DK143413B - PROCEDURE FOR PREPARING AMINOCYCLITOL COMPOUNDS OR ACID ADDITION SALTS - Google Patents

Description

(19) DENMARK

| J | oz) PRESENTATION WRITING ου 1 3 B

PATENT AND TRADEMARKET DIRECTORATE

(21) Application No. 1772/79 (51) IntCI.3 C 12 P 19/48 (22) Filing date 30 Apr. 1979 C H 15/22 (24) Race day 17 · Feb. 1976 (41) Aim. available Apr 30 1979 (44) Posted 17 Aug. 1981 (86) International application no. "(86) International filing day" (85) Transfer day "(62) Master application no. 631/76

(30) Priority 22. s ep. 1975, 615593 * US

(71) Applicant STERLING DRUG INC., New York, US.

(72) Inventor Sol Jacob Daum, US: Robert La Grone Clarke, US.

—R (74) Hofman-Bang & Bout Patent Office Agent.

(64) Process for the preparation of aminocyclitol compounds or acid addition salts thereof.

The invention relates to a process for the preparation of amino-cyclitol compounds having the general formula I or acid addition salts thereof according to the preamble of claim 1, which process is characterized by the characterizing part of claim 1.

n 2 US Patent No. 3,669,838 discloses a process for preparing certain aminocyclitol compounds. This method consists in cultivating a nutrient medium containing carbohydrates, a source of assimilable nitrogen, necessary salts and an added λ added aminocyclitol derivative, especially streptamine or 2-epistreptamine, § 2 U 3 A 1 3 in the presence of a suitable mutant microorganism which is unable to synthesize the said aminocyclitol itself, but is able to incorporate this subunit into an antibiotic when the aminocyclitol is added to the nutrient medium.

It has now been found that a novel mutant strain of Micro-monospora purpurea, namely M. purpurea ATCC 31164, is capable of incorporating in an antibiotic of formula I an aminocyclitol of formula H0 \, NH2 V __ ^ 2 ' _j1 ... iia 2 '3' 1 '

wherein R and R are each hydrogen or hydroxy, and R

is amino or hydroxy, or Schiff bases of the compounds of formula IIa with benzaldehyde having the formula: H0-N / = CHC6H1> 1V___r2 '_tV \ HO ^ -R1 * ... Ilb' II Of wherein R is hydroxy or benzylidenimino (CgHgCH = N) and R and R4 have the above meaning. Hereby compounds of formula I are prepared in which R and R are each independently hydrogen or methyl, R and R are individually hydrogen or hydroxy and R

* 2 O.

R 1 and R are all hydrogen.

13 8

The compounds of formula I wherein one of R, R 4 and R is a ω-amino-α-hydroxy lower alkanoyl group are prepared by the method described in U.S. Patent No. 3,780,018 and which consists of reacting the compound obtained with the culture of η -zq of formula I, wherein R, RJ and R are all hydrogen, with an N-hydroxy-succinimide ester of formula 3

o L

/ VcH, oy -NHCH9 (CH,) CHOHCO-O-N

Wherein n means 0 or 1. The resulting mixture of compound 1 * 8 is of formula I wherein one of R 1 and R is 3 (N-benzyloxy-carbonyl) amino-α -hydroay-lower-allcanoyl group: µl / 1 'yr_CH2O-C'-NHCH2 (CH2) nCHOHCO- and the other two are hydrogen, then hydrogenolysis of the benzyloxycarbonyl group with hydrogen is subjected to a catalyst.

In the acylation reaction, a mixture of the three possible isomeric monoamides is obtained in which the R 1, R 2 and R 8 amine hydrogen atom are respectively replaced by the u) - (N-benzyloxycarbonyl) amino-ct-hydroxy lower alkanoyl group. . Of course, when individual characterization and examination of these products is desired, they must be separated from one another. The acylation reaction is carried out by reacting molar equivalent amounts of the compound of formula I and the N-hydroxysuccin imide ester, preferably at a temperature of about -10 to about 10 ° C and in an aqueous solution of an inert organic solvent, e.g. tetrahydrofuran, dioxane, ethylene glycol dimethyl ether, dimethylacetamide, dimethylformamide, propylene glycol dimethyl ether and the like.

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The hydrogenolysis of the benzyloxycarbonyl group is carried out over a palladium-on-carbon catalyst in an inert, water-miscible organic solvent, e.g. methanol, ethanol, dioxane, tetrahydrofuran, ethylene glycol dimethyl ether, propylene glycol dimethyl ether and the like.

The compounds of formula I, namely the gentamicins C1 and Cla and hence analogs, have been tested by a standard successive dilution test and have been shown to have antibacterial activity; some of the analogs also to gentamicin-resistant organisms. Thus, the compounds are useful as anti-bacterial agents.

The compounds of formula I are intended primarily for oral, topical or parenteral administration and may be prepared for use with a pharmaceutical carrier by suspension, either in the form of their free bases or as pharmaceutically acceptable, non-toxic acid addition salts, in an inert carrier such as polyethylene glycol. , or by tableting or encapsulation for oral administration either alone or with suitable additives, or they may alternatively be formulated with conventional creams or vaselines for topical application. The molecular structures of the compounds of formula I were established on the basis of their chromatographic properties determined by thin-layer chromatographic analysis, their magnetic-nuclear resonance (NMR) and mass spectrum, by decomposition into known compounds, and according to calculated and found values for elemental analyzes of the compounds.

