DE2342946C3 - Process for the preparation of 1-N - ((S) -alpha-substituted omega-aminoacyl) neamines or -ribostamycins and 1-N - ((S) -atpha-hydroxy-gamma-amino-nvaleryl) -ribostamycin; 1-N - ((S) -alpha-hydroxy-gamma-amino-n-butyryl) -neamine; 1-N-C (S) - and (SR) -alpha-hydroxy-betaamino-n-propionyl) -ribostamycin - Google Patents

Description

(S)
. NHCOCHR ₃

\ j I

(CH ₂ ).

OH

(IV)

NR ₁ R,

in which R ', R ₁ , R ₂ , R ₃ , η and Y have the meaning given, the protective groups R ^ and R, as well as Y, if they are present, are split off in a manner known per se and the reaction product in the usual way, as by chromatography, purifies.

2. The method according to claim 1, characterized in that that the starting material is general Formula III uses compounds which are obtained by reacting 1 mole of neamine or ribostamycin with four times the molar amount of trifluoroacetic acid and the usual esterification of the obtained neamine or ribostamycin trifiuortetraacetats with an (S) - «- substituted- <i-aminocarboxylic acid the general formula

(S)
R ₁ -RiN (CH ₂ I _n -CHR ₃ COOH (V |

in which R \ and R ' ₂ together a phthaloyl group, R ₃ a hydroxyl group, amino group (- NH ₂ ), phthalimido group or acylamino group - NHR ₄ , where R _{4 is} an acyl group, preferably an alkanoyl group with 1 to 4 carbon atoms, and η a means an integer from 1 to 4 have been obtained.

3.1 -N - ((S) - (i-Hydroxy-n-amino-n-valeryl) -ribostamycin.

4.1 -N - ((S) - (/ - Hydroxy -] '- amino-n-butyryl) -neamine.

5. 1-N - ((SR) and (S) - «- hydroxy-, ·> '- aminon-propionyl) -riboslamycin.

It was the task of the invention. to find a method with which certain l-N - ((S) - «- substitute "'.-AminoacyK-ncaminc or -ribostamycine can ultimately be produced semi-synthetically from neamine or ribostamycin, as assumed it could be found that the 1 - N - ((S) - «- substituted-aminoacylJ-derivatives of neamine or ribostamycin even against some neamine-resistant bacteria and ribostamycin-resistant bacteria be.

Neamine (that's neomycin A) is a well-known aminoglycosidic antibiotic. Is ribostamycin also a well-known aminoglycosidic antibiotic, which was originally used as an antibiotic

SF-733 has been designated (viii. »The Journal of

Antibiotics «. Vol. 23, No. 3, pp. Ix ^ to 161 [1970] and No. 4. pp. 173 to 183 [1970] and Japanese Patent Publication 17 150 70).

Neamin and Ribostamycin can be given by the general formula

40

45

NH,

OH

are represented in which R is hydrogen or a, / - D-Ribofuranosy'gruppc of the formula

The invention relates to a process for the preparation of 1 -N - ((S) - «- substituted <·> - aminoacyl) - neamines or ribostamycins and lN - ((S) -« - hydroxy -; '- amino-n- valeryl) ribostamycin; 1-N - ((S) - , ι - Hydroxy- γ- amino - η - butyryl) - neamine: 1 - N - (S) - and (SR) - «- Hydroxy - // - amino- η - propionyl ) - ribostamycin.

It is known that butyrosines are aminoglycosidic antibiotics that are produced with the help of a microorganism of the Strcptomyces genus can be produced. They are against some kanamycin-resistant bacteria as well as effective against ribostamycin-resistant bacteria. Butyrosine B could already be used as l-N - ((S) - «- Hy-CH, 0H

Ha)

OH OH

means. The given formula 1 corresponds to the neamine when R is a war.serstoffatom. and the

droxy-; '- amino-n-butyryl) -ribostamycin identifies 65 ribostamycin when R is a, .'- n-ribofuranosyl group (cf. "Tetrahedron Letters", vol. 28 p. 2617 represents.

to 2628 [1971] as well as German patent application. The subject of the invention is now a new

"''" drive to the production of l-N-üSH-substituted-

-aminoacyl) -neamincn or -ribostamycinen of the general formula

(S)
NHCOCHR ₃

HO

(CH ₂ ), NH,

NH,

in which R is a hydrogen atom or a /; - n-Ribofuranos \! group of the formula

CH ₂ OH O

OH OH

and R _{3 is} an OH or NH _r group or an NHR ₄ - from 1 to 4. which is characterized by a group where R _{4 is} an alkanoyl group with 25, that one is an O - ((S) -. i-substituted c-e> -aminoacy!) - 1 to 4 carbon atoms, and η is an integer neamine or -ribostamycin of the general formula

YO

NH,

(S)
O-COCHR ₃ (CH ₂ ^ NR ₁ R ₂

in which R 'is hydrogen or a substituted or unsubstituted C // - D-ribofuranosyl group of the general formula

CH ₂ OY

! ο

(purple)

OY OY

R, and R ₂ either each represent hydrogen or _so and Y is a hydrogen atom or a protecting group

is a protective group acting as acyl, alkylosy- acting group, at preferably 50 to

carbonyl, aralkyloxycarbonyl or aryloxycarbonyl 100 C with hydrazine in ethanol or a strong

group or together a phthaloyl group treated bedcu-basic anion exchanger, in the

d k d lli Fl

ten. R ₃ and η have the preceding meaning

CH ₂ NH ₂

holding general formula product

YO

NH,

- O "Ν

NH,

(S)
NHCOCHR ₃

(CH ₂ J _n NR ₁ R ₂

in which R '. R ₁ . R ₂ , R ₃ . / 1 and Y have the meaning given, the protecting groups R and R ₂ and Y, if they are present, in a manner known per se

cleaved and the reaction product in the usual manner, such as by chromatography, purified. The symbol (S). which in the general For

mcl II is used is a term for sterile marriage Configuration of organic compounds (cf. R. S. C ahn. C. K. I η e ο 1 d and V. P r e 1 ο si in "Fxperientia". Vol. 12, pp. 81 to 94 [1956]).

In the inventive method, the starting material, ie, the O-USJ-n-subsliluicrte '■ i-aminoacyl) -neamin or -ribostamycin of general formula 111 (hereinafter abbreviated as O- (ANA ARBM) hereinafter) in ethanol with hydrazine in Temperatures of preferably 50 to 100 C implemented, using a solution of the starting material in water or an organic solvent. Lower alkanols with 1 to 4 carbon atoms, for example methanol, ethanol, butanol or propanol, can be used as organic solvents; these solvents can be either aqueous or anhydrous. If the starting material of the general formula II! is exposed to the action of hydrazine or the strongly basic anion exchanger in the manner described, the group

-COCHR ₃ (CH ₂ I _n NR ₁ R ₂

those by an ester bond to the hydroxyl group in the 6-position of the neamine or ribostamycin molecule of the starting material is bound. released and to the adjacent amino group in 1-position of the neamine or ribostamycin molecule shifted so that the acyl rearrangement product of the general formula IV is obtained. In this implementation you can see. that in general such acyl groups. which are bonded to a hydroxyl group through an ester formation, which are in the neighboring position to an amino group and is in position to this amino group, under the stated conditions is easily shifted from the hydroxyl group to the amino group. This fact is the basis for the present invention.

It is known that in general acyl groups are more likely to be bonded to a hydroxyl group under acidic conditions and to an amino group under basic conditions, so that the acyl group migrates from the hydroxyl group to the amino group if the character of the surrounding medium in which the various groups are located, is shifted from the acidic area to the basic area - and vice versa. This phenomenon is known as the so-called acyl rearrangement (cf. "Organic Reactions", Vol. 12, p. 173. Verlag John Wiley Sons, 1962). However, the acyl group has never been used to introduce an acyl group into an amino group which is present in a certain position in a molecule which contains several amino groups; this is the case for the first time with the method of the present invention.

