IE40437B1 - Novel pseudotrisaccharides and methods for their production - Google Patents

Abstract

1473733 Pseudo trisaccharides SCHERICO Ltd 1 Aug 1974 [6 Aug 1973 19 March 1974 (2)] 33968/74 Heading C2C The invention comprises 1-N substituted derivatives of the 4,5-di-(aminoglycosyl)-1,3- diaminocyclitols gentamicin A, gentamicin B, gentamicin B 1 , gentamicin C 1 , gentamicin C 1a , gentamicin C 2 , gentamicin C 2a , gentamicin C 2b , gentamicin X 2 , sisomicin, vendamicin, tobramycin, Antibiotic G-418, Antibiotic 66-40B, Antibiotic 66-40D, Antibiotic J1-20A, Antibiotic J1-20B, Antibiotic G-52, mutamicin 1, mutamicin 2, mutamicin 4, mutamicin 5 and mutamicin 6, where the substituent is -CH 2 X with X being hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, hydroxyalkyl, aminoalkyl, N-alkylaminoalkyl, aminohydroxyalkyl, N-alkylaminohydroxyalkyl, phenyl, benzyl or tolyl, said aliphatic radicals having up to seven carbon atoms and, if substituted by amino and hydroxy, bearing the substituents on different C atoms and pharmaceutically acceptable acid addition salts thereof. The compounds of the invention may be prepared by treating one of the above named 4,6-di-(aminoglycosyl)-1,3- diaminocyclitols which may have aminoprotecting groups at any position other than position 1, with an aldehyde of formula X<SP>1</SP>-CHO, with X<SP>1</SP> being a group as defined for X above wherein any amino or hydroxy group present may be protected in the presence of a hydride donor and reducing agent and, if required, removing all protecting groups present in the molecule the last process step being followed by isolating the derivative as such or as a pharmaceutically acceptable acid addition salt. [GB1473733A]

Description

40437 This invention relates to novel pseudotrlsaccharides and methods for their production, and to methods for their use as antibacterial agents.

More specifically, this invention relates to novel, anti-bacterially active 1-N-substltuted derivatives of 4,6-di-(aminoglycosyl)-l,3-diaminocyclitols, to methods for their production, to pharmaceutical compositions comprising said 1-N-substltuted derivatives of 4,6-dl-(aminoglycosyl)-l,3-diamlnocyclltols and to methods for their use in treating bacterial infections.

Known in the art are broad spectrum antibacterial agents which may be classified chemically ar. '»,6-dl-(aminoglycosyl )-l»3-diaminocyclitols. Valuable antibacterial agents of this group are those wherein the aminocyclitol is 2-deoxy-streptamine or a derivative thereof having amino functions at positions 1 and Particularly valuable antibacterials of the 4,6-di-(aminoglycosyl)-2-deoxystreptamines are those wherein the aminoglycosyl group at the 6-position is a garosaminyl radical. Within the class of 4-aminoglycosyl-6-garocaminyl-2-deoxvstreptamines are antibiotics such as gentamicins ^ ^1' ^la* ^2* ^*2a* ^2b sisomicin^ verdamicin, Antibiotic C-'»l8, Antibiotic G-52, Antibiotic JI-20A and Antibiotic JI-POB. 40437 This Invention relates to compounds, wherein tho amino group at the 1-pooitlon of the 2-deoxystreptamlne or derivative thereof In a 4,6-di-(aminoclycosyl )-l,."5-diarnino-cyclltol is selectively N-subr.tltuted. The 1-N-nubsti tuted derivatives of the 4,6-dl-(aiulnoglycosyl)-2-dcoxyctreptamincs or derivatives thereof are valuable broad spectrum antibacterial agents.

