IE42807B1 - Aminocyclitol antibiotics and processes therefor - Google Patents

Abstract

1529376 Aminocyclitol antibiotics STERLING DRUG Inc 9 Feb 1976 [18 Feb 1975 22 Sept 1975] 04973/76 Heading C2C Novel aminocyclitol antibiotics of the general Formula I wherein R 1 , R 3 and R 8 are hydrogen, or one of R 1 , R 3 and R 8 is H 2 NCH 2 (CH 2 ) n CHOHCO-, wherein n is 0 or 1, and the others are hydrogen; R 2 is H or OH; R 5 is hydrogen, hydroxy or halogen, except that when R 2 is hydrogen, R 5 is not hydroxy cis to the amino groups at the 1- and 3- positions; and R 6 and R 7 are each hydrogen or methyl, and acid addition salts thereof are prepared (a) when R 1 ,R 4 and R 8 are each hydrogen, by culturing a nutrient medium containing carbohydrates, a source of assimilable nitrogen, essential salts and a corresponding aminocyclitol of the general formula wherein R 1 , R 2 , R 3 and R 5 are as defined above, except that R 1 and R 3 may also form a single bond between the nitrogen atoms to which they are attached, in the presence of Micromonospora purpurea ATCC 31,119, and isolating the desired product from the medium; (b) when R 1 , R 3 and R 8 are each hydrogen and R 5 is as defined above with the exception of halogen, by culturing a nutrient medium containing carbohydrates, a source of assimilable nitrogen, essential salts and a cyclitol of the general formula wherein R is hydrogen or acetyl, R1 3 is oxo or hydroxy and R1 2 and R1 5 are each hydrogen or OR, in the presence of Micromonospora purpurea ATCC 31,164; (c) when R 1 , R 3 and R 8 are each hydrogen and R 5 is as defined above with the exception of halogen, by culturing a nutrient medium containing carbohydrates, a source of assimilable nitrogen, essential salts and an aminoclitol of the general formula wherein R1 2 and R1 5 correspond to R 2 and R 5 and R1 1 is amino or hydroxy, in the presence of Micromonospora purpurea 31,164; and (d) when one of R 1 , R 3 and R 8 is by reacting the product of (a), (b) or (c) with an N-hydroxysuccinimide ester of the general formula followed by hydrogenolysis of the resulting N- benzylorycarbonyl derivative with hydrogen and a catalyst; followed optionally by salification of the product. dl-Deoxyinosose is prepared by microbiological oxidation of dl-viboquercitol with Acetobacter suboxydans. dl - 2,3,4,6 - Tetrahydroxy - cyclohexanone- (2,4,6-cis) is prepared by reducing dl-epiinosose with hydrogen and PtO 2 and oxidizing the resulting di-epi-quercitol with Acetobacter suboxydans, 2,4,5-Tri-hydroxycyclohexanone(2,4-cis) is prepared by treating 4-cyclohexene-1α,2#-diol with 3-chloroperbenzoic acid and heating the resulting 4,5-epoxycyclohexane-1α,2#-diol with BF 3 etherate. 2,5-Dideoxy-5-iodostreptamine is prepared by treating 2-deoxy-1,6 : 3,4-dicarbonylstreptamine with methanesulphonyl chloride, treating the resulting 5-O-methanesulphonyl derivative with Na1, refluxing the resulting 5-deoxy- 5-iodo derivative with HCl, treating the resulting 2,5-dideoxy-5-iodostreptamine dihydrochloride with acetic anhydride and sodium acetate and hydrolysing the resulting N,N1-diacetyl-2,5- dideoxy-5-iodo-streptamine with aqueous HCl. 2,5-Dideoxy-5-fluorostreptamine is prepared by heating 2-deoxy-5-O-methanesulphonyl-1,6 : 3,4-dicarbonylstreptamine with HCl, reacting the resulting 2-deoxy-5-O-methanesulphonylstreptamine dihydrochloride with aqueous NaOH and then benzyl chloroformate, treating the resulting N,N1- di - carbobenzoxy - 2 -deoxy - 5-O- methanesulphonylstreptamine with KF and hydrolysing the resulting N,N1-dicarbobenzoxy- 2,5-dideoxy-5-fluorostreptamine with aqueous mineral acid. Pharmaceutical compositions having antibacterial activity comprise, as active ingredient, an aminocyclitol antibiotic (I) or an acid addition salt thereof, together with a pharmaceutical carrier.

Description

This invention relates to aminocyclitol antibiotics and in particular compounds having the formula: where R^, Rg and Rg each represent hydrogen, or one of R^, Rj and Rg represents an ω-amino-a-hydroxy-lower-alkanoyl group having the formula: H2NCH2(CH2)nCHOHCOwhere n is zero or 1, the others of R^> Rg and Rg being hydrogen; R2 represents hydrogen or hydroxy; Rg represents hydrogen, hydroxy or halogen (i.e. fluorine, chlorine, bromine and iodine), except that when Rj is hydrogen, Rg is -242807 not hydroxy cis to the amino groups at the 1- and 3-positions; and Rg and R^ each represent hydrogen or methyl, and to acidaddition salts thereof. The present invention also relates to a pharmaceutical composition which comprises a compound of the Formula I, or an acid-addition salt thereof, and a pharmaceutical carrier.

Compounds of Formula I where Rp Rg and Rg are hydrogen are prepared by the method described in Shier et al. United States Patent Specification No. 3,669,838. This method comprises culturing a nutrient medium containing carbohydrates, a source of assimilable nitrogen, essential salts and an added aminocyclitol derivative having the formula : where Rp Rp Rg and Rg have the meanings given above, and where Rg and Rg can in addition represent a single bond joining the two amino nitrogen atoms together, in the presence of a suitable mutant microorganism, said mutant microorganism being incapable of synthesizing said aminocyclitol (II) itself but being capable of incorporating said aminocyclitol into the compound of Formula I under the above conditions, specifically a mutant of Micromonospora purpurea designated Mlcromonosproa purpurea ATCC 31,119, and isolating the product from the culture medium. The compounds of Formula I where both Rg and Rg are hydrogen are produced when the aminocyclitol of Formula II where Rg and Rg represent a single bond is used. In accordance with the procedure described by Shier et al., the nature of the mutant is such that it is incapable of synthesizing the aminocyclitol subunit from a nutrient medium to thereby produce the antibiotic, but is capable of incorporating the latter into an antibiotic when the aminocyclitol is added to the nutrient medium.

Moreover, studies with radiolabelled compounds have demonstrated that'non-nitrogen-containing cyclitols are probable biogenetic precursors of aminocyclitols of the type represented by Formula II, such as streptamine and deoxy5 streptamine [Rinehart et al., J. Am. Chem. Soc. 96, 22632265 (1974); Walker et al., Biochem. 8, 763-770 (1969)? Demain et al., Bacteriol. Rev. 34, 1-19 (1970)]. Nevertheless, although it is known that aminocyclitols can be incorporated into aminocyclitol antibiotics either by culture LO of a nutrient medium lacking the aminocyclitol subunit as such and in the presence of an appropriate microorganism [e.g., the incorporation of deoxystreptamine into neomycin by Waksman et al., Science 109, 305-307 (1949)] or by culture of a nutrient medium containing an aminocyclitol subunit in 'St, L5 the presence of a microorganism mutant which is incapable of biosynthesizing the aminocyclitol as such but which is capable of incorporating the aminocyclitol into the antibiotic [e.g., the incorporation of streptamine or epistreptamine into the hybrimicin antibiotics by Shier and Rinehart, United States Patent 3,669,838, patented June 13, 1972] as described above, the direct incorporation of cyclitols into aminocyclitol antibiotics has not been heretofore achieved.

In fact, efforts to incorporate either myo-inositol, a probable biogenetic precursor of streptamine, or a hexahydr25 oxy—cyclohexane monomethyl ether (quebrachitol) into aminocyclitol antibiotics using the Rinehart/Shier method were completely unsuccessful [Testa et al., J. Antibiotics 27, 917-921 (1974)].

Yet a process that would permit the use o£ cyclitols, instead of aminocyclitols, for incorporation into aminocyclitol· -442807 type antibiotics by microorganism mutants using the Rinehart/Shier method would provide a very significant advance in the aminocyclitol antibiotic art, because the method would afford, by judicious selection of the microorganism and the cyclitol subunit, a certain degree of biogenetic tailoring of the resultant antibiotic molecule. Moreover, since the aminocyclitols are invariably much more expensive than the nonaminated cyclitols, significant savings in costs of the final products could be realized. (For example, streptamine, at present prices, costs about $1 per gram, whereas its probable biogenetic precursor, scyllo-inosose, can be obtained in about 80% yield by fermentative oxidation of myo-inositol, which only costs about 2 cents per gram at present).

In accordance with an improved process, which is the subject of our Application, it has been surprisingly found that aminocyclitol antibiotics of the streptamine, deoxystreptamine or dideoxystreptamine type having the general structure of Formula I above can be produced by culturing a nutrient medium containing carbohydrates, a source of assimilable nitrogen, essential salts and a cyclitol of the 2-R2'-S-Rg'-3,4,6-trihydroxycyclohexan-3-one or 2-R2'-5-R5'-l,3,4,6-tetrahydroxycyclohexane class represented by the formula: or of the 5-Rg'-3,4,6-trihydroxycyclohexene class repre- -543807 where, in either case, R is hydrogen or acetyl; Rg' is oxo (=0) or hydroxy; and R2' and Rg1 each are hydrogen or OR, in the presence of mutants of a microorganism which is incapable of biosynthesizing the cyclitol unit but which is capable of incorporating the cyclitol into the antibiotic molecule as an aminocyclitol unit.

The above process can be used for preparing the novel gentamicin-type antibiotics having the Formula I above where Κθ and R-j each represent hydrogen or methyl; R2 and Rg each represent hydrogen or hydroxy; and Rj, Rg and Rg represent hydrogen; which process comprises culturing a nutrient medium containing carbohydrates, a source of assimilable nitrogen, essential salts and a cyclitol having the Formula Ilia or Illb above in the presence of such a microorganism. The specific strain of a microorganism for carrying out the above process is M. purpurea ATCG 31, 164.

Xt is also significant that the same mutant, M. purpurea ATCC 31, 164, is also capable of incorporating into the antibiotics of Formula I above an aminocyclitol having the formula: HO (Xla) where R2' and Rg' are each hydrogen or hydroxy and Rj' is amino or hydroxy.