In the process according to the invention, the action of the hydrazine or the strongly basic anion exchanger takes place. migration of the (S) - «- substituted ei-aminoacyl group. d. h the group —COCHR ₃ (CH ₂ ) _n NR ₁ R ₂ from the 6-position hydroxyl group to the {position amino group of the neamine or ribostamycin molecule of the starting compound of the general formula III. If this starting compound also has acyl groups for R ₁ , R ₂ . Contains R * and / or Y (which will be discussed below), the acyl group can be obtained from the rearrangement product of the general formula IV during the process or by continuing the heating of the reaction mixture in the same basic medium without isolating the acyl rearrangement product of the general formula IV, so that the conversion of the —OY groups into hydroxyl groups and the conversion of the aylamino group —NR ₁ R ₂ or —NHR ₄ into an amino group —NH. take place in the same reaction mixture, whereby the end product of general formula 11 is obtained immediately.

When the starting material of the general formula contains 111 other Schulz groups for the Y-group as the acyl group and other protecting groups for R ₁ and R ₂ or as the acyl groups, it is necessary to further treat the Acylumlagerungsprodukt of the general formula IV. so that the R ₁ -. R ₂ - and Y groups of the acyl rearrangement product are converted into hydrogen atoms: the conversion takes place in a manner known per se. For example, when the protecting group Y is a benzyl group, R ₁ and or R _{2 are} protecting groups such as alkyloxycarbonyl. Aralkvloxycarbonyl or arvloxycarbonyl. especially carobenzoxy. are, the acyl rearrangement product of the general formula IV obtained from the starting compound of the general formula III still contains these Schui / groups. so that further treatment is required. to convert these protecting groups into hydrogen atoms. Appropriate methods for carrying out such conversions are known to the person skilled in the art. For example, an acyl rearrangement product of the general formula IV. Which contains the benzyl group as a substituent and carbobene / oxy groups for the radicals R ₁ and R ₂ , can be subjected to simultaneous dcbenzylation and decarbobenzylation by either adding the product to a mixture of dioxane. Acetic acid and water dissolve and then hydrogenated in the presence of palladium carbon or by dissolving the product in liquid ammonia and treating the ammoniacal solution with metallic sodium.

The O-lANA ARBMI of the general formula III.

which is used as the starting compound for the first embodiment of the process according to the invention can have been obtained from neamine or ribostamycin in various ways. For example, neamine or ribostamycin can first be reacted with a reagent which is usually used in peptide synthesis, and protective groups can thus be introduced into the four amino groups of neamine or ribostamycin.

The introduction of protective groups on the amino

groups of neamine or ribostamycin are easily obtained by acylation. Carboalkoxylation or carbobenzoxylation of the starting material is achieved. For example, Neamine or Ribostamycin with acetyl chloride. Ethyl chloroformate or benzyl chloroformate are reacted, whereby one the Tetra-N-acetylderivate, the Tetra-N-äthoxv carbonyl derivatives or - the tetra-N-carbobenzoxvderi- yate of neamine or ribostamycin.

The tetra-N-acetyl derivative, tetra-N-ethoxycarbo-

nyl derivative or tetra-N-carbobenzoxy derivative of neamine or ribostamycin. which as described has been made. can then be further treated in a manner known per se, so that all hydroxyl

groups other than those in the 6-position by known protective groups such as acyl. Isopropylidene, cyclohexylidene. Benzylidene or benzyl. As an example of a tetra-N-carbobenzoxydcnvate of ribostamycin. in which all hydroxyl groups except the 6-position hydroxyl group by a known protecting group are protected, the retra-N-carbobenzoxy-3 ', 4', 2 ", 3" -dicyclohcxyliden-5 "-O- (l -methoxycyclohcxyl) -ribostamycin mentioned whose production is published in the "Journal of Antibiotics", Vol. 2 ?. No. 10, p. 61 "3 to 616 (1972) is. The derivative with the protected hydroxyl groups obtained in this way is then substituted with an (S) -u- -.- aminocarboxylic acid of the general formula

(S) "5

R ₁ R ₂ N (CH ₂ I ₁₁ CHR ₁ COOH (V)

in which R ₁ . R ₂ . R ₃ and η have the meaning given above, reacted so that a 6-0- (S) -asubstituted <i-aminoacyl derivative of neamine or ribostamycin is obtained, the four amino groups and the hydroxyl groups in the neamine or ribostamycin molecule additionally being obtained corresponding protecting groups are substituted. If this 6-O- (S) - "- substituted r-aminoacyl derivative is treated in a manner known per se so that the protective groups for the amino groups and, if desired, all or some of the protective groups for the hydroxyl groups are converted into hydrogen atoms, so one obtains the O- (S) - «- substituted« »-aminoacylneamine or -ribostamycin of the general formula III.

If intended. a connection that both Contains amino groups as well as hydroxyl groups in the molecule, preferably on the hydroxyl group acylate, it has already been proposed, the amino group by reaction with a strong organic acid, such as trifluoroacetic acid. The hydroxyl group can then preferably be acylated by adding acetyl chloride or acetic anhydride or mixed anhydrides used as acylating agents (cf. J. B e 1 1 o and J. R. Vino- «R a d in» Journal Am. Chem Soc ". Vol. 78, p. 1369 [1956]).

If this method is used to protect the amino group with a strong organic acid such as trifluoroacetic acid. the O- (ANA ARBM) of the general formula III can also be prepared as follows : The neamine or ribostamycin (in the form of the free base) is dissolved in trifluoroacetic acid while cooling with ice. The solution obtained is mixed with ethyl ether so that the trifluoroacetic acid salt of neamine or ribostamycin, in which all four amino groups of the neamine or ribostamycin molecule are protected by trifluoroacetic acid molecules, precipitates. The neamine or ribostamycin trifluoroacetate thus formed is then dissolved with an (S) - «- substituted f» -aminocarboxylic acid of the general formula V in dimethylformamide (DMF), acetone or tetrahydrofuran, while cooling with ice and in the presence of a strong acid or a dehydrating agent, such as Dicyclohexylcarbodiimide, esterified or condensed. The simplest acid used is an (S) -a-substituted ω-N-phthalimido acid, ie an acid of the general formula V in which R ₁ and R ₂ together form a phthaloyl group. In the reaction (esterification) of the neamine or ribostamycin trifluoroacetate with the acid of the general formula V, all hydroxyl groups, including the 6-position hydroxyl group of the neamine or ribostamycin molecule, are attacked and O-aminoacylated by the acid of the general formula V, so ti.ass a mixture from mono-O-aminoacylated, di-O-aminoacylated. tri-O-aminoacylated and tetra-O-aminoacylated neamines in the form of the trifluoroacetates or a mixture of mono-O-aminoacylated to hexa-O-aminoacylated ribostamycins in the form of the trifluoroacetates. If this mixture of O-aminoacylated neamines or ribostamycins in the form of trifluoroacetates is neutralized with a base, mixed O-aminoacylated neamines or ribostamycins are obtained. which contain a 6-0- (ANA / ARBM) of the general formula III as an intermediate. From the mentioned mixed O-aminoacylated neamines or ribostamycins, the 6-0- (ANAARBM) can be obtained by chromatography, for example over cellulose powder, silica gel or a molecular sieve, which consists of a three-dimensional dextran network. available under the trade name "Sephadcx G-15". The mentioned mixed O-aminoacylated neamines or ribostamycins, which contain the O- (ANA ARBM) as such, can be used immediately as starting material for the process according to the invention, before or after the trifluoroacetic acid has been released by treatment with a base in the manner described above is. If the mixed O-aminoacylated neamines or ribostamycins are reacted in the manner according to the invention, mixed N-aminoacylated neamines or ribostamycins, including the desired 1-N- (ANA, ARBM) of the general formula II, are formed as acyl rearrangement products. The mixed N-aminoacylated neamines or ribostamycins which have been formed as acyl rearrangement products are then separated from the reaction mixture and dissolved in water so that an aqueous solution with a pH of about 8 is obtained with them. This solution can then be subjected to chromatographic separation in a CM-Sephadex C-25 *) column (ammonium form) in order to obtain the desired 1-N- (ANA ARBM). The active fractions of the eluate withdrawn from the column, which contain the 1-N- (ANA ARBM), can be determined by an oxidation test with periodic acid, since the presence of an 1N-acyl substituent can be recognized by the test mentioned (cf. »Tetrahedroc Letters ", Vol. 28, pp. 2624 to 2628 [1971]).