The following embodiments serve to illustrate in detail the process of the invention and the preparation of the starting compounds thereof.

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MTATIONSEREMGANGSMIdER

In the following procedures, various media composite were used as follows:

Medium_1: N-Z Amine

Glucose 10

Soluble Starch 20 Yeast Extract 5 N-Z-Amine Type A (Difco) 5

CaCO3 1

Agar 15

Medium_2: Sprout medium (in distilled water) Meat extract $ 0.3

Trypton $ 0.5

Dextrose 0.1 $

Soluble Starch $ 2.4 Yeast Extract $ 0.5

CaCO4 0.4 #

Medium__3: So Jabean Glueose g ^ i

Soybean meal 30

Dextrose (Cerelose) 40

CaCO3 t

Medium_4: TGE g / i

Irypticase glucose extract 5.0 Irypticase peptone 3.0

Glucose t, 0

Agar 15.0 143413 6

Medium_5: Production medium Meat extract 0.3 in »Yeast extract °, 5 $

Soybean meal $ 0.5

Maltose 0.1 $

Starch $ 2.4

Casamino acid 0.1 $

CaCO5 0.4 i

Co012'6H20 1 mg / liter

Medium 6: Streptomicin Test Agar sZi Meat Extract 1> 5 Yeast Extract 3.0

Pepton 6.0

Agar 15.0

The organism Micromonospora purpurea was obtained from the U.S. Dept. of Agriculture as NREL 2953 and held on N-Z-amine oblique substrates (Medium 1). Submerged ferments were carried out in flasks containing germination medium (Medium 2) for 4 days at 37 ° C on a rotary shaker. At this first stage, a 10 per cent transfer was made. graft material to the germinating medium (Medium 2) and fermentation was continued as above at 28 ° C for 7 days.

In order to establish the organism's ability to biosynthesize gentamicin in the absence of added deoxystreptamine, a third-stage fermentation was performed using a 5 per cent. graft in a 10 liter fermentor in a soybean glucose medium (Medium 3) at 28 ° C, stirring at 200 rpm and aerated at 2 liters / minute with filtered air. After 6 days, the tank contents were acidified to pH 2.0 with 6N sulfuric acid, filtered, and a 500 ml portion neutralized with ammonium hydroxide and passed through an "IRC-50" ion exchange resin (Na + form). The column was then rinsed with water. and eluted with 2N sulfuric acid In accordance with the procedure described in U.S. Patent No. 3,091,572, a 300 mg sample of crude gentamicin was found which was found to be biologically active and contained three components corresponding to gentamicin-C ^, -Cg and -C ^ a by thin layer chromatographic examination.

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For mutation of the organism, liquid cultures were grown in Medium 2 (37 ° C for 3 days) and the resultant cells harvested by centrifugation, washed and resuspended in buffered saline. This suspension was treated with the mutagenic agent, N-methyl-N'-nitro-N-nitrosoguanidine. Samples of the mutagenic culture were placed on plates in Medium 4 at 37 ° C until colonies were evident (usually about a week). Colonies were seeded on similar plates (Medium 4), one set of which was poured with a spore suspension of B. subtilis. After incubation at 37 ° for 18-20 hours, the "seeds" showing no inhibition zone on the B. subtilis plate were transferred from the main plate (no B. subtilis) to Medium 1 oblique substrates and incubated until full growth was clear.

These potentially non-producing mutant strains were then exposed to deoxystreptamine in an attempt to stimulate antibiotic biosynthesis as follows. Stock cultures of the potential mutant strains were plated as bands on the surface of Medium 4 plates and incubated at 37 ° C until growth was evident (about 3-4 days). Pellet paper slices were then dipped in a solution of deoxystreptamine (500 µg / ml) and placed on top of charcoal. After 24 hours of incubation, the surface of the plate was seeded with B. subtilis by the overlay technique and the incubation was continued for an additional 18-20 hours. Isolates showing inhibition zones around the disc were designated as deoxystreptamine mutants. One such mutant, designated mutant VIB, and deposited in the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852, such as Mieromonospora purpurea ATCC 31119, can be used to produce gentamicin-type antibiotics.

The latter organism was itself subjected to the same mutation procedure as described above, and samples of the mutagenic culture were placed on plates in Medium 4 at 37 ° C until colonies were evident (usually about a week). Colonies were seeded on similar plates containing Streptomicin test agar (Medium 6).

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St sets served as the main plate for later recovery, while the second set contained 25 µg / ml streptamine sulfate and an overlying spore suspension of B. subtilis as the test organism. Piece plates were incubated at 37 ° C for 24 hours and examined for inhibition zones. Be, which showed the largest zones, was transferred from the main plate onto N-Z-amine oblique substrates (Medium 1) and incubated for one week at 37 ° C.