The process, which can prepare compounds of the Formula I where Rg and Ry each represent hydrogen or methyl; R2 and Rg each represent hydrogen or hydroxy; and R^, R^ and Rg each represent hydrogen, is carried out in the presence of M. purpurea ATCC 31, 164 using the procedure described above and comprises culturing a nutrient medium containing carbohydrates, a source of assimilable nitrogen, essential salts and an aminocyclitol having the Formula Ila in the presence of M. purpurea ATCC 31, 164. This process is considered to be within the purview of the present invention. This process also comprises optionally converting a free base obtained to an acid-addition salt thereof.

As described above, preparation of the compounds of Formula I by culturing a nutrient medium containing carbohydrates, a source of assimilable nitrogen, essential salts and an aminocyclitol having the Formula IX above where Rj_ and Rg are hydrogen; Rg is hydrogen or hydroxy; and Rg is hydrogen, hydroxy or halogen, can also be carried out in the presence of the microorganism mutant, Micromonospora purpurea ATCC 31,119. However, as indicated above, the aminocyclitols required as substrates in that process are LO generally rather expensive, and thus the cost of the final products produced is correspondingly high. The process comprising use of cyclitols in the presence of mutant M. purpurea ATCC 31,164 for incorporation into aminocyclitol antibiotics thus provides an advantage over the first L5 described process of the invention.

In either case, whether the non-nitrogen-containing cyclitols of Formulas Ilia or Illb or the aminocyclitols of Formulas II or Ila are used as substrates, the compounds of Formula I are prepared by culturing a nutrient medium containing carbohydrates, a source of nitrogen, essential salts and an added cyclitol of Formulas Ilia or Illb or an aminocyclitol of Formula Ha in the presence of the mutant, M. purpurea ATCC 31,164, and isolating the product from the culture medium.

The compounds of Formula I, where one of Rp Rg and Rg represents an ω-amino-a-hydroxy-lcwer-alkanoyl group, are prepared by the method described by Konishi et al., United States Patent 3,780,018,which comprises reacting the compound of Formula I where each of R^, Rg and Rg is hydroiO gen with an N-hydroxysuccinimide ester having the formula: -842807 where n has the meanings given above. The resulting mixture of the compounds of Formula I where one of Rg, Rg and Rg is the ω-(N-benzyloxycarbonyl)amino-a-hydroxy-lower-alkanoyl group: s CH2O-C-NHCH2(CH2)nCHOHCOthe other two being hydrogen, is then subjected to hydrogenolysis of the benzyloxycarbonyl group with hydrogen over a catalyst.

As indicated above, when a compound of Formula I where each of Rg, R-j and Rg is hydrogen is used as starting material in the acylation reaction, a mixture of the three possible isomeric mono-amides is obtained in which one of the Rg, Rg or Rg amine hydrogen atoms is replaced by the ω(N-ben2yloxycarbonyl)amino-a-hydroxy-lower-alkanoyl group. When individual characterization and study of these products are desired, they must of course 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-hydroxysuccinimide ester, preferably at a temperature from -10°C. to about 10°C., and in an aqueous solution of an inert organic solvent, for example tetrahydrofuran, dioxane, ethylene glycol dimethyl ether, dimethylacetamide, dimethylformamide or propylene glycol dimethyl ether.

Hydrogenolysis of the benzyloxycarbonyl group is -93807 carried out over a palladium-on-charcoal catalyst in an inert, water-miscible organic solvent, for example methanol, ethanol, dioxane, tetrahydrofuran, ethylene glycol dimethyl ether, or propylene glycol dimethyl ether.

The aminocyolitols of Formula II where R]_, Rg and I Rg are hydrogen and Rg is fluorine or iodine are novel compounds and are prepared as described hereinbelow.

Other aminooyclitols of Formula II, which are also useful in the practice of the present invention, are known compounds. These are: streptamine sulfate [Peck et al., J. Am. Chem. Soc. 68, 776 (1946)]; 2-epistreptamine dihydrochloride [Suami et al., J. Org. Chem. 33, 2831 (1968)]; 2,5-didgoxystreptamihe dihydrochloride, m.p. >300°C.; and 6,7-diazabicyclo[3.2.1]octahe-2,4-diol (exo, exo), πί.ρ. 185-1934C. [Both the latter two compounds disclosed by Testa et al., J. Antibiotics 27, 917-921 (1974)].

The N-hydroxysuccinimide esters of Formula IV are a generally known class of compounds.

The compounds of Formula I have been tested in a standard serial dilution antibacterial test and have been found to have antibacterial activity, particularly against gentamicin-resistant organisms. The compounds are thus useful as antibacterial agents.

The compounds of Formula I are primarily intended for oral, topical or parenteral administration and can be prepared for use with a pharmaceutical carrier by suspension, either ih the form of their free bases or as pharmaceutically acceptable, non-toxic acid addition salts, in an inert -1043807 carrier such as polyethylene glycol, or by tabletting or encapsulation for oral administration either alone or with suitable adjuvants, or alternatively they can be formulated with conventional creams or jellies for topical application.

The molecular structures of the compounds of the I invention were assigned on the basis of study of their chromatographic characteristics determined by thin layer chromatographic (tic) analysis; their nuclear magnetic resonance (nmr) and mass spectra; by degradation to known compounds; by comparison of the products prepared by fermentation with mutant M. purpurea ATCC 31,119 using aminocyclitols of Formulas II or Ila as substrates with products prepared by fermentation with mutant M. purpurea ATCC 31,164 using cyclitols of Formulas Ilia or IHb and by the correspondence between calculated and found values for elementary analyses for the elements.

The following specific examples are illustrative of the manner of making the compounds and carrying out the process of the invention without the latter being limited thereto.

PREPARATION OF NOVEL INTERMEDIATES Preparation 1 dl-Viboquercitol [dl-1,2,3,4,5-cyclohexanepentol (1,2,4-cis)] [McCasland et al., J. Am. Chem. Soc. 75, 4020 (1953)] (0.40 mole) was subjected to microbiological oxidation by Acetobacter suboxydans using the procedure described by Posternak, Helv. Chim. Acta 33, 1594-1596 (1950). To the resulting broth was added 5 g. of lead acetate in 60 ml. of water, the solution was filtered through a filter aid, and the filtrate was passed over 180 g. of Dowex (Registered Trade Mark) resin 50-124. 4 3 8 0,7 The resulting eluate was concentrated to a volume of about 150 ml., diluted with an equal volume of ethanol and cooled. The material which separated was collected to give 15.1 g. of dl-deoxyinosose [dl-2,3,4,5-tetrahydroxy-l-cyclohexanone (2,4-cis)], m.p. 221-222°C. Further concentration of the filtrate from the first crop afforded three additional crops totalling 28.7 g., m.p. 220-221°C.

Preparation 2 A solution of 9.5 g. (0.053 mole} of dl-epi-inosose 10 [Posternak, Helv. Chim. Acta 19, 1333 (1936)] in 300 ml. of 0.1N hydrochloric acid was reduced with hydrogen over 6 g. of platinum oxide at an initial hydrogen pressure of about 56 p.s.i. The product was isolated by filtration of the reaction mixture, concentration of the filtrate to dryness and recrystallization of the residue from aqueous methanol.

There was thus obtained dl-epi-guercitol, m.p. 214-215°C.

The latter was subjected to microbiological oxidation by Acetobacter suboxydans using the procedure described by Posternak recorded above in Preparation 1, and the pro20 duct was isolated as described in Preparation 1 and recrystal -1242807 lized from ethanol to give dl-2,3,4,6-tetrahydroxy-l-cyclohexanone (2,4,6-cis), m.p. 175-177°C.

Preparation 3 To a solution of 22.5 g. (0.11 mole) of 3-chloroperbenzoio acid in 150 ml. of methylene dichloride was added 11.4 g. (0.1 mole) of 4-cyclohexene-la,28-diol [McCasland et al., J. Org. Chem. 28, 898 (1963)], and the resulting solution was stirred with cooling while maintaining the temperature below 30°C. The mixture was stirred for one hour, diluted with 150 ml. of diethyl ether, stirred for another three hours, and then diluted with 150 ml. of water. The product was isolated from the organic layer in the conventional manner to give 9.1 g. of crude material which was recrystallized from ethanol/ether to give two crops totalling 4.75 g. of 4,5-epoxycyclohexane-la,26-diol, m.p. 80°C. and 70-75’C.

A solution of 6.63 g. (0.051 mole) of the latter in 20 ml. of dimethylsulfoxide was treated with 0.06 ml. of boron trifluoride etherate. The resulting solution was heated on a steam bath for about twenty hours, an additional 0.03 ml. of boron trifluoride etherate added, and the solvent removed in vacuo. The residue was treated with 50 ml. of ethanol, the resulting solid separated, and the filtrate concentrated to dryness to give 7.75 g. of a reddish brown oil, 1.1 g. of which was chromatographed on silica gel plates, eluting with tetrahydrofuran, to give 550 mg. of 2,4,5-trihydroxycyclohexanone (2,4-cis) whose mass spectrum gave mass peaks at 144 and 145 and whose infrared spectrum showed a strong peak at 1723 cm-·'·. -1342807 Preparation 4 2-Deoxy-l,6:3,4-di-N,0-carbonylstreptamine [Umezawa et al., Bull. Chem. Soc. (Jap.) 44, 1411-1415 (1971)] (47 g., 0.21 mole) was suspended in 500 ml. of pyridine and the stirred suspension treated with 45 ml. of methanesulfonyl chloride. After cooling, the mixture was diluted with about 3 liters of methanol and the product filtered and dried to give 39 g. of 2-deoxy-5-0-methane-sulfonyl-l,6;3,4-di-N,0carbonylstreptamine, m.p. 264-266°C.