In a preferred embodiment that Process according to the invention for the preparation of 1-N- (ANA / ARBM) of the general formula II se carried out that one uses such compounds as starting material of the general formula III the reaction of 1 mole of neamine or ribostamycin with four times the molar amount of trifluoroacetic acid and the usual esterification of the neamine or ribostamycin tetraacetate with a (SJ-a-substituted lu-aminocarboxylic acid of the general form

(S)
RiRiN (CH ₂ J _n -CHR ₃ COOH

(V)

*) CM-Sephadex C-25 is the trade name for eini Ion exchanger that acts as a molecular sieve and essentially! consists of a crosslinked dextran, which is carboxymethyl contains groups as weakly acidic functional groups.

in which R 'and R' ₂ together represent a phthaloyl group, R, a hydroxyl group, amino group (-NH ₂ ), phthalimido group or acylamino group (-NHR ₄ , where R _{4 is} an acyl group, preferably an alkanoyl group with 1 to 4 carbon atoms, such as acetyl or phthaloyl) and π is an integer from 1 to 4. In such a case the starting material has the general formula III '

CH ₁ NH ₁ ■ Z

NH, Z

--- Os

OY '

Y'O

NH ₁ Z

to you

in which R ₁ ', R ₂ , R, and η have the meaning already given, R "Y' or a substituted /) '- i) -Riboruranosylrcst of the general formula

CH ₁ OY '

OY 'OY'

Y 'is a hydrogen alom or group
-COCHR ₃ (CH ₂ I _n NR ₁ ¹ R;

and Z represents trifluorocacetic acid. That at the subsequent reaction according to the invention obtained acyl rearrangement is from the Reaction mixture isolated in the usual way or from the other acyl rearrangement products by chromatography severed.

NH ₁ · Z

(S)
0-CO-CHR ₃

(CH ₂ I,

no; r ₂

In the esterification of neamine or ribostamycin trifluoroacetate with the Sä'uredcrivai of the general formula V according to the preferred described Embodiment of the invention, it has proven to be beneficial, 1 mol of neamine or ribostamycin trifluoroacetate with 3 to 7 times the molar amount of acid of the general formula V for the reaction bring to.

If the compound of general formula III is exposed according to the invention to the action of a hydrazine solution in aqueous ethanol, the migration of the O- (S) - <i-substituted-N-phthalimidoacyl substituent from the hydroxyl group to the amino group, advantageously simultaneously Release of trifluoroacetic acid such as the phthaloyl group as protective group R ₁ and R ₂ . The rearrangement reaction is preferably performed at 50 to 10O ⁰ C.

The method according to the invention including the production of the starting material wire by the reaction scheme below. For the sake of simplicity, the introduction of the protective groups is and their secession has been omitted.

NH,

HO

HO

(S)
R ₁ R ₂ N (CH ₂ J _n CHR ₃ COOH

Esterification

NH ₂

O- OCHR ₃ (CHj) ₁₁ NR ₁ R ₂

Acyl rearrangement

CH ₂ NH ₂ NH ₂ -O\
\ I. / "—— \ CH OR > - ■ () ■

Acyl rearrangement

(S)

NHCHRj (CH ₂ I ₁₁ NR ₁ K ₂

HO

NH,

OH I release of R ₁ - and
R ₂ groups

NH,

HO

(S)
NHCOCHR., (CH ₂ ) "NH ₂

NH,

OH

Among the aminocarboxylic acids of the general 25 (S) - 'i-hydroxy- f.-N-phthalimidocarboxylic acid ode

Formula V. which can be used according to the invention can be used as (S) - </ - substituted <., - amino carboxylic acid. in which R ₁ and R, not hydrogen and R, represent a hydroxyl group, for example use (S) - <! - Hydroxy-ei-N-carbobenzoxyaminocarboxylic acid. Fine (SJ-n-Hydroxy-iiN-phthalimidocarboxylic acid of the general formula

CO

HOOC -CH (OH) - (CH,) ,, - N Ü I

\ / V

co

is obtained by reaction of the hydrochiride of a (Sl-./..■i--Diaminocarhonsäure of the alluemcinen formula

(Si
HOOC --CH (NHOlCH ₂ I ₁ -NH, ■

in which m is 1 or 2, with a basic copper carbonate in an aqueous-alkaline solution. Reaction of the copper salt obtained with N-carbethoxyphthalimide, treatment of the copper salt: the (St-a-amino-ci-N-phthalimidocarboxylic acid of the general formula

CO

(S)

j - OOC - CH (NH ₂ ) (CH ₂ I _n - N

t ^y co '

v \

with dilute hydrochloric acid in methanol and reacting the (Sj-a-amino-ru-N-phthalimidocarboxylic acid hydrochloride obtained with sodium nitrite in aqueous acetic acid Acetic anhydride or acetyl chloride acetylated in a manner known per se, the corresponding (S) -oN-acetyiamino-f ') - N-pnthalimido acid is obtained, which is the (S) - «- substituted <·> - ammocarboxylic acid of the general formula V, in which R ₁ and R ₂ together form a phthaloyl radical and R ₃ an acylamino group - NHR ₄ , can be used. If an (S) -a-substituted <-i-aminocarboxylic acid of the general formula V, in which R ₁ and R _{2 are} hydrogen atoms and R _{3 is} only one amino group ^ -NH ₂ , is used, it is easiest to use this amino acid to implement in a manner known per se to the corresponding (SJ-ao-diphthaloylammocarboxylic acid which is then used for the esterification of the neamine or Ri-

bostaruycins can be used.

If an (S) -a-substituted m-aminocarboxylic acid of the general formula V, in which R ₁ and R _{2 are} hydrogen atoms, are used, it is good if the amino groups -NH ₂ simply pass through

Reaction with a strong organic acid, such as trifluoroacetic acid, can be seen; in this way a solubility in organic solvents is achieved and, at the same time, a decrease in the reactivity of the amino groups.

The 1-N- (ANA / ARBMX with the help of the invention Process can be obtained as 1-N-aminoacylated derivatives of neamine or Ribostamvcins thereby iHt »ntifi-7i <» w «« ..- λ «.., j-r" »

During oxidation with periodic acid and subsequent hydrolysis with 6N-hydrochloric acid, 2-deoxyätreptamine are obtained.
Some examples of 1-N (ANA ARBM) that start with

Table 1

connection

1-N - ((S (- «- Hydroxy -; - aminon-butyryl (-neamine

1-N - ((S) - «- Hydroxy - ') - aminon-valeryl (-neamin

l-N - ((S) - «- hydroxy -; - aminon-caproyl (-neamine

I -N - ((S) -! (.; '- Diamino-n-butyryl) -neamine

lN - ((S) - "- N-acetyl-".; ^p -diaminon-butyryl (-neamine

l-N - ((S) - «. ^ - Diamino-n-valeryl (-. 'leamine

l-N - ((S) - <i.i-diamino-n-caproyl (-neamine

lN - ((SR) - "- Hydroxy -," - aminon-propionyl (-ribostamycin
IN - ((S) - «- Hydroxy -,; - aminon-propionyl (-ribostamycin

l-N - ((S) -n-hydroxy -; - amino-

n-butyryl (-ribostamycin

l-N - ((S) - (i-Hydroxy-f) -amino-

n-valeryl) ribostamycin

l-N - ((S) - «- Hydroxy -> - amino-

n-caproyl (-ribostamycin

l-N - ((S) -ii.y-Diamino-n-buryryl) -

ribostamycin

l-N - ((S) - «- N-Acetyl -«.; - diamino-

n-butyryl) - ribostamycin

*) With decomposition.