35e nutrients which incorporated low levels of streptamine sulfate were then sieved with streptamine and scylloinosose at 500 µg / ml son base, storage cultures were transferred to flasks containing Medium 2 plus the above intermediates and incubated at 37 ° C on a rotary shaker. 7 days. Flasks were periodically tested for antibiotic activity by the disk diffusion assay using B. subtilis as the test organism. Isolates showing inhibition zones around the disc were designated as streptamine or scylloinosose mutants. One such mutant, designated mutant 7ΓΒ-3Ρ, and deposited in The American Type Culture Collection, 12301 Parklawn Brive, Rockville, Md. 20851 as Micromonospora purpurea ATCC 3Hb4, was used to prepare the streptamine, deoxystreptamine, and dideomystreptamine-type aminocyclitol antibiotics as described below.

BIOSYNTHESES WITH M. PURPUREA ATCC 31164 Example 1

The mutant organism was kept on N-Z-amine agar slant substrates (Medium 1), from which transfers were made to flasks containing 500 ml of germ medium (Medium 2). The flasks were incubated at 28 ° C for 4 days on a rotary shaker (5 cm punch) at 225 rpm.

10% by volume graft stage seed material was aseptically transferred to 14 liter fermentors containing 9 liters of sterile germ medium (Medium 2). Bits were stirred at 450 rpm at 28-29 ° C and aerated with filtered air at 5 liters / minute. At the inoculation time, 200 mg / liter of streptamine sulfate was added as a suspension in sterile distilled water. The fermentation was continued for 8 days.

9 liters of 24 liter old seed material prepared as above were aseptically transferred to 130 liter fermentors containing 70 liters of sterile germ medium (Medium 2) and 0.31 g / liter of streptamine sulfate suspended in sterile distilled water was added. Aero fermentation was performed at 29 ° C for 7 days.

The ferments were terminated by adding 10N sulfuric acid to pH 2.0 and the fermentation medium was filtered using a filter aid for removing mycelia. The filtered liquid was adjusted to pH 7.0 and 1.56 g of oxalic acid was added per ml. grams of calcium carbonate present in the medium to remove calcium. The mixture was allowed to stand overnight and the clarified liquid was peeled off and passed through "Bio-Rex 70" (weak cation exchange resin) in the Na + form using about 14 g of resin per minute. liter of liquid. The column was then washed with distilled water and eluted with 2N sulfuric acid. All fractions exhibiting antibiotic activity were combined, neutralized and concentrated in vacuo at below 50 ° C to the point where salt crystallization became apparent (about one-third volume). The pB value was then adjusted to 10.5, and 4 volumes of acetone were added to precipitate inorganic material which remained. removed by filtration, the filtrate was adjusted to pH 5.0 with 6N sulfuric acid, concentrated in vacuo to about 1/20 of the original volume and cooled.

A white crystalline material precipitated, which was collected by filtration and melted at over 300 ° 0. Based on its thin-layer chromatographic properties and its infrared spectrum, it was found to be identical to streptamine sulfate. from two 10 liter fermentations, processed as above, a total of 0.7 g of streptamine sulfate was obtained, and from two 80 liter of fermentations a total of 21 g of streptamine sulfate was obtained in this step.

the filtrate was further concentrated and 10 volumes of methanol were added to give the first crude solid antibiotic. from two 10 liter fermentations 7 g was obtained and from two 80 liter fermentations 24 g was obtained.

In order to identify antibiotic components during the purification procedures, chromatographic mobility values obtained by paper chromatography and thin layer chromatography for gentamicin-C ^, -C₂ and -C ^α and for each of the corresponding amino-cyclitol analogs prepared as above were determined. where the chromatographic mobility (hereinafter referred to as CM) is expressed as: π w = the distance of the component from the origin * * gentamicin-C1 distance from the origin

Chromatographic systems used were as follows:

System 1:

Whatman No. 1 paper saturated with 0.95M sulfate hydrogen sulfate and developed downwards in 80 per cent. aqueous ethanol + 1.5 # NaCn. and subsequent bioautography using B. subtilis as the test organism.

System_2:

Silica gel Έ 254 plate developed in the lower phase of 1: 1: 1 chlorophyll worm / methanol / concentrated ammonium hydroxide ($ 28). The components were localized with a ninhydrin spray after heating.

The CM values of the predominant antibiotic components obtained according to the invention, as compared to a reference gentamicin complex, all relative to gentamicin C-1, are shown in Table I, where the compounds designated component 1, component 2 and component 5 is understood to be respectively: 0-3-deoxy-4-C-methyl-5-methylamino-p-arabinopyranosyl- (O- [2-amino-6-methylamino-6-C-methyl-2,3) 4,6-tetradeoxy-αB-ery-thro-giucopyranosyl- (1-4)] -B-streptamine; Q-3-deoxy-4-C-methyl-3-methylamino-P1 ~ arabinopyranosyl- (1 ** 6) -O- [2,6-diamino-6-C-methyl-2,3,4,6-tetradeoxy-αB-erythroglucopyranoyl- (1-4)] -B-streptamine; and 0 -3-Deoxy-4-C-methyl-3-methylamino-p-arabinopyranosyl- (1- * 6) - 2,6-diamino-2,3,4,6-tetradeoxy-αB-erythro-glucopyranosyl- (1 - * 4)] - B-streptamine.