A mixture of 2 g. (0.0069 mole) of 2-deoxy-5-0methane-sulfony1-1,6:3,4-di-N,0-oarbonylstreptamine, described above, and 4.8 g. (0.032 mole) of sodium iodide in 70 ml. of dimethylformamide was heated at 125°C. for twenty-four hours and then taken to dryness. The crude 2,5-dideoxy-5-iodo1,6:3,4-di-N,0-carbonylstreptamine was mixed with 30 ml. of 6N hydrochloric acid, the mixture refluxed for two and a half hours and then cooled and evaporated to dryness in vacuo. The crude 2,5-dideoxy-5-lodostreptamine dihydrochloride was dissolved in 30 ml. of acetic anhydride, the solution treated with 5.25 g. of sodium acetate and the mixture refluxed for two and a half hours. The mixture was then cooled, poured into 200 ml. of water and extracted with chloroform. The chloroform extracts, on washing once with sodium thiosulfate solution, once with brine and once with water and evaporation to dryness, afforded an oil which was crystallized from ethanol to give two crops totalling 1.1 g. of N,N'-diacetyl-2,5-dideoxy-5-iodostreptamine, m.p. 256-258°C Hydrolysis of the latter by refluxing with aqueous hydrochloric acid and isolation from a basic medium affords 2,5dideoxy-5-iodostreptamine.

Preparation 5 2-Deoxy-5-0-methanesulfonyl-l,6:3,4-di-N,0-carbonylstreptamine (39 g., 0.12 mole), described in Preparation 4 above, was suspended in approximately 200 ml. of 6N hydrochloric acid, the mixture warmed on a stream bath for two hours, evaporated to dryness in vacuo, mixed with 200 ml. of isopropyl alcohol and evaporated to dryness once again. The residual oil was triturated with methanol, cooled and the solid collected and recrystallized from methanol to give 2-deoxy-5-0-methanesulfonylstreptamine dihydrochloride, m.p. 208-210°C.

Anal. Calc'd for C^gNgOgS^HCl: C,26-84f H, 5.79; N, 8.94 Found: C,26.77; H, 5.76; N, 9.17.

A solution of 28.7 g. (0.09 mole) of 2-deoxy-5-0methanesulfonylstreptamine in 45 ml. of water and 90 ml. of 2N sodium hydroxide was cooled in an ice bath and treated dropwise with stirring with a solution of 45 ml. of benzyl chloroformate in 80 ml. of toluene added from one dropping funnel and with 160 ml. of 2N sodium hydroxide from another. When addition was complete, the mixture was stirred for an additional fifteen minutes, diluted with about 50 ml. of toluene, stirred for three hours and filtered. Recrystallization of the solid from ethanol afforded 4.0 g. of N,N'-dicarbobenzoxy-2-deoxy-5-0-methanesulfonylstreptamine, m.p. 198201°C.

Anal. Calc'd for C23H28N2°9S: C, 54.32; H, 5.55; N, 5.51 Found: C, 54.75; H, 5.61; N, 5.60.

Reaction of the latter with potassium fluoride in benzene or acetonitrile containing a crown ether, e.g., 1,4,7,10,13,16-hexaoxacyclooctadecane, using the procedure described by Liotta et al., J. Am. Chem. Soc. 96.' 2250-2252 48807 (1974), affords N,N'-dicarbobenzoxy-2,5-dideoxy-5-fluoro- streptamine which, on hydrolysis with aqueous mineral acid. affords 2,5-dideoxy-5-fluorostreptamine. Multation Processes Xn the following procedures, various media constituted as follows were employed. Medium 1: N-Z Amine g./i. Glucose 10 Soluble starch 20 Yeast extract 5 N-Z-Amine-Type A (Difco, Trade Mark) Registered 5 CaCOj 1 Agar 15 Medium 2: Germination Medium (in distilled water) per cent Beef extract 0.3 Tryptone 0.5 Dextrose 0.1 Soluble starch 2.4 Yeast extract 0.5 CaCO^ 0.4 Medium 3: Soybean-Glucose g./i. Soybean meal 30 Dextrose (Cerelose, Registered Trade Mark) 40 CaCOj 1 - 16 Medium 4: 43807 TGE ?.-/1Trypticase glucose extract... 5.0 Trypticase peptone........... 3.0 Glucose...................... 1.0 Agar.........................15.0 Medium 5: Production Medium Medium 6: Beef extract.................

Yeast extract................

Soybean meal.................

Maltose......................

Starch.......................

Casamino acid................

CaCO,........................

CoCl2-6H20................... 0.3% 0.5% 0.5% 0.1% 2.4% 0.1% 0.4% mg./liter Streptomicin Assay Agar Beef extract.................

Yeast extract................

Peptone......................

Agar......................... 1.5 3.0 6.0 .0 The organism Micromonospora purpurea was obtained from the U.S. Dept. of Agriculture as NRRL 2953 and maintained on N-Z amine slants (Medium 1). Submerged fermentations were conducted in flasks containing germination Medium 2 for four days at 37 eC. on a rotary shaker. From this first stage seed, a 10% inoculum was transferred to the germination medium (Medium 2), and fermentation was continued as above at 28°C. for seven days.

For purposes of establishing the capability of the organism to biosynthesize gentamicin in the absence of added deoxystreptamine, a third stage fermentation using a 5% inoculum was carried out in a 10 liter fermentor in a soybeanglucose medium (3) at 28°C., agitating at 200 rpm and sparging at 2 liters/minute with filtered air. After six 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 2807 4.

(Na form). The column was then rinsed with water and eluted with 2N sulfuric acid. Following the procedure described in United States Fatent Specification No. 3,091,572, there was isolated a 30Ctrg. sample of crude gentamicin, which was found to be biologically active and which contained three components similar to gentamicin Cj, C2 and by TLC examination.

For purposes of mutating the organism, broth cultures were cultivated in medium 2 (37eC. for three 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 mutagenized culture were plated in Medium 4 at 37°C. until colonies were evident (usually about One week). Colonies were picked to duplicate plates (Medium 4), one set of which was overlaid with a spore suspension of B. subtilis and the other set of which provided a master plate of mutants. After incubation at 37°C. for frcm eighteen to twenty hours, the picks which shewed no zone of inhibition on -the B. subtilis plate were transferred frcm the master plate (no B. subtilis) to medium 1 slants and incubated until full growth was evident.

These potential non-producing mutants were then challenged with deoxystreptamine in an attempt to stimulate antibiotic biosynthesis as follows. Stock cultures of the potential mutants were streaked as bands on the surface of medium 4 plates and incubated at 37°C. until growth was evident (about three to four days). Filter paper discs were then dipped into a solution of deoxystreptamine (500 meg./ml.) and placed on top of the culture streak. After incubation for twenty-four hours, the surface of the plate was inoculated with B. subtilis using the overlay technique, and incuba18 42807 tion was continued for an additional eighteen to twenty hours. Isolates showing zones of inhibition surrounding the disc were designated as deoxystreptamine mutants. One such mutant, coded mutant VIB and deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 as Micromonospora purpurea ATCC 31,119, was used for the production of the gentamicin-type antibiotics as described below.

The latter organism was itself subjected to the same mutation procedure described above and samples of the mutagenized culture were plated in Medium 4 at 37’C. until colonies were evident (usually about one week). Colonies were picked to duplicate plates containing streptomicin assay agar (Medium 6).

One set served as a master plate for later recovery while the second set contained 25 pg./ml. of streptamine sulfate and an overlay spore suspension of B. subtilis as a challenge test organism. These plates were incubated at 37°C. for twenty-four hours and examined for zones of inhibition. Those showing the largest zones were transferred from the master plate onto N-Z amine slants (Medium 1) and incubated for one week at 37°C.

Those mutants incorporating low levels of streptamine sulfate were then screened with streptamine and scylloinosose at 500 pg./ml. as base. Stock cultures were transferred to flasks containing Medium 2 plus the above intermediates and incubated at 37°C. on a rotary shaker for seven days. Flasks were i^eriodically assayed for antibiotic activity via the disc diffusion assay method using B. subtills as test organism. Isolates showing zones of inhibition 2807 surrounding the disc were designated as streptamine or scyllo-inosose mutants. One such mutant, coded mutant VIB-3P and deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20851 as Micromonospora purpurea ATCC 31,164, was used for the production of the aminocyclitol antibiotics of the streptamine, deoxystreptamine and dideoxystreptamine-type as described below.

BIOSYNTHESES WITH M. PURPUREA ATCC 31,119 Example 1 The mutant organism was maintained on N-Z amine agar slants (Medium 1) from which transfers to flasks containing 500 ml. of germination Medium 2 were made. The flasks were incubated at 28°C. for four days on a rotary shaker (2 stroke) at 225 rpm.

A 10% (y/ν) inoculum from the germination stage was aseptically transferred to 14 liter fermentors containing 9 liters of sterile germination Medium 2. These were agitated at 450 rpm at 28-29°C. and sparged with filtered air at 5 liters/minute. At time of inoculation, 200 mg./liter of streptamine sulfate was added as a suspension in sterile distilled water. Fermentation was continued for eight days.

A twenty-four hour, 10 liter inoculum prepared as above was aseptically transferred to 130 liter fermentors containing 70 liters of sterile germination Medium 2, and 0.31 g./liter of streptamine sulfate suspended in sterile distilled water was added. Aerobic fermentation was carried out at 29°C. for seven days.

Fermentations were terminated by addition of 10N sulfuric acid to pH 2.0 and filtration using a filter aid to remove mycelia. The filtered broth was adjusted to pH 7.0, and 1.56 g. of oxalic acid per gram of calcium carbonate present in the medium was added to remove calcium. This was allowed to stand overnight, and the clarified broth was decanted and passed over Bio-Rex 70 (Registered Trade Mark) (weak cation exchanger) resin in the Ifo+ form using about 14 g. of resin per liter of broth. The column was then washed with distilled water and eluted with 2N sulfuric acid. All fractions displaying antibiotic activity were combined, neutralized and concentrated under vacuum below 50°C. to the point where salt crystallization became evident (about 1/3 volume). The pH was then adjusted to 10.5, and four volumes of acetone were added to precipitate inorganifas which were removed by filtration.

The filtrate was adjusted to pH 5.0 with 6N sulfuric acid, concentrated under vacuum to approximately 1/20 of the original volume and chilled. A white crystalline crop melting over 300°C. was collected by filtration which was found, from its thin layer chromatography properties and its infrared spectrum, 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 fermentations, a total of 21 g. of streptamine sulfate was recovered at this step.

The filtrate was further concentrated and 10 volumes of methanol added yielding the first crude antibiotic solid. From two 10 liter fermentations, 7 g. was obtained, and from two 80 liter fermentations, 24 g. was obtained.