With the aid of the method according to the invention, together with their chemical-physical properties, are in the following table 1 compiled.

Look Melting point [■ Ο ·· PPC *) 0.44 (Cl 0.50 White dust 130-198 + + 42- ρ
^ Neamin 0.50 White dust 130-195 + + 41 ρ
Neamin 0.64 White dust + 40 Neamin 0.59 White dust ρ
Neamin 0.5 White dust ** Neamin 0.44 White dust D.
Neamin , "0.43 White dust - D.
Neamin , "0.43 White dust 160-198 + - "■ RihnManuc , "0.47 White dust 195 + `` Riboslanuc , "0.47 White dust 130 191 + + 34 ", 0.50 White dust 130-195 + + 33 ρ ,,, 0.6I light brown powder - + 30 ρ
¹ ^ Rihostaimi: 0.59 White dust - ρ
Riboslannc White dust ρ
¹ ^ Ritx ^ laniM-

In the above table 1 7_cigcn in the Column "PPC" numbers given the ratio of the distance in the migration of the spots each Connection to that of neamine (this ratio is called RNeamine) or that of ribostamycin (this ratio is known as RRibostamycin). if the compound in a paper chromatogram using n-butanol-pyridine-acetic acid-water (6: 4: 1: 3) has been developed as a mixed solvent.

The compounds prepared according to the invention are characterized by a broad antibacterial properties Effective range and low toxicity and are therefore suitable for chemotherapy of Infections caused by various bacteria.

The compounds listed in Table 1 include all compounds except for the 1-N - ((S) -u-hydroxy -; '- amino-n-butyryl) -ribostamycin (this is butyrosine B) new, d. h .. it concerns connections, which have not yet been described in the literature.

lN - ((S) -K-Hydroxy- ₍ ; -amino-n-propionyl) -ribostamycin,

l-N - ((SR) - «- hydroxy -, - i-amino-n-propionyl) -so ribostamycin.

1-N-I (S) -. «- Hydroxy-amino-n-butyryl) -neamine and

1-N - ((S) - «- Hydroxy-i> -amino-n-valeryl ribostamycin

are particularly valuable therapeutic agents because they have a broad antibacterial action range and a low toxicity, so that they are particularly suitable for the treatment of various bacterial infections. They are against gram-positive bacteria, gram-negative bacteria, and acid-resistant bacteria as well as against bacteria that are opposed to known antibiotics such as kanamycin. Neamine and ribostamycin resistant are effective. They have a low toxicity and bes: .t / .en ι D ₅₀ -WCrIe from 6s 400 to 100 mg kg with intravenous injection in mice.

The superior properties of these compounds are shown in Table 2.

609 685 290

Table 2

Tesivcrhindun ^ n

Minimum inhibitory concentration (mcg / ml)

Staphylo-Eschcri-Pseudomonas aeruginosa

coccus chia coli

aureus W 677 Aj No. 12 H-II

According to the present invention

1. l-N-dSI-d-Hydroxy-'Vamino-n-valeryD-ribostamycin 3.12

2. l-N - ((S) - <i-Hydroxy -; - amino-n-butyryl) -neamine -

3. 1 -N - ((SR) - «- hydroxy - / .'-amino-n-propionyl) -ribostamycin -

A. IN - ((S) - '/ - Hydroxv - /' - amino-n-propionyl) -ribostamycin -

5. Comparison

Butyrosine B (that is ambutyrosine B) 6.25

The LD _{50 of} the compounds I to 4 corresponds approximately to the LD _{50 of} the compound 12.5

3.12
3.12

6.25

6.25 3.12

The compounds prepared can be administered orally or intraperitoncally in any of the pharmaceutical forms of administration administered in the same manner as kanamycin. Examples of pharmaceutical Application forms that are suitable for oral administration are powders, capsules, tablets. Syrups and solutions.

1CR **) series mice. in groups of 10 animals each, which are 4 weeks old and male Were of sex and had an average body weight of 22.5 g were determined by intraperitoneal Injection with Pseudomonas aerusiinos IAM-1007 or Eschcrichia coli K-12 IAM 1254 infused. Immediately after infection, the infected mice were given an intramuscular injection administered to a compound according to the invention. A dose of 25 to 100 mgkg l-N - ((S) - «- hydroxy -] - amino-n-butyryl) -neamine found 90 to 100% survivors in the P. aeruginosa infected mice at the end of the 6th day after infection and 90 to 100% survivors in those infected with E. coli Mice at the end of the 7th day post infection. In contrast, the number of survivors was untreated infected mice at zero. For comparison purposes, those infected with P. aeruginosa were used Mice also use neamine or ribostamycin in an amount of 25 to 100 mg'kg intramuscularly injected, the number of survivors in the treated mice was also zero.

Then mice of the ICR-JCL ***) series, also in groups of 10 animals each, were the 4 weeks old, male and weighed an average of 20.8 g inoculated intraperitoneally with Escherichia coli K-12 IAM 1264. Immediately after vaccination the infected mice were given an intramuscular dose of 12.5 to 100 mg / kg 1-N - ((SH.-hydroxy - / »- amino-n-valeryl-ribostamycin injected. The percentage

**) The expression "mice of the ICR-Rcihc" refers to one Mouse lamb. obtained by pure breeding of mice by the Institute Of Cancer Research, abbreviated ICR.

···) The expression »Mause of the ICR-JCL-Rcihc« denotes a mouse lamb, which was selected by re-breeding a Pair of mice of the ICR series by the Japan Cancer Laboratory, abbreviated to JCL. has been received.

tual percentage of survivors of those treated in this way, E. coli infected mice was 90 to 100%. whereas the infected but not treated Only about 10% of the mice survived at the end of the 7th day after infection.

Mice of the ICR **) series, again in groups of 10 animals each, 4 weeks old and male Were of sex and had an average body weight of 21.0 g were by intraperitoneal injection with Pseudomonas aeruginosa IAM 1007 or EscherichiacoliK-12IAM 1254 infected. Immediately after infection, the infected mice were given an intramuscular dose of 25 to 100 mg / kg 1-N - ((SR) - «- hydroxy -, <- arninon-propionyl) -ribostamycin injected.

Those infected with Pseudomonas aeruginosa or E. coli and then in the manner described treated mice survived 100%, while the percentage of those who did not treated infected mice at the end of the 7th day after infection was only 10%. During a treatment At a dose of 12.5 mg / kg of the compound, the survivors were those with E. coli infected and treated mice at 100%.

Mice of the ICR **) series, in groups of 10 animals each, 4 weeks old and male and had an average body weight of 21.8 g were determined by intraperitoneal Injection with Pseudomonas aeruginosa IAM 1007 or Escherichia coli K-12 IAM 1254 infected. Immediately after infection, the infected mice were injected intramuscularly at a dose of 25 to 100 mg / kg 1-N - ((S) -ri - Hydroxy - / i - amino - η - propionyl) - ribostamycin treated.

Those infected with Pseudomonas aeruginosa or Escherichia coli and treated as described Mice survived 100%, while the number of survivors in the infected and afterwards did not treated animals at the end of the 7th day after infection was zero.

