethyl-BM123y 0.38

Fermentation, a process leading mainly to the antibiotics BM123 / 3 and BM123y

Cultivation of Nocardia sp. NRRL 8050 can be conducted in many ways in liquid environments. Environments that are useful for making antibiotics include an assimilable carbon source such as starch, sugar, molasses, glycerin, etc .; an assimilable nitrogen source such as proteins, protein hydrolyzate, polypeptides, amino acids, corn steep liquor, etc .; and inorganic anions and cations such as potassium, magnesium, calcium, ammonium, sulfate, carbonate, phosphate, chloride, etc. Trace elements such as boir, molybdenum, copper, etc .; the latter are added as contaminants to other media components. The aeration of tanks and containers is carried out by pushing sterile fermentation air. or on its surface. Mixing in tanks is performed by mechanical stirrer. If desired, an anti-foaming agent such as Hodag FD82 may be added.

Preparation of inoculum for antibiotics BM123 / 3 and ΒΜ123χ

First shake with Inoculum · Nocardia sp. The NRRL 8050 is prepared by incorporating 100 ml of sterile liquid medium in a 500 ml flask with a scraper or wash containing spores from an agar slant culture. Typically, the following environments are used:

Bacto-trypton 5 g yeast extract 5 g beef extract 3 g glucose 10 g water ad 1000 ml

The flasks are incubated at temperature. cd 25 to 29 ° C, preferably 28 ° C, and stirred vigorously on a rotary shaker for 30 to 48 hours. The inocula are then transferred to the culture tubes with the closures and stored below 18 ° C. This vegetative inoculum source is used instead of slant agar scraping to inoculate additional shake flasks to prepare this first stage of inoculum.

Flasks with this first stage inoculum are used to inoculate 121 liter batches of the same medium in 2 liter liter fermenters. The inoculum seed is aerated with sterile. air while growth continues for 30 to 48 hours.

The 121-liter batches of the second inoculum stage are used to inoculate tank phenmentors containing 300 liters of the following liquid medium to produce the third and final inoculum stages:

Meat soluble extract 15 g ammonium sulphate 3. g potassium phosphate 3 g calcium carbonate 1 g magnesium sulfate heptahydrate 1.5 g glucose 10 g water ad 1000 ml

The glucose is sterilized separately.

The third stage of the inoculum is aerated by the amount *. 0.4 to 0.8 L of sterile air per liter of nutrient medium per minute, and the fermentation mixture was stirred with a 150-3 * 0.0 stirrer per minute. The temperature is maintained at 25-29 ° C, usually at 28 ° C. Growth is continued for 48 to 72 hours, after which time the inoculum is used to inoculate fermentation in 300 * 1 * tanks.

Fermentation of BM123fi and BM123y antibiotics in a tank

For the production of the antibiotics BM123-3 a. BM1, 23y it uses the following fermentation medium *:

Meat soluble extract 30 g ammonium sulfate 6 g phosphate, potassium 6 g, calcium carbonate 2 g magnesium sulfate heptahydrate 3 g glucose 20 g water * ad 1000 mil

The glucose is sterilized separately.

Each tank is inoculated with 5-10% of the third stage of the inoculum prepared as described in the inoculum production section. Fermentation broth is maintained at 25 to 28 ^C, conveniently at 26 ° C. The broth is aerated with sterile air * at a rate of 0.3 to 0.5 liters of sterile air in 1 * liter of ferment and mixed with a stirrer * at 70 to 100 rpm. The fermentation is allowed to continue for 65-9 hours and then the fermentation is isolated.

The invention will be described in more detail in connection with the following specific examples.