In one of its product aspects, the Invention relates to 1-N-substltuted derivatives of the 4,6-di-(aminoglycosyl)-1,3-diarainocyclitols gentamlcin A, gentamlcin B, gentamicin Bj, gentamicin C^, gentamlcin C1&, gentamlcin Cg, gentamlcin C2a* EGhtamicin gentamlcin Xg, sicomicin, verdamicin, tobramycin. Antibiotic G-418, Antibiotic 66-40B, Antibiotic 66-40D, Antibiotic JI-20A, Antibiotic JI-20B, Antibiotic G-52, mutamicin 1, mutamicin 2, mutamicin 4, mutamicin 5 and mutamicin 6; wherein the substitu&nt is -CHgX with X being hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, hydroxyalkyl, aminoalkyl, N-alkylaminoaJkyl, aminohydroxy-alkyl,N-alkylaminohydroxyalkyl, phenyl, benzyl or tolyl, said aliphatic radicals having up to neven carbon atoms and, if subr.tituted by amino and hydroxy, bearing the sub-stitucnts on different carbon atoms, and the pharmaceutical^ acceptablc acid addition salts thereof. 40437 Included nmon;', the intbst I IttrutQ oont r-.nplatwl IVr U»o moiety CIl^C in tho novol compound:; nrc; Htralght and branched t chain alkyl groups such as ethyl, n-propyl, n-butyl, 3-methylpropyl, n-pentyl, P-roethylbutyl, 7-methylbutyl and 3,3-dimcthylpropyl; n-hexyl, £-methylpentyl, p-ethyl-butyl, 7-ethylbutyl, n-heptyl, 6 -methylheptyl, 3-ethyl-pentyl, 7-ethylpentyl, S-ethylpentyl, 7-propylbutyl, n-octyl, iso-octyl, 3-ethylhexyl, £-ethylhcxyl, C -ethylhexyl, P-propylpcntyl, 7-propylpontyl; alkcriyl croup:; such as P-proponyJ, P-mcthylpropenyl, P-butonyl, P-mcthyl-P-but.onyl, P-ethyl-P-hoxenyl; cyclic /'.roups such as cyclopropylmethyl, cyelopentylrnethyl, eyclohexy]methyl and cyclopentylethyl; aromatic groups such as o-, rn—, £-methylbenzyl; hydroxy substituted straight and branched chain alkyl groups such as £-hydroxypentyl, P-hydroxy-7-mctliylbutyl, 3-hydroxy-P-rnethylpropyl, S-hydroxybutyl, P-hydroxypropyl, 7-hydroxypropyl, GJ-hydroxyoctyl; amino substituted straight and branched chain alkyl groups such as € -aminopentyl, P-aminopi-opyl, 7-arninopropyl, S-ax.ino-bufcyl, P-amino-7-melhylbutyl and CJ-aminooctyl and rnorio-N-alkylated derivatives thereof such as the N-methyl, N-ethyl anu N-propy] derivatives, e.g. £ -methylaminoperityl, 0-iuethyl-aminopropy], P-otliylarninoprop.y 1, £-iiieUiylaminobuty I, P-niol'.hylariiino-7-ni'M.liyl butyJ and (*) -mcthy Jam J nohiit.y J; amino and hydroxy disubstituted .-.traight and branched chain alky] 40437 groups such as P-hydroxy—£. — aminopentyl, 7-hydroxy-7- methyl- $-aminobutyl, P-hydroxy- S-aminobutyl, 0-hydroxy- / s 7-aminopropyl, and 0-hydroxy-0-mothyl-7-aminopropyl; and mono-N-alkylatcd derivatives thereof such as p-hydroxy-5 £ -mcthy lamlnopentyl, 7-hydroxy-7-methyl- £ -methylamino- butyl, P-hydroxy- S-methylaminobutyl, P-hydroxy-7-ethyl- amlnopropyl and P-hydroxy-p-methyl-7-methylaminopropyl. 1 1 i i The compounds arc preferably 1-N-CIlgX-derivatlves containing garosaminyl as 6-amlnoglycoslde radical and, in a more 10 preferred feature, 2-deoxystreptamine as 1,3-diaminocyclitol.