For purposes of identifying antibiotic components during purification procedures, chromatographic mobility values obtained on paper chromatography and thin layer chromatography were determined for gentamicins C^, C2 and Cj_a and 07 for each of the corresponding aminocyclitol analogs prepared as indicated above, where the chromatographic mobility (hereinafter designated C.M.) is expressed as: _ distance component from origin * ’ - distance gentamicin £rom origin The chromatography systems used were as follows: System 1 - Whatman (Registered Trade Mark) Ko. 1 paper saturated with O.95M sulfate-bisulfate and developed in descending fashion in 80% aqueous ethanol + 1.5% NaCl and subsequent bioautography using B. subtilis as test organism.

System 2 - Silica gel F 254 plate developed in lower phase of chloroform(1):methanol(1):concentrated (28%) ammonium hydroxide(1). Components were located with a ninhydrin spray on heating.

The C.M. values of the major antibiotic components of the present invention in comparison with a reference gentamicin complex, all relative to gentamicin Cj, are shown in Table X, where the compounds designated Component 1, Component 2 and Component 3 are to be understood to be, respectively: 0-3-deoxy-4-C-methyl-3-methylamino-8-L-arabinopyranosyl(1+6)-0-[2-amino-6-methylamino-6-C-methyl-2,3,4,6-tetradeoxya-D-erythro-glucopyranosyl-(1+4)]-D-streptamine; 0-3-deoxy-4-C-methyl-3-methylamino-8-L-arabinopyranosyl(1+6)-0-t2,6-diamino-6-C-methyl-2,3,4,6-tetradeoxy-a-D-erythro gluoopyranosyl-(1+4)] -D-streptamine; and 0-3-deoxy-4-C-methvl-3-methylamino-8- -L-arabinopyranosyl- (1+6)-0-[2,6-diamino- 2,3,4,6-tetradeoxy-a· -D-erythro-gluco- pyranosyl-(1+4)]-D-streptamine. Table I C.M. System I C.M. System 2 Gentamicin C·^ 1 1 Gentamicin Cj 0.89 0.83 Gentamicin Ci , 0.50 0.67 Component 1 (Major) 0.96 0.92 Component 2 (Minor) 0.76 0.75 Component 3 (Minor) 0.50 0.61 From the 10 liter fermentors, the crude solid (7 g.), which displayed antibacterial activity, was suspended in 200 ml. of methanol and 10 ml. of concentrated (28%) ammonium hydroxide, and the mixture agitated for thirty minutes and filtered. This was repeated two additional times, and the filtrates were combined and concentrated under vacuum yielding a pale yellow oil weighing 0.9 g.

The spent salts were essentially devoid of antibiotic activity.

The oily base was mixed with 4 g. of silica gel (Davison grade 923, 100-200 mesh) and charged 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 (1):chloroform(2):17% aqueous ammonium hydroxide(1). The column was developed with this solvent and 50 ml. fractions collected.

Fraction? 8 and 9 contained a single ninhydrinpositive component which yielded 20 mg. as a pale yellow oil on removal of solvent. The mass spectrum of this material, designated Component 1, showed a molecular ion and major fragments each 16 mass units (i Reference gentamicin C-, : M+ 477, 420, 360, 350, 347, 322, 319, 304 Component 1: M+ 493, 436, 376, 366, 363, 338, 335, 320 This material was converted to its sulfate salt by dissolving in ethanol and adding a few drops of ethanol containing sulfuric acid. The resulting white solid was collected and dried to yield 22 mg. of Component 1, 0-3-deoxy-4-Cmethvl-3-methylamino-6-L-arabinopvranosyl- (l-*-6) -0- [2-amino23 6-methvl-amino-6-C-methvl-2,3,4,6-tetradeoxy-a-D-erythro£28«7 glucopyranosyl-(l->4)]-D-streptamme as the di-base'hepta sulfate·decahydrate, m.p. >300°C.

Anal. Calc'd for (c21h43n5°8 >2‘7H2SO4’1°H2O: Found: C, 27.22; H, 6.53; N, 7.55; S, 12.09 C, 27.15; H, 6.67; N, 7.79; S, 12.76.

Fractions 10-13 yielded a more polar ninhydrin component designated 0-3-deoxy-4-C-methyl-3-methylamino-gL-arabinopyranosyl-(l-»6)-0-[2,6-diamino-6-C-methyl-2,3,4,6tetradeoxy-a-D-erythro-glucopyranosyl- (l->-4) ] -D-streptamine, Component 2, which displayed antibiotic activity.

Fractions 15-26 yielded a third more polar component displaying antibiotic activity. The mass spectrum of this component showed characteristic sugar peaks corresponding to gentamicin C^a at 129 (purpurosamine) and 160 (garosamine) and is designated 0-3-deoxy-4-C-methyl-3-methylamino-g-Larabinopyranosyl-(1+6)-0-[2,6-diamino-2,3,4,6-tetradeoxy-aD-erythro-glucopyranosyl-(1+4)]-D-streptamine, Component 3.

Alternatively, the new Component 1 was isolated as follows: a mixture of crude antibiotic base obtained as above (0.9 g.) was dissolved in 7 ml. of water and the pH adjusted to 4.5 with IN sulfuric acid. The solvent was passed over a strong anion exchange column (IRA 401) in the 0H~ form (bed measurement = 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% aqueous ammonium hydroxide : isopropyl alcohol:chloroform (1:1:2). The solvent was decanted and concentrated under vacuum leaving an oily residue weighing 140 mg. The mass spectrum of this material corresponds to that from fractions 8 and 9 above, i.e., M+ 493, 436, 376, 366, 363, 338, 335, 320.

The nuclear magnetic resonance spectrum for Component 1 was also consistent for the proposed structure corresponding to 0-3-deoxy-4-C-methyl-3-methylamino-f5-Larabinopyranosyl-(1+6)-0-[2-amino-6-methylamino-6-C-methyl2,3,4,6-tetradeoxy-a-D-erythro-glucopyranosyl-(1+4)]-Dstreptamine and is summarized in Table II.

Table II .87, 5.60 .22 3.09, 3.15 2.9- 4.8 1.9- 2.6 1.72 Integration Assignment_ anomeric OCHO's exchangeable H NCH3 x 2 -CHO x 6, -CHN X 5, -CHgO -CH2CH26 CHgC-, CHgCH Alternatively, a 4 g. sample of the crude antibiotic salt was dissolved in 50 ml. of water and passed over an anion exchange resin AG1X8 (resin bed 1 x 27 cm.). The antibiotic was eluted with water and all active fractions combined and evaporated under vacuum. Further desalting was' carried out on the residue by extraction with methanolic sodium hydroxide. The liberated base was mixed with 7 g. of silica gel and charged on a 50 g. silica gel column. This column was developed with chloroformzmethanol:concentrated(28%)ammonium hydroxide (3:4:2) and 25 ml. fractions colle.cted. Early fractions yielded the new less polar Component 1 but admixed with several less polar impurities as evidenced by thin layer chromatography. These fractions were combined and the concentrate charged on a 50 g. silica gel column as above. This column was developed with chloroformxmethanol:concentrated (28%)ammonium hydroxide (5:3:1) and 25 ml. fractions collected. Fractions 6-12 contained the desired component free of obvious impurities. On removal of solvent, the resulting oil was converted to the sulfate salt in ethanolic sulfuric acid as previously described yielding 0.124 g. of Component 1 as the di-base·heptasulfate*decahydrate, m.p. >300°C. described above.

By culturing an appropriate aminocyclitol with mutant Micromonospora purpurea ATCC 31,119, in germination Medium 2 and isolation of the products as described above in Example 1, the following compounds of Formula I are similarly prepared: Example 2 0-3-Deoxy-4-C-methvl-3-methvlamino-g-L-arabinopyranosyl- (1-6) -0- [2-amino-6-methvlamino-6-C-methvl-2,3,4,6tetradeoxy-a-D-erythro-glucopyranosyl-(1-4) ] epistreptamine (C.M. relative to gentamicin Cg: System 1 = 0.59, System 2 = 0.63); 0-3-deoxy-4-C-methyl-3-methylamino-B-L-arabinopyranosyl- (l-*-6) -0- [2,6-diamino-6-C-methyl-2,3,4,6-tetradeoxy-ct-Derythro-glucopyranosyl-(1-4)1epistreptamine; and 0-3-deoxv4-C-methyl-3-methylamino-8-L-arabinopyranosyl-(1-6)-0- [2,6diamino-2,3,4,6-tetradeoxy-a-D-erythro-glucopyranosyl-(1-4)]epistreptamine obtained by use of epistreptamine in place of streptamine in the fermentation procedure.

Example 3 0-3-Deoxy-4-C-methyl-3-methylamlno-8-L-arabinopyranosyl- (1-6) -0- [2-amino-6-methylamino-6-C-methyl-2,3,4,6tetradeoxy-a-D-erythro-glucopyranosyl- (1-4) ] -2,5-dideoxystreptamine (C.M. relative to gentamicin Cg: System 1 = 0.95, System 2 = 0.98); 0-3-deoxy-4-C-rnethyl-3-methylamino-g-Larabinopyranosyl-(1+6)-0-[2,6-diamino-6-C-methyl-2,3,4,6-tetradeoxy-a-D-erythro-glucopyranosyl-(1+4)]-2,5-dideoxystreptamine (C.M. relative to gentamicin System 1 = 0.66, System 2 = 0.73); and 0-3-deoxy-4-C-methyl-3-methylamino-B-L-arabinopyranosyl-(1+6)-0-[2,6-diamino-2,3,4,6-tetradeoxy-a-Derythro-glucopyranosyl-(1+4)]-2,5-dideoxystreptamine obtained by use of dideoxystreptamine in place of streptamine in the fermentation procedure.

The same 0-3-deoxy-4-C-methyl-3-methylamino-B-Larabinopyranosyl-(1+6)-0-[2-amino-6-methylamino-6-C-methyl2,3,4,6-tetradeoxy-a-D-erythro-glucopyranosyl-(1+4))-2,5dideoxystreptamine; and 0-3-deoxy-4-C-methyl-3-methylaminoβ-L-arabinopyranosyl-(1+6)-0-[2,6-diamino-6-C-methyl-2,3,4,6— tetradeoxy-g-D-erythro-glucopyranosyl-(1+4)]-2,5-dideoxystreptamine described above and having the same C.M. values as given above were obtained by use of 6,7-diazabicyclo[3.2.1]octane-2,4-diol (exo, exo) in place of streptamine in the fermentation procedure.

