CA1061731A - Aminocyclitol antibiotics and processes therefor - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
Aminocyclitol analogs of gentamisin C1, C2 and C1a and the corresponding compounds acylated on the 1-, 3- and 2'-amino groups with an .omega.-amino-.alpha.-hydrowy-lower-alkanoyl group are prepared by culturing a nutrient medium containing carbo-hydrates, a source of assimilabe nitrogen, essential salts and an added cyclitol or aminocyclitol with mutants of Micromonospora purpurea and acylating the product with an ester of an .omega.-N-benzyloxycarbonyl) amino-.alpha.-hydroxy-lower-alkanoic acid followed by catalytic hydrogenolysis of the benzyloxycarbonyl group.

Description

1 0 ~ 3 1 This invention relates to aminocyclitol antibiotics and in particular compounds having the formula:

I\ H-NH-R6 L~
R8-N~ ~
d~__ ~ ~ H-R3 ~R2 R5~ ~\
~IH-R
I"--~ .,, H~ \ :
CH3N~ \~ 3 .:
where Rl, R3 and R8 each represent hydrogen, or one of Rl, R3 and R8 represents an ~-amino--hydroxy-lower-alkanoyl group having the formula:

H2NCH2 (CH2 ) ncHoHco-where n is zero or l, the o~her of Rl, R3 and R8being h~x~en;
R2 represents hydrogen or hydroxy; R5 represents hydrogen, hydroxy or halogen (including fluorine, chlorine, bromine and iodine), except that when R2 is hydrogen, R5 i~

-2-;1'7~
not hydroxyj and R6 and R7 each reprcsent hydrogen or methyl.
Compounds of Formula I where Rl R3 and R8 are hydrogen are prepared hy the method described in Shier et al.
United States Patent 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:
HO ~ ~H-R3 R \ ~ R2 5 ~ \
HO ~__N~-Rl ...II
where Rl R2 R3 and R5 have the meanings given above and where Rl and R3 can in addition represent a single bond join-ing the two amino nitrogen atoms to~ether in the presence of a suitable mutant microorganism said mutant microorganism ~ being incapable of synthesizing said aminocyclitol itself but being capable of incorporating said aminocyclitol into the compound of Formula I specifically a mutant of Micro-monospora purpurea designated Micromonospora purpurea ATCC
31 119 and isolating the product from the culture medium.
The compounds of Formula I where both Rl and R3 are hydrogen are produced when the aminocyclitol of Formula II where Rl and R3 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 amino-cyclitol subunit from a nutrient medium to thereby produce 2S the antibiotic but is capable of incorporating the latter into an antibiotic when the aminocyclitol is added to the nutrient medium.

-3-` ` ' ' ~, ':

Moreover, studies with radiolabelled compounds have demonstrated that non-nitrogen containing cyclitols are prob-able biogenetic precursors of aminocyclitols of the type represented by Formula II, such as streptamine and deoxy-streptamine [Rinehart et al., J. Am. Chem. Soc. 96, 2263-2265 (1974); Walker et al., Biochem. 8, 763-770 (1969);
De~ain et al., Bacteriol. Rev. 34, 1-19 (1970)]. Neverthe-less, although it is known that aminocyclitols can be in-corporated into aminocyclitol antibiotics either by culture o~ 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 the presence of a microorganism mutant which is incapable of bioqynthesizing the aminocycli~ol as such but which is capable of incorporating the aminocyclitol into the anti-biotic [e.g., the incorporation of streptamine or epistrept-amine 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 hexahydr-2S oxy cyclohexane monomethyl ether (quebrachitol) into amino-cyclitol antibiotics using the Rinehart/Shier method were completely unsuccessful lTesta et al., J. Antibiotics 27, 917-921 (1974)].
Yet a process that would permit the use of cyclitols, instead of aminocyclitols, for incorporation into aminocyclitol-

-4-lV~731 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 amino-cyclitols are invariably much more expensive than the non-aminated cyclitols, significant savings in costs of the final products could be realized. (For example, streptamine, at preqent prices, costs about $1 per gram, whereas its probable biogenetic precursor, scyllo-inosose, can be obtained in about 80~ yield by fermentative oxydation of myo-inositol, which only costs about 2 cents per gram at present).
In accordance with an improved process of the pres-ent invention, it has been surprisingly found that aminocycli-tol antibiotics of the streptamine, deoxystreptamine or dideoxy-streptamine type having the general structure of Formula I
above can be produced by culturing a nutrient medium contain-ing carbohydrates, a source of assimilable nitrogen, essential salts and a cyclitol of the 2-R2'-5-R5'-~,4,6-trihydroxy-cyclohexanone or 2-R2'-5-R5'-1,3,4,6-tetrahydroxycyclohexane class xepresented by the formula:
RO

~_ R2 \
. RO ~ OR ... IIIa;
or of the 5-R5'-1,4,6-trihydroxycyclohex-2-ene class repre-9ented by the formula:

R
RO R ... IIIb

-5- .

: .

7~1 where, in either case, R is hydrogen or acetyl; R3~ i8 OXO
(-01 or hydroxy; and R2l and R5~ each are hydrogen, hydroxy or OR, in the presence of mutants of organisms which are incapable of biosynthesizing the cyclitol unit but which S are capable of incorporating these cyclitol unit into the antibiotic molecule as an aminocyclitol unit.
Illustrative aminocyclitol antibiotics which can be obtalned by the present process, and the organisms whose mutants are used to produce them, are represented by the following types:
Antiabiotic Microorganism hybrimicin Streptomyces fradiae gentamicin Micromonospora pur~urea and Micromonospora echlnospora tobramicin Streptomyces tenebrarius ribostamicin ~ ribosidificus sisomicin Micromonospora inyoensis .. ~ .
kanamicin Streptomyces kanamyceticuæ
butirosin Bacillus circulans The above process can be used for preparing genta-micin type antibiotics having the Formula I above where R6 and R7 each represent hydrogen or methyl; R2 and R5 each represent hydrogen or hydroxy; Rl, R3 and R8 represent hydrogan; which comprises culturing a nutrient medium con-~aining carbohydrates, a source of a~similable nitrogen, essential salts and a cyclitol having the Formula IIIa or IIIb above. A specific strain of a microorganism for carrying out the process is M.E~urpurea ATCC 31,164.
: - .

U~

It is also significant that the same mutant, ~
purpurea ATCC 31,164, is also capable of incorporating into the antibiotics of Formula I above an aminocyclitol having the formula:

HO ~ R ' R ' I \
HO ~ _Rl' ...IIa where R2' and R5' are each hydrogen or hydroxy and Rl' is amino or hydroxy, or Schiff bases of the compounds of Formula IIa, for example Schiff bases of benzaldehyde and the aminocyclitols of Formula IIa, and having the formula:
Ho~ N=CHC6H5 I \~R2 R5A~ ~ Rl' ...IIb where Rl' is hydroxy or benzylideneimino (C6H6CH=N), and R2' and R5' have the same meanings as in Formula IIa. The pro-cess, which can prepare compounds of the Formula I where R6 ~ and R7 each represent hydrogen or methyl; R2 and R5 each `~ 15 represent hydrogen or hydroxy; and Rl- R3 and R8 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 carbo-hydrates, a source of assimilable nitrogen, essential salts and an aminocyclitol having the Formula IIa or IIb in the presence of N. purpurea ATCC 31,164. This process is con-~idered to be within the purview of the present invention.

, . ~ .
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~.~. . .

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 II above where Rl and R3 are hydrogen; R2 is hydrogen or hydroxy; and R5 is hydrogen, hydroxy or halogen, can also be carried out in the presence of the microorganism mutant, Micromonospora pur~ rea ATCC 31,119. However, as indicated above, the aminocyclitols required as substrates in that process are generally rathex 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.
~EEgE~ ATCC 31,164 for incorporation into aminocyclitol antibiotics thus provides an advantage over the ~irst described process of the invention.
In either case, whether the non-nitrogen contain-ing cyclitols of Formulas IIIa or IIIb or the aminocyclitols of Formulas II, IIa or IIb are used as substrates, the com-pounds of Formula I are prepared by culturing a nutrient medium containing carbohydrates, a source of nitrogen, Rssential salts and an added cyclitol of Formulas IIIa or IIIb or an aminocyclitol of Formulas IIa or IIb in the presence of the mutant, M. pu_purea ATCC 31,164, and isolat-ing the product from the culture medium.
The compounds of Formula I, where one of Rl, R3 and R8 represents an ~-amino-~-hydroxy-lo~!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 Rl, R3 and R8 is hydro-gen with an N-hydroxysuccinimide ester having the formula:

.. . . ~ . .