Ex .1 .a d 1

Inoculum preparation for BM.123 '; and BM123y

The typical medium used for the growth of the first and second inoculum was prepared as follows:

Baoto-trpton yeast extract beef * glucose water extract. ad g 5 g 3 g 10 g

100 * 0 ml

In a 500 ml flask containing 100 ml each of the above sterile medium, the inoculum was 5 ml of a frozen vegetative inoculum from Nocardia species NRRL 8050. The flasks were placed on a shaker and shaken vigorously for 48 hours at 28 ° C. The inoculum formed in the flask was transferred to a 10 liter glass feeder, a mentor containing 12 liters of the above sterile medium. The ferment was aerated with sterile. air for 48 hours for * growth time: then * the content was used to inoculate a 400 liter feeder tank containing> 309 liters of sterile liquid medium of the following composition:

Meat soluble extract 15 g ammonium sulphate 3 * potassium phosphate 3 g calcium carbonate 1. g, heptahydrate. magnesium sulphate 1.5- g glufcose 10 g, water ad * 10 * 00. mil

Glucose is sterilized separately.

The third stage of the inoculum was aerated with sterile air introduced into the fermenter at 0.4 L per liter of mass per minute. Stirring was accomplished by centrifugation with a 240-rpm stirrer: per minute. The mass of the inoculum was maintained at 28 ° C. After 48 hours of growth, the inoculum was used to inoculate 3009 L of fermentation.

Example 2

Fermentation using Nocardia * NRRL 8050 and media affecting production of BM123 / 3 and BM123y.

The fermentation medium was prepared as follows:

meat soluble extract 30 g ammonium sulphate 6 g potassium phosphate 6 g calcium carbonate. 2 g * Broccin sulphate heptahydrateor · 3'g glucose 20 g science ad 1000 ml

Glucose was sterilized separately.

Fermentation * ^! µm was sterilized at 120 ° C, with steam, for a period of time. 60 minutes; Value*. pH of medium *, po; sterilization * was 6.9. 3000 liters of sterile medium in: 4600, in a fermentation tank were inoculated with 3 * 0 liters of inoculum as described in Example 1 and fermiehtaee was fed. at 26 ^{t 1 £> G.:} Hodag FD32 use. as an antifoaming agent. Aeration was; performed at a rate of 0.35 liters of sterile air per liter of inoculum mass per minute: a 70-72 revolutions per minute, * isolage was performed after 67 hours of fermentation?

Example 3

Isolation of BM123 / 3 and BM123y .3000 liters of fermentation broth, prepared as described in Example 2, with a pII value of 4.3, was adjusted to a value of 7.0 with sedium hydroxide and a filtering solution. * using * * 5 '% ^s diatomaceous earth as a filter. The cake was washed by about 100 L of water and discarded. The soldered filtrates and wash water were pumped onto three 2 * 0.8 cm x 122 cm stainless steel columns each containing 15 liters of Sefadex C-25 sodium (cross-linked dextraepichlorohydrin iRaticmt exchange gel).

The packed columns were ipiroimyte with a total of 390 liters of water, followed by elution of 200 liters. 1% aqueous sodium chloride solution, followed by elution with 560 liters of 5% aqueous sodium chloride solution. An aqueous solution of 50% sodium chloride was clarified by filtration through celite. and the clarified filtrate would be spilled with glass. column, measuring 23 cm x 150 cm containing. 25 liters of granulated Dairco G-60 (Granular activated carbon) The packed column was washed with 120 liters of water and developed with 120 liters of 15% aqueous methanol, then 340 pounds of 50% aqueous methanol and then with 120 liters of 50% aqueous acetone. the methanolic eluate was concentrated in vacuo to about 7 liters of the aqueous phase, and the pH was adjusted from 4.5 to 6.0 Amber Lite IR-45 (011% (weakly basic polystyrene-polyanvine-anion-type anion exchange resin). filtration and the filtrate was concentrated under vacuum to about 1 liter and then freeze dried to obtain 38 g of a material consisting mainly of BM123 / 3, together with a small amount of BM123 / (especially BM123 / 2) pH 50 The aqueous methanol eluate was adjusted from 4.65 to 6.0 with Amberliteim® IR-45 (OH-) resin The resin was removed by filtration and the filtrate was concentrated in vacuo to about 6.3 L and then lyophilized to give 213 gm The value of 50% aqueous acetone eluate was adjusted from 4.9 to 6.0 with Amber Litera IR-45 (OH-) resin. The resin was removed by filtration and the filtrate was concentrated in vacuo to about 1.5. 1 and then lyophilized. 56 g of impure ΒΜ1.23χ were obtained.