11 1A 3 Λ1 3

W I

C.M. System 1 C.M. System2

Gentamicin-C1 1 V

Gentamicin-C2 0.89

Gentaraicin-C1a 0.50 0.67

Component 1 (predominantly) 0.96 0.92

Component 2 (minor) 0.76 0.75

Component 3 (minor) 0.50 0.61

From 10 liters of fermentorerae, the crude solid (7 g) showing antibacterial activity was suspended in 200 ml of methanol and 10 ml of concentrated ammonium hydroxide (28 ¢) and the mixture was stirred for 30 minutes and filtered. This was repeated twice. to, and the filtrates were combined and concentrated in vacuo to give a pale yellow oil weighing 0.9 g. The "spent salts" were essentially free of antibiotic activity.

The oily base was mixed with 4 g of silica gel (Davison grade 923, particle size 0.074 - 0.149 mm) and placed on a silica gel column measuring 1.8 x 28 cm. The column was prepared as a slurry using the lower phase of isopropyl alcohol / chloroform / 17 per cent. aqueous ammonium hydroxide (1: 2: 1). The column was developed with: this solvent and 50 ml fractions were collected.

Fractions 8 and 9 contained a single ninhydrin positive component which gave 20 mg as a pale yellow oil after removal of the solvent. The mass spectrum of this material, designated component; 1 showed a molecular year ion and major fragments, each of which were 16 mass units (i.e., an oxygen atom) larger than that obtained from gentamicin-0 as follows:

Reference gentamicin M + 477, 420, 60, 350, 347y 322, 319, 304 Component 1: M + 493, 436, 376, 366, 363, 338, 335, 320.

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Seed material was converted to its sulfate salt by dissolving in ethanol by adding a few drops of ethanol containing sulfuric acid. The resulting white solid was collected and dried to give 22 mg of component 1, o-3-deoxy-4-C-netbyl-3-methylamino-PI-arabinopyranosyl- (1- * 6) -0- [ 2-Amino-6-methyl-amino-6-C-methyl-2,3,4,6-tetradeoxy-α-D-erythro-glucopyranosyl (1-4)] -D-streptamine as di-base * heptasulfate * decahydrate, m.p.

> 3C0 ° C.

Calcd for C 21 3 N 5 ° 8 4 H 2 S 4 O: C 27.22 - 6.53 - N 7.55 - S 12.09

Pound: C 27.15 - H 6.67 - N 7.79 - S 12.76.

Fractions 10-13 yielded a more polar ninhydrin positive component, designated component 2, 0-3-deoxy-4-C-methyl-3-methylamino-pI-arabino-pyranosyl- (1-6) -0- [2 , 6-Diamino-6-C-methyl-2,3,4,6-tetradeoxy-αD-eryihro-glucopyranosyl- (1-H)] D-streptamine, which showed antibiotic activity.

Reactions 15-26 provided a third more polar component that exhibited antibiotic activity. The mass spectrum of this component showed characteristic sugar maxima similar to gentamicin-C. at 129 (purpurosanine) and 160p (garosamine), and it is designated component 3,. 0-3-rdeox7-4-C-methyl-3-methylamino-P1-arabinopyranosyl- (1-6) -O-L2, b-diamino-2,3,4,6-tetradeoxy-αD-erythro-glucopyranosyl - (1- * 4)] - D-streptamine.

Alternatively, the new component 1 was isolated as follows: A mixture of crude antibiotic base, prepared as above (0.9 g), was dissolved in 7 ml of water and the pH was adjusted to 4.5 with 1N sulfuric acid. The solution was passed through a strong anion exchange column ('IRA 401') in the OH 'form (layer's size = 0.7 x 10 cm). The column was eluted with water and the eluate evaporated in vacuo at 35 ° C.

The resulting residue was triturated with 50 ml of the lower phase of a solvent composed of 17 per cent. aqueous ammonium hydroxide / isopropyl alcohol / chloroform (1: 1: 2). The solution was decanted off and concentrated in vacuo to give an oily residue weighing 140 mg. The mass spectrum of this material corresponds to the spectrum of fractions 8 and 9 above, i.e.

M + 493, 436, 376, 366, 363, 338, 335, 320.

The magnetic nuclear resonance spectrum of component 1 also corresponded to the assumed structure corresponding to 0-3-deoxy-4-C-methyl-3-methylamino-PL-arabinopyranosyl- (1-3 * 6) -O- [2-amino] 6-Methylamnor 6-C-methyl-2,3,4,6-tetradeoyl-αD-erythro-glucopyranosyl- (1-H)] -D-streptamine and is summarized in Table XI.

ΧΑΒΕΙ. II

_§_ Integration Attribution_ 5.57; 5.60 2 anomeric OCHO 'groups

5.22 12 interchangeable H

3, C3; 3.15 6 NOH 3 x 2 2.9 - 4.8 13 -CHO x 6, -CHN x 5, -CH 2 1.9-2.6 4 -CH 2 CH 2 -

1.72 6 -CH2 O-, OH ^ QH

Alternatively, a 4 g sample of the crude antibiotic salt was dissolved in 50 ml of water and passed through an anion exchange resin "AGIXS" (resin layer 1 x 27 cm). The antibiotic was eluted with water and all active fractions combined and evaporated under vacuum. Further desalting was performed on the residue by extraction with methanolic sodium hydroxide. The released base was mixed with 7 g of herring gel and placed on a 50 g of silica gel column. This column was developed with cZaloroform / methanol / concentrated (28 $) ammonium hydroxide (3 * 2: 1) and 25 ml fractions were collected. Early fractions gave the new minor polar component 1, but mixed with several minor polar impurities as detected by thin layer chromatography. These fractions were combined and the concentrate was put on a 50 g silica gel column as above. This column was developed with chloroform / methanol / concentrated ($ 28) ammonium hydroxide (5: 3: 1) and 25 ml fractions were collected. Fractions 6-12 contained the desired component free of obvious impurities. After removal of the solvent, the resulting oil was converted to the sulfate salt in ethanolic sulfuric acid as described above to give 0.124 g of component 1 as the di-base * heptasulfate * decahydrate, m.p. > 300 ° C, as described above.