Example 4 0-3-Deoxy-4-C-methyl-3-methylamino-B-L-arabinopyranosyl-(1+6)-0-[2-amino-6-methylamino-6-C-methyl-2,3,4,6tetradeoxy-g-D-erythro-glucopyranosyl-(1+4)]-5-iodo-2,5-dideoxystreptamine; 0-3-deoxy-4-C-methyl-3-methylamino-B-Larabinopyranosyl-(1+6)-0-(2,6--diamino-6-C-methyl-2,3,4,6tetradeoxy-a-D-erythro-glucopyranosyl-(1+4)]-5-iodo-2,5-dideoxystreptamine ; and 0-3-deoxy-4-C-methyl-3-methylamino-BL-arabinopyranosyl-(1+6)-0-[2,6-diamino-2,3,4,6-tetradeoxyq-D-erythro-glucopyranosyl-(1+4)]-5-iodo-2,5-dideoxystreptamine obtained by use of 5-iodo-2,5-dideoxystreptamine in place of streptamine in the fermentation procedure. ^28 0 7 Example 5 0-3-Qeoxy-4-C-methy1-3-methylamino-g-L-arabinopyranosyl- (l-»-6) -0- [2-amino-6-methylamino-6-C-methyl-2,3,4,6tetradeoxy-g-D-erythro-glucopyranosyl-(l->4)]-5-fluoro-2,55 dideoxystreptamine; 0-3-deoxy-4-C-methyl-3-methylamino-g-Larabinopyranosyl-(l-»6)-0-[2,6-diamino-6-C-methyl-2,3,4,6tetradeoxy-a-D-erythro-glucopyranosyl-(l->4)]-5-fluoro-2,5dideoxystreptamine; and 0-3-deoxy-4-C-methyl-3-methylaminog-L-arabinopyranosyl-(1+6)-0-[2,6-diamino-2,3,4,6-tetradeoxy10 g-D-erythro-glucopyranosyl-(l->4)]-5-fluoro-2,5-dideoxystreptamine obtained by use of 5-fluoro-2,5-dideoxystreptamine in place of streptamine in the fermentation procedure.

BIOSYNTHESES WITH M. PURPUREA ATCC 31,164 A. Incorporation of Cyclitols Example 6 The mutant organism, M. purpurea ATCC 31,164, was maintained on N-Z amine agar slants (Medium 1), and a first stage seed was prepared by inoculating a loopfull from the slant to 50 ml. of germination medium (Medium 2) and allowed to incubate for four days on a rotary shaker at 27-28eC. A 5% inoculum was then transferred to 500 ml. of germination medium, and this was incubated for three days as above. One liter of this second stage seed was used to inoculate nine liters of production medium (Medium 5) in tanks agitated at 400 r.p.m. and sparged with filtered air at 5 liters/minute at 28-29°C. for forty-eight hours. Pinally the growth from this third stage seed was used to inoculate seventy liters of production medium containing 16 g. of scyllo-inosose. Fermentation was carried out for four days, agitating at 400 r.p.m. and aerating at 1.5 cubic feet per minute at 29-30°C.

The tank contents were acidified to pH 2.0 with ION sulfuric acid and filtered through a filter aid to remove mycelia. The filtrate was adjusted to pH 6.0 and passed over an 8 x 50 cm. cation exchange resin bed (Bio-Rex 70, sodium ion form, 20-50 mesh). The eluate was checked for antibiotic activity via bio-assay and found to be inactive against B. subtilis. The column was then eluted with 2N sulfuric acid, and 500 ml. fractions were collected. The fractions were bio-assayed as above against B. subtilis, and all fractions displaying antibiotic activity were combined and neutralized to give a final volume of 10 liters.

This was concentrated to about 5 liters under vacuum, the pH was adjusted to 10.5 with 10N sodium hydroxide, and five volumes of acetone were added with vigorous agitation. The inorganic salts which had separated were removed by filtration, and the filtrate was concentrated in vacuo after adjusting to pH 7.0 with dilute sulfuric acid. When the volume was about 4 liters, the pH was readjusted to 4.5, and the sample was further concentrated to 150 ml. The pH was then adjusted to 10.5, and five volumes of acetone were added for an additional desalting step as described above. The filtrate was concentrated, adjusted to pH 4.5 and further concentrated to 10 ml. On addition of 100 ml. of methanol, a crude antibiotic solid was obtained weighing P g., which was dissolved in 10 ml. of water and extracted with five 50 ml. portions of the lower phase of a solvent composed by volume of chloroform(2)sisopropanol(1):17% ammonium hydroxide(1).

The extracts from two such runs were combined and concentrated in vacuo to give an oily residue weighing 300 mg.

The sample was mixed with 5 g. of silica gel (10029 200 mesh) and placed on a 100 g. silica gel column (2.5 x 45 cm.) and developed with the chloroformzisopropanol:ammonium hydroxide solvent described above. Fractions were collected, subjected to tic analysis, and selected fractions were combined and concentrated in vacuo to a pale yellow oil weighing 0.210 g. as base. This was converted to the sulfate salt to give 0.288 g. of 0-3-deoxy-4-C-methyl-3-(methylamino)β-L-arabinopyranosyl-(1+6)-0-(2-amino-6-(methylamino)-6-Cmethyl-2,3,4,6-tetradeoxy-g-D-erythro-glucopyranosyl-(1+4)]D-streptamine as the bis-base-pantasulfate-tetrahydrate. Anal. Calc'd for: C21H43N50g-2 1/2 H2SO4*2H2O: C, 32.55; H, 6.76; N, 9.04; S, 10.35 Found: C, 32.49; H, 6.91; N, 9.36; S, 9.75 The identity of the product with the compound of the same name, designated Component 1 in Example 1 above, was established by the following: First, the chromatographic mobilities of the compound in two solvent systems determined by tic analysis were identical with the chromatographic mobilities in the same systems of Component 1. The chromatographic mobilities of the compound identified above and designated Example 6, Component 1 of Example 1 above, and gentamicin C^ are shown in the following table.

Gentamicin Cg Component 1 Example 6 System 1 Rf RfCi’ 0.39 1.00 0.30 0.77 0.30 0.77 System 2 Rf RfCl* 0.70 1.00 0.63 0.90 0.63 0.90 System 1 - silica gel 60F254, lower phase of chloroform (1):methanol(1):30% ammonium hydroxide(1), plate sprayed with ninhydrin.

System 2 - Watman (Registered Trade Markjipl paper-solvent system same as System 1 - bioautography using B. subtilis, *RfCl - Mobility relative to gentamicin C^. - 30 42807 Second, the mass spectrum of a sample, in the form of the free base, showed a molecular ion at 493 and fragments at 477, 476, 463, 457, 436, 418, 405, 401, 383, 376, 366, 363, 344, 335, 321, 318, 277, 261, 160 and 157.

Third, the nuclear magnetic resonance spectrum showed signals attributable to two NCH·, groups and one CH,CH group and was thus consistent with Component 1.

Fourth, a 10-20 mg. sample of the product obtained by fermentation as described above in 0.3 ml. of 6N hydrochloric acid in a 0.5 mm. capillary tube was heated in refluxing 6N hydrochloric acid for six hours. The mixture was allowed to stand at room temperature for two days, then diluted with 1.5 ml. of ethanol. The resulting clear supernatant liquid was decanted from the solid residue, and the solid was dissolved in water and chromatographed on silica gel plates using a chloroform(3)imethanol(4):concentrated ammonium hydroxide(2) system. Comparative samples of authentic streptamine and deoxystreptamine were chromatographed simultaneously. The identity of the hydrolysis product with streptamine was shown by the identity of the chromatographic mobilities for these two samples (0.1) as compared with the chromatographic mobility for deoxystreptamine (0.2).

As further proof that the degradation product from the above hydrolysis was streptamine, an authentic sample of Ν,Ν-diacetylstreptamine tetraacetate was prepared by reacting 810 mg. of streptamine sulfate with 50 ml. of acetic anhydride in the presence of 500 mg. of sodium acetate. After refluxing for one hour, the mixture was cooled to room temperature, evaporated to dryness and extracted with chloroform/water to give 440 mg. of material having m.p. 252-256°C., 4 2807 (melts with recrystallization), decomposes 334-336°C. [Lit.: melts partially at 250°C. with transition to long needles, melts >300eC. Peck et al. J. Am. Chem. Soc. 68, 776 (1946)].

Similar acetylation of the degradation product 5 obtained above with 5 mg. of sodium acetate in 1 ml. of acetic anhydride afforded 5.6 mg. of material having m.p. 250-257°C. (melts with recrystallization), decomposes at 336-339°C. The mixed melting point between the known and the reference sample was undepressed.

Finally, vapor phase chromatographic comparisons between the Ν,Ν-diacetylstreptamine tetraacetate and the corresponding oompound prepared from the degradation product were compared along with Ν,Ν-diacetyldeoxystreptamine triacetate. The streptamine derivative and the degradation sample were shown to be identical (R.T. = 10.6 minutes) but different from that of Ν,Ν-diacetyldeoxystreptamine triacetate (R.T. - 9.1). 2 3 0 7 Example 7 Scyllo-inosose pentaacetate [2,3,4,5,6-pentahydroxycyclohexanone pentaacetate (2,4,6-cis)] [Kluyver et al., Rec. trav. chim. Pays-Bas 58, 956 (1939)] (500 ug./ml.) was incubated with mutant M. purpurea ATCC 31,164 for four days in germination Medium 2 and the resulting broth containing the streptamine analog of gentamicin was found to be antibiotically active by the disc diffusion assay method against B. subtilis as test organism.

Example 8 dl-2,3,4,6-Tetrahydroxy-l-cyclohexanone (2,4,6cis) described above in Preparation 2 was incubated with mutant M. purpurea ATCC 31,164 for four days in germination Medium 2, and the resulting broth containing a mixture of 0-3-deoxy-4-C-methyl-3-(methylamino)-g-L-arabinopyranosyl(1-)-6)-0-[2-amino-6-(methylamino)-6-C-methyl-2,3,4,6-tetradeoxy-tt-D-erythro-glucopyranosyl-(1Ή)]-D-5-deoxystreptamine; 0-3-deoxy-4-C-methvl-3-(methylamino)-g-L-arabinopyranosyl10 (1-)-6)-0-[2,6-diamino-6-C-methyl-2,3,4,6-tetradeoxy-g-D-erythro-glucopyranosyl- (1-)-4) ] -p-5-deoxystreptamine; and 0-3-deoxy-4-C-methyl-3- (me thy lamino)-β-L-arabinopyranosyl- (1-)-6) 0-[2, 6-diamino-2,3,4,6-tetradeoxy-a-D-erythro-glucopyranosyl(1-)-4) j -D-5-deoxystreptamine was found to be antibiotically 15 active by the disc diffusion assay method against B. subtilis a9 test organism.