Example 4

Further cleaning ΒΜ123χ

A suspension of CM Sefadex C-25 (NH 4 +) in 2% aqueous ammonium chloride was poured into a 2.6 cm diameter glass column up to about 62 µm resin. The excess 2% aqueous ammonium chloride solution was removed and 5.0 g of the ΒΜ123χ sample prepared as described in Example 3 was dissolved in about 10 ml of a 2% aqueous ammonium chloride solution and poured onto the column. The column was then eluted with 6 liters, 2% and 6 liters of a 4% aqueous solution of ammonium chloride. Fractions of about 75 ml were automatically collected after 15 minutes. The antibiotic · BM ', 123 · / was exposed in eluates from the column in µf. by light and bioautography of moistened paper discs on large agar plates inoculated with Klebsiella pneumoniae strain AD? Most of the antitrotic BM123y was in fractions 71-107.

13Ό ml of granulated Darco G-60 was suspended in water, transferred to a glass column, allowed to settle and excess water was discarded: Fractions 84-96 of the above-mentioned OM S & with granulated, active carbon .. · the packed column was washed with 600 ml of water and then developed with 1 liter 5O0 / ₀ aqueous flowable acetone ,. The eluates containing BM123 were concentrated in vacuo to the aqueous phases and a total of 886 mg of BM123 was obtained as the hydrochloride after lyophilization. However, by this process, a sample for microanalysis was obtained.

Antibiotic BM123 / does not have a defined melting point, in the vicinity of 200 ° C gradual decomposition occurs. Microanalysis of a sample maintained in an atmosphere containing 23% relative humidity for 24 hours at 22 ° C gave the following data: C: 39.44%; H, 6.10%; N: 16.19%; chlorine (ionic): 11,54 drying loss ·: 8,19%. The antibiotic, BM123y showed uf in water. absorption maximum at 286 nm · E? 4 = 250. The position of this maximum did not change with pH. Antibiotic · ΒΜ123χ produced a specific rotation [a] _D ²⁵ = = + 7-Γ (c = 0,97, in water):

Antibiotic ΒΜ123χ exhibited characteristic infrared absorption 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 .

Example 5

Isolation of antibiotic BM123 / 1

A suspension of CM Sefadex C-25 (Na 2) in 2% aqueous sodium chloride solution was poured into a 2.6 cm diameter glass column to a height of at least 70 cm resin. The excess 2% sodium chloride solution was removed and poured into water. a 4.11 g sample containing mainly the antibiotic BM123 / together with the admixture of the antibiotic BM123y2 and the other contaminants was prepared as described in Example 3, which was dissolved in about 10 ml of 2% aqueous chloride and is applied to the column.

The column was. screed tin 41 2o / _o and 4 1 4% defective sodium chloride solution. About 75 miles of fraction were collected every 15 minutes. The present antibiotic (BM123) was detected in ultraviolet, light and bioautography on moistened paper discs, on large agar plates inoculated. Klebsiellou · pneumonlae strain AD. The majority of the antitrust was in fractions 64-90; the initial fractions (64-80) contained mixtures of antibiotics BM123 / 1 and BM123 / 2, whereas the last fractions (81-90) contained substantially pure antibiotic BM123 / 1.

100 ml of granulated Darco-G-60 was suspended in water, transferred to a glass column, allowed to settle, and excess water was drained. Fractions 81-90 of the above CM Sephex Chiromatography were combined and applied to a granulated charcoal column. The packed column was washed with 500 ml of water and developed with 500 ml of 10% aqueous methanol, then with 1 liter of 1% methanol. The pH of the eluate obtained by 50% methanololeim, which contained most of the antibiotic BM123? 1, was nastaivein from 5.9 to 6.0 with Amlberlit IR-45 (OH ^- ) resin. The resin was removed by filtration, and the filtrate was concentrated in vacuo to an aqueous phase and lyophilized to yield 294 mg of white amorphous antibiotic BM123 / 1 as the hydrochloride.