~:H~O-C~ C~2 (CH2) nCHOHCO-O-N~ . . . IV

where n has the meanings given above. The resulting mixture of the compounds of Formula I where one of Rl, R3 and R8 is the ~-(N-benzyloxycarbonyl)amino-~-hydroxy-lower-alkanoyl group:

~CH20-C-NHCN2 (C~12)nCHONCO-the other two being hydrogen, is then subjected to hydrogen-olysis of the benzyloxycarbonyl group with hydrogen over a catalyst.
As indicated above, when a compound of ~ormula I
where each of Rl, R3 and R8 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 Rl, R3 or R8 amine hydrogen atoms is replaced by the ~-(N-benzyloxycarbonyl)amino-~-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, preferàbly at a temperature from -10C. to about 10C., and in an aqueous solution of an inert organic solvent, for example tetrahydrofuran, dioxane, ethylene glycol dimethyl ether, dimethylacetamide, dimethyl-formamide, propylene glycol dimethyl ether, and the like.
Hydrogenolysis of the benzyloxycarbonyl group is _g_ ~ ~

lV~17;~

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, propylene glycol dimethyl ether, and the like.
The aminocyclitols of Formula II where Rl, R2 and R3 are hydrogen and R5 is fluorine or iodine are novel com-pounds which are considered to be within the purview of the instant invention, and are prepared as described hereinbelow.
Other aminocyclitols of Formula II, which are also useful in the practice of the present invention, are known compounds.
~hese are:
~y~byc~ ylfate ~Peck et al., J. Am. Che~ Soc.
6B, 77~ tl946)];
~ ide [Suami et al., . rg. Chem. _, 2831 (1968)];
2,5-dideoxystreptamine dihydrochloride, m.p. >300C.;
an 4-diol (exo, exo), m.p. - 3 C. I~ rt~r'r;~E~ two compounds disclosed by ~esta et al., J. Antibiotics 27, 917-921 (1974)].
~he N-hydroxysuccinimide esters of Formula IV are a generally known class of compounds.
The compounds of Formula I have been tested in a ~tandard serial dilution antibacterial test and have been found to have antibacterial acti~ity, particularly against gentamicin reQistant organisms. The compounds are thus use- ;
ful as antibacterial agents.
The compounds of Formula I are primarily intended ~ -for oral, topical or parenteral administration and can be prepar~d for use with a pharmaceutical carrier by suspension, either in the form of their free bases or as pharmaceutlcally j~; -acceptable, non-toxic acid addition salts, in an inert -10- ' :

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carrier such as polyethylene glycol, or by tabletting or en-capsulation 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 invention were assigned on the basis of study of their chromatographic characteristics determined by thin layer chromatographic (tlc) analysis; their nuclear magnetic resonance ~nmr) and mass spectra; by degradation to known compounds; by comparison of the products prepared by fermen-tation with ~utant M. purpurea ATCC 31,119 using amino-cyclitols of Formulas II, IIa or IIb as substrates with pro-ducts prepared by fermentation with mutant M. E~urea ATCC
31,164 using cyclitols of Formulas IIIa or IIIb 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 proce~s 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)] t0.40 mole) was subjected to microbiological oxida- -tion by Acetobacter suboxydans using the procedure describ-ed by Po~ternak, 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 filter aid, and B 30 the filtrate was passed over 180 g. of Dowex resin 50-124.

~* ~e~. 7;/'1. -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-deox~inosose [dl-2,3,4,5-tetrahydroxy-1-cyclohexanone (2,4-cls)], m.p. 221-222C. Further concentration of the filtrate from the first crop afforded three additional crops totalling 28.7 g., m.p. 220-221C.
Preparation_2 A solution of 4.5 g. (0.031 mole) of 2,5-dideoxy-streptamine in 20 ml. of benzaldehyde was warmed on a steam bath, and the mixture was flushed with nitrogen and set aside for about eight hours. The solution was then diluted with 150 ml. of benzene, cooled, and the solid which sepa-rated was 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-~benz~lideneamino)-1,3-cyclohexanediol (1,3-cis), m.p. 151-154C.
Preparation 3 A solution of 9.5 g. (0.053 mole) of dl-epl-inosose [Posternak, Helv. Chim. Acta 19, 1333 (1936)] in 300 ml. of O.lN 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 re-àction mixture, concentration of the filtrate to dryness and recrystalli2ation of the residue from aqueous methanol.
There was thus obtained dl-epi-quercitol, m.p. 214-21~C.
The latter was subjected to microbiological oxi-dation by Acetobacter ~ using the procedure describ-ed by Posternak recorded above in Preparation 1, and the pro-duct was isolated as described in Preparation 1 and recrystal-; -12-:' ..
, lized from ethanol to give dl-2,3,4,6-tetrahydroxY-l-cvclo-hexanone (2,4,6-cis), m.p. 175-177C.
PreParation 4 To a solution of 22.5 g. (0.11 mole) of 3-chloro-perbenzoic acid in 150 ml. of methylene dichloride was added11.4 g. (0.1 mole) of 4-cyclohexene-1~,2~-diol ~McCasland et al., J. Org. Chem. 28, 898 (1963)], and the resulting solution was stirred with cooling while maintaining the tempQrature below 30C. 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 con-ventional manner to give g.l g. of crude material which was recrystallized from ethanol/ether to give two crops totalling lS 4.75 g. of ~ , m.p. 80C. and 70-75C.
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 heat-ed on a steam bath for about twenty hours, an additional 0.03 ml. of boron trifluoride etherate added, and the solvent re-moved in vacuo. The residue was treated with 50 ml. of ethanol, the resulting solid separated, and the filtrate con-centrated 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. o 2,4,5-tri-whose mass spectrum gave mass peaks at 144 and 145 and whose infrared spectrum showed a strong peak at 1723 cm 1, Preparation 5 2-Deoxy-1,6:3,4-dicarbonylstreptamine [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 ~ liters of methanol and the product filtered and dried to give 39 g. of 2-deoxy-S-methane-sulfony~ 6.3~4-di carbo ~ trept_mine, m.p. 264-266C.
A mixture of 2 g. (0.0069 mole) of 2-deoxy-5-methane-sulonyl-1,6:3,4-dicarbonylstreptamine, described above, and 4.8 g. (0.032 mole) of sodium iodide in 70 ml.
of dimethylformamide was heated at 125C. for twenty-four hours and then taken to dryness. The crude 2,5-dideoxy-5-iodo-1,6:3,4-dicarbonylstre~tamine 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 ~acuo. The crude 2,5-dideoxy-5-iodostreptamine dihydro-chloride was dissol~ed in 30 ml. of acetic anhydride, the solution treated with 5.25 g. of sodium acetate and the mix-ture refluxed for two and a half hours. The mixture was than cooled, poured into 200 ml. of water and extracted with chloroform. The chloroform extracts, on washing once with ~x~um thiosulfate soluti~n, once with brme, once with watex and evapora-~ion to dryness, afforded an oil which was crystallized ~rom ethanol to give two crops totalling 1.1 g. of N,N'- -, m.p. 256-258~C.
Hydrolysis of the latter by refluxing with aqueous ffl dro-chloric acid and isolation from a basic medium affords 2,5-dideoxy~;5-iodostr.epta.mine. -: ' .; ' .

2-Deoxy-5-methanesulfonyl-1,6:3,4-dicarbonyl~trept-amine (39 g., 0.12 mole), described in Preparation 5 above, was suspended in approximately 200 ml. of 6N hydrochloric acld, the mixture warmed on a steam bath for two hours, evaporated to dryness in va¢uo, mixed with 200 ml. of i~opropyl alcohol and evaporated to dryness once again. The residual oil was triturated with methanol, cooled and the ~olid collected and recryqtallized from methanol to give 2-deoxy-$-methanesulfo_-yl8treptamine dihydrochloride~ m.p. 208-210C.
Anal- Calc'd for C7H16N2O5S-2HCl: C, 26.84; 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-methanesulfonylstreptamine in 45 ml. of water and 90 ml. of lS 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. o 2N sodium hydroxide from another.
When addition was complete, the mixture was stirred for an additional fifteen minute~, diluted with about 50 ml. of toluene, ~tirred for three hour3 and filtered. Recry~talliza-tion of the solid from ethanol afforded 4.0 g. of N,N'-di-, m.p. 198-201C.
Anal. Calc d for C23H28N2OgS 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-hexaoxacycloo¢tadecane, using the procedure dQscribed by ~iotta et al., J. Am. Chem. Soc. 96, 2250-2252 (1974) affords N,N'-dicarbobenzoxy-2,5-dideoxy-5-fluoro-streptamine which, on hydrolysis with aqueous mineral àcid, affords 2,5-dideoxy-5-fluorostreptamine .

MUTATION PROCESSES
In the following procedures, various media con-stituted as follows were employed.
Medium 1: N-Z Amine g ./1 .
Glucose 10 Soluble starch20 Yeast extract** 5 N-Z-Amine-Type A (Difco) 5 CaC03 Agar 15 Medium 2: Germination Medium (in distilled water) Beef extract0.3%
Tryptone 0.S%
Dextrose 0.1%
Soluble starch2.4%
Yeast extract0.5%
CaCO3 0.4%
: Medium 3: Soybean-Glucose g ./1 .
Soybean meal 30 Dextrose (cerelose) 40 CaC03 :
.

** Reg. T.M.

~B
... .. . . .. ........... . . ..
.... .. ... . .
~ ~. - . . .. ~.