When treated with a dose of 12.5 mg / kg of the compound, those with E. coli survived infected and treated mice to 100%.

The following examples serve to explain

modification of the invention. In the examples, abbreviations are used for frequently recurring terms used, namely the following:

DMF = dimethylformamide.

XHF = tetrahydrofuran.

DCC = dicyclohexylcarbodiimide.

XFE = trifluoroacetic acid.

XFA = trifluoroacetate

NA = Neamine.

RBM = ribostamycin.

example 1 a) starting material

To 30 g of RBM (in the form of the free base) were added 127 ml of TFE, using ice cooling and stirring; then the mixture was allowed to stand at ambient temperature overnight. The present thereafter, amber solution was mixed _m it 400 ml of ethyl ether. The mixture was cooled, with the formation of 69 g of a precipitate which is the salt of the RBM with TFE; the salt was filtered off. 20 g of this RBM-TFA were dissolved in 250 ml of THF together with 16.4 g of (S) - (- hydroxy-; · -N-phthalimido-n-butyric acid of the formula

(S)
HOOC ■ CH (OH) - (CH ₂ J ₂ -N

CO

CO

eclösl. The solution was cooled with ice. A solution 36.4 g of DCC in 146 ml of THF were prepared while cooling with ice. Bicdc solutions were then combined with ice-cooling and stirring. The combined solutions were at ambient temperature 2 Stirred up to 3 hours and then treated with 8 ml of glacial acetic acid in order to decompose the excess of DCC. It 3286 mg of dicyclohexylurea were formed, which settled and was filtered off. The filtrate was concentrated to dryness, whereby 43 g of a pale yellow colored, solid body obtained from the mixed TFE salts of the O - ((S) - «- hydroxy -.- N- phthalimido- η - bulyryl - ribostamycin consisted.

b) Invention

The solid substance was added to 800 ml of a liquid mixture of hydrazine hydrate and 80% strength aqueous ethanol (1:10, v / v) and dissolved. The so eewonnene solution was heated for 1 hour and 30 minutes "^ an oil bath at 90 to 100 ⁰ C, with the N - ((S) -« - hydroxy-y-amino-n-butyryl) -RBM with liberation of the phthaloyl The reaction mixture was then cooled, a precipitate settling out, which was filtered off. The precipitate was washed with 100 ml of ethanol. The filtrate and the washing solutions were combined and concentrated to a solid syrup. This syrup was extracted with 100 ml of ethanol, then evaporated to dryness and extracted again with 180 ml of ethanol. The solid residue remaining after the ethanol extractions was mixed with the aforementioned precipitate. The solid substance was then extracted with 100 ml of water. The ethanol extract and the aqueous extract were combined and diluted with water to a volume of 1000 ml (pH 8.0 to 8.2). This solution was then passed into a column (38 χ 276 mm; about 300 m!) containing 40 g of a weakly acidic

Ion exchanger, known under the trade name "CM-Sephadcx C-25", contained. It essentially consists from a three-dimensional gel network of dextran with carboxymethyl residues as functional Groups. The product »C 25« is more closely linked

ίο as the product »C 50«. The first part from the Column was discarded and the column successively with 1000 ml of water and 41 0.1 N aqueous Ammonia washed. Thereafter, the column was made using 0.2N aqueous ammonia

■ 5 eluted, and the eluate was divided into 20 ml fractions caught. Fractions 80 to 156 were combined, and the solution was concentrated under reduced pressure. This gave the 1 -N - ((S) - </ - hydroxy -; ■ - amino - η - butyryl) - RBM (compound 1)

as a crude product in a yield of 1.7 g. This crude product was redissolved in 770 ml of water (pH value 8.8 to 9.0) and the aqueous solution in a corresponding manner over a column of 40 g "CM-Sephadex C-25" headed. After the column

with 1000 ml of water and 41 0.1 N aqueous ammonia Was washed with the eluent using 0.2N aqueous ammonia began. The eluate was collected in 20 ml fractions; Fractions 68 to 128 turned out

than the active factions. These active fractions were combined and concentrated to give 1336 mg of the above compound 1 was obtained as a pure product.

Elemental analysis for C ₂₁ H ₄₁ N ₅ O ₁₂ · 2H ₂ O:

Calculated ... C 42.7, H 7.63, N 11.85%: found .... C 42.67, H 7.18, N 10.54%.

The antibacterial spectrum of activity of this product against various microorganisms is given in Table 3 below.

Table 3 Incubation medium Mindcsl Test- inhibitory
focus Microorganism lration (mcg mil Heart infusion 6.25 Escherichia broth coli NIHJ Heart infusion 6.25 Escherichia broth coli A 3 Heart infusion 3.13 Escherichia broth coli K-12 ML-1629 Heart infusion 0.1 Staphylococcus broth aureus 209 P Heart infusion 3.13 Proteus vulgaris broth OX-19 Heart ir.fusion 3.13 Pseudomonas broth aeruginos IAM 1007 Heart infusion 0.2 D ipl ococcus broth pneumoniae

continuation

Test microorganism

Incubation medium

Escherichia
coli K 12
Staphylococcus
aureus 209 P
Pseudomonas
aeruginosa A 3
Pseudomonas
aeruginosa No. 12
Pseudomonas
aeruginosa H 9

Nutrient agar
Nutrient agar
Nutrient agar
Nutrient agar
Nutrient agar

Minimum concentration

(meg ml)

1.56
6.25

25th
12.5
6.25

"5

When the product of this example (in the form of the free base) was injected intravenously into mice in order to determine the acute toxicity of the compound, the LD ₅₀ value was found to be 690 mg / kg.

Example 2
a) Production of the starting material

2 g of the RBM-TFA, which had been prepared as described in Example 1, was dissolved in 35 ml of a mixture of THF and DMF (10: 1: VV). This solution was mixed with 1.74 g of (S) -n-hydroxy-Λ-N-phthalimido-n-valeric acid in 16 ml of THF. 3.62 g of DCC in 16 ml of THF were added to the solution while cooling with ice. Thereafter, the mixture was stirred with further ice cooling for 30 minutes and then for a further 1 hour and 25 minutes at ambient temperature in order to achieve the esterification of the hydroxyl groups of the RBM with the substituted valeric acid. The reaction mixture was then treated with 0.8 ml of glacial acetic acid and stored at room temperature overnight in order to decompose the excess DCC. The dicyclohexylurea formed and precipitated as a by-product was filtered off. The filtrate was concentrated to give a solid substance composed of the mixed TFF.-S;>'zrn des O - ((S) - n -hydroxy - /' sN-phthalimido-n-valerylJ-RBM.

SO

b) Invention

The solid substance obtained in this way was dissolved in 50 ml of a mixture of hydrazine hydrate and 80% strength aqueous ethanol (1:10, W) and the solution was heated to 83 ° C. in an oil bath for 1 hour and 30 minutes. The reaction mixture was then concentrated to dryness and the residue extracted with 80 ml of water. The insoluble substances were bottled off. and the filtrate (the aqueous extract) with a pH of 8.2 to 8.4 was immediately passed through a column with 15.5 ml (about 2 g) of a weakly acidic ion exchanger, known under the trade name "CM-Sephadex C-25". directed. It consists essentially of a three-dimensional gel network of dextran with carboxymethyl residues as functional groups. The column was then washed successively with 100 ml of water and 600 ml of 0.1N aqueous ammonia. The F.luicren was stirred in the same manner as in Example 1 using 250 ml of 0.2 N aqueous ammonia. In this way, 82.4 mg] - N - '((S) - ί - hydroxy-A-amino - η - valcryl) - RBM (compound 2) were obtained as crude product. This crude product was redissolved in water, so that 20 ml of an aqueous solution (pil 8.4) were obtained, which in a column with 15.5 ml (about 2 g) of a weakly acidic ion exchanger, known under the trade name CM-Sephadex C -25 «. was directed. It essentially consists of a three-dimensional gel network of dextran with carboxymethyl residues as functional groups. The column was washed successively with 50 ml of water and 100 ml of 0.1 N aqueous ammonia. 0.2 N aqueous ammonia was used for elution. The eluate was collected in 10 ml fractions. The desired product was contained in fractions 29 to 44 of the fluate. These fractions were combined and concentrated: Compound 2 was obtained in this way as a pure product in a yield of 66.8 mg.