The antibiotic BM123yi has a defined melting point of about 200 ^{° C} C, however, begins a gradual decomposition. The microanalysis of the sample maintained for 24 hours (under a nitrogen atmosphere at 22 ° C containing 60% relative humidity) gave: C: 37.84%; H: 5.73%; N: 15.58%; Cl (ionic) Loss on drying: 10.45% In methanol, the absorption maximum at 286 mm Eicm = 225 was found. The position of this maximum did not change with pH The antibiotic ·12 · 3χι had a specific rotation of + 55 ° (c). = 0.803 in water).

The antibiotic, BM123 / 1, exhibited characteristic infrared absorption 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 .

Example

Isolation of antibiotic BM123 / 2

25 g sample containing mainly antibiotic! BM123 / 2 and the antibiotic BM123 / 3, prepared as described in Example 3, were dissolved in 120 ml of a 2% aqueous solution of sodium chloride and apilicovines on a column containing 1800 ml of CM Sefadex C-25 (Na ⁺ ) in 2% aqueous sodium chloride solution. The column was then eluted sequentially with 20 L of 21% and 20 L of 4% aqueous sodium chloride solution. The first 12 L of collected eluate was discarded. Then, ^one every 40 minutes were ishromíažďovány automatically fractions of about 800 ml. The presence of BM123 / in the column fractions was determined by µf. lights. Most of the antibiotic BM123y was localized in fractions 7-18; fractions (7-15) contained mainly pure BM123 / 2 and other fractions (16-18) contained a mixture of antibiotics BM123yi and BM123 / 2.

600 ml of granulated Dairco G-60 was dispersed in water, transferred to a glass column, allowed to settle and excess water was drained. Fractions 7-15 of the above CM Sephexex Chromatography were pooled and applied to a granulated charcoal column. The packed column was washed with 3 L of water and then developed with 3 L of 10% aqueous methanol, followed by developing 6 L of 50% aqueous methanol. The pH of the 10% aqueous methanol eluate was adjusted from 5.8 to 6.0 with Amlberlit IR (OH) resin. The resin was removed by filtration and the filtrate was concentrated in vacuo to an aqueous phase and after lyophilization gave 595 mg of white amorphous antibiotic BM123 / 2 as the hydrochloride. 50% aqueous methanolic eluate. the pH was adjusted from 4.6 to 6.1 with Amiberlitemi IR 45 resin in (OH ^- ) form. The resin was removed by filtration and the filtrate was concentrated in vacuo to the aqueous phase, after lyophilization, 3.645 g of a slightly less pure white amorphous antibiotic ΒΜ123χ2 was obtained as the hydrochloride salt.

Antibiotic BM123 / 2 does not have a defined melting point, and gradually decomposes near 200 ° C. Mibroanalysis of the sample stored for 24 hours at 22 ° C in a nitrogen atmosphere with a relative humidity of 60% gave the following values: C: 36.14 ^! %; H: 5.67%; N: 15.1%; Cl (ionic): 11.11%; loss on drying was 10.87%. The antibiotic BM123 / 2 provided uf in methanol. absorption spectrum at 286 nm E ί ™ = 220. The position of this maximum did not change with pH values. The antibiotic BM123 / 2 had a specific rotation of + 60 ° (c = 0.851 in water).

The antibiotic, BM123 / 2, exhibited characteristic infrared absorption at wavelengths: 770, 830, 870, 950, 980, 1035, 1110, 1175, 1225, 1345, 1380, 1470, 1515, 1560, 1605, 1660, 1755, 2950 and 3350 cm ^-1 .