'731 Medium 4: TGE
g.~l.
Trypticase glucose extract..... 5.0 Trypticase peptone............. 3.0 Glucose........................ 1.0 Agar........................... 15.0 Medium 5: Production Medium Beef extract................... 0.3%
Yea~t extract.................. 0.5%
Soybean meal................... 0.5%
Maltose........................ 0.1%
Starch~........................ 2.4 Casamino acid.................. 0.1~
caco3----------~ 0,4%
CoC12~6H2O..................... 1 mg.~liter Medium 6: Streptomicin Assay Agar Beef extract Yeast extract.................. 3.0 Peptone........................ 6.0 Agar................................... 15.0 The organism Micromono~pora ~urpurea was obtained from the U.S. Dept. of Agriculture as NRRL 2953 and maintain-ed on N-Z amine slants (Medium 1). Submerged fermentations were conducted in flasks containing germination Medium 2 for four days at 37C. on a rotary`shaker. From thi~ first stage se~d, a 10% inoculum was transferred to the germination medium (Medium 2), and fermentation was continued as above -at 28C. for seven days.
For purposes of establishing the capability o thê
organism to biosynthesize gentamicin in the absence of addsd deoxyqtreptamine, a third stage fermentation using a 5%
inoculum was carried out in a 10 liter fermentor in a soybean-glucose medium (3) at 28C., agitating at 200 rpm and sparg-ing at 2 liters/minute with filtered air. After six day3, the tan~ contents were acidified to pH 2.0 with 6N sulfuri~
acid, filtered, and a 500 ml. portion neutralized wi~h ammonium ,~
hydroxide and passed through an IRC-50 ion ex~hange re31n .
.

7;~

(Na+ form). The column was then rinsed with water and eluted with 2N sulfuric acid. Following the procedure described in United States Patent 3,091,572, there was isolated a 300 mg.
sample of crude gentamicin, which was found to be biological-ly active and which contained three components similar to gentamicin Cl, C2 and Cla by TLC examination.
For purposes of mutating the organism, broth cultures were cultivated in medium 2 (37C. for three days) and the resultant cellc harvested by centrifugation, wa~hed and resuspended in buffered saline. This suspension was treated with the mutagenic agent, N-methyl-N'-nitro-N-nitrOso-guanidine. Samples of the mutagenized culture were plated in Medium 4 at 37C. until colonies were evident (usually about one week). Colonies were pi.cked to duplicate plates (Medium 4), one set of which was overlaid with a spore suspen-sion of B. subtilis. After incubation at 37C. for from eighteen to twenty hours, the "picks" which showed no zone of inhibition on the B.jsubtilis plate were transferred from the master plate (no B. subtilis) to medium 1 slants and incubated until ull growth was evident.
These potential nonproducing mutants were then challenged with deoxystreptamine in an attempt to stimulate antibiotic biosynthesis as follows. Stock cultures of the poten~ial mutants were streaked as bands on the surface of madium 4 plates and incubated at 37C. until growth was ;evident (about three to four days). Filter paper di~cs were then dipped into a solution of deoxystreptamine (500 mcg./ml.) and placed on top of the culture streak~ After incubation for twenty-four hours, the surface of the plate was inoculat-ed with B. subtilis using the overlay technique, and incuba-~, .

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tion was continued for an additional ei~hteen to twenty hours. Isolates showing zones of inhibition surrounding the disc were designated as deoxystreptamine mutants. On~ such mutant, coded mutant VIB and deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 as Micromonospora pur~urea ATCC 31,119, was used for the production of the gentamicin-type antibiotics as described below.
The latter organism was itself sub~ected to the same mutation procedure described above and ?amples of the mutagenized culture were plated in Medium 4 at 37C. 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 ~g./ml. of streptamine sulfate and an overlay spore suspension of B. subtilis as a challenge test organism. These plates were incubated at 37C. for twenty-four hours and examined for zones of inhibi-tion. Those showing the largest zones were transferred from the master plate onto N- Z amine slants (Medium 1) and incubat-ed or one wee~ at 37C.
Those mutants incorporating low levels of strept-amine sulfate were then screened with streptamine and scyll_-inosose at`500 ~g./ml. as base. Stock cultures were trans-ferred to flasks containing Medium 2 plus the above inter-mediates and incubated at 37C. on a rotary shaker for seven days. Flasks were periodically assayed for antibio~ic activity via the disc diffusion assay method using B. subtilis as test organism. Isolates showing ~ones of inhibition ~q . ? . ~

surrounding the disc were designated as streptamine or scyllo-inosose mutants. One such mutant, coded mutan* VIB-3P
and deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20851 as MicromonosPora 5 purpurea ATCC 31,164, was used for the production of the aminocyclitol antibiotics of the streptamine, deoxystrept-amine and dideoxystreptamine-type as described below.
BIOSYNTHESES WITH M. PURPUREA ATCC 31,119 .
ExamE~le 1 The mutant organism was maintained on N-Z amine agar slants ~Nedium 1) from which transfers to flasks con-taining 500 ml. of germination Medium 2 were made. The flasks were incubated at 28C. for four days on a rotary shaker (2"
stroke) at 225 rpm.
A 10% (v/v) inoculum from the germination stage was aseptically transferred to 14 liter fermentors containing 9 liters of sterile germination Medium 2. These were agitat-ed at 450 rpm at 28-29C. and sparged with filtered air at 5 liters/minute. At time of inoculation, 200 mg./liter of ~-20 streptamine sulfate was added as a suspension in sterile distilled water. Fermentation was continued for eight days.
~ 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 25 0.31 g./liter of streptamine sulfate suspended in sterile distilled water was added. Aerobic fermentation was carried ~ -out at 29C. for seven days.
Fermentations were terminated by addition of 10N
sulfuric acid to pH 2.0 and filtration using a filter aid to 30 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 wa3 allowed to stand overnight, and the clarified broth was de-canted and passed over sio-Rex 70l(weak cation exchanger) resin in the Na+ 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 concentrat-ed under vacuum below 50C. to the point where salt crystal lization became evident (about 1/3 volume). The pH was then adjusted to 10.5, and four volumes of acetone were added to precipitate inorganics 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 melt-ing over 300C. was collected by filtration which was found, from its thin layer chromatography properties and its infra-red spectrum, to be identical to streptamine sulfate. From two 10 liter fermentations processed as above, a total of 0.7 ~. 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 chroma-tography were determined for gentamicins Cl, C2 and Cla and ~a,~

for each of the corresponding aminocyclitol analogs prepared as indicated above, where the chromatographic mobility (here-inafter designated C.M.) is expressed as:
C M = d1stance component from origin lstance gentamicin Cl rom orlg n The chromatography systems used were as follows:
Syst_m 1 - Whatman No. 1 paper saturated with 0.95M sulate-bisulfate and developed in descending fashion ln 80% aqueous ethanol + 1.5~ NaCl and subsequent bioauto-graphy using B. subtilis as test organism.
System_2 - Silica gel F 254 plate developed in lower phase of chloroform~l):methanol(l):concentrated (28%) ammonium hydroxide(l). Components were located with a ninhydrin spray on heating.
The C.M. values of the ma~or antibiotic components ~
of the present invention in comparison with a reference ~ -gentamicin complex, all relative to gentamicin Cl, are shown in Table I, 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-~-L-arabinopyranosyl- :
~1~6)-0-12-amino-6-methylamino-6-C-methyl-2,3,4,6-tetradeoxy-a-D-erythro-glucopyranosyl-tl~4)]-D-streptamine;
0-3-deoxy-4-C-methyl-3-methylamino-~-L-arabinopyranosyl-

6)-0-[2,6-diamino-6-C-methyl-2,3,4,6-tetradeoxy-a-D-erythro-glucopyranosyl-(1~4)]-D-streptamine; and 0-3-deoxy-4-C-methvl-3-methylamino-~-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 Cl Gentamicin C2 0.89 0.83 Gentamicin Cl 0.50 0.67 Component 1 l~ajor) 0.96 0.92 Component 2 (Minor) 0.76 0.75 Component 3 (Minor) 0.50 0.61 ~, ' .

.. . .

3~.

From the 10 liter fermen~or~, 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~
chloroform~2):17% aquecus ammonium hydroxide(l). The column was developed with this solvent and 50 ml. fractions collected.
Fractions 8 and 9 contained a single ninhydrin-- positive component which yielded 20 mg. as a pale yellow ; oil on removal of solvent. The mass spectrum of this mate-rial, designated Component 1, showed a molecular ion and major fxagments each 16 mass units (i.e., one oxygen atom) greater than that obtained from gentamicin Cl as follows:
Reference gentamicin Cl: M+ 477, 420, 360, 350, 347, 322, 319, 304 Component 1: M+ 493, 436, 376, 366, 363, 338, 2~ 335, 320 ~ his material was converted to its sulfate salt by dis~olving in ethanol and adding a few drops of ethanol con-taining sulfuric acid. The resulting white solid wa~ collect-ed and dried to yield 22 mg. of Component 1, ~ -~ _ _~ :
. ~ .

.. . . ...

~0~17~1 glucop~ranosyl~ 4?]-D-streptamine as the di-base-hepta-sulfate decahydrate, m.p. >300C.
Anal. Calc'd for (C21H43N5O8)2 7H2SO4-10H2O: C, 27.22; H, 6.53; N, 7.55;
S, 12.09 Found: C, 27.15; H, 6.67; N, 7.79;
S, 12.76.
Fractions 10-13 yielded a more polar ninhydrin component designated 0-3-deox~-4-C-m~ 3-methylamino-~-I.-arabinopyranosy~ 6)-o-~2~6-diamino-6-c-methyl-2~3~4~6 tetradeoxy-~-D-~ thro-~lucopyranosy~ 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 Cla at 129 (purpurosamine) and 160 (garosamine) and is designated 0-3-deoxy-4-C-methyl-3-methylamino-~-L- --arabinopyranosyl-(1~6)-0-[2,6-diamino-2,3,4,6-tetradeoxy-a-D-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 t0.9 g.) was dissolved in 7 ml. of water and the pH
adjusted to 4.5 with lN sulfuric acid. The solvent was passed over a strong anion exchange column (IRA 401) in the OH ~orm (bed measurement = 0.7 x 10 cm.). The column was elutQd with water and the eluate evaporated in vacuo at 35C.
The resulting residue was triturated with 50 ml. of the lower phas`e of a solvent composed of 17% aqueous ammonium hydr-oxide:isopropyl alcohol:chloroform (1:1:2). The solvent was decanted and concentrated under vacuum leaving an oily resid~e weighing 140 mg. The mass spectrum o~ this material Y , , -'.
~ ~ .