Elemental analysis for C ₂₂ H ₄₁ N ₅ O ₁₂ ■ 2 H ₂ O:

Calculated ... C 43.63. H 7.82, N 11.56%;
found .... C, 44.34. H 7.28. N 11.67%.

The spectrum of antibacterial activity of the product of this example is given in Table 3 below anüCizcben.

Table 3 Incubation medium Mtndcsi- Test- hcmm- Microorganism kon / cn- tration Imcü mil Heart infusion 6.25 Escherichia broth coli NlHJ Heart infusion 6.25 Escherichia broth coli A 3 Heart infusion 6.25 Escherichia broth coli K-12 ML-1629 Heart infusion 0.1 Staphylococcus broth aureus 209 P Heart infusion 6.25 Proteus vuluaris broth OX-19 Heart infusion 12.5 Pseudomonas broth aerudnosa IAM 1007 Heart infusion 0.39 Diplococcus broth pneumoniae Nutrient agar 3.13 Escherichia coli K 12 Nutrient agar 3.13 Staphylococcus aureus 209 P Nutrient agar 100 Pseudomonas aeruginosa A 3 Nutrient agar 50 Pseudomonas aeruginosa No. 12 Nutrient agar 12.5 Pseudomonas aeruginosa H 9

/ Ί

When the product of this example (in the free base form) was injected intravenously into mice, they died none of the mice at a dose of 600 mg / kg.

Example 3
a) Production of the starting material

2 g of the RBM-TFA prepared as in Example 1 were in 35 ml of a mixture of THF and DMF (10: 1, V / V) solved. This solution was mixed with a further solution of 1.9 g of (S) - «- hydroxy - / - N-phthalimido-n-caproic acid mixed in 18 ml of THF. A solution of 3.62 g of DCC in 16 ml of THF was then added to the mixture while cooling with ice. The The resulting mixture was left under ice cooling for 30 minutes and then an additional 1.5 hours at ambient temperature stirred to esterify the hydroxyl groups of the RBM with the substituted To achieve caproic acid. 0.8 ml of glacial acetic acid was added and the reaction mixture became at ambient temperature Stopped overnight to decompose the excess DCC. The formed dicyclohexylurea T precipitate was filtered off. The filtrate was evaporated to dryness, whereby a solid substance obtained from a mixture of the TFE salts of O - ((S) - «- Hydroxy-i-N-phthalimidon-caproyl) -RBM duration.

b) Invention

This solid substance was dissolved in 80 ml of a mixture of hydrazine hydrate and 80% strength aqueous ethanol (1:10. V / V), and the resulting solution was heated to 83 ° C. in a water bath for 1.5 hours. The reaction mixture was then evaporated to dryness and the residue extracted with 80 ml of water; the insoluble material was filtered off. The filtrate (the aqueous extract) was passed at pH 8.4 through a column with 15.5 ml (about 2 g) of a weakly acidic ion exchanger, known under the trade name "CM-Sephadex C-25". It essentially consists of a three-dimensional gel network of dextran with carboxymethyl residues as functional groups. The column was washed with 100 ml of water and then with 500 ml of 0.1 N aqueous ammonia. Thereafter, elution was carried out using 300 ml of 0.2 N aqueous ammonia. wherein the 1 -N - (, (S) -u-Hidroxy-i-amino-n-caproyl) - RBM could be obtained as a crude product in an amount of 5 mg.

The spectrum of antibacterial activity against "various microorganisms" is in Table 4 given. -,

Table 4 Incubation
medrum - Test microorganism Nutrient
broth
Nutrient
broth minimum
bemm-
icon
tration
(mcg / ml) Escherichia coli
communis
Escherichia coli K-12 25th
6.25

Test microorganism

Obligations iTU'dium

Minck inhibitor concentration

I mcg ml)

IAM 1264 !O B e i s ρ Nutrient 6.25 Staphylococcus aureus broth ¹⁰ 209 P. Nutrient more than Pseudomonas broth 100 aeruginosa IAM 1007 Nutrient more than Pseudomonas broth 100 15 aeruginosa H-11 Nutrient 6.25 Klcbsiella pneumoniae broth Nutrient 12.5 Proteus vulgaris OX-19 broth iel 4

a) Production of the starting material

774 mg of neamine (NA) in the form of the free base were dissolved in 7.7 ml of TFE while cooling with water solved. The solution was stirred for about 30 minutes and then mixed with 80 ml of ethyl ether, wherein 1545 mg of a precipitate formed which consisted of NA-TFA and was filtered off. 636 mg of this Precipitation and 615 mg (S) - <i-Hydroxy-; · -N-phthalimido-n-butyric acid were rubbed together and in 20 ml of a mixture of THF and DMF (9: 1. V V) solved. A second solution of 1360 mg DCC in 5.44 ml THF was then added to this solution given under ice cooling. The mixture was ice-cooled for 30 minutes and then for another 2 hours and stir for 15 minutes at ambient temperature to prevent the esterification of the hydroxyl groups of the To effect neamines with butyric acid. 0.3 ml of glacial acetic acid was then added to the reaction mixture, to decompose the excess DCC. The mixture stayed overnight so that the Decomposition product dicyclohexylurea could settle. The precipitate was filtered off, and that The filtrate was concentrated to a solid substance composed of a mixture of the TFE salts of O - ((S) - «<- Hydroxy-y-N-phthalimido-n-buiyryl) -nearnine duration.

■ ·

b) Invention

The solid substance was dissolved in 30 ml of a mixture of hydrazine-hydral and 80% aqueous ethanol (1:10, V / V) and the resulting solution was heated to 83 ° C. for 1 hour and 30 minutes in a water bath The reaction mixture was evaporated to dryness and the residue was extracted with 40 ml of water, the insoluble material being filtered off. The filtrate (the aqueous extract) was passed at pH 8.0 into a column with 11 ml of a weakly acidic ion exchanger, known under the trade name "CM-Sephadex C-25". It essentially consists of a three-dimensional gel network of dextran with carboxymethyl residues as functional groups. The column was filled with 50 ml of water and then with 200 ml

409 685/290

Washed 0.1-η aqueous ammonia. Subsequently the column was eluted using 0.2 N aqueous ammonia. The eluate was in 10 ml fractions collected with the desired Product was contained in fractions 6 to 12. These active fractions were pooled and concentrated; in this way one obtained the l-N - ((S) - «- hydroxy - '> - amino-n-butyryl) -neamine in a yield of 46.95 mg.

Elemental analysis for C ₁₆ H ₃₃ N ₅ O ₈ · 2H ₂ O:

Calculated ... C43.5, H 7.93. N 15.86%:
found .... C 42.8, H 7.50, N 14.86%.

The antibacterial spectrum of this product against various microorganisms is shown in Table 5.