Example 1 a d 7

Paper chromatography and thin-layer chromatography of antibiotics BM123 / S and BM123 /

Antibiotics of the BM123 family can be distinguished by paper chromatography. For this purpose, an aqueous or methanolic solution of the substances was applied to the Whatman No. 1 paper strip and allowed to equilibrate the phases for 1-2 hours. The bands were developed overnight with the lower (organic) phase obtained by mixing 90% phenol: m-cresol: acetic acid: pyridine: water (100: 25: 4: 4: 75, v / v). The paper strips were then removed from the chromatography chamber and dried for 1-2 hours, washed with ether to remove residual phenol, and bioautographed on large agair plates inoculated with Klebsiella pneumioniae strain AD. The corresponding R _f values are given in Table VII below:

j »

TABLE VII Component Rf BM123y 0.85 BM123 / 3 0.5, 0.7 jS-Sloaka se turned out to be in this system

a mixture of two antibiotics. The antibiotic BM123 / 3 was composed of a main antibiotic (R _f = 0.50) called ΒΜ123βι and a minor antibiotic (R _f = 0.70) called the antibiotic BM123 / S2.

Antibiotics of the BM123 family can also be distinguished by thin-layer chiromatography. For this purpose, an aqueous solution of the substance to be chromatographed (about 20 to 40 .mu.g per spot) was applied to the cellulose F coated plates (layer thickness 0.1 m). The plates were, developed overnight with a mixture of 1-butanol:

: water: pyridine: acetic acid; the components were in a ratio of 15: 12: 10: 1, by volume. The deisics were then removed from the chromatography chamber and air dried for 1 hour. Antibiotics were exposed either by a standard ninhydrin procedure or by spraying with Sakagujchi reagent. Corresponding Rj values are shown in Table VII below.

TABLE VIII

Folder R _f

BM123 / 0.17, 0.23

BM123 / 1 0.08, 0.14

Both antibiotics BM123 /? and BM123 / were in this system a mixture of two components. The antibiotic BM1230 was composed of the main component (R _f = 0.08) of the antibiotic BM123 / 3i and the minor component (R _f = 0.14) of the antibiotic ΒΜ123β2. The less polar component of the antibiotic ΒΜ123χ (R _t = 0.23) was the antibiotic, BM123 / 1 and the more polar component (R _f = 0.17) was the antibiotic · BM123 / 2. Example 8

General insurance of reductive alkylation of antibiotic BM123 /

To a stirred solution of 100 mg of BM123 / 20 ml of methanol is added 5 ml (or 5 g) of the corresponding aldehyde or ketone and 100 mg of sodium cyanoboirol hydride. The pH of the resulting solution is maintained at about 7.0 with a 1N methanolic hydrogen chloride solution for 3 to 24 hours. The reaction is monitored by thin layer chromatography by disappearance of the antibiotic BM123 /. Then the reaction mixture is filtered and the filtrate is evaporated to dryness. The residue is triturated with 3 ml of methanol and filtered. The filtrate was diluted with 50 ml of acetone and the precipitate that formed was removed by filtration and dried. The methanolic solvent can be replaced by a million of water when the aldehyde or ketone is water soluble.

Example 1 a d 9

Preparation ίΐδορηοργ1-ΒΜ123χ

To a solution of 200 mg BM123y in 30 ml methanol was added 5 ml acetone. To this solution was added 139 µg of sodium cyanoborohydride and the reaction mixture was stirred at room temperature for 30 minutes. The pH of the reaction mixture was maintained between 7.4 and 7.8 by the addition of 0.1N methanolic hydrochloric acid solution. A small amount of the precipitate that formed was removed by filtration and the filtrate was evaporated to dryness in vacuo. The residue was triturated with 2 ml of methanol. and filtered. The filtrate was diluted with 100 ml of acetone and the solid product formed was removed by filtration and dried; yield: 184 mg.