The 2-deoxystreptamine Is present in all the above listed compounds of tho invention except the mutamicins. The 1,3-diaininocyclitol nucleus in each of the 1-N-CHgX-mutarnicins 1» 2, ;l, 5 and 6 are streptamine, 2,5-dldeoxystreptamine, 15 2-epl-streptamine, 5-amino-2,5-didcoxystreptamine and 3-epl-2-deoxystreptaminc, respectively.

The l-N-CllgX-4 • ai.iinoglycosyl-6-garosarninyl-2-deoxystrcptamines of this invention are the derivatives from gcntamicin D, gentamiciri D^, gcntamicin C^, gcntamicin C^&, gentamlcin Cg, 20 gentamlcin C2&, gcntamicin Cgb, gcntamicin Xg, sisomicin, verdamicin, Antibiotic G-4l8, Antibiotic JI-20A, Antibiotic JI-20H and Antibiotic G-52 YJhich compounds are defined by the following structural formula I: 40437 HCII^X wherein X is as defined hereinabove, and wherein Y is an aminogli'conyl__£unction selected from" ch2nh2 (in 1-N-CH2X-gentamicin B) (in l-N-CH X-gentiimiciri B^) cii3nii nil., _ (in l4M;[!^X-grnLnmiciri C^) CH2OTI2 fi nl gonlniiiicin ) l.'i Nfl„ 40437 cn2Niini3 cii2nh2 cii2oii (in 1-H-CH-X- |\OH gcntamicin HO (in l-n-cii^x-verdamiciri) (in 1-N-CH-X-Antibiotic HO JI-20A) nii_ ,cii2nhcii3 (in 1-N-OH-X-gentamicin X2) ch, ho- (in 1-N-CH X-Antibiotic HO G-52) and. (in 1—N-CH.^X-sioomicin") (in 1-N-CH9X-Antibiotic JI-20B) Q- (in 1-N-CH X- Antibiotic G-413) nh, 40437 Other useful .l-N-t'll^X—l (;imiiuxjlyeo.;yl) —;?• deoxy-strcptamincs of this invention include. 1-H-C1]2X-tobramycin of the follov/ing formula XI: 'caJm2 NHCH2X II wherein X is a.*; '.lereinabove def ined; l-N-CH2X-Anfcibiotic 66-40D of the follov/ing formula III (which are among the preferred compounds of this invention) : CII0NH_ III 10 wherein X is nrs hereinabove defined; - 0 40437 and l-N-CII2X-gentflinicin A and l-N-CfJ2X-Antibiotic 66-40B of tho following formula IV: Nil, iich-x O iv OU whoroin X is as hereinabove defined and Y' i cii20ii A o 5 ho nh, 2 and Y' is C,H2mi2 in 1 -K!-CII^X-Antibiotic 6G-4023 nil, 2 Cj .. 40437 The! l-K-cn^X-mutaraicins of thin invention in- whorcin X is as hereinabove defined, and in 1-N-CII2X-mutajnj ein 1, V2 and V7,. are hydrogen and W2 and arc hydroxy — - in l-N-CH2X-mutamic.in 2, W2» V2> W,. and are hydrogen; in l-N-CII^X-mutomicin 4, W2 and VI ^ are hydrogen and V2 and arc hydroxy: in 1-N-CIIpX-mutamicin 5, W2» V2 and W,. are hydrogen and Vj is amino; and in l-N-CII_X-mutamicin 6, W_, V_ and V_ are — ii /. & !> hydrogen while Wg is hydroxy.

(In th» structural famliw Mt forth htrala, tha ualatlgBaM aubstltuanta at tha band t*mnal« ar* understood to b« bjtro&n atou). - lo 40437 Alco included wJ thin tho Invention are pharmaccutically j aoooptui>le uciU addition r.nllr, of the l-N-CJIgX-^fi-di-(nminonlycooyl )-l,2-diamlnoeyclitol.s cuch a3 defined by formulae I, II, III, IV and V, which cults are made accord-f, inn to known proccdurco ouch as by neutralizing the free base with the appropriate acid usually to about pH 5.