Table 5

I est microorganism at Incubation Minimum medium inhibitory focus tration (meg ml) Escherichia coli K-12 Nutrient agar 6.25 Staphylococcus aureus Nutrient agar 12.5 209 P Pseudomonas Nutrient agar 1Z5 aeruginosa A 3 Pseudomonas Nutrient agar 12.5 aeruginosa No. 12 Pseudomonas Nutrient agar 6.25 aeruginosa H-9 game 5

a) Production of the starting material

567 mg of RBM-TFA, which had been prepared in the same manner as described in Example 1 were, in a solution of 708 mg (S) - <v / -Di-N-phthaloyl-2,4-diaminobutyric acid dissolved in 7 ml of THF. The resulting solution was ice-cooled and this ice-cold solution was then mixed with another ice-cooled solution of 1030 mg DCC in 4 ml THF with further ice cooling and "mixed with stirring. The obtained mixture was cooled with ice for 30 minutes and thereafter for 2 hours and 45 minutes stirred at ambient temperature to prevent the esterification of the hydroxyl groups of the RBM with the To effect butyric acid. 0.25 ml of glacial acetic acid were used then added to decompose the excess DCC. The insoluble dicyclohexylurea formed was filtered off and the filtrate was concentrated. In this way a solid substance was obtained which from a mixture of the TFE salts of O - ((S) -n, y-Di-N-phthaloyl-2,4-diamino-butyryl) -RBM duration.

b) Invention

This solid substance was dissolved in 27 ml of a mixture of hydrazine hydrate and 80% aqueous ethanol (1:10, V / V) solved, and the one afterwards Solution was heated to 83 ° C. in a water bath for 1.5 hours. Thereafter, the reaction mixture became evaporated to dryness and the remaining residue was extracted with 30 ml of water; the insoluble Material was filtered off. The filtrate (the aqueous extract) was in a column of 15 ml a weakly acidic ion exchanger, known under the trade name »CM-Sephadex C-25 ". It essentially consists of a three-dimensional gel network of dextran with carboxymethyl residues as functional groups. After the column has been washed with 50 ml of water

ίο was eluting with 150 ml of 0.1 N aqueous Ammonia carried out. The l-N - ((S) - «, y-diaminon-butyryl) -RBM was obtained as a crude product in a yield of 302 mg. This product inhibited the growth of Escherichia coli communis on a nutrient agar plate.

Example 6 a) Production of the starting material

zo 562 mg of that described in Example 1 RBM-TFA obtained in this way were dissolved in a solution of 536 mg (S) - «- N-acetyl - / - N-phthaloyl-2,4-diaminobutyric acid dissolved in 7 ml of THF. The resulting solution was ice-cooled and then with a

ice-cold solution of 1018 mg DCC in 4 ml THF mixed, with stirring and further ice cooling. The mixture was ice-cooled for 30 minutes and then at ambient temperature for 1 hour stirred to the esterification of the hy-

To effect hydroxyl groups of the RBM with the substituted butyric acid. The reaction mixture was then 0.25 ml of glacial acetic acid was added to decompose the excess DCC. The failed insoluble Dicyclohexylurea was filtered off. The

Filtrate was concentrated to give a solid residue, consisting of a mixture of TFE-salts of O - η - - ((S) N - Acetyl-N- -γ phthaloy 1 to 2.4 - diaminobutyrylHlBM existed.

b) Invention

This solid substance was dissolved in 26 ml of a mixture of hydrazine hydrate and 80% aqueous ethanol (1:10, V / V) and the resulting solution heated in a water bath at 83 ° C for 2 hours. The

The reaction mixture was evaporated to dryness as such and the residue with 30 ml of water extracted; the insoluble material was filtered off

^^^ Wet, 1 ** annt linier deni Trade name ^

^a ^ ^? m ^ thin ^

nut Cjtfbo ^ et ^^

¹ ^ column was washed with 50 M water ^ ^^ ß ³¹¹ ^ ^^ mawcadung of 150 ml ft $ p

A little ammonia and 100 ml of 0.2N aqueous? ^

Anunomak elutes the active fractions of the ElUaIp

^w ^ r (th captured and concentrated on this weäsf k fio ^ei W? '■'?% * £ ** ^^^ - ^ N-Acetyl-o ^ -dianmiqS H-OUt ^ I) ₇ RBM; as a crude product in 4ner yield φ

of 330 mg: This product stunted growth!

of Escherichiacoli on a nutrient plate:; Ä

jj · '' ..V -;>. ■; ■. · " ^: ': .. ^B ; e35! piel.7' - ..; '_; , ■ - / ^ (M ^ ^Her ^ ^lun ^ of ^the starting material 'f | * "Jg RBMVTFAv which was described in the same manner as in Example 1,

and the TFA of (SR) - «- hydroxy- / i-amino-n-propionic acid, made from 533 mg (SR) - <i-hydroxy- / i-amino-n-propionic acid, were dissolved in a mixture of 10 ml of THF and 3.8 ml of DMF. The resulting solution was ice-cooled and then with a likewise ice-cold solution of 2928 mg DCC dissolved in 10 ml THF, being cooled with ice and was stirred. The mixture was stirred for a further 2 hours with ice cooling and then for a further 3 hours Stirred at ambient temperature. Thereafter, the reaction mixture was left over at ambient temperature Turned off overnight and finally treated with 1 ml of water and a further 9 hours at room temperature touched. Finally, it was turned off again at ambient temperature overnight. The mixture became filtered and the filter cake was washed with 20 ml of THF. The filtrate was washed with THF combined, whereupon the solution was concentrated to dryness. The remaining solid matter existed from a mixture of the TFE salts of the 0 - ((SR) -U-hydroxy- / f-amino-n-propionyl} -RBM. The solid substance was extracted with 30 ml of water and then again with 10 ml of water; the insoluble solid substance was filtered off. The filtrate (the aqueous extract solution) was mixed with 20 ml of ethanol.

b) Invention

The solution obtained in this way, which contained the mixed TFE salts of O - ((SR) - «- Hydroxy - ji - aminon-propionyl) -RBM, was mixed with about 30 ml of the strongly basic anion exchange resin" Amberlite IRA 400 " . This anion exchange resin consists of a crosslinked polystyrene with quaternary ammonium groups

-N- (CHj) ₃ OH

The resin corresponds to the OH cycle. The mixture was stirred for 4 hours at ambient temperature in order to achieve the conversion of the O - ((SR) - «- hydroxy- / i-arninon-testionyl) - RBMe to the N - ((SR) -« - hydroxy - / * - amino -n-propionyl) -RBMe under the action of the basic anion exchange resin. The reaction mixture was then filtered and the filtration residue was washed with 50 ml of 40% strength aqueous ethanol. The filtrate was combined with the washing water and the solution obtained was concentrated to dryness under reduced pressure. Thus, 677.8 mg of a solid substance were obtained. This solid substance was dissolved in 100 ml of water. The resulting aqueous solution was passed through a column with 20 ml of a weakly acidic ion exchanger, known under the trade name "CM-Sephadex C-25". It consists essentially of a three-dimensional gel network of dextran with carboxymethyl residues as functional groups. The column was washed with water and eluted with 0.2 mm aqueous ammonia. The eluate was collected in 5 * nl fractions, and fractions 7. to 16 were combined and concentrated to dryness under reduced pressure a weakly acidic ion exchanger chromatography, known under the trade name "CM-Sephadex C-25". It consists essentially of a three-dimensional gel network of dextran with carboxymethyl residues as functional groups. 0.1 N aqueous ammonia was used for development. The eluate was collected in 5 ml fractions and fractions 14-19 were pooled. The solution was evaporated to dryness. 289.1 mg of des! -N - ((SR) - «- Hydroxy- / i-amino-n-propionyl) -RBM as crude product. For purification, the crude product was dissolved in 50 ml of water. The aqueous solution became weak through a 19 ml column

ίο acidic ion exchanger, known by the trade name »CM-Sephadex C-25« chromatographies, with 0.05-N aqueous ammonia for development has been used. The weakly acidic ion exchanger consists essentially of a three-dimensional one Gel of dextran with carboxymethyl residues as functional groups. The eluate was collected in 5 ml fractions. As soon as fraction no. 64 of the eluate has been collected 0.1 N aqueous ammonia was used as the solvent in place of 0.05 N aqueous ammonia Develop used. Fractions 69 to 72 were combined and under reduced pressure concentrated to dryness. This gave 30.03 mg of 1 -N-ifRi-H-hydroxy-zi-arnino-n-propionyl-RBM.