Example 10

Preparation of 1,3,3-trimethylbutyl-BM123y

To a solution of 200 mg of the antibiotic BM123 / in 50 µl of methanol was added 3 ml of 4,4-dimethyl-2-pentanomide and 108 g of sodium cyanohorohydride. The pH of the reaction solution was maintained at 7 by addition of methanolic hydrogen chloride solution. The reaction mixture was stirred at room temperature after. for 18 hours and filtered. The filtrate was evaporated to dryness in vacuo. The residue was dissolved in 3 mL of methanol, diluted with 50 mL of acetone and filtered; yield: 125 mg.

Example 11

Preparation of 1-methylphenethyl-BM123y

To a solution of 200 mg of antibiotic ΒΜ1 · 23χ in 50 ml of methanol was added 5 ml of phenylacetone. To this solution was added 170 mg of sodium cyainoborohydride and the reaction mixture was stirred at room temperature for 3.5 hours. The pH of the reaction mixture was maintained at 7.0 with methanol saturated with hydrogen chloride gas during this time. The reaction mixture was concentrated to about 5 mL volume, diluted with · 2 mL methanol and filtered. The filtrate was poured into 100 ml of acetone, the solid product collected by filtration and dried; yield: 233 mg.

Example 12

Preparation of 1-methylnonyl-BM123y

100 µg of sodium cyanoborohydride was added to the solution of antibiotic BM123y, prepared from 200 mg of antibiotic. ml 2-de208178 cannon · and 40 ml methanol. The pH of the solution was maintained at 7.0 ± 0.2 by adding 0.1 N methanolic hydrogen chloride solution, if necessary. After 19.5 hours, the reaction mixture was filtered and the filtrate was concentrated in vacuo at 30 ° C. The residue was suspended in 5 ml of methanol and filtered. The filtrate ⁵ was added to 50 ml acetone. Created almost colorless. the precipitate was collected by filtration; washed with acetone and dried in vacuo. The yield of crude 1-methylnonyl-BM12'3y was 107 µg.

Example .13

Preparation of 1,3-dimethylbutyl-BM123y

To a solution of 210 mg of BM1-23 antibiotic in 50 mL of methanol was added 5 mL of methyl isobutyl ketone. To this solution was added 1-6 mg of sodium cyanoborohydride and the reaction mixture was stirred under stirring. of room temperature for 5 hours. During; at this time, the pH was maintained at 7.0 by addition. methanolic hydrogen chloride. The reaction mixture was triturated with two ml of methanol and filtered. The filtrate was diluted with 100 mL of acetone and the solid product was removed by filtration and dried; yield: 210 mg.

Example 14

Preparation of iisopropyl-BM1.23yi

A mixture of 50 mg BM123 / 1, 5 ml acetone and 60 mg sodium cyanoborohydride in 35 ml methanol was stirred at room temperature for 40 minutes. The pH of the solution was maintained at 7 by dropwise addition of a toethanol solution of hydrogen chloride. The mixture was evaporated to dryness in vacuo. The residue was triturated with 5 ml of methanol and the resulting solution was diluted with 50 ml of acetone; yield: 49 mg.

Example 15

Preparation. isopropyl-BM123yi

A mixture of 41 mg of ΒΜ123χ antibiotic, 5 ml of acetone and 50 mg of sodium cyanoborohydride in 35 ml of methanol is stirred at room temperature for 40 minutes. The pH of the solution was maintained at 7 by dropping saturated. methanolic hydrogen chloride. The mixture was filtered and evaporated to dryness in vacuo. The residue was triturated with 5 ml of methanol and the resulting solution was diluted with 50 ml of acetone; yield: 46 mg.

Example 16

Preparation of 1-methyl-2-phenyl-ethyl-BM123 / 2

A mixture of 200 mg antibiotic BM123 / 2, 5 ml phemylaceitone and 170 µg sodium cyanoborohydride in 50 ml methanol was stirred at room temperature for 3 hours 45 minutes. The pH of the reaction mixture was maintained at 7 during this time by adding saturated methanolic hydrogen chloride solution. The mixture was evaporated to dryness in vacuo. The residue was stirred with 5 mL of methanol and the resulting solution was diluted with approximately 50 mL of acetone; yield: 233 mg.