Example 9 2,4,5-Trihydroxycyclohexanone (2,4-cis) (500 pg./ml.), described above in Preparation 3,was incubated with mutant M. purpurea ATCC 31,164 for four days in germination Medium 2, and the resulting broth containing a mixture of 0-3-aeoxy-4-C-methyl-3-(methylamino)-g-L-arabinopyranosyl(1-)-6)-0- [2-amino-6- (methylamino) -6-C-methyl-2,3,4,6-tetradeoxy-a-D-erythro-glucopyranosyl- (l-»-4) 3-D-2,5-dideoxystrept25 amine; 0-3-deoxy-4-C-methyl-3-(methylamino)-g-L-arabinopyranosyl-(l-»6)-0-[2,6-dlamino-6-C-methvl-2,3,4,6-tetradeoxyg-D-erythro-glucopyranosyl- (l->-4) 1 -D-2,5-dideoxystreptamine; and 0-3-deoxy-4-C-methy1-3-(methylamino)-g-L-arabinopyranosyl (1-)-6) -0- [2,6-diamino-2,3,4,6-tetradeoxy-a-D-erythro-gluco30 pyranosyl-(1+4)]-D-2,5-dideoxystreptamine was found to be antibiotically active by the disc diffusion assay method against B. subtilis as test organism.

B. Incorporation of Aminocyclitols Example 10 Following a procedure similar to that described in Example 6,using 0.31 g./liter of streptamine sulfate in eight 10 liter fermentations, two in production Medium 5 plus 0.1% added phytone, three in production Medium 5 in which tryptose was. substituted for soybean meal and 3% cerelose was substituted for starch, and three in production Medium 5 in which proteose peptone was substituted for soybean meal and 3% cerelose was substituted for starch in the presence of M. purpurea ATCC 31,164, and isolation of the product as before gave 2.4 g. of a viscous oily residue which was chromatographed on silica gel plates to give 0.56 g. of a pale yellow solid which was shown by tic analysis to be identical with Component 1 described in Example 1 above, namely 0-3-deoxy-4-C-methyl-3-(methylamino)-β-Larabinopyranosyl-(1+6)-0-[2-amino-6-(methylamino)-6-C-methyl2.3.4.6- tetradeoxy-a-D-erythro-glucopyranosyl-(1~*4)]-D-streptamine, m.p. 119-123’C., and 0.996 g. of a pale yellow solid which was shown to be identical with Component 2 described in Example 1 above, namely 0-3-deoxy-4-C-methyl-3-(methylamino) -B-L-arabinop/ranosyl-(1+6)-0-[2,6-diamino-6-C-methyl2.3.4.6- tetradeoxy-x-D-erythro-glucopyranosyl-(1+4)]-D-streptamine, m.p. 115-119’C., [a]?5 (0.2% Hn0) = +137.1°. β z A 200 mg. sample of the free base was converted to the bis-base·pentasulfate·hexahydrate to give 256 mg. of the latter, m.p. 228-230°C. £3807 Anal. Calc'd for 1/2 H2SO4’3H2O: C, 30.85; H, 6.73; N, 8.99; S, 10.29 Found: C, 30.45; H, 6.53; N, 9.03; S, 10.14. The nuclear magnetic resonance spectrum of the base was also consistent with the assigned structure and is summarized as follows: δ_ Integration Assignment .82, 5.94 1 '0 5.59 1 /° CHX 10 5.20 13 0 NH2x4, NHxl, OHx4 3.0-4.6 13 -CHN-x5, -CHO-X6, 3.09 3 ch3-n 1.73 3 ch3-c 1.72 3 ch3-ch 15 1.90-2.5 4 CH2x2 2807 Example 11 Following a procedure similar to that described in Example 6,using 0.10 g./liter of 2,5-dideoxystreptamine in two 80 liter fermentations and six 10 liter fermentations in production Medium 5 in which 0.5% tryptone was substituted for soybean meal and 2% cerelose was substituted for starch in the presence of M. purpurea ATCC 31,164, and isolation of the product as before gave two residues, 0.68 g. and 0.13 g., which were combined and chromatographed on silica gel plates to give three bands whose mass spectra showed them to be, respectively, Component Cg: Q-3-deoxy-4-C-methyl-3-(methylamino) -g-h-arabinopyranosyl-(1-6)-0-[2-amino-6-(methylamino)6-C-methyl-2,3,4,6-tetradeoxy-ct-D-erythro-glucopyranosyl(1-4)]-D-2,5-dldeoxystreptamine; Component C2: O-3-deoxv-415 C-methvl-3-(methylamino)-6-L-arablnopyranosyl-(1^6)-ύ-t2,6dlamlno-6-C-methyl-2,3,4,6-tetradeoxy-a-D-erythro-glucopyranosyl-(1-4)1-D-2,5-dldeoxystreptamine; and Component Cg&: 0-3-deoxy-4-C-methyl-3-(methylamino)-g-L-arabinopyranoayl(1-6)-0-[2,6-diamino-2,3,4,6-tetradeoxy-a-D-erythro-qluco20 pyranosyl-(1-4)]-D-2,5-dideoxystreptamine. The mass spectra for the three above-indicated components Cg, C2 and Cga, showed mass peaks as follows: Component Cg: 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 Cla: M+ 433, M++l 434, 404, 334, 316, 306, 303, 288, 275, 160 and 129.

The Rf values, on tic analysis on silica gel plates using the lower phase of chloroform(l)smethanol(1)concentrated ammonium hydroxide(1) as developing solvent, were 0.43, 0.37 and 0.29, for Components Cg, C2 and cla' respectively. 38 0 7 Example 12 Following a procedure similar to that described in Example 6,using 0.50 g./liter of 2-amino-l,3,4,5,6-cyclohexanepentaol (1,3,5-cis) in three 10 liter fermentations in production medium 5 plus 0.1% added phytone in the presence of M. purpurea ATCC 31164 and isolation of the product as before gave 214 mg. of material which was shown, from its chromatographic mobility and from its mass spectrum, to be identical with Component 1 described in Example 1 above, namely 0-3-deoxy-4-C-methvl-3-(methylamino)-B-L-arabinopyranosyl- (l->-6)-0- [2-amino-6- (methylamino) -6-C-methyl2,3,4,6-tetradeoxy-a-D-erythro-glucopyranosyl- (l->-4) ] -Dstreptamine. The mass spectrum showed mass peaks as follows M+ 493, 457, 436, 383, 376, 366, 363, 346, 344, 338, 335, 321, 318, 261, 160 and 157. - 38 42807 Example 13 A solution of 270 mg. (0.54 millimole) of 0-3deoxy-4-C-methyl-3-methylamino-6-L-arabinopyranosyl-(1+6)0-[2-amino-6-methylamino-6-C-methyl-2,3,4,6-tetradeoxy-aD-erythro-glucopyranosyl-(1+4)]-D-streptamine, described above in Example 1 (Component 1) , dissolved in 5 ml. of 50% aqueous tetrahydrofuran was cooled to 5°C. in an ice bath and treated with 208 mg. (0.59 millimole) of the N-hydroxysuccinimide ester of S-(-)-Y-(N-benzyloxycarbonyl)amino-ahydroxybutyric acid (Konishi et al. O.S. Patent 3,780,018), and the mixture was stirred at 5°C. for twenty hours. The mixture was then concentrated to 10 ml. in vacuo. n-Butanol (25 ml.) and water (10 ml.) 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-(Nbenzyloxycarbonyl)-amino-a-hydroxybutyric acid as before.

Work up of the reaction mixture afforded an additional 435 mg. of crude product which was combined with the 513 mg. previously obtained and chromatographed on seven 40 x 20 cm. silica gel plates,1 mm. thick. The system was developed with chloroform:methanol:concentrated (28%)ammonium hydroxide (3:1:1) (lower phase). Seven passes in this solvent system were necessary, and after eluting the product band which was ultraviolet visible, 229.5 mg. of a crude mixture of the three monoacylated products was obtained.

The mixture of acylated products was put on 4280 three 40 χ 20 cm. silica gel plates, 1 mm. thick, and the plates developed five times with chloroform:isopropanol:concentrated(28%)ammonium hydroxide (4:1:1) (lower phase), twice with chloroform:isopropanol:concentrated(28%)ammonium hydroxide (3:1:1) (lower phase) and nine times with chloroform:methanol:concentrated(28%)ammonium hydroxide (4:1:1) (lower phase). Three distinct bands visible under ultraviolet irradiation were obtained which were separately cut out and eluted from the silica gel with chloroform:methanol:10 concentrated(28%)ammonium hydroxide (1:1:1) (lower phase) affording three components: A, 90.9 mg.; B, 59.1 mg.; and C, 48.5 mg., which are the S-(-)-γ-(N-benzyloxycarbonyl)amino-a-hydroxybutyric acid amide derivatives at the 2'-, 1- and 3-positions, respectively, of 0-3-deoxy-4~C-methyl15 3-methylamino-B-L-arabinopyranosyl-(1+6)-0-[2-amino-6-methylamino-6-C-methyl-2,3,4,6-tetradeoxy-a-D-erythro-glucopyranosyl (1+4)]-D-streptamine. The R^ values for components A, B and C, when developed five times on silica gel with a chloroform:methanol:concentrated(28%)ammonium hydroxide (4:1:1) (lower phase) system, were: A - 0.48 B - 0.S2 C - 0.70 Component B (the 1-amide, 56.1 mg.), dissolved in ml. of 50% aqueous ethanol,and 20 mg. of 10% palladiumon-charcoal were shaken on a Parr shaker at 55 p.s.i. for five and a half hours,after which time the catalyst was removed by filtration through a filter aid. Evaporation of the solvent afforded 34.3 mg. of a white glass which was dis30 solved 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-N- [S-(-)-γ-amino-g-hydroxybutyryl] -03-deoxy-4-C-methyl-3-methylamino-0 -L-arabinopyranosyl- (I1- 6)0-[2-amino-6-methylamino-6-C-methyl-2,3,4,6-tetradeoxy-aD-erythro-glucopyranosyl- (1+ 4)]-D-streptainine as the pentasulfate salt (32 mg.), m.p. 230-235’C. (decomp.); tic, = 0.18 (silica gel, chloroform:methanol;concentrated(28%) ammonium hydroxide:water, 1:4:2:1,- R^ gentamicin C.^ standard = 0.73) .