Ted culture of an appropriate aminocyclitol with the mutant strain Micromonospora purpurea ATCC 31164 in germ medium (Meediium 2) and isolation of the products as described above in Example 1 are similarly prepared by the following compounds of formula I:

Example 20 0-3'-Deoxy-4-C-methyl-3-methylamino-β-L-arabinopyranosyl- (1-6) -O-2-amino-6-methylamino-6-C-methyl-2,3 4) 6-tetradeoxy-αD-erythro-glucopyranosyl- (1-M-)] epistreptamine (CM relative to gentamicin-C₂: System 1 = 0.59, System 2 = 0.63); 0-3-Deoxy-4-C-methyl-3-methylamino-p-arabinopyranosyl- (1- * 6) -O- [2,6-diamino-6-C-methyl-2,3,4,6- tetradeoxy - αD-erythro-glucopyranosyl- (1-M)] epistreptamine; cg O- "3-Sxyxy-4-C-methyl-3-methylamino-pI-arabinopyranosyl- (1- * 6) -O-Γ 2, c-diamino-2,3,4,6-tetradeoxy-αD- erythro-glucopyranosyl (1-M-)] -epistreptamine obtained by using epistreptamine instead of streptamine in the fermentation procedure.

Example 3

One. procedure similar to that described in Example 1 using 0.10 g / liter of 2-deoxystreptamine in a 10 liter fermentor containing production medium (medium 5) in the presence of M. purpurea AIC-C 31164 and isolating the product which previously gave 0.51 g of crude material which, by chromatography on silica gel as its main component, gave a material which, by the identity of the chromatographic mobility, was shown to be identical to gentamicin-C, namely 0-3-de-oxy-4 C-methyl-3- (methylamino) -p-arabinopyranosyl- (1-> 6) -O- [2-amino-6- (methylamino) -6-C-methyl-2,3,4,6-tetradeoxy- αE-erythro-gluco-pyranosyl (1-> 4) JE-2-deoxystreptamine.

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Example 3

A procedure similar to that described in Example 1 using 0.10 g / liter of 2,5-dideoxystreptamine in two 80 liter fermenters and six 10 liter fermenters containing production medium (medium 5) in which the soybean meal was replaced. with 0.5 1 tryptone and the starch with 2 $ cerelose in the presence of M. purpurea ATCC 31164 and isolation of the product which previously gave two residues of 0.68 g and 0.13 g, respectively, which were combined and chromatographed on silica gel plates, thereby obtaining three bands whose mass spectra showed that they consisted of respectively

Component C1: 0-3-Deoxy-4-O-methyl-3- (methylamino) -PL-arabino-pyranosyl- (1- * 6) -0- [2-amino-6- (methylamino) -r6-C -methyl-2,3,4,6-tetradeoxy-uD-erythro-glucopyranoyl- (1 * 4)] -E-2,5-dideoxystreptamine;

Component C2: 0-3-Deoxy-4-C-methyl-3- (methylamino) -p-1-arabino-pyranosyl- (1 * 6) -O- [2,6-diamino-6-C-methyl] 2,3,4,6-tetradeoxy-αE-erythro-glucopyranosyl- (1 * 4 $ -1) -2,5 · dideo cystreptamine; and

Component G1-: 0-3-Deoxy-4- [0-methyl-3- (methylamino) -PI] -arabino-pyranosyl- (1 * 6) -O- [2,6-diamino-2,3,4,6 -tetradeol yaE-erythro-glucopyranosyl- (1-4)] -D-2,5-dideoxystreptamine.

The mass spectra of the three components C, Cn and C mentioned above

at 2 ° 1a the mass maxima as follows:

Component M + 461, 404, 344, 334, 331, 306, 303, 288, 160 and 157.

Component C2: M + 447, 404, 334, 330, 317, 306, 288, 160 and 143.

Component ϋ1 &: M + 433, M ++ 1 434, 404, 334, 316, 306, 303, 288, 275, 160 and 129.

The Rf values by thin layer chromatographic analysis on silica gel plates using a solution system consisting of the lower phase of a mixture of chloroform / methanol / concentrated ammonium hydroxide (1: 1: 1) as the developing agent was 0. 43, 0.37 and 0.29 for component C 1, C 6 and C 1, respectively.

Example 5

A procedure similar to that described in Example 1 using 0.50 g / liter of 2-amino-1,3,4,5,6-cyclohexane pentol (1,3,5-cis) in three 10 liters fermentors containing production medium (medium 5) plus 0.1 µl of phyton added in the presence of M. purpurea ATCC 31164 and isolation of the product which previously gave 214 mg of material which, from its chromatographic mobility and from its mass spectrum, was shown to be identical with the compound of Example 1 described above, namely, 0-3-deoxy-4-C-me thy 1-3- (me thy lamino) - β -L-ar abino-pyranosyl- (1- > 6) -O-Γ 2-amino-6- (methylamino) -6-C-methyl-2,3,4 »6-te-tradeoxy-α-11-erythro-glucopyranosyl- (1 · 4 D The strep amine showed mass maxima as follows: M + 493, 457, 436, 383, 376, 366, 363, 346, 344, 338, 335, 321, 318, 261, 160 and 157.