Example 17

Preparation of (2-ethylcyclopemtyl) -BM123 /

Solution 200 mg antibiotic ika1Ζ3χ, 3 ml

2-ethylcyclopentanone and 101 mg of sodium cyanoborohydride in 50 ml of methanol were removed for 16 hours at room temperature. During this time, the solution was maintained at 7 by adding a saturated solution of hydrogen chloride in methanol. The reaction mixture was evaporated to dryness. The residue was triturated with 3 mL of methanol; filtered and the filtrate was diluted with 40 ml of acetone, yield 157 mg.

Example 18

Preparation of 3,5-Dimethylcyclohexyl-BM123

A solution of 200 mg of antibiotic ·1 · 23χ, 5 ml of 3,5-dimethylcycloehexanone and · 20 µg of sodium cyanoborohydride in 50 ml of methanol was allowed to stand at room temperature for 1 hour. The pH value during this. The reaction was maintained at 7 by adding a saturated solution of hydrogen chloride in methanol. The reaction mixture was triturated with 3 mL of methanol, filtered, and the filtrate was diluted with 40 mL of acetone, yield 200 mg.

Example 19

Preparation of 2,4-dimethyleyklopeintyl B'M123 ⁱ⁾ A solution of 206 mg of antibiotic BM123 /, 3 ml of 2,4-diimetihylcyklopentanonu and 104 'kyanoborohydíridu mg of sodium in 50 ml methanol was weaned at room temperature for 6 h ·. The pH of the solution was maintained during this time by addition of saturated methanolic hydrogen chloride solution. The reaction mixture was triturated with 3 ml of methanol, filtered and the filtrate was diluted with 40 ml of acetone, yield 101 mg.

Example 20

Preparation of ^{2-cyclohexyl-L} etihylcyk BM123y

Solution 200 mg antibiotic BM123 · /, 5 ml

2-ethylcyclohexanome and 213 mg of sodium cyanoborohydride in 50 ml of methanol were allowed to stand at room temperature for 3 hours. During this time, the pH of the solution was maintained at 7 by addition of a saturated solution of hydrogen chloride in methanol. The reaction mixture was triturated with 3 mL of methanol, filtered, and the filtrate was diluted with 40 mL of acetone, yield 200 mg.

Example 21

Preparation of 3-methylcyclohexyl-BM 12 ⁱ 3y

Solution 200 mg antibiotic ΒΜ1: 23χ, 1.5 ml

3-methylcyclohexainone and 200 µg of sodium cyanoborohydride in 50 ml of methanol were allowed to stand at room temperature for 2 hours. During this time, the reaction pH was maintained at 7 by addition of a saturated solution of hydrogen chloride in methanol. The reaction mixture was triturated with 3 mL of methanol, filtered, the filtrate diluted with 40 mL of acetone, yield 200 mg.

Example 22

Preparation of 2,4,4-trimethylcyclopentyl-BM123y

Solution 200 mg antibiotic BM123 / 5 ml

2,4,4-tri (ethylcyliclopentainone and 179 mg sodium cyanoborohydride in 50 ml methanol was allowed to stand at room temperature for 24 hours, during which time the pH of the solution was maintained at 7 by adding a saturated solution of hydrogen chloride in methanol. with 3 ml of methainol, filtered, the filtrate diluted with 40 ml of acetone, yield 176 mg.

Example 23

Preparation of 2-propylcyclohexyl-BM123y

A solution of 200 µg of B.M123y, 3 ml of 2-piropyleyclohexanone and 157 mg of sodium cyanoborohydride in 50 ml of methanol was left at room temperature for 4 hours. During this time, the reaction pH was maintained at 7 by addition of a saturated solution of hydrogen chloride in methanol. The reaction mixture was triturated with 3 ml of methanol. filtered and the filtrate was diluted with 40 mL acetone, yield 57 mg.