Elemental analysis for C ₂₀ H ₃₉ N ₅ O ₁₂ - 2H ₂ O:

Calculated ... C 41.6, H 7.46, N 12.1%;
found C 41.68, H 6.46, N 11.75%.

The antibacterial spectrum of activity of this product goes against various microorganisms from the following table 6.

Table 6

_

Test microorganism 40 Escherichia coli NlHJ Bcbnitirags- Mmdcsl Escherichia coli K-12 racdium inhibitory ⁴⁵ Staphylococcus aureus focus 209 P 1 ration Pseudomonas (mcg ml aeruginosa A 3 Nutrient agar 3.12 ₅ o Pseudomonas Nutrient agar 1.56 aeruginosa No. 12 Nutrient agar 6.25 Klebsiella pneumoniae PCI 602 Nutrient agar 50 Nutrient agar 6.25 Nutrient agar 3.12

When the product of this example (in the form of the free base) was injected intravenously into mice to determine the acute toxicity of the compound, the LD ₅₀ value was found to be 650 mg / kg.

. Example 8

a) Production of the starting material

1560 mg of RBM-TFA prepared in the same way as described in Example I, and the TFA of (S) -a-hydroxy-0-amino-n-propionic acid, prepared from 540mg (S) -ci-hydroxy- / i-ammo-n-propionic acid, were dissolved in a mixture of 12 ml of THF and 4 ml of DMF. The resulting solution

was ice-cooled and then mixed with a likewise ice-cooled solution of 3 (X) 2 mg DCC in 11 ml THF with stirring and further ice-cooling. The mixture was stirred for 2 hours with ice-cooling and then for 3 hours at ambient temperature. The reaction mixture was then left to stand overnight at room temperature and then mixed with 1 l of water, whereupon it was stirred for a further 9 hours at ambient temperature and then again turned off over Nachi at ambient temperature. The mixture was then filtered and the filter cake washed with 20 ml THF. The filtrate was combined with the THF washing solutions and the solution thus obtained was evaporated to dryness. In this way, a solid substance was obtained which consisted of the mixed TFE salts of O - (S) - a- hydroxy - /, '- amino-η-propionyl) -RBM. The solid substance was extracted with 30 ml of water and then with 10 ml of water; the insoluble solid material was filtered off. The filtrate (the aqueous extract solution) was mixed with 21 ml of ethanol.

b) Invention

The sd recovered solution that the mixed TFE salts of O - ((S) - «- Hydroxy - /; - amino-n-propion \ I) -RBM was mixed with about 32 ml of a strongly basic anion exchange resin, which consists essentially of a polystyrene with qualifying ammonium groups

45

-N (CHj) ₃ OH

duration. This anion exchange resin is known under the trade name "Amberlite IRA 4 <X1". The resin corresponds to the OH cycle. The mixture was left for J-hours at ambient temperature stirred in order to convert the O - ((S) - <i-Hydrox \ - .. '- amino - η - propionyl) - RBM to the N - ((Si -. < - Hydroxy-, .'-amino-η-propionyl) -RBM under the action of the basic anion exchange resin to effect.

The reaction mixture was filtered and the filtration residue was washed with 60 ml of 40% strength aqueous ethanol. The filtrate was combined with the washing solutions. The resulting solution was evaporated to dryness under reduced pressure to obtain 685 mg of a solid substance. This was dissolved in 100 ml · water. The resulting aqueous solution was passed through a column with 20 ml of a weakly acidic ion exchanger, known under the trade name "CM-Sephadex C-25" (ammonium form). The column was washed with water and eluted with 0.2 N aqueous ammonia. The eluate was collected in 5 ml fractions. Fractions 7 to 18 were combined and evaporated to dryness under reduced pressure. The remaining solid residue was redissolved in 100 ml of water. This solution was chromatographed again in the same manner via a column with a weakly acidic ion exchanger, known under the trade name "CM-Scphadcx C-25", 0.1 N aqueous ammonia being used as the solvent for developing. The elual is collected in 5 ml fractions. The fractions I: to 20 were combined. The solution obtained in this way was concentrated to dryness, 275 ni {I - N - ((S) - «- hydroxy - // - amino - η - propionyl) - R BN 'being obtained as a crude product.

For purification, the crude product was dissolved in 50 ml of water and the aqueous solution was again used: a column with 20 ml of a weakly acidic knee exchanger, known by its trade name! "CM-Sephadex C-25", chromatographies, wöbe 0.05-n aqueous ammonia as solvent to be π Develop was used. The eluate was collected in 5 ml fractions. As soon as parliamentary group 6 ( of the eluate had been removed, O.l-r Aqueous ammonia as a solvent instead of: 0.05-N aqueous ammonia for developing vcr turns. Fractions 70 to 73 were combined and dried to dryness under reduced pressure narrowed. In this way 42.1 m were obtained; 1 -N - ((S) -u-hydroxy - /, '- amino-n-propionyl) -RBM

Elemental analysis for C ₂₀ H ₃₉ N ₅ O ₁₂ 2H _: O:

Calculated ... C 41.6, H 7.46. N 12.1%:
found .... C 41.71, H6.81. N 7.81%.

The spectrum of antibacterial activity of this product against various microorganisms is compiled in Table 7.

Table 7

Test microorganism

cherichia coli NIHJ
Escherichia coli K-12

Staphvlococcus aureus
209 P '

Pseudomonas
aeruginosa A 3
Pseudomonas
aeruginosa No. 12

Klebsiella pneumoniae
PCI 602

If the product of this example (in the form dei free base) was injected intravenously mice to determine uir the acute toxicity of the compound, se a LDO value yielded by Λ ^ Π / ΐ

Breeding
mcdium Minimum
inhibitory
kon? cn-
tration (meg mi Nutrient agar
Nutrient agar
Nutrient agar 3rd! 2
1.56
3.12 Nutrient agar 50 Nutrient agar 6.25 Nutrient agar L56

Claims (1)

Patent claims: 1. Process for the preparation of l-N - ((S) - <«- substituted - <..- aminoacyl) -neamines or -ribostamycins the general formula HO CH ₂ NH,
t
i
■ ^L - ° \ > - ο— < NH, \ NHCOCHR ₃ OH /
y / Χι / (CH ₂ ). -Γ Γ
NH, NH ₇ (H) in which R is a hydrogen atom or an o-ribofuranosyl group of the formula CH ₂ OH O OH OH (Ia) and R ₃ denotes an OH or ΝΗ, group or an NHR ^ group, where R _{4 is} an alkanoyl group having 1 to 4 carbon atoms, and η denotes an integer from 1 to 4 », characterized in that an O - ((S) - «- substituted- <·> - aminoacyl) - neamine or -ribostamycin of the general formula YO NH, (III) NH, (S)
O-COCHr ₃ (CHANR ₁ R ₂ in which R 'is hydrogen or a substituted or unsubstituted, / - D-ribofuranosyl group of general formula (III a) OY OY R ₁ and R _{2 are} either hydrogen or an acyl, alkyloxycarbonyl, aralkyloxycarbonyl or aryloxycarbonyl group acting as a protective group or together represent a phthaloyl group, R ₃ and; i have the above meaning and Y is a hydrogen atom or a group acting as a protective group is, at preferably 50 to 10O ⁰ C with hydrazine in Ethanol or a strongly basic anion exchange treated, in the product obtained the general formula CH ₂ NH ₂ OY YO NH, NH,