Suitable acids for this purpose include acids such as hydrochloric, sulfuric, phosphoric, nitric, hydrobromic, acctlc, propionic, maleic, ascorbic, citric and the like. 10 The physical embodiments of the acid addition salts of the 1 -N-CUgX, 6-di - (aminoglycosy 1) -1,2-diaminocycli tol s of this invention are characterized by bcinr: white solids which are soluble in water sparingly solubl* la aost polar solvents 15 tad ianolubla la bob-polar orRanlo solnnta( The 1 -N-CH,,X-'l, f>-di- (aminoc,.lycosyl )-l, jj-diaminocycli tols of this invention such as defined by formulae I, II, III, IV and V and their non-toxic pharinaci.-utically acceptable acid ziddltion salts, in f.oi.oral, exhibit broad spectrum 2o antibacterial activity . Pai ticular-ly the 1-N-lower alkyl derivatives possess Improved a-it.1 bacterial activities ':onipaiv;d to th« parent antibiotic?; which is apcei finally cinnl in the enhanced activity of the compounds against orfvini : .m:- resistant to tho parent, oon.uound. Thus, for 25 ^xainp] r, the compounds are i.ioro act. iV'- ornarii s^is 40437 which inactivate the par.-iil antlbioUfie by noetylati on of the >-amino (iroup and/or by adenylylation or tho 2M-hydroxyl r.roup, Of tho no, oomo aloo exhibit anti-protozoal, antl-amoobio and anthelmintic properties.

Fj> A proforred croup of oompounds of tho invention are tho l-N-aubntitut.od derivative of tho A-aminoplycocyl-6-C.aroaaminyl-P-deoxyotreptairtinoB (jcntamicin 13, gcntamicin B^, r.crt.imiein Cj, r.entamiolri gentamlcin Cg, gentamlcin Xg, r.i«omicin, vordnmicin, Antibiotic JI-20A, Antibiotic JI-20B, .10 Antibiotic C-'jP and Antibiotic f»-'llb, of which the derivatives of r.entamie in C^, r.entamlcJ n Cja, sloomicin, verda-micln and Antibiotic G~'jP art; moat preferred. Other particularly useful compounds arc tho l-N-substituted derivatives of Antibiotic 66-40D. 15 In the 1-N-subctJtuent X is preferably selected from hydrogen, alkyl, hydroxya]):yl, aminoalkyl, amlnohydroxy-alkyl, phenyl ox- benzyl. :u\Ju aliphatic radicals having up to seven carbon atom;-, If substituted by amino and hydroxy, be.*i ri nr. the sub::!. •i.uentr. on different carbon atom:;. f'O 01' l.hi.:::<: the preferred radicals art- hydroi'.cri, alkyl, aminoalkyl and hydroxy.-*Ikyl wi I'i uj> to seven carbon atoms and aminohydrox.yal ky.l with up to thre e carbon atoms and bearing the 1 tuents on different carbon atoms. 40437 Particularly uucTul compounds of the; invention arc those whercJn X is Jiydrogen, methyl, ethyl and propyl and preferably methyl and ethyl. A particularly valuable group are the l-N-CHgX-;l-aminoglyco5yl-6-garosaminyl-2-dcoxyKtrcptamineo of formula I wherein X is a lower alkyl having 1 to J carbon atoms, particularly the 1-N-lower alkyl derivatives of gcntamicin C^, gentamlcin Cla, gentamlcin Cg, gcntamicin C2a> gentamlcin Cgb, sisomlcln, vcrdamicln, and Antibiotic G-'j2 as wall as 1-N-lower alkyl-Antibiotic 66-40D of formula III, which derivatives are broad spectrum antibacterial agents, being active against gram-positive bacteria (e.g. Staphylococcus aureus) and gram-negative bacteria (e.g. Escherichia coll and Pscudomonas aerurt nosa) as determined by standard dilution tests, including bacteria resistant to the 1-N-unsubstituted precursors. Particularly useful are 1-N-ethylverdamicin, and 1-N-lowcr alkylsisomicins, e.g. 1-N-methylsisomicin, l-N-(n-propyl )-r.ioomicin, l-N-(n-butyl )-sisomicin and, preferably, l-N-ethylsir,omicin which exhibit activity against grom-negatJve organisms which are rcr.istant to their 1-N-unr.u)»:;titutod precursors. Other particularly useful compound:-. are 1-N-c-tliy.lgentainlcin 1-N-ethylgentainLcin C^, 1 -N-cthylantibiotLc G-|>?, l-N-(n-propyl )-vcrdamicln, 1-N-(S--*»iiiir)obutyl )-:;ir.omicin, 1 -N-mcUiylverdaniicin, 1-N- (n-buty 1 )vordainj cln, l-N-(S-2-hydroxy —'l-am.i.nobutyl )-gentamicin 1 -M-(£5-2-hyd i-oxy-'l n obi it yli fomioin and l-H-(S-2-hy cU\; x y - >1 - ;u i; i n o b u ty 1) - v;: i • c t n mi c .1 n. -13- 40437 In another of its product aspects the invention relates Jto 1-N-acylated derivatives of the .'l,6-di-(aminor:lycosyl )-l,3-dlnminocyclitols, i.e. compounds of formulae I, II, III, 9 IV I- 40437 In a more limited manner tho amid on may be defined as 1-H-substl tuted derivatives of the 4,6-di-(aminof:lycosyl )-l,."5-diatninocyclitols listed above wherein Z is hydrogen, alkyl, alkcnyl, cycloalkyl, cycloalkylulkyl, hydroxyalkyl, 5 nminoalkyl, N-alkylaminoalkyl, aminohydroxyalkyl with at least 'j carbon atoms, N-alkylaminohydroxyalkyl, phenyl, benzyl or tolyl, said aliphatic radicals havinp up to seven carbon atoms and, if substituted by amino and hydroxy, henrJnp th'.; :;ul>:;l.it.tit!iitr. on different carbon atonn>. 10 InrJ tided amour, the substituonts contemplated for the moiety 9 -C-2 Jn tho novel umido compemnds arc most of tho radicals listed above* for the moiety -CII^X, wherein tho terminal methylene rroup is replaced by carbonyl, such as formyl, acetyl, propionyl, propenoyl, butyryl, isobutyryl, cycloid propylcarbonyl, benzoyl, hydroxyacetyl, aminoacetyl, 4-hydroxybutyryl, 4-aminobutyry.l and the like.