Example 24

Preparation of 2-methylcyclopentyl-BM123y

Roizteik 211 mg of antibiotic BM123y, 3 ml of 2-methylcyclopentanone and 98 mg of sodium cyanoborohydride in 50 ml of methanol was allowed to stand at room temperature for 3.5 hours. During this time, the pH of the solution was maintained at 7 by addition; saturated solution of hydrogen chloride in methanol. The reaction mixture was triturated with 3 mL of methanol, filtered, and the filtrate was diluted with 40 mL of acetone, yield 157 mg.

Claims (4)

SUBJECT OF THE INVENTION Process for the preparation of compounds of the group of antibiotics of the general formula I, trans R C- W- & (t) where 7 ' R is a residue of formulas II or III,. R 2 denotes C1-C 3-alkyl, R 3 denotes C 1 -C 6 -alkyl, N, N-dipropylaminomethyl, 2-chloroethyl, benzyl, styryl, cyclohexyl, 4-methylpenten-3-yl, 1,3-dimethylhexyl, β -indolylmethyl, cyclopenten-2-ylmethyl, n-heptyl , in-octyl, (o-tolylcarbamoyl) methyl, propenyl, methoxymethyl, cyclobexylmethyl, 2- (4-hydroxyphenyl) ethyl, n-noinyl, m-dodecyl, methoxyvinyl, 1-methyl-2-hydroxyethyl, 3,4-dimethoxybenzyl, Ν , Ν-diethylaminoethyl, carbethoxychlorraethyl, 1-methyl-1-hydroxyethyl, 4-chlorobenzyl, (tert-butylcarbamoyl) methyl, dimethoxymethyl, 3-methoxybenzyl, femoxymethyl, ethinyl, Ν, Ν-dimethylaminomethyl, N, N-di-thylaiminoO | P ' R 5 is methyl, cyclopropyl, 2,2-dimethoxyethyl, 2-methoxybenzyl, acetonyl, 5-phenynylethyl, 3-hydroxypropyl, 2-chlorobenzyl, 3-chlorobenzyl, buiten-3-yl, 1-acetamidoethyl, 3-pyridyl, icarbebhoxytmaityl, 5-nitro-riborinein-2-yl, cyclopentyl, 1-carboethoxyethyl, 1-hydroxyoxyethyl and 3-hydroxypropyl, R 2 and R 5 together with the attached methylidine group mean cyclobutyl, cyclopentyl, monomethylcyclopentyl, dimethylcyclopentyl, trimethylcyclopeintyl, cyclohexyl, monomethylcyclohexyl, dimethylcycyclohexyl, trimethylcyclohexyl, cyclohepityl, cyclooctyl, cycloocyl, 2,2,4,4-tetraimethyl, 2,2,4,4-tetraimethyl .pentytylcyclopentyl, 2- (1-cyclohexenecyclohexyl, 2-cyclohexylcyclohexyl, 2-ethylcyclohexyl, 2-propyleyclohexyl, 4-tetrahydrothiopyra, n, 1,2-ethynyl-tilentyl, 3-methyl-6-isopropyl-cyclohexyl, 3,3-dimethyl-cyclohexen-a-yl, 2-phenyl-cyclohexyl, 2-cyclin-hexenyl, 4-tert-butyl-cyclohexyl, 2-ethyl-ethylideneyph-hexyl, decahydro- 1-methyl-2-naphthyl, decahydro-3-methyl-2-naphthyl-1-benzyl-4-piperidyl, 1-methyl-4-piperldyl, bicyclo [3.2.1] octan-2-yl, 3- quinuclidinyl, 5-methoxy-2-tetralyl, adamantyl or 2-norbornyl, as well as their physiologically acceptable acid addition salts, characterized in that the amine of formula IV is alkylated, R • trans Q C17-C-NM-tCH4NU-tCN4N1N (IV) wherein R is as defined above, with a ketone; of formula V, O R2-C-R3, (V) in m R 2 and R 3 are as defined above, in the presence of a reducing agent, in a solvent inert to the reactants, maintaining the reaction mixture at a pH of 6.0 to 8.0 for the duration of the reaction and at a temperature of +10 to -35 ° C.