1~)ti1~7;~1 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 cons~stent for the proposed structure corresponding to 0-3-deoxy-4-C-methyl-3-methylamino-~-L-arabinopyranosyl-(1~6)-0-[2-amino-6-methylamino-6-C-methyl-2,3,4,6-tetradeoxy-~-D-erythro-glucopyranosyl-(1~4)]-D-streptamine and is summarized in Table II.
Tab ~ Integration Assig_ment 5.87, 5.60 2 anomeric OCHO's S.22 12 exchangeable H
3.09, 3.15 6 NCH3 x 2 2.9-4.8 13 -CHO x 6, -CHN x 5, -CH20 1.9-2.6 4 -CH2CH2-1.72 6 CH3C-, C_3CH
Alternatively, a 4 g. sample of the crude antibiotic salt was dissolved in 50 ml. of water and passed over an anion exchange resin AGlX8 (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 libarated base was mixed with 7 g. of silica gel and charged on a 50 g. silica gel column. This column was developed with chloroform:methanol:concentrated(28%)ammonium hydroxide ~3:4:2) and 25 ml. fractions collected. 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 con- ~ -centrate charged 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 collect-ed. 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. ~300C. de-scribed above.
By culturing an appropriate aminocyclitol with mutant ~ ~e9~ ATCC 31,119, in germination Nedium 2 and isolation of the products as described above in Example 1, the following compounds of Formula I are similar-ly prepared:
Example 2 0-3-DeoxY-4-C-methYl-3-methylamino-~-L-arabi-o-pyranos~l-(1~6)-0-~2-amino-6-methYlamino-6-c-methYl-2,3,4,6-tetradeoxY--D-erY_hro-~lucoPYranosy~ 4)]epistreptamine (C.M. relative to gentamicin Cl: System 1 = 0.59, System 2 =
0.63); 0-3-deoxY-4-C-methy,1-3-methYlamino-~-L-arabinoPyran ~ _ ~ ; and 0-3-deoxY-4-C-methYl-3-methYlamino-3-L-arabinoPyranosyl-(l)6)-o-[2~6 diamino-2,3,4,6-tetradeoxY-~-D-erYthro-~lucoPYranosyl~ 4 ; ~ obtained by use of epistreptamine in place of ~treptamine in the fermentation procedure.
~xample 3 abino-~ 6 _~~ ' :' strePtamine (C.M. relative to gentamicin Cl: System 1 ~ 0.95, ~

~ . .

1()~1 7 ~

System 2 = 0.98); 0-3-deoxY-4-C-methyl-3-methylamino-~-L-arabinopyranosyl~ 6)-o-[2~6-diamino-6-c-methyl-2~3~4~6-tetra deoxy-a-D-erythro-glucopyranosyl~ 4)]-2,5-dideox~ystreptamine tC.M. relative to gentamicin Cl: System 1 = 0.66, System 2 =
0.73); and 0-3-deoxy-4-C-methy~-3-methYlamino-~-L-arabino-pyranosyl-(1+6)-0-[2,6-diamino-2,3,4,6-tetradeoxy-~-D-~ obtain-ed by use of dideoxystreptamine in place of streptamine in the fermentation procedure.
Tha same 0-3-deoxy-4-C-methYl-3-met_ylamln-o-~-L-arabinopYranosyl-(1~6)-0-L~amino-6-methYlamino-6-C-methYl-2,3,4,6-tetradeoxy-~-D-erYthro-glucopYranosyl-~1'4)]-2,5-; and 0-3-deoxy-4-C-methyl-3-metXYlamino-~ 6-tetradeox~-~-D-erythro-glucopyranosYl-(1l4~]-2~5-dideoxY-streptamine described above and having the same C.M. values as given above were obtained by use of 6,7-diazabicyclo-13.2.1]octane-2,4-diol (exo, exo) in place of streptamine in the $ermentation procedure.
Example 4 0-3-Deoxy-4 C-methyl-3-methylamino-~-L-arabino-pyranosyl~ 6)-0-~2-amino-6-methylamino-6-C-methyl-2,3,4,6-tetradeoxy-a-D-erythro-glucopyranos~Yl-(1~4)1-5-iodo-2,5-di-deox _t eptamine; 0-3-deoxy-4-C-meth~-3-methylamino-~-L-arabinopyranosyl-(1~6)-0-[2,6-diamino-6-C-methyl-2,3,4,6-tetradeoxY-a-D-erythro-glucopyranosyl-(l)4)]-5-iodo-2~5-di-; and 0-3-deoxy-4-C-methyl-3-methylamino-~-_ ____ ~_ amine obtained by use of 5-iodo-2,5-dideoxystreptamine in place of streptamine in the fermentation procedure.

~, . . . .

~i7;~1 Exam~l_ 5 0-3-Deoxy-4-C-methyl-3-methylamino-~-L-arabinO-pyranosy~ >6)-o-[2-amino-6-methylamino-6-c-meth~l-2~3~4~6 tetradeoxy-a-D-erythro-~ucopyranosyl~ 4)?--5-fluoro-2~5-dideoxystrePtamine; 0-3-deoxY-4-C-methyl-3-methvlamino-~-L-arabinopyranosyl-(1~6j-0-[2~6-diamino-6-C-methyl-2~3,4,6-tetradeQxy-a-D-erythro-glucopyranosyl- (1 ~ 4? ] -5-fluoro-2,5-dideoxystreptamine; and 0-3-deoxY-4-C-methYl-3-methYlamino-~_ 10 ~ ~ ~ ~ -amine 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 ~9~
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 27-28C. 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 28-29C. for forty-eight hours. Finally 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 29-30C.
.B

17;~1 The tank contents were acidified to pH 2.0 with 10N sulfuric acid and filtered through filter aid to remove mycelia. The filtrate was adjusted to pH 6.0 and pas~ed over an 8 x 50 cm. cation exchange resin bed (Bio-Rex 70, ~odium ion form, 20-50 mesh). The eluate was checked for anti-biotic 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 com-bined and neutralized to give a final volume of 10 liters.
This was concentrated to about S 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 filtra-tion, and the filtrate was concentrated in vacuo after adjust-ing 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 fil-trate was concentrated, adjusted to pH 4.5 and further concen-trated to 10 ml. On addition of 100 ml. of methanol, a crude antibiotic solid was obtained weighing ~ g., which was dis-solved in 10 ml. of water and extracted with five 50 ml.
portion~ of the lower phase of a solvent composed by volume of chloroform(2):isopropanol(1):17% ammonium hydroxide(l).
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 (100-.

1l)~1731 200 mesh) and placed on a 100 g. silica gel column (2.5 x 45 cm.) and developed with the chloroform:isopropanol:-ammonium hydroxide solvent de~cribed above. Fractions were collected, subjected to tlc analysis, and ~elected 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 O=3-deoxy-4-C-meth~1-3-(methylamino)-~-L-ara ~ ranosyl-(1~6)-0-~2-amino-6-(methYlamino)-6-C-~ _ _ D-streptamine as the bis-base-pentasulfate-tetrahydrate.
Anal. Calc'd for C21H43N$O8 2 1/2 H2SO4 2H2 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 lS the same name, designated "Component 1" in Example 1 above, was established by the following:
First, the ohromatographic mobilities of the com-pound in two solvent systems determined by tlc analysis were identical with the chromatographic mobilities in the same Qystem~ of "Component 1". The chromatographic mobilities of the Qompound identified above and designated Example 6, "Component 1" of Example 1 above, and gentamicin Cl are 8hown in the following table~
_System 1 System 2 Rf RfCl* Rf RfCl*
Gentamicin Cl 0.39 1.00 0.70 1.00 Component 1 0.30 0.77 0.63 0.90 Example 6 0.30 0.77 0.63 0.90 Sy8tem 1 - ~ilica gel 60F254, lower phase of chloroform (l):methanol(1):30~ ammonium hydroxide(l), plate sprayQd with ninhydrin.
Sy8tem 2 - Watman ~1 ~aper-solvent system same as System 1 -bioautogrlaphy using B. subtilis.
~RfCl - Mobility relative to gentamicin Cl.

e9~ T. ~

lU~1'7;~1 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 NCH3 groups and one CH3CH 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 hydro-chloric acid in a 0.5 mm. capillary tube was heated in re-fluxing 6N hydrochloric acid for six hours. The mixture was allowed to stand at room temperature ~or two days, then diluted with 1.5 ml. of ethanol. The resulting clear super-natant liquid was deaanted from the solid residue, and the ~olid was dissolved in water and chromatographed on silica gel plates using a chloroform(3):methanol(4):concentrated ammonium hydroxide(2) system. Comparative samples of authentic streptamine and deoxystreptamine were chromatographed simul-taneously. 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 N,N-diacetylstreptamine tetraacetate was prepared by react- -ing ~10 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 temp-erature, evaporated to dryness and extracted with chloroform/-water to give 440 mg. of material having m.p. 252-256C., -~2-.