A preferred /-.roup of amide compounds are derivatives of the 4-.-iri:i.no/:1yeosyl-6-;';.-irosaminyl-?-deo;<.yutreptamlnes gentamicin H, pent ami c: in pen t ami e: in C^, pentamicin C^a, pentamicin 20 C0, pentamicin C.ir , pentamicin C0. , pentamicin X~, sisomicin, C £D t* verdamieln, Antibiotic (J-'llL, AntibiotJe; J1-20A, Antibiotic JI-J'OD and Antibiotic 0-52 or which the more preferred derivatives are tho:.c of i oatrmicin 0, pentamicin C, , J id uir.o:i::i e: in, vcrei:>:.)it: in and y\s11■' ;>.LoLjc (I-S2. 40437 On account of their antibacterial activity, tho derivatives of sisomlcin, vcrrin.mic.in and Antibiotic 66-40J) and especially the acetyl, proplonyl and butyryl derivatives are most preferred. 1-N-Acetylsisomicin, 1-N-acetyl-verdamicin, 1-N-acetylantibiotic G-52 and 1-N-acetyl-I'.entarnicin Cla are ex ampler, of particularly useful compounds .

Tlx? invention also cmbrae«::: Uie pharmaceutical^ acceptable acid addition salts of the 1-N-acylatcd compounds s.ueh as those formed with hydrochloric, sulfuric, phosphoric, propionic, maleie, tartaric, benzoic, phenyl-acetic, cyclopropane carboxylic acids or the like.