Example 6

A solution of 4.5 g (0.031 mol) of 2,5-dideoxystreptamine in 20 ml of benzaldehyde was heated on a steam bath and the mixture was rinsed with nitrogen and set aside for about 8 hours. The solution was then diluted with 150 ml of benzene, cooled and the precipitated solid collected and dried to give 8.5 g of crude product which was recrystallized from acetonitrile to give 4.23 g of 4,6-bis- benzylideneamino) -1,3-cyclohexanediol (1,3-cis), mp: 151 - 154 ° C.

This compound (500 µg / ml) was incubated with the mutant strain M. purpurea ATCC 31164 for four days in germ medium (medium 2) and the resulting liquid containing the 2,5-dideoxystreptamine analog to gentamicin with physical constants similar to Example 4 above. was found to be antibiotically active by the disk diffusion assay method against B. subtilis as a test organism.

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SYNTHESIS OF AMINO-HYDROXY-LOWERE-ALKANOYL DERIVATIVES

Example 7

A solution of 270 mg (0.54 millimoles) of 0-3-deoxy-4-C-methyl-3'-methylamino-β-L-arabinopyranosyl- (1-6) -0- [2-amino-6- methylamino-6-C-methyl-2,3,4,6-tetradeoxy-αD-erythro-glucopyranosyl (ht4)] -D-streptamine prepared and described above in Example 1 (component 1), dissolved in 5 ml of 50% aqueous tetrahydrofuran, cooled to 5 ° C in an ice bath and treated with 206 mg (0.59 mil) of the N-hydroxysuccinimide ester of S- (-) -) f- (N-benzyloxycarbonyl) amino-α-hydroxybutyric acid (Konishi et al., U.S. Pat.

3,780 018) and the mixture was stirred at 5 ° C for 20 hours. The mixture was then concentrated to 10 ml in vacuo. 25 ml of n-butanol and 10 ml of water were added and the layers were separated. The aqueous layer was washed again with 10 ml of n-butanol. The combined organic layers were evaporated leaving a residue of 513 mg of crude product which was set aside.

The aqueous layer was evaporated to dryness to give 304 mg of residue, which was dissolved in 25 ml of 50% aqueous tetrahydrofuran and treated with an additional 208 mg of the N-hydroxysuccinimide ester of S _ (_) _ Y- (N- benzyloxycarbonyl) -amino-α-hydroxybutyric acid as before. Workup of the reaction mixture yielded an additional 435 µg of crude product which was combined with the previously obtained 513 cloths and chromatographed on 7 1 mm thick 40 x 20 cm silica gel plates. The system was developed with a solution system consisting of the anterior phase of a mixture of chloroform / methanol / concentrated (28 µl) ammonium hydroxide (3 * 1 * 1). Seven passes in this solvent system were necessary and after elution of the ultraviolet visible product band, 229.5 mg of a crude mixture of the three monoacylated products was obtained.

The breath of acylated products was placed on three 1 mm thick silica gel plates of 40 x 20 cm and the plates were developed five times with dsn lower phase of chloroform / isopropanol / co-centered (28%) ammonium hydroxide (4: 1: 1) twice. the lower phase of chloroform / isopr opanol / concentrated (28%) ammonium hydroxide (3: 1: 1) and nine times with the lower phase of chloroformartinethanol / concentrated (28%) ammonium hydroxide (4 * 1 * 1). Three distinct bands were obtained which were visible under ultraviolet "irradiation" and which were excised separately and eluted from the silica gel with the lower phase of chloroform / methanol / concentrated (28 fo) ammonium hydroxide (1: 1: 1), whereby three components were obtained: A 90.9 mg; B, 59.1 mg; and C 48.5 mg which is S - (-) - γ- (N-henzyloxycarbonyl) amino-α-hydroxybutyric acid amide derivatives in the 2'-, 1- and 3-position of 0-3-deoxy, respectively -4-O-methyl-3-rethylamino-; 3-l-arabinopyranosyl- (1-> 6) -O- [2-amino-6-methylamino-cC-methyl-2,3,4,6-tetradeoxy- α-D-erythro-glucopyranosyl (1Ή)] -B-streptamine. The R ^ values of components A, B and C after development five times on silica gel with the lower phase of a system consisting of chloroform / methanol / concentrated (28 f) ammonium hydroxide (4: 1: 1) were: A - 0.48 B - 0.62 C - 0.70

Component 3 (the 1-amide, 56.1 mg) dissolved in 25 ml of 50 µl of aqueous ethanol and 20 mg of 10 µl of palladium-on-charcoal was shaken on a Parr shaker and kept at a hydrogen pressure of 3 85 kp / cm for 5 1/2 hours, after which the catalyst was removed by filtration through filter aid. Evaporation of the solvent gave 34.3 mg of a white glassy substance which was dissolved in 2.5 ml of water and treated with 11.4 mg of sulfuric acid in 0.1 ml of water. Addition of 10 ml of ethanol precipitated 1-S - (-) - γ-amino-α-hydroxybutyryl] -0-3-deoxy-4-C-methyl-3-methylamino-3-1-arabinopyranosyl- (1-> 6) -O- [2-amino-6-methylamino-6-C-methyl-2,3,4,6-tetradeoxy-α-D-erythro-glucopyranosyl (1-M)] -D-streptamine as the pentasulfate salt (32 mg), mp, 230 - 235 ° C (decamp); thin layer chromatography, R f = 0.18 (silica gel, ehloro-forn / nethanol / concentrated (28 f>) ammonium hydroxide / water 1: 4: 2: 1; R f, gentanicin C. standard = 0.73).