... . . . . .. ....

(melts with recrystallization), decomposes 334-336C. [Lit :-melts partially at 250C. with transition to long needles, melts >300C. Peck et al. J. Am. Chem. Soc. 68, 776 (1946)].
Similar acetylation of the degradation product obtained above with 5 mg. of sodium acetate in 1 ml. of acetic anhydride afforded 5.6 mg. of material having m.p.
250-257C. (melts with recrystallization), decomposes at 336-339C. The mixed melting point between the known and the reference sample was undepressed.
Finally, vapor phase chromatographic compari~ons between the N,N-diacetylstreptamine tetraacetate and the corresponding compound prepared from the degradation product were compared along with N,N-diacetyldeoxystreptamine tri-acetate. The streptamine derivative and the degradation sample were shown to be identical (R.T. = 10.6 minutes) but diferent from that of N,N-diacetyldeoxystreptamine tri-acetate (R.T. - 9.1).
Example 7 Following a procedure similar to that described in Example 6 above using 0.50 g.~liter of dl-epi-inosose-2 12,3,4,5,6-pentahydroxycyclohexanone (2,3,4,6-ci~)] in three 10 liter fermentations in medium 5 plus 0.1% added phytone in the presence of M. purPurea ATCC 31,164, and isolation of th~ product as before gave 0.148 g. of an oily product which, from comparison of its chromatographic mobility with known samples and from its mass spectrum, was shown to be identical with gentamicin Cl, namely ~ -~-~ . The mass spectrum showed-a ,~

. ~ - - ,, :

molecular ion at 477 and major fragments identical to genta-micin Cl at 420, 360, 350, 347, 322, 319, 304, 160 and 157.
Example 8 Following a procedure similar to that described in Example 6 above using 0.50 g./liter of 3,4,5,6-tetra-hydroxycyclohexene (3,5-cis) in three 10 liter fermentations in Nedium 5 plus 0.1~ added phytone in the presence of M.
ATCC 31,164, and isolation of the product as before gave 0.68 g. of material which was shown, from tlc analysis and the chromatographic mobilities, to be identical with gentamicin Cl, C2 and Cla mixture. Moreover, a 2 milligram sample of the material was spotted on a tlc plate along with a sample of commercial gentamicin and the plate developed with chloroform(l):methanol(l):`concentrated ammonium hydr-oxide~l). The resulting three bands from the experimental sample, whose chromatographic mobility values (Rf) corre-sponded identically to the three bands from the commercial gentamicin, were removed, eluted with the developing solvent and submitted for mass spectral analysis. ~he spectra of the first two bands having Rf values of 0.37 and 0.29, re-spectively, were consistent with the mass spectra of genta-micin Cl and gentamicin C2, respectively, the mass peaks for the two ~amples being as follows:
Gentamicin Cl: N+477, M++l 478, 420, 360, 350, 347, 322, 319, 304, 160, 157.
Gbntamicin C2: M+(none), 42Q, 350, 346, 333, 322, 305, 304 160, 143.
The third weaker band from the experimental sample, having an ~ of 0.22~ provided an inQufficient sample, on elution to permit mass spectral analysis.

~, . . . - .

g~U~

Example 9 dl-Deoxyinosose [dl-2,3,4,5-tetrahydroxy-1-cyclo-hexanone] (500 ~g./ml.) described above in Preparation 1 was incubated with mutant M. ~urpurea ATCC 31,164 for four days in germination medium 2, and the resulting broth was found to be antibiotically active by the disc diffusion assay method against B. subtilis as test organism. In addi-tion, tlc o a crude isolate from the broth showed, vla bioautography using B. subtilis as test organism, three antibacterial components corresponding to gentamicins Cl, C2 and Cla at Rf's of 0.37, 0.31 and 0.26, respectively, in System 1 described in Example 6 above.
Example 10 Scyllo-inosose pentaacetate [2,3,4,5,6-pentahydr-oxycyclohexanone pentaacetate (2,4,6-cis)] [Kluyver et al., Rec. trav. chim. Pays-Bas 58, 956 (1939)] (500 ~g./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 anti-biotically active by the disc diffusion assay method against B. subtilis as test organism.
Example 11 dl-Viboquercitol [dl-1,2,3,4,5-cyclohexanepentol ~1,2,4-cis)] [McCasland et al., J. Am. Chem. Soc. 75, 4020 (1953)] (500 ~g./ml.) was incubated with mutant M. purpurea ATCC 31,164 for seven days in germination Medium 2, and the resulting broth containing gentamicin was found to be anti-biotically active by the disc diffusion assay method against B. subtilis as test organism.

3~
-3~-. .

7;~1 Example_12 dl-2,3,4,6-Tetrahydroxy-l-cyclohexanone (2,4,6-cls) described above in Preparation 3 was incubatèd 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-meth~yl-3-(methylamino~-L-arabinoPyranosvl-(1~62-0-[2-amino-6-~methYlaminol -6-C-methYl-2~3,4~6-tetra-deoxY-~-D-erYthro-glucoPYranosYl-(l~ 4 L~ -D-5-deoxYstrePtamine;
0-3-deoxy-4-C-methYl-3-(methYlamino)-~-L-arabinoPyranosYl-(1~ ~ [2,6-diamino-6-C-methYl-2,3,4,6-tetradeoxv-~-D-erY-t_r~a~ucopYranosyl-(1~4)]-D-5-deoxYstreptamine; and 0-3-de-oxy-4-C-methYl-3-~methYlamino)-~-L-arabinoPyranosyl-~l~6 0-[2,6-diamino-2,3,4,6-tetradeoxY-~-D-erYthro-qlucoPYranosY
(1~4)~-D-5-deoxYstrePtamine was found to be antibiotically 15 active by the disc diffusion assay method against B. subtilis as test organism.
Example 13 2,4,5-Trihydroxycyclohexanone (2,4-cis) (500 ~g./-ml.) described above in Preparation 4 was incubated with mutant M. purpurea ATCC 31,164 for four days in germination .
M~dium 2, and the resulting broth containing a mixture of O-3-deoxY-4-C-methYl-3-(methyla~mino)-~-L-arabinopYran tl~6)-0-~2-amino-6-(methYlamino)-6-c-methyl-2~3~4~6-tetra-~ t- .
amine; ~ bino-and 1-(1.6)-0 ~L6-diamino-2~3~4~6-tetradeoxY-a-D-erythro-q pYranosyl-(l~4)3-D-2~5-dideoxystreptamine was found to be `

~U~7;~1 antibiotically active by the disc diffusion assay method against B. subtilis as test organism.
B. Incor~oration of Aminoc~clitols ExamPle 14 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 O.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 soy-bean meal and 3~ cerelose was substituted for starch in the presence of M. pur~urea 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 tlc analysis to be identical with "Component 1" described in Example 1 above, namely 0-3-deoxv-4-C-methyl-3-(methylamino) -~-L-arabinoPyranosy~ 6)-o-[2~amino-6-(methylamino)-6-c-meth 2,3,4,6-tetradeoxy-a-D-erYthro-qlucopyranosyl-(l)4~]-D-strept amine, m.p. 119-123C., and 0.996 g. of a pale yellow solid which was shown to be identical with "Component 2" described in Example 1 above, namely O-3-deoxy-4-C-meth_1-3-(methYl-amino)-~-L-arabinopyranosvl-~1~6)-0-~2~6-diamino-6-C-methYl-2~3~4~6-tetradeoxY-~-D-ervthro-~lucoPyranosY-l-(l~4)l-D-strept amine, m.p. 115-119C., [~]25 ~0.2% H20) = +137.1.
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. 228-230C.
~B. _ ~6 .
.

Anal. Calc~d for C20H41N508 2 1/2 H2S04 3H20 C, 30.85; H, 6.73; N, 8.99; S, 10.29 Found: C, 30.45; ~, 6.53; N, 9.03; S, 10.14.
The nuclear magnetic re~onance spectrum of the base was also consistent with the assigned ~tructure and i~
~ummarized as follows:
~ Integration AssignmQnt 5.82, 5.94 1 CH\

~0 5.59 1 C~

5.20 13 NH2x4, N~xl, OHx4 3.0-4.6 13 -CHN-x5, -CHO-x6, -CH20-3.09 3 CH3-N
1.73 3 CH3-C
1.72 3 CH3-CH
1.90-2.5 4 CH2x2 ~.
Following a procedure similar 'o that described ln Example 6 u~ing O.10 g./liter of 2-deoxystreptamine in a 10 liter fermentation in production Medium 5 in the pres-~nce of ~. purpurea ATCC 31,164, and i~olation o the pro-d~t a~ ba~oro gavo 0.51 g. of crude material which, on .~ :
~` ~hromatography on ~lll¢a gel, afforded, as a ma~or ~omponent m~t rlal whlch wa~ shown, by the ldentity o the chromato-graphlc mobll~tle~, to be ~dentical with gentamicin Cl, 2S nam~ly, ~ _ _~_ E ~ A

'", ~ , ; ' '' ' ' ' ' '. , ', ~ ' , 173~

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 or 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 ~o give three bands whose mass spectra showed them to be, respectively, Component Cl: 0-3-deoxy-4-C-methyl-3-(methYl-amino)-~L-arabinopvranosYl-(1)6)-0-[2-amino-6-(methYlamino)-6-C_~5by~L3~4~6-tetradeoxv-a-D-erythro-qlucopyranosyl-(1~4L~ D-2,5-dideoxYstrePtamine; Component C2: 0-3-deoxY-4-C-methyl-3-(methylamino)-~-L-arabinoPyranosYl-(1~6)-O-[2,6-diamino-6-C-methyl-2,3,4,6-tetradeoxy-a-D-erYthro-qluco-Pvranosyl-(l+4)]-D-2~s-dideoxvstreptamine; and Component Cla:
0-3-deoxy-4-C-methvl-3-(methYlamino)-~-L-arabinoPYranosYl-(1~6)-0-12,6-diamino-2~L~,6-tetradeoxY-~-D-erYthro-qluco-pyranosyl-(1~4~ -2!5-dideoxystrePtamine. The mass spectra for the three above-indicated components Cl, C2 and Cla, showed mass peaks as follows: Component Cl: M+ 461, 404, 344, 334, 331, 306, 303, 288, 160 and 157. Component C~:
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 tlc analysis on silica gel plates using the lower phase of chloroform(l):methanol(l):concentrat-ed ammonium hydroxide(l) as developing solvent, were 0.43, 0.37 and 0.29, for Components Cl, C2 and Cla, respectively.
i.~ ' . .