Anal. Calc'd for C25H50O10Ng·5H2SO4: C, 27.67; H, 5.57; N, 7.75 Found: C, 27.50; H, 5.58; N, 7.42. Components A and C were treated in a similar fashion. A afforded 47 mg. of 2'-N-fS- (-) -γ-amino-ct--hydroxybutyryl ]-0-3-deoxy-4-C-methyl-3-methylamino-8-L-arabinopyranosyl- (1 6) -0- [2-amino-6-methylamino-6-C-methyl-2,3,4,6tetradeoxy-ct-D-erytliro-glucopyranosyl- (1--4) ] -D-streptamine as the bis-base-tetrasulfate-heptahydrate, m.p. 237-241°C. (decomp.); tic, Rj - 0.33 [silica gel, chloroform:methanol:concentrated(28%)ammonium hydroxide:water, 1:4:2:1,- Rj gentamicin C·^ standard = 0.73].

Anal. Calc'd for (C25H50N6O10)2-4h2SO4-7H2O: C, 35.16; H, 7.20; N, 9.84 Found: C, 35.38; H, 7.08; N, 9.49. Component C afforded 26 mg. of 3-N-Cs-(-)-γ-amino-Khydroxybutyryl)-0-3-deoxy-4-C-methyl-3-methylamino-g-Larabinopyranosyl-(1 -6)-0-[2-amino-6-methylamino-6-C-methyl2,3,4,6-tetradeoxy-'i-D-erythro-glucopyranosyl-(1+4))-Dstreptamine as the bis-base-pentasulfate-trihydrate, m.p. 220-230°C. (decomp.;,· tic. Rj = 0.30 [silica gel, chloroform:methanol:concentrated(28%)ammonium hydroxide:water, 1:4:2:1; Rf gentamicin C·^ standard = 0.733 .

Anal. Calc'd for (^25Η5θΝ6θ·^ο) '3^0: C, 34.64; H, 6.74; N, 9.67 Found: C, 34.35; H, 6.38; N, 8.60.

Proceeding in a manner similar to that described in Example 13 using the respective antibiotics described in Examples 2, 3, 4 and 5 and either the N-hydroxysuccinimide ester of S-(-)-γ-(N-benzyloxycarbonyl)amino-a-hydroxybutyrio acid or the pentafluorophenyl ester of N-(benzyloxycarbonyl)10 (S)-isoserine (S)-8- amino-a-hydroxypropionic acid , described by Haskell et al., Carbohydrate Research, 28, 273-280 (1973), the following compounds of Formula I are similarly prepared: Example 14 1-N-Γ(S)-8-Amino-a-hydroxypropionyl~| -0-3-deoxy-4-C15 methyl-3-methylamino-g-L-arabinopyranosyl-(1+6)-0-f2,6-diamino-6-C-methyl-2,3,4,6-tetradeoxy-a-D-erythro-glucopyranosyl(1+4)1epistreptamine and 2 *-N-Γ(S)-8-amino-a-hydroxypropionyll0-3-deoxy-4-C-methyl-3-methylamine-8-L-arabinopyranosyl- (1+6) 0- [~2,6-diamino-6-C-methyl-2,3,4,6-tetradeoxy-a-D-erthro20 gluoopyranosyl-(1+4)]epistreptamine.

Example 15 1-N-fS—(-)-y-Amino-a-hydroxybutyrylT-0-3-deoxy-4-Cmethyl-3-methylamino-8-L-arabinopyranosyl-(1+6)-0-[2,6-diamino-2,3,4,6-tetradeoxy-a-D-erythro-glucopyranosyl-(1+4)1 25 2,5-dideoxystreptamine and 21-N-fs-(-)-γ-amino-o-hydroxybutyryl]-0-3-deoxy-4-C-methyl-3-methylamino-8-L-arabihopyranosyl(1+6)-0-f2,6-diamino-2,3,4,6-tetradeoxy-a-D-erythro-glucopyranosyl-(1+4)3-2,5-dideoxystreptamine.

Example 16 1-N-Γ(S)-8-Amino-a-hydroxypropionyll-0-3-deoxy-4-C42 42807 methyl-3-methylamino-β-L-arabinopyranosyl-(1+6)-0-[2,6-diamino-2,3,4,6-tetradeoxy-a-D-erythro-glucopyranosyl-(1+4)]5- iodo-2,5-dideoxystreptamine and 2 1 - N- [(S)-g-amino-a-hydroxypropiony1]-0-3-deoxy-4-C-methy1-3-me thylamino-g-L-arabinopyranosy1-(1+ 6)-0-[2,6-diamino-2,3,4,6-tetradeoxy-a-Derythro-glucopyranosyl-(1+4)]-5-iodo-2,5-dideoxystreptamine.

Example 17 1-N-fs-(-)-Y-Amino-a-hydroxybutyryl]-0-3-deoxy-4-Cmethyl-3-methylamino-B-L-arabinopyranosyl-(1+6)-0-[2-amino6- methylamino-6-C-methyl-2,3,4,6-tetradeoxy-a-D-erythroglucopyranosyl-(1+4)]-5-fluoro-2,5-dideoxystreptamine and 21 - N-Es-(-)-y-amino-a-hydroxybutyryl1-0-3-deoxy-4“C-methyl-3methylamino-g-L-arabinopyranosyl-(1+6)-0-[2-amino-6-methylamino-6-C-me thyl-2,3,4,6-tetradeoxy-a-D-erythro-glucopyranosyl(1+4)]-5-fluoro-2,5-dideoxystreptamine.

ANTIBACTERIAL TEST RESULTS The 0-3-deoxy-4-C-methyl-3-methylamino-g-L-arabinopyranosyl-(1+6)-0-[2-amino-6-methylamino-6-C-methyl-2,3,4,6tetradeoxy-a-D-erythro-gluoopyranosyl-(1+4)]-D-streptamine, described above in Example l,and designated Component 1 was tested in comparison with gentamicin against a number of microorganisms according to the following procedure.

Stock solutions of each compound containing 200 meg./ml. base were prepared in distilled water and filtersterilized. Cultures of the test organisms were grown for twenty-four hours at 37°C. in 10 ml. tubes of tryptose phosphate or Mueller-Hinton broth. Each culture was adjusted with broth to 0.1 optical density on a Spectronic 20 (Registered Trade Mark) (approximately 108 cells/ml.). The adjusted cultures were diluted 1:500 in broth for use as inoculum (final cell concentration 42007 approximately 2 χ 105 cells/ml.). The test compounds were tested for antibacterial activity by a single-row tube dilution method. Master two-fold serial dilutions were made in broth from the stock drug solutions, and 0.2 ml. of each drug concentration was placed in seventeen 13 x 100 mm. tubes. All tubes were inoculated with 0.2 ml. of the appropriate diluted culture (final cell concentration per tube = 10$ cells/ml.). Minimum inhibitory concentrations (lowest drug concentration showing no visible growth) were read after sixteen hours incubation at 37°C.

The results are given in Table III below. The compounds were considered inactive at inhibitory concentrations greater than 100 meg./ml.

Table III* Organism Minimal Inhibitory Cone. (meg./ml.) Component 3 Gentamicin Staphylococcus aureus Smith 0.78 0.195 Escherichia coli Vogel 3.13 3.13 20 Escherichia coli W677/HJR66 50 Inactive Escherichia coli JR 35 3.13 6.25 Escherichia coli JR 76.2 6.25 100 Escherichia coli JR 89 50 50 Escherichia coli K12 ML 1629 3.13 1.56 25 Enterobacter cloacae A-20960 1.56 25 Klebsiella pneumoniae 39645 1.56 0.78 Klebsiella pneumoniae A-20636 3.13 50 Proteus mirabilis MGH-1 6.25 1.56 Providencia 164 100 Inactive 30 Providencia stuartii A-20894 100 100 Pseudomonas aeruginosa MGH-2 3.13 0.39 Pseudomonas aeruginosa A-20717 12.5 4:12.5 Pseudomonas aeruginosa A-20741 Inactive Inactive Pseudomonas aeruginosa A-20897 12.5 Inactive 35 *Cultured in tryptose phosphate broth.

The same Component 1 described above in Example 1, 0-3-deoxy-4-C-methyl-3-methylamino-8-L-arabinopyranosyl-(1+6)0-[2-amino-6-methylamino-6-C-methyl-2,3,4,6-tetradeoxy-a-Derythro-glucopyranosyl-(1+4)]-D-streptamine, was tested in comparison with gentamicin complex (C-^, C2 and C^a) and gentamicin Cg, and the 1-, 3- and 2'-(S-(-)-Y-amino-ot-hydroxybutyryl] amides of Component 1, described above in Example 21, and designated, respectively, Component 1 (1-HABA), Component 2 (3-HABA) and Component 1 (2'-HABA), were tested in comparison with the corresponding 1-, 3- and 2'-[S-(-)-Y-aminoα-hydroxybutyryl]amides of gentamicin Cj_ (all described by Konishi et al. United States Patent 3,780,018, patented December 18, 1973) and designated, respectively, Cg (1-HABA), Cg (3-HABA) and Cg (2'-HABA) . These results in minimum inhibitory concentrations (meg./ml.) are given in Table IV below where test organisms 1,2,3,4,5 and 6 indentify B. subtilis ATCC 6633, S. aureus Smith, E_.coli JR 76.2, Ent. cloacae A-2O96Q, K pneumoniae A-20636 and Ps. aeruginosa Compound A-20897, respectively. Table IV* 6_ 1 Test Organisms 2_ 3_ 4_ 5_ Gentamicin Cl' c2' cla <0.024 0.39 50 12.5 25 >100 Gentamicin Ci 0.049 0.78 50 12.5 25 >100 Component 1 0.098 1.56 6.25 0.78 3.13 25 Ci (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 Cx (3-HABA) 3.13 25 >100 100 >100 >100 Component 1 (3-HABA) 6.25 50 100 50 50 >100 Cx (2’-HABA) 6.25 50 100 25 50 >100 Component 1 (2 -HABA) 1.56 25 50 25 50 >100 *Cultured in Mueller-Hinton broth.