Analysis calculated for Cg ^H ^ QO₂Ng · 5HgSO ^: 027.67 - H 5.57 - N 7.75 Pound: C 27.50 - H 5.58 - N 7.42.

19 143413

Components Α and C were treated in the same manner. A gave 47 mg of 2 '- [S-> (-) -y-amino-: c-hydroxybutyryl] -0-3-deaxy ~ 4 ~ C-methyl-3-methylamino-pL-arabinopyranosyl- (1-> 6 ) -O- [2-methylno-6-methylamino-6-C-methyl-2,3,4,6-tetradeoxy-α-D-erythro-glucopyrioliosyl- (1- * 4)] - D-sireptamine as bis -bass-tetrasulfate · heptahydrate, emp. 237 - 241 ° 0 (decomp.); thin layer chromatography, Rf = 0.33 (silica gel, chloroform / methanol / concentrated (28 f =) ammonium hydroxide / water 1: 4: 2: 1} Rf geneamycin-C = standard = 0.73).

Analysis calculated for (C 25 H 50 N 6 ° 10 2 · 2 H 2 S 4 ’H 2 O: B 35.16 - H 7.20 - N 9.84

Found: C 35.38 - H 7.08 - N 9.49.

Component C gave 26 mg of 3- [S - (-) - Y-amino-α-hydroxybutyryl] -0-3-deoxy-4-C-methyl-3-methylamino-pL-axabinopyriolsyl (1-6) ) - * O- [2-Amino-6,6-methylamino-6-C-methyl-2,3,4,6-tetradeoxy-α'-β-erythrQ-glucopyranoyl- (1-4)] - D -streptamine as bis-base «pentaeulfate · trihydrate, amp. 220 DEG-230 DEG C. (decomp.); thin layer chromatography, R f = 0.30 (silica gel, chloroform / methanol / concentrated (28 $) ammonium hydroxide / water 1: 4: 2: 1; R 1 gentamicin C 2 standard = 0.73).

Analysis calculated for (Cg ^H ^NOOOig · 5¾¾ * 5 5 ^: C 34.64 - H 6.74 - N 9.67

Found: C, 34.35; H, 6.38; N, 8.60.

ANTIBACTERIAL TEST RESULTS

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The compounds of Example 1 prepared 2-hydroxygentamicin-C1, 2-hydroxygentamicin-C2 and 2-hydroxygentamicin-C1- / C2-complex (hereinafter referred to as Oxy-C-, Oxy-C / C2) and the compounds of Example 3 prepared 5-deoxygentamicin-R Ci and 5-deoxygentamicin-CL / C2 / Cia complex (hereinafter referred to as Deoxy-Ci and Beoxy-C O ^) was tested in comparison with gentamicin-C1, gentamicin-C2, and gentamicin-C1 / C2 / Cia'-complex (hereinafter referred to as GC2 G-C2 and GCL / C2 / Cla respectively) against a number of microorganisms after the following procedure, 20 143 Λ13

Stock solutions of each compound containing 200 µg / ml base were prepared in distilled water and filter sterilized. Cultures of the sample organisms were grown for 24 hours at 37 ° C in 10 ml glass with tryptose phosphate or Miller-Hinton liquid. Each culture was adjusted with optical density liquid 0.1 on a Spectronic 20 (approximately 10 cells / ml). The set cultures were diluted 1: 500 in liquid for use as graft material (final cell concentration approximately 2 x 10 6 cells / ml). The test compounds were tested for antibacterial activity by a single series of dilution methods. lobe dilution rows were prepared in liquid from the stock solutions and 0.2 ml of each antibiotic concentration was placed in 17 13 x 100 n® glass. All glasses were seeded with 0.2 ml of the appropriately diluted culture (final cell concentration per glass = 10 5 cells / ml). Minimum inhibitory concentrations (lowest antibiotic concentration showing no visible growth) were read after 16 hours of incubation at 37 ° C.

The results are listed in Table III below. The compounds were considered inactive at inhibitory concentrations above 100 p.g / ml.