~J~.~'7~i1 Example 17 Following a procedure similar to that described in Example 6 using 0.50 g./liter of 2-amino-1,3,4,5,6-cyclo-hexanepentol ~1,3,5-cis) in three 10 liter fermentations in production medium 5 plus 0.1% added phytone in the presence of M. pur~urea 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, 10namely 0-3-deoxy-4-C-methyl-3-(met~hylamino)-~-L-arabin pYranosyl-(1~6)-0-~2-amino-6-(methylamino)-6-C-methyl-2,3,4,6-tetradeoxy-a-D-erythro-glucopyranosyl-(1~4)¦-D-`
treptamine. The mass spectrum showed mass peaks as follows:
M+ 493, 457, 436, 383, 376, 366, 363, 346, 344, 338, 335, - 15321, 318, 261, 160 and 157.
Example 18 ; ~-4,6-Bis(benzylideneamino)-1,3-cyclohexanediol (1,3-cis) (500 ~g./ml.) described above in Preparation 2 was incubated with mutant M. purpurea ATCC 31164 for four 20days in germination medium 2, and the resulting broth con-taining the 2,5-dideoxystreptamine analog of gentamicin was found to be antibiotically active by the disc diffusion assay method against B. subtilis as test organism.
Synthesis of Amino-hydroxy-lower-alkanoyl Der~vatlves `
Example 21 A solution of 270 mg. (0.54 millimole) of 0-3- ;
~, .

t~
. . . ~ .
, ' ' ~' ., ` ~ . , . , . , . ~

10~

deoxy-4-C-methyl-3-meth~lamino-~-L-arabinopy~anosyl-(1~6)-O-~2-amino-6-methylamino-6-C-methyl-2,3,4,6-tetradeoxy-a-D-erythro-glucopyranosyl-(1~4)~-D-streptamine, described above in Example 1 (Component 1), dissolved in 5 ml. of 50 aqueous tetrahydrofuran was cooled to 5C. in an ice bath and treated with 208 mg. (0.59 millimole) of the N-hydroxy-succinimide ester of S-(-) -r- (N-benzyloxycarbonyl)amino-~-hydroxybutyric acid ~Xonishi et al. U.S. Patent 3,780,018), and the mixture was stirred at 5C. for twenty hours. The mlxture 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 S13 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~ a~ueous tetrahydrofuran and treated with an additional 208 mg. of the N-hydroxysuccinimide ester of S- (-)-r- (N-benzyloxycarbonyl)-amino-~-hydrox~butyric acid as before.
Work up of the reaction mixture afforded an additional 435 mg.
of crude product which was combined with the 513 mg. pre-viously obtained and chromatographed on seven 40 x 20 cm.
silica gel plates 1 mm. thick. The system was developed with chloroform:methanol:concentrated (28%) G onium 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 ~D
`U
~.

three 40 x 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 chloro-form:methanol:concentrated(28%)ammonium hydroxide (4:1:1) (lower phase). Three distinct bands visible under ultra-violet irradiation were obtained which were separately cut out and eluted from the silica gel with chloroform:methanol:-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-(-)-y-(N-benzyloxycarbonyl)-amino-~-hydroxybutyric acid amide derivatives at the 2'-, 1- and 3-positions, respectively, of 0-3-deoxy-4-C-methyl-3-methylamino-~-L-arabinopyranosyl-(1)6)-O-[2-amino-6-methyl-amino-6-C-methyl-2,3,4,6-tetradeoxy-~-D-erythro-glucopyranosyl-(1~4)]-D-streptamine. The Rf 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 - 0.62 C - 0.70 Component B (the l-amide, 56.1 mg.) dissolved in 25 ml. of 50~ aqueous ethanol and 20 mg. of 10~ palladium-on-charcoal was shaken on a Parr shaker at 55 p.s.i. for five and a half hours after which time the catalyst was re-moved by filtration through filter aid. Evaporation of the solvent afforded 34.3 mg. of a white glass which was dis-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 17~1 ethanol precipitated l-[S-(-)-Y-amino~ -hvdroxybutYr~l]-O-3-deoxy-4-C-methyl-3-methylamino4 -L-arabinoPyranosyl-(1'6~-O-t2-amino-6-methylamino-6-c-methvl-?~3~4~6-tetradeoxy~x-_ D-erythro-glucopyranosy~ 4)}D-streptamine as the penta-sulfate salt (32 mg.), m.p. 230-235C. (decomp.); tlc, Rf = 0.18 (silica gel, chloroform:methanol:concentrated(28~) ammonium hydroxide:water, 1:4:2:1; Rf gentamicin Cl stan-dard = 0.73).
Anal. Calc'd for C25H5010N6 5H2 4 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'-[S-(-)-y-_mino-a-hydroxy-butyryl]-0-3-d~ y-4-C-methyl-3-methylamino-~-L-arabino-pyranos~ 6)-O-[2-amino-6-methylamino-6-C-methyl-2,3,4,6-as the bis-base tetrasulfate heptahydrate, m.p. 237-241C.
(decomp.); tlc, Rf = 0.33 [silica gel, chloroform:methanol:-concentrated(28~)ammonium hydroxide:water, 1:4:2:1; Rf gentamicin Cl standard = 0.73].
Anal~ Calc'd for (c25H5oN6olo)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-[S-(-)-y-amino-a-hYdroxybutyrYl]-0-3-deoxy-4-C-methyl-3-methylamino-~-L-streptamine as the bis-base pentasulfate trihydrate, m.p.
220-230C. (decomp.); tlc. Rf = 0.30 [silica gel, chloroform:-methanol:concentrated(28%)ammonium hydroxide:water, 1:4:2:1;
. ~
~ `~; - .

. . . . .. , ` .. : .. . ~ .

Rf gentamicin Cl standard = 0.73].
Anal. Calc'd for (C25H50N6olo)2 5H2S4 H2 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 21 using the respective antibiotics described in Examples 2, 3, 4 and 5 and either the N-hydroxysuccinimide ester of S~ -Y-(N-benzyloxycarbonyl)amino-~-hydroxybutyric acid or the pentafluorophenyl ester of N-(benzyloxycarbonyl)-(S)-isoserine [(S)-~-amino-~-hydroxypropionic acid], describ-ed by Haskell et al., Carbohydrate Research, 28, 273-280 (1973)], the following compounds of Formula I are similarly prepared:
Exam_le 22 ~~ '.
methyl-3-methYlamino-~-L-arabinopyranosyl-(l~6)--o-[2~6-di-amino-6-C-me~1-2~3~L6-tetradeoxy-a.-D-erythro-~ucopyranosyl- -` ~ and ~ _ 0-3-deoxy-4-C-methyl-3-methylamino-~-L-arabinoPyranosyl-(1~6)-O-12~6-diamino-6-C-methyl-2,3,4,6-tetradeoxy-~-D-erYthro-glucop~ranosyl-(1~4)]epistreptamine.
Exam~le 23 l-~S-(-~-y-Amino-~-hy_rox but ryl]-0-3-deoxy-4-C-methyl-3-methylamino-~-L-arabinoPyranosyl-(1)6~-O-[2,6-di-amino~2,3,4,6-tetradeoxy-~-D-e ~ -2,5-dideoxystreptamine and 2'-[S-(-)-y-amino-a-hydroxybutyr-~ .
~_ Example 24 _ ~3 ~B : ``

3~

amino-2,3,4,6-tetradeoxy-~-D-erythro-glucoPYranosyl~ 4)}
5-iodo-2,5-dideoxystreptamine and ~ -ro ion 1]-0-3-deoxy-4-C-methyl-3-methylamino-~-L-arabino-P P Y
pyranosyl-(1~6)-0-[2,6-diamino-2,3,4,6-tetradeoxY-~-D-erythro-giucopyranosy~ 4)]-5-iodo-2~5-dideox~streptamine.
Example 25 l-[S-~-)-y-Amino-~-hyd-rox~butyryl]-o-3-deoxy-4 methyl-3-methylamino-~-L-arabi-n-op-yranosy~ 6)-o-[2-amin 6-methylamino-6-C-methyl-2~3~4~6-tetradeoxy-~-D-erythro-glucopyranosyl-(1~4)]-5-fluoro-2,5-dideoxystr-ptamine and 2'-[S-(-)-~-amino-~-hydroxYbutyrYl]-0-3-deoxy-4-C-methvl-3-methylamino-~-L-arabinopYranosy~ 6)-o-t2-amino-6-meth amino-6-C-met~yl-2~3~4~6-tetradeoxy-~-D-erythro-glucopyran ~

The 0-3-deoxy-4-C-methyl-3-methylamino-~-L-arabino-pyranosyl-(1~6)-0-12-amino-6-methylamino-6-C-methyl-2,3,4,6-tetradeoxy--D-erythro-glucopyranosyl-(1~4)]-D-streptamine described above in Example 1 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 mcg./ml~ base, were prepared in distilled water and filter sterilized. Cultures of the test organisms were grown for twenty-our hours at 37C. 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 (approx-imately 108 cells/ml.). The adjusted cultures were diluted 1:500 in broth for use as inoculum (final cell concentration ~`..