Claims (27)

1. CLAIMS:1. A compound having the formula I (herein) where R^, Rg and Rg each represent hydrogen, or one of R·^, Rg and Rg represents an ω-amino-ctj hydroxy-lower-alkanoyl group having the formula: H 2 NCH 2 (CH 2 ) n CHOHCOwhere n is zero or 1, the others of Rj, Rg and Rg being hydrogen; R 2 represents hydrogen or hydroxy; Rg represents hydrogen, hydroxy or halogen, except that when R 2 hydro.0 gen, Rg is not hydroxy cis to the amino groups at the 1and 3-positions; and Rg and Ry each represent hydrogen or methyl.

2. A compound according to claim 1, where Rg_, Rg and Rg each represent hydrogen. .5

3. A compound according to claim 1, where one of R^, Rg and Rg represents an ω-amino-a-hydroxy-lower-alkanoyl group*having the formula: H 2 NCH 2 (CH 2 ) n CHOHCOwhere n is zero or 1, the others of R^, Rg and Rg being 0 hydrogen.

4. A compound according to claim 3, where R 2 and Rg each represent hydroxy.

5. 0-3-Deoxy-4-C-methyl-3-methylamino-B-L-arabinopyranosyl-(1+6)-0-[2-amino-6-methylamino-6-C-methyl-2,3,4,6 5 tetradeoxy-a-D-erythro-glycopyranosyl-(1+4)]-D-streptamine.

6. 2'-N-[S-(-)-γ-Amino-a-hydroxybutyryl]-0-3-deoxy-446 C-methyl-3-methylamino-g-L-arabinopyranosyl-(1+6)-0-[2-amino6-methylamino-6-C-methyl-2,3,4,6-tetradeoxy-a-D-erythroglucopyranosyl-(1+4)]-D-streptamine.

7. 1-N-[S-(-)-γ-Amino-a-hydroxybutyryl] -0-3-deoxy-4-Cmethyl-3-methylamino-g-L-arabinopyranosyl-(1+6)-0-[2-amino6-methylamino-6-C-methyl-2,3,4,6-tetradeoxy-a-D-erythroglucopyranosyl- (1-,4) J-D-streptamine.

8. A process for preparing a compound according to claim 1, where R^, Rg and Rg each represent hydrogen, and Rg, Rg, Rg and Ry have the indicated meanings, which comprises culturing a nutrient medium containing carbohydrates, a source of assimilable nitrogen, essential salts and a corresponding added aminocyclitol having the Formula II (herein) where R^ R 2 , Rg and Rg have the meanings given above, and where R^ and Rg can in addition represent a single bond joining the two amino nitrogen atoms together, in the presence of a Micromonospora purpurea ATCC 31, 119, and isolating the product from the culture medium, a compound of Formula I where both R^ and Rg are hydrogen being produced when R·^ and Rg in Formula II represent a single bond.

9. A process for preparing a compound according to claim 1, where R x , Rg and R g each represent hydrogen and R 2 , Rg, Rg and Ry have the indicated meanings; except that R 5 does not include halogen, which comprises culturing a nutrient medium containing carbohydrates, a source of assimilable nitrogen, essential salts and a cyclitol having the Formula Ilia or Illb (herein) where, in either case, R is hydrogen or acetyl; Rg 1 is oxo or hydroxy; and Rg' and Rg' each are hydrogen or OR, in the presence of Micromonospora purpurea ATCC 31, 164.

10. A process for preparing a compound according to claim 1, where R^, R 3 and Rg each represent hydrogen and Rg, Rg, Rg and Ry have the indicated meanings, except that Rg does not include halogen which comprises culturing 5 a nutrient medium containing carbohydrates, a source of assimilable nitrogen, essential salts and an aminocyclitol having the formula Ila (herein), where R 2 1 and Rg' correspond to R 2 and Rg, respectively, and Rj' is amino or hydroxy in the presence of Micromonospora purpurea ATCC 10 31, 164.

11. A process for preparing a compound according to claim 1, where one of R^, Rg and Rg represents an ω-aminoα-hydroxy-loweralkanoyl group, which comprises reacting the compound of Formula I obtained according to any one of 15 claims 8 to 10 with an N-hydroxy-succinimide-ester having the - 48 42807 Formula IV (herein) where n is zero or 1, and subjecting the resulting product to hydrogenolysis with hydrogen over a catalyst.

12. A process according to claim 9, for preparing 0-3-deoxy-4-C-methy1-3-(methylamino)-β-Larabinopyranosyl-(1+6)-0-[2-amino-6-(methylamino)-6-Cmethy1-2,3,4,6-tetradeoxy-a-D-erythro-glucopyranosyl-(1+4)]D-streptamine,wherein the added cyclitol is scyllo-inosose.

13. A process according to claim 9, for preparing 0-3-deoxy-4-C-methy1-3-(methylamino)-β-Larabinopyranosyl-(1+6)-0-[2-amino-6-(methylamino)-6-C-methyl2.3.4.6- tetradeoxy-a-D-erythro-glucopyranosyl-(1+4)]-D-5deoxystreptamine; 0-3-deoxy-4-C-methyl-3-(methylamino)-BL-arabinopyranosyl-(1+6)-0-[2,6-diamino-6-C-methyl-2,3,4,6tetradeoxy-a-D-erythroglucopyranosyl-(1+4)]-D-5-deoxystreptamine; or 0-3-deoxy-4-C-methy1-3-(methylamino)-β-L-arabinopyranosy1-(1+6)-0-(2,6-diamino-2,3,4,6-tetradeoxy-a-D-erythroglucopyranosyl-(1+4)]-D-5-deoxystreptamine,wherein the added cyclitol is dl-2,3,4,6-tetrahydroxy-l-cyclohexanone (2,4,6cis) .

14. A process according to claim 9, for preparing 0-3-deoxy-4-C-methyl-3-(methylamino)-β-Larabinopyranosyl- (1+6) -0-[2-amino-6-(methylamino)-6-C-methyl2.3.4.6- tetradeoxy-a-D-erythro-glucopyranosyl-(1+4)]-D-2,5dideoxystreptamine; 0-3-deoxy-4-C-methyl-3-(methylamino)-βL—arabinopyranosyl—(1+6)—0—[2,6—diamino—6— C-methyl-2,3,4,6-tetradeoxy-a-D-erythro-glucopyranosyl(1+4)]-D-2,5-dideoxystreptamine; or 0-3-deoxy-4-C-methyl3-(methylamino)-β-L-arabinopyranosyl-(1+6)-0-[2,6-diamino2,3,4,6-tetradeoxy-a-D-erythro-glucopyranosyl-(1+4)]-D-2,549 4 28 07 dideoxystreptamine,wherein the added cyclitol is 2,4,5-trihydroxycyclohexanone (2,4-cis).

15. The process according to claim 10, for preparing 0-3-deoxy-4-C-methy1-3-(methylamino)-β-L5 arabinopyranosyl-(1-6)-0-[2-amino-6-(methylamino)-6-C-methyl 2.3.4.6- tetradeoxy-a-D-erythro-glucopyranosyl-(1-4)-D-strept amine and 0-3-deoxy-4-C-methyl-3-(methylamino)-β-L-arabinopyranosyl-(1-6)-0-[2,6-diamino-6-C-methy1-2,3,4,6-tetradeoxy a-D-erythro-glucopyranosyl-(1-4)]-D-streptamine wherein the 10 added aminocyclitol is streptamine.

16. A process according to claim 10, for preparing 0-3-deoxy-4-C-methyl-3-(methylamino)-β-Larabinopyranosyl-(1+6)-0-[2-amino-6-(methylamino)-6-C-methyl 1 2.3.4.6- tetradeoxy-a-D-erythro-glucopyranosyl-(1+4)]-D-2,515 dideoxystreptamine; 0-3-deoxy-4-C-methyl-3-(methylamino)-β-L·· arabinopyranosyl-(1+6)-0-[2,6-diamino-6-C-methyl-2,3,4,6tetradeoxy-a-D-erythro-glucopyranosyl-(1-4)]-D-2,5-dideoxystreptamine or 0-3-deoxy-4-C-methy1-3-(methylamino)-β-Larabinopyranosyl-(1+6)-0-[2,6-diamino-2,3,4,6-tetradeoxy-B20 D-erythro-glucopyranosyl-(1-4)]-D-2,5-dideoxystreptamine, wherein the added aminocyclitol is 2,5-dideoxystreptamine.

17. A process according to claim 10, for preparing 0-3-deoxy-4-C-methyl-3-(methylamino)-β-Larabinopyranosyl-(1+6)-0-[2-amino-6-(methylamino)-6-C-methyl· 25 2,3,4,6-tetradeoxy-a-D-erythro-glucopyranosyl-(1-4)-D-streptamine ,wherein the added aminocyclitol is 2-amino-l,3,4,5,6cyclohexanepentaol (1,3,5-cis)

18. A process according to any one of claims 8 to 17, which includes converting a free base obtained to an acid30 addition salt thereof. 50 42807

19. An acid-addition salt of a compound according to any one of claims 1 to 7.

20. A process for preparing a compound according to claim 1, or an acid-addition salt thereof, substantially 5 as herein described with reference to the Examples.

21. A process for preparing a compound according to claim 1, or an acid addition salt thereof, substantially as herein described with reference to Examples 1 to 5 and 13 to 17. 10

22. A process for preparing a compound according to claim 1, or an acid-addition salt thereof, substantially as herein described with reference to Examples 6 to 12.

23. A compound prepared by the process according to any one of claims 8 to 18 and 20 to 22. 15

24. A compound according to claim 1, substantially as herein described with reference to the Examples.

25. A compound according to claim 1, substantially as herein described with reference to Examples 1 to 5 and 13 to 17. 20

26. A compound according to claim 1, substantially .is herein described with reference to Examples 6 to 12.

27. A pharmaceutical composition which comprises a compound according to any one of claims 1 to 7, 19 and 23 to 26 and a pharmaceutical carrier.