These salts may be prepared by dissolving the free nitrogen base in water, adjustinr. the solution of the antibacterial ar.ent to pH 4.0 and lyophilifcinp the resultant solution. 40497 Most of the aforementioned l-lj-unsubstituted 4,6-0i-(nminoglyconyl)-1,3-diaminocyclitol antibiotics from vhich the 1 --N-subntituted derivatives of the invention nay bo mndc arc known. Of the gentaraicins, the starting 5 compound referred to herein as gcntamicin is also known in the art as gcntamicin X. The starting compound referred to herein as gcntamicin C2a iu isolated and characterized as set forth herein in Preparation 1.

The starting compound referred to herein as lo gentamicin C2b* *s *-so^atc^ an<* characterized as set forth in Preparation 2, and having the structural formula shown herein, is named in some prior art as gentamicin C2a.

The isolation,properties, and planar configuration of gcntamicin C2 is described in U.S. Patent No. 15 3,651, 042.

Antibiotics 65-^OB and GG-40)), their preparation, .isolation, propertie:-., and conf.igu.rntion, are described in Uelgian Patent No. B.ll,37<>. The antibiotics arc co-produccd with sicomicin, the major product of fermentation 20 of Micrornononnora inyoen.'jj r. (described in Patent Specification No. 33,174) and may bo separated from the fomenta tion medium by applying rpecial ehr cs^atogr,.ph ic separation techniques. - 1.7 - 40437 Mutamicino 1, 2, 4, 5, and 6, the configuration of which ia shown abovo, may be prepared by cultivating a mutant strain of Micromononpora invoonsis heroin designated Micrrraonospora invoonni« strain 1550P-1G in an aqueous nutrient medium. This mutant strain is incapable of producing an antibiotic when cultivated under submerged aerobic conditions in an aqueous nutrient mediuan absent tho 1,3-diaminocyclitol building block. However, when certain of such compounds are added to the fermentation medium, the mutamicins are produced. When 2-deoxystrepta-mine in added to the fermentation, tho known antibiotic siuomicin is produced.

The requisite 1,3-diaminocyclitols which must bo present during fermentation in orclor to obtain the n.utnmicinn are as follows: stroptnmine for mutamirin 1 2,5-didooxyn Lreptaniinc for mutcuTJ.icin 2 2-cpistreptain:!no for luutrwnicin 4 2,5-d.ideoi:y-5-;*j1iinoctn ptaminc for mutamicin 5 5-o»i-2-deo.;y:;Lr«-ptyiriino for mut;>i::i nr- a known co.,i;>o>mrl, 2, c; i oooJiy:;!:I'c.'.no n«* may )jc ;• rod aei.-orcling to the proc -vui ;• r.et forth in PrcparalJon G by jsnbstitu;-. ino hydras.j rif for rr.ono:,Ltliyl.-hytlrar.i in:. 'i.:iv.c:t>1:nu* i w i.i.- y br. i■;by I i:r 40437 described by Tetsue Suami et al. in tho Journal of Organic Chemistry 33, No. 7, 2831-2834 (1968). 2,5-Dideoxy-S-nminostreptranino may be prepared by i) cleaving an N,N-diacyl derivative of 2-deoxyctrcptamine with periodate, ii) treating the so-obtained dialdehyde with nitrcmiethane under alkaline conditions which effects ring closure and the insertion of a nitro group at the 5-position, iii) reducing the nitro group to an amino group followed by hydrolysis of the N,N-diacyl functions to the free amine. 5-Kpi-2-deoxystreptamine may be prepared from 1,3,4,6-te trace tyl-5-epi -2 -deoxystreptamine (prepared by the method of Hnsagawn and Sable, Tetrahedron, 25, 35-67 (1969)) by hydrolyzing the ester and amide groups with 6 W hydro-chloric acid.