- : , . : ~ .
. .

1'7~31 approximately 2 x 105 cells/ml.). The test compounds were tested for antibacterial activity by a single-row tube dilu-tion 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 ln seventeen 13 x 100 mm.
tubes. All tubes were inoculated with 0.2 ml. of the appro-priate diluted culture ~final cell concentration per tube - 105 cells/ml.). Minimum inhibitory concentrations (lowest drug concentration showing no visible growth) were read a~ter sixteen hours incubation at 37C.
The results are given in Table III below. The compounds were considered inactive at inhibitory concentra-tions greater than 100 mcg./ml.
; Table III*

Minimal Inhibitory Conc.
(mcg./ml.) Orqanism Component 1Gentamicin Staphylo-coccus aureus Smith 0.78 0.195 Escherichia coli Vogel 3.13 3.13 Escher1ohi~ coli W677/HJR66 50 Inactive Escherichia c _ 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 Enterobacter cloacae A-20960 1.56 25 Klebsiella pneumoniae 39645 1.56 0.78 __ Klebsiella pneumoniae A-20636 3.13 50 Pro~eus mirabilis MGH-l 6.25 1.56 Providencia 164 100 Inactive .
Providencia stuartii A-20894 100 100 Pseudomonas aeru~_nosa MGH-2 3.13 0.39 Pseudomonas aeruginosa A-20717 12.5 ~12.5 Pseudomonas aeruginosa A-20741 Inactive Inactive -~
Pseudomonas aeruginosa A-20897 12.5 Inactive = = . . .
*Cultured in tryptose phosphate broth.
; ~ ~s .

,: .

11)~17;~1 The same Component 1 described above in Example 1, 0-3-deoxy-4-C-methyl-3-methylamino-~-L-arabinopyranosyl-(1'6)-O-[2-amino-6-methylamino-6-c-methyl-2~3~4~6-tetradeoxy-x-D
erythro-glucopyranosyl-(1~4)]-D-streptamine, was tested in comparison with gentamicin complex (Cl, C2 and Cla) and genta-micin Cl, and the 1-, 3- and 2'-~S-(-)-y-amino-~-hydroxy-but~ryl]amides of Component 1, described above in Example 21, and designated, respectively, Component 1 (l-HABA), Compon-ent 2 (3-HABA) and Component 1 (2 ' -HABA), were tested in com-parison with the corresponding 1-, 3- and 2'-~S-(-)-Y-amino-~-hydroxybutyryl]amides of gentamicin Cl (all described by Xonishi et al. United States Patent 3,780,018, patented December 18, 1973) and designated, respectively, Cl (l-HABA), Cl (3-HABA) and Cl (2l-HABA). These results are given in Table IV below where 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. aerug_nosa A-20897, respectively.
Table IV*
Test Organisms Com~ound 1 2 3 4 5 6 Gentamicin Cl, C2, Cla ~0.024 0~39 50 12.525 ~100 Gentamicin ~1 0-049 0.78 50 12.525 ~100 Component 1 0.098 1.56 6.25 0.783.13 25 C~ HABA) 0.39 3.13 12.5 3.13 6.25>100 Component 1 (l-HABA) 0.78 6.25 12.5 6.25 12.5>100 Cl (3-HABA) 3.13 25 ~100 100 >100>100 Component 1 (3-HABA) 6.25 50 100 50 50>100 Cl (2'-HABA) 6.25 50 100 25 50>100 C(o2,mponen)t 1 1.56 25 50 25 50 >100 *Cultured in Mueller-Hinton broth.

Claims (28)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A process for preparing a compound having the Formula where R1, R3 and R8 each represent hydrogen, or one of R1, R3 and R8 represents an .omega.-amino-.alpha.-hydroxy-lower-alkanoyl group having the formula:

H2NCH2(CH2)nCHOCHO-where n is zero or 1, the other of R1, R3 and R8 being hydrogen;
R2 represents hydrogen or hydroxy; R5 represents hydrogen, hydroxy or halogen, except that when R2 is hydrogen, R5 is not hydroxy, and R6 and R7 each represent hydrogen or methyl, or a pharmaceutically acceptable acid-addition salt thereof, which comprises culturing a nutrient medium containing carbohydrates, a source of assimilable nitrogen, essential salts and a corresponding added aminocyclitol or a cyclitol as defined below in the presence of a suitable mutant microorganism as defined below, and isolating the product from the culture medium wherein there is used:
a) an aminocyclitol of the Formula wherein R1 and R3 represent hydrogens or together represent a single bond joining the two amino nitrogen atoms together and R2 and R5 have the above meanings in the presence of a mutant micro-organism incapable of synthesizing said aminocyclitol itself but being capable of incorporating said aminocyclitol into the com-pound of Formula I, under the above conditions, wherein a com-pound of Formula I where R1, R3 and R8 each represent hydrogen, and R2, R5, R6 and R7 have the indicated meanings is produced;
b) a cyclitol having the Formula IIIa or IIIb where, in either case, R is hydrogen or acetyl; R3' is oxo or hydroxy; and R2' and R5' each are hydrogen or OR, in the presence of a strain of a microorganism which is incapable of bio-synthesizing the cyclitol unit but which is capable of in-corporating the cyclitol into the antibiotic molecule as an aminocyclitol unit, wherein a compound of the Formula I where R1, R3 and R8 each represent hydrogen and R2, R5, R6 and R7 have the indicated meanings; except that R5 does not include halogen, is produced; or c) an aminocyclitol having the Formula IIa or IIb (herein) where, in either case, R2' and R5' are each hydrogen or hydroxy, and R1' is amino or hydroxy in Formula IIa and hydroxy or benzylideneimino in Formula IIb, in the presence of Micromonospora purpurea ATCC 31,164, wherein there is produced a compound of the Formula I where R1, R3 and R8 each represent hydrogen; R2, R5, R6 and R7 have the indicated meanings, except that R5 does not include halogen;
and, in order to produce a compound where one of R1, R3 and R8 represents an .omega.-amino-.alpha.-hydroxy-lower-alkanoyl group, reacting the compound of Formula I obtained according to (a), (b) or (c) with an N-hydroxy-succinimide ester having the Formula IV (herein) where n is zero or 1 and subjecting the benzyloxycarbonyl group in the resulting product to hydrogenolysis with hydrogen over a catalyst.

2. A process according to claim 1, for preparing a com-pound of Formula I therein, where R1, R3 and R8 each represent hydrogen, and R2, R5, R6 and R7 have the indicated meanings, which comprises culturing a nutrient medium containing carbo-hydrates, a source of assimilable nitrogen, essential salts and a corresponding added aminocyclitol having the Formula II
(herein) where R1, R2, R3 and R5 have the meanings given above, and where R1 and R3 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 itself but being capable of incorporating said aminocyclitol into the com-pound of Formula I, under the above conditions, and isolating the product from the culture medium, a compound of Formula I
where both R1 and R3 are hydrogen being produced when R1 and R3 in Formula II represent a single bond.

3. A process according to claim 2, wherein the strain of the microorganism is Micromonospora purpurea ATCC 31,119.

4. A process according to claim 1, for preparing a com-pound of the Formula I therein, where R1, R3 and R8 each represent hydrogen and R2, R5, R6 and R7 have the indicated meanings;
except that R5 does not include halogen, which comprises cul-turing a nutrient medium containing carbohydrates, a source of assimilable nitrogen, essential salts and a cyclitol having the formula IIIa or IIIb where, in either case, R is hydrogen or acetyl; R3' is oxo or hydroxy; and R2' and R5' each are hydrogen, hydroxy or OR, in the presence of Micromonospora purpurea ATCC
31,164.

5. A process according to claim 1, for preparing a com-pound of the Formula I therein, where R1, R3 and R8 each represent hydrogen and R2, R5, R6 and R7 have the indicated meanings, except that R5 does not include halogen which comprises culturing a nutrient medium containing carbohydrates, a source of assimilable nitrogen, essential salts and an aminocyclitol having the Formula IIa or IIb where, in either case, R2' and R5' are each hydrogen or hydroxy, and R1' is amino, or hydroxy in Formula IIa and hydroxy or benzyl-ideneimino in Formula IIb, in the presence of Micromonospora purpurea ATCC 31,164.

6. A process according to claim 1, which includes con-verting a free base obtained to a pharmaceutically acceptable acid-addition salt thereof.