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In the field of antibiotics, where microorganisms develop resistance to antibiotic therapy by mutation, progress is measured by the ability of new antibiotics to control microorganisms that are, for some reason, resistant to known antibiotics. Comparisons of the test results in the above Table III between 2-hydroxygentamicin-C 2 and gentamicin-2, 2-hydroxygentamicin-C 2 and gentamicin-Cg 2-hydroxygentamicin-C 2 / C 2 complex and gentamicin-C 2 / C 2 / Cla, respectively. Complex, 5-deoxygentamicin and gentamicin-C samt, and S-deoxygentamicin-C ^ / C ^ / C--complex and gentamicin-C - C / C₂ complex show that the compounds of the invention . are active against from 4 to 8 microorganisms that are resistant to the corresponding gentamicin. These comparisons are summarized in Table III-A below, where the organisms listed in Table III are numbered in sequence and identified by the numbers assigned to the sole, and where better activity for a particular designated 2-hydroxy or 5-deoxygentamicin or complex thereof in comparison with the corresponding gentamicin or complex thereof is indicated by +.

23 U3413

TABLE III-A

Organism Oxy-C1 Oxy-C2 Oxy-C / C Dcoxy-C, Dooxy-C, / CjC

——, - - „-„ -, - Λ * · __L _1 2 la 2 3 +++ 4 5 + + + 6 + 7 8 + + + + 9 + +. + + 10 11 12 + + + + 13 + + + + 14 + + + + 15 + 16 17 18

The same component 1 as described above in Example 1, 0-3'-de-oxy-4-C-methyl-3-methylamino-pL-arabinopyranosyl- (1-6) -O- [2-amino-6-methylamino -6-C-methyl-2,3,4 »6-tetradeoxy-αD-erythro-glucopyranoyl- (1-4)] - D-streptamine, was tested in comparison with gentamicin complex (C1, C2 and C ^a) and gentamicin-C ^; and the 1-, 3- and 2 '- [S - (-) - Ύ-amino-α-hydroxy-butyryl] amides of component 1, described above in Example 7, and designated component 1 (1-HABA), component 1, respectively (3-HABA) and component 1 (2'-HABA) i were tested in comparison with the corresponding 1-, 3- and 2 '- [S - (-) - γ-amino-α-hydroxybutyrylamides of gentamicin (All disclosed by Konishi et al. In U.S. Patent No. 3,780,018 issued December 18, 1973), "designated (1-HABA), (3-HABA) and (2'-HABA, respectively) .

Various results are given in Table IV below, where the test organisms 1, 2, 3, 4, 5 and 6 identify B. subtilis ATCC 6633, S. aureus Smith, E. coli JR 76.2, Ent. cloacae A-20960, K. pneumoniae A-20636 and Ps. aeruginosa A-20987.

ΡΑΒΕΙ IV *

Test organisms

Compound 1 23456 G-entamicin-C ^, -O ^, 0.024 0.39 50 12.5 25> 100 & entamicin-C ^ 0.049 0.78 50 12.5 25> 100

Component 1 0.098 1.56 6.25 0.78 3.13 25 01 (1-HABA) 0.39 3.13 12.5 3.13 6.25> 100

Component 1 (1-HABA) 0.78 6.25 12.5 6.25 12.5> 100 C., (3-HABA) 3.13 25> 100 100> 100> 100

Component 1 (3-HABA) 6.25 50 100 50 50> 100 C1 (2'-HAM) 6.25 50 100 25 50> 100

Component 1 (2 * -HAM) 1.56 25 50 25 50> 100 # Uncultivated in Mueller-Hinton Case.

Claims (5)

25 143413 A process for the preparation of aminocyclitol compounds of the general formula: nJ. CH-NH-R6 RP -NH> 4 O_ NH-R5 y \ Z_ ~~ r2 CH3NH —ch3 OH. ... In "12 O * 1 wherein R, R * 'and R are either all hydrogen or one of R, R O and R R are a 1b-amino-α-hydroxy-alkanoyl group of the formula HgN-CH ((CH)) n-CH-C-OH 0 1 * 20 where n is 0 or 1 and the other two of R R 'and R ° each represent hydrogen, R og and R hver each represent hydrogen or hydroxy, AV and R og and R' each represent hydrogen or methyl, or acid addition salts thereof, characterized in that the microorganism strain Micromonospora purpurea ATCC 31164 is grown in a nutritional medium containing carbohydrates, a source of assayable nitrogen, necessary salts and an aminocyclitol of the formula: TV-, "Ha I lb i" wherein R is amino or hydroxy of formula IIa and hydroxy 2 '5' or benzylideneimino of formula IIb, and R and RJ each represent hydrogen or hydroxy, whereupon the compound formed is reacted, if desired, with an N-hydroxysuccinimide ester of the formula: L 2 -CH 2 O-C-NHCH 2 is 0 or 1 and the benzyloxycarbonyl group of the resulting compound is subjected to hydrogenolysis with hydrogen over a catalyst to form a compound of formula I wherein one of R, R 4 and R is a w-amino-α-hydroxyalkanoyl group and, if desired, a formed free base is transferred into an acid addition salt thereof. A process according to claim 1 for the preparation of 0-3-deoxy 4-C-methyl-3- (methylamino) -β-L-arabinopyranosyl- (L-> 6) -0- [2-amino-6- (methylamino) -6-C-methyl-2,3,4, 6-tetradeoxy-oc-D-erythroglucopyranosyl- (1-4)] - D-streptamine or 0-3-deoxy-4-C-methyl-3- (methylamino) -β-L-arabinopyranosyl- (L 6) -O- [2,6-di-amino-6-C-methyl-2,3,4,6-tetradeoxy-oc-D-erythroglucopyranosyl (1-4)] -D-streptamine, characterized know that the added aminocyclitol is streptamine.