The jjreparntion of the-, mutamicins is set forth heroin in Preparation 10. - 19 40437 Taxoncvn»;nt, B.lochcmic;;l .-.nd Morpholepj.ca.1 Proportion off Micromononporn in vomit i r. Strain 15SOP-IC (NUUT. 57*12) Tho parent microorganism (Mi oromononpora invoonoin) io deposited with U.S. Department of Agriculture, Northern Utilization Research and Development Division, Peoria, Illinois, where it was assigned the numerical designation NRRL 3292, and is described in the Journal of Antibiotics (Japan) Vol. XXIII, No. 11, pages 551-558 (1970) in a publication by M. J. Weinstein, et al.

Micromonospora nyoensi s strain 1550F-1G exhibits grov/th characteristics that are similar to Microraonospora inyoensi:? (the microorgani&tn from which it is derived) . A culture of Mj_ inyoennis .«?train 1550F-13 is also deposited with the above-mentioned depository under the numerical designation URRL 5742.

The: following tables so!, forth a number of t;o: — onpmic<"il, biochcmi cal and i.>orpholoc)ic;• i prc.-pcrtj.os of the microorganism. In describinv; the color; formations the following system .■•vp'J oyed: Tho color ck-signati.i a consists of two designates. The first is a color na:ae t_nl-.cn i r<,ir. U:c 'Descriptive Color Name Dictionary, " 40437 by 1'iiylor, Knocho and Granville, pnblir;hcd by the Container Corporation of Anirrica, 1050 (U.S.A.), with a color chip numlx;r corresponding to the color name; said chip number taken from tho "Color Harmony Manual", 4th edition, 1958, 5 published by the Container Corporation of Amcrica# U.S.A.

The sccond designate, consists of a color-name and number Which refers to tho synonym or near synonym found in the National Bureau of Standard, Circular 553, November 1, 1955 (U.S.A.). 10 When cultivated on an agar medium comprising 3% Kii nminc Type A (a peptone resulting f.voin the enzymatic digestion of casein and serving as a source of nitrogen), 1% dextrose and 1.5% agar the microorganism ML invoonnjb strain 1550F-1G NRJilj 5742 shows such a poor growth that 15 it is insufficent for characterization. - 21 - 40437 VAMI.K T i'oJ o!\y. JJty'iqi'J.|V°.1}.L}f-.." • • •. t .'|;i>!«>;.p. >) a_ Jj.i.w.-;:";•.! A1. i?j.}f* NHKIi S7-1 t.m Various M»«li;i or condition it Medium or Condition Obncrval .ions Sucrose Utilised Temperature Grows well at 28° and 37°C No growth at 50 C Acrobic or Anacrobic Aerobic Czapeks Medium (Gluco.se) Growth fair to poor, plicate -membranous, no diffusible pigment g3ic light amber - dark orange yellow 72 Anparagino- Glucose Medium Growth fair to poor, flat to membranous, no diffusible pigment g3ic light amber - dark orange yellow 72 Calcium Malate Agar Growth poor, flat, no diffusible pigment g3ic light amber - dark orange yellow 72 Nitrate Reduction Variable Ordinary Agar (Water Agar) Growth poor, insufficient for description Nutrient Agar Growth fair to poor, flat to slightly wrinkled g4ne luggage tan -strong brown 55 to g4pn chocolate brown -dark brown 5?) l^.f/lcr's Serum Medium (Diuo) Crov/th fco.v, trii'cr part ially 1 italic J.'icd l'C tcrr.i<•(.»; !...i -strong brovn 55 - - «« I .T.o.V?(! J.