7. A process according to claim 2, for preparing a com-pound of the Formula I, where one of R1, R3 and R8 represents an .omega.-amino-.alpha.-hydroxy-lower-alkanoyl group, which comprises reacting the compound of Formula I obtained according to said process with an N-hydroxy-succinimide ester having the Formula IV where n is zero or 1 and subjecting the benzyloxycarbonyl group in the resulting product to hydrogenolysis with hydrogen over a catalyst.

8. A process according to claim 4, for preparing 0-3-deoxy-4-C-methyl-3-(methylamino)-.beta.-L-arabinopyranosyl-(1?6)-0-[2-amino-6-(methylamino)-6-C-methyl-2,3,4,6-tetradeoxy-.alpha.-D-erythro-glucopyranosyl-(1?4)]-D-streptamine wherein the added cyclitol is scyllo-inosose.

9. A process according to claim 4, for preparing 0-3-deoxy-4-C-methyl-3-(methylamino)-.beta.-L-arabinopyranosyl-(1?6)-0-[2-amino-6-(methylamino)-6-C-methyl-2,3,4,6-tetradeoxy-.alpha.-D-erythro-glucopyranosyl-(1?4)]-D-5-deoxystreptamine; 0-3-deoxy-4-C-methyl-3-(methylamino)-.beta.-L-arabinopyranosyl-(1?6)-0-[2,6-diamino-6-C-methyl-2,3,4,6-tetradeoxy-.alpha.-D-erythroglucopyranosyl-(1?4)]-D-5-deoxystreptamine; or 0-3-deoxy-4-C-methyl-3-(methyl-amino)-.beta.-L-arabinopyranosyl-(1?6)-0-[2,6-diamino-2,3,4,6-tetra-deoxy-.alpha.-D-erythro-glucopyranosyl-(1?4)]-D-5-deoxystreptamine wherein the added cyclitol is d1-2,3,4,6-tetrahydroxy-1-cyclo-hexanone (2,4,6-cis).

10. A process according to claim 4, for preparing 0-3-deoxy-4-C-methyl-3-(methylamino)-.beta.-L-arabinopyranosyl-(1?6)-0-[2-amino-6-(methylamino)-6-C-methyl-2,3,4,6-tetradeoxy-.alpha.-D-erythro-glucopyranosyl-(1?4)]-D-2,5-dideoxystreptamine; 0-3-deoxy-4-C-methyl-3-(methylamino)-.beta.-L-arabinopyranosyl-(1?6)-0-[2,6-diamino-6-C-methyl-2,3,4,6-tetradeoxy-.alpha.-D-erythro-glucopyranosyl-(1?4)]-D-2,5-dideoxystreptamine; or 0-3-deoxy-4-C-methyl-3-(methylamino)-.beta.-L-arabinopyranosyl-(1?6)-0-[2,6-diamino-2,3,4,6-tetradeoxy-.alpha.-D-erythro-glucopyranosyl-(1?4)]-D-2,5-dideoxystreptamine wherein the added cyclitol is 2,4,5-trihydroxycyclohexanone (2,4-cis).

11. A process according to claim 5, for preparing 0-3-deoxy-4-C-methyl-3-(methylamino)-.beta.-L-arabinopyranosyl-(1?6)-o-[2-amino-6-(methylamino)-6-C-methyl-2,3,4,6-tetradeoxy-.alpha.-D-erythro-glucopyranosyl-(1?4)]-D-streptamine or 0-3-deoxy-4-C-methyl-3-(methylamino)-.beta.-L-arabinopyranosyl-(1?6)-0-[2,6-diamino-6-C-methyl-2,3,4,6-tetradeoxy-.alpha.-D-erythro-glucopyranosyl-(1?4)]-D-streptamine wherein the added aminocyclitol is streptamine.

12. A process according to claim 5, for preparing 0-3-deoxy-4-C-methyl-3-(methylamino)-.beta.-L-arabinopyranosyl-(1?6)-0-12-amino-6-(methylamino)-6-C-methyl-2,3,4,6-tetradeoxy-.alpha.-D-erythro-glucopyranosyl-(1?4)]-D-2,5-dideoxystreptamine; 0-3-deoxy-4-C-methyl-3-(methylamino)-.beta.-L-arabinopyranosyl-(1?6)-0-[2,6-diamino-6-C-methyl-2,3,4,6-tetradeoxy-.alpha.-D-erythro-gluco-pyranosyl-(1?4)]-D-2,5-dideoxystreptamine or 0-3-deoxy-4-C-methyl-3-(methylamino)-.beta.-L-arabinopyranosyl-(1?6)-0-[2,6-diamino-2,3,4,6-tetradeoxy-.alpha.-D-erythro-glucopyranosyl-(1?4)]-D-2,5-dideoxystreptamine, wherein the added aminocyclitol is 2,5-dideoxystreptamine.

13. A process according to claim 5, for preparing 0-3-deoxy-4-C-methyl-3-(methylamino)-.beta.-L-arabinopyranosyl-(1?6)-O-[2-amino-6-(methylamino)-6-C-methyl-2,3,4,6-tetradeoxy-a-D-erythro-glucopyranosyl-(1?4)-D-streptamine wherein the added aminocyclitol is 2-amino-l,3,4,5,6-cyclohexanepentol (1,3,5-cis).

14. A compound of the Formula I as defined in claim 1 or an acid-addition salt thereof, when prepared by the process according to claim 1 or 6 or by an obvious chemical equivalent thereof.

15. A compound of the Formula I as defined in claim 2, when prepared by the process according to claim 2 or 3 or by an obvious chemical equivalent thereof.

16. A compound of the Formula I as defined in any one of claims 4, 5 or 7, when prepared by the process according to claim 4, 5 or 7, respectively, or by an obvious chemical equivalent thereof.

17. A streptamine according to claim 8 when prepared by the process according to claim 8 or by an obvious chemical equivalent thereof.

18. A 5-deoxystreptamine as defined in claim 9, when prepared by the process according to claim 9 or by an obvious chemical equivalent thereof.

19. A 2,5-dideoxystreptamine according to claim 10 when prepared by the process according to claim 10 or by an obvious chemical equivalent thereof.

20. A streptamine according to claim 11 when prepared by the process according to claim 11 or by an obvious chemical equivalent thereof.

21. A 2,5-dideoxystreptamine according to claim 12 when prepared by the process according to claim 12 or by an obvious chemical equivalent thereof.

22. A streptamine according to claim 13 when prepared by the process according to claim 13 or by an obvious chemical equivalent thereof.

23. A process for preparing an aminocyclitol antibiotic of the streptamine, deoxystreptamine or dideoxystreptamine-type which comprises culturing a nutrient medium containing carbo-hydrates, a source of assimilable nitrogen, essential salts and a cyclitol of the 2-R2'-5-R5'-3,4,6-trihydroxy-cyclohexanone or 2-R2'-5-R5'-1,3,4,6-tetrahydroxy-cyclohexane class represented by the Formula IIIa or of the 5-R5'-1,4,6-trihydroxycyclohex-2-ene class represented by the Formula IIIb where, in either case, R is hydrogen or acetyl: R3' is oxo or hydroxy; and R2' and R5' each are hydrogen, hydroxy or OR, in the presence of Mioromonospora purpurea ATCC, 31,164.

24. A process according to claim 23, for preparing a com-pound of the Formula where R1, R3 and R8 each represent hydrogen;R2 and R5 each represent hydrogen or hydroxy and R6 and R7 each represent hydrogen or methyl and where there is used a cyclitol of Formula IIIa or IIIb where R is hydrogen or acetyl; R3' is oxo or hydroxy; and R2' and R5' each are hydrogen, hydroxy or OR, and a strain of a microorganism which is incapable of biosynthesizing the cyclitol unit but which is capable of in-corporating the cyclitol into the antibiotic molecule as an aminocyclitol unit.

25. A process for preparing a compound of the Formula ...I

where R1, R3 and R8 each represent hydrogen and R2 and R5 each represent hydrogen or hydroxy; and R6 and R7 each represent hydrogen or methyl; which comprises culturing a nutrient medium containing carbohydrates, a source of assimilable nitrogen, essential salts and an aminocyclitol having the Formula IIa or IIb where in either case R2' and R5' are each hydrogen or hydroxy, and R1' is amino or hydroxy in Formula IIa and hydroxy or benzylideneimino in Formula IIb, in the presence of Micro-monospora purpurea ATCC 31,164.

26. A process according to claim 23, for preparing 0-3-deoxy-4-C-methyl-3-(methylamino)-.beta.-L-arabinopyranosyl-(1?6)-0-[2-amino-6-(methylamino)-6-C-methyl-2,3,4,6-tetradeoxy-.alpha.-D-erythro-glucopyranosyl-(1?4)]-D-2-deoxystreptamine wherein the added cyclitol is epi-inosose-2.

27. A process according to claim 23, for preparing gontamicin wherein the added cyclitol is 3,4,5,6-tetrahydroxycyclo-hexono (3,5-cis).

28. A process according to claim 25, for preparing gontamicin C1, 0-3-deoxy-4-C-methyl-3-(methylamino)-.beta.-L-arabino-pyranosyl-(1?6)-0-[2-amino-6-(methylamino)-6-C-methyl-2,3,4,6-tetradeoxy-.alpha.-D-erythro-glucopyranosyl-(1?4)]-D-2-deoxystreptamine, wherein the added aminocyclitol is 2-deoxystreptamine.