Jto.jJ i V«li\ OV Cot »rl i 11 On OhsorvntJonp Potato Plug No growth Prpl.orM! Glucose Agar Growth J«.1r lc» poor, flstL to slightly furrowed, no diffusiblo pigment produced g4ne luggage tan - strong brown 55 Kgg Agar (Dorset Egg Medium-Di£co) Growth poor, insufficient for characterization Gelatin Medium Growth fair to poor, flat to slightly ridged, no diffusible pigment produced, gelatin weekly hydrolyced g41o turf tan - light brown 57 Sttirch Agar Growth fair, flat, no diffusible pigment, starch weakly hydrolysed only directly under colony g31e yellow maplo - strong yellowish brown 74 to black Tyrosine Medium Growth fair to poor, flat, slight darking of medium g41e turf tan -light brown 57 Litmus Milk (Difco) Peptonized, acid reaction Cellulose Medium Cellulose poorly decomposed (hydrolysis of cellulose weak) Hennett 's Agar ):ucr::on' s Agar Growth good, membranous -plicate, no diffusible", pigment black Growth Hydrjde-donor reducing agents useful in this process include dialkylaminoboranes (e.g. dimethylamino-boranc, diethylaminoborane and preferably morpholinoborane), tetraalkylarunonium cyanoborohydride (e.g. tetrabutylsmmonium cyanoborohydride), alkali metal borohydride (e.g. sodium borohydridc) and preferably, alkali metal cyanoborohydride (e.g. lithium cyanoborohydride and sodium cyanoborohydride).

The process is conveniently carried out in an inert solvent. By "inert solvent" is meant any organic or inorganic solvent in which tho 4,6-di-(aminoglyooc.yl) -1,3-diaminocyclitol starting compounds r>nd the reagents are soluble, and which will not interfere with the process 4043? under the reaction conditions thereof so thcro are produced a minimum of competing sido reactions. Although anhydrous aprotic-solvents may sometimes advantageously be employed in the process (such as tetrahydrofuran when utilizing morpholinoboreine as hydride donor-reducing agent) the process is usually carried out in protic solvents, e.g. in a lower alkanol or, preferably, in water or in an aqueous lower alkanol (e.g. aqueous methanol, aqueous ethanol), although other water-miscible co-solvent systems may be employed such as aqueous dimethylformamide, aqueous hexamethylphosphoramide, aqueous tetrahydrofuran and aqueous ethylene glycol dimethyl ether.

The process is conveniently carried out at a pH in the range of from 1 to 11, preferably from 2 to 5 and proceeds best in the range of from 2.5 to 3.5. The acidic medium which is preferred may be obtained by adding an organic or inorganic acid to the 4,6-di-(aminoglycosyl) -1,3-diaminocyclitol. Thereby acid addition salts of the compounds are formed. Any organic acid such as acetic acid, trifluoroacetic acid, or £.-toluene-sulfonic acid or inorganic acid such as hydrochloric acid, sulfuric acid, phosphoric acid, or nitric acid may be used. It is most convenient to use sulfuric acid. In the preferred feature of the process, it is also convenient to prepare the acid addition salt starting compound in situ by adding i 40437 the desired acid (e.g. sulfuric acid) to a solution or suspension of the 4#6-di-(aminoglycosyl)-l,3.-diaroino-cyclitol (e.g. sisoraicin) in a protic solvent (e.g. water) until the pll of the solution is adjuctcd to tho desired pll.

Typical aldehydes of the formula X'CBO wherein X' is as above defined which are useful in the process include straight and branched chain alkyl aldehydes juch as formaldehyde, acctaldehyde, n-propanal, n-butanal, 2-methyl-propanal, rj.-pcntanal, 2-methylbutanal, 3-methylbutanal# 2,2-diraethylpropanal, n-hcxanal# 2-ethylbutanal, n-heptanal and n-octanal» alkenyl aldehydes such as propenal, 2-methyl-propenal, 2-butenal# 2-methyl-2-butcnal, 2-ethyl-2-hcxenal; cyclic aldehydes such as cyclopropanecarbaldehyde, cyclo-pentanccarbaldehyde, cyclopentaneacetaldehyde, cyclohexane-caxbaldehydoj bcnzaldehyde, o, m, and jj-toluenecarbaldehydes and phcnylncotnldehyde; hydroxy substituted straight and branched chain alkyl aldehydes such as 5-hydroxypentanal, 2 -hydroxy-3 -methylbutanal, 2 -hydroxy-2 -raethylpropana 1, 4-hyOroxybutanal, 2-hydroxypropanal and 8-hydroxyoctanalj amino substituted straight and branched chain alkyl aldehydes such as 5-aminopcntanal, 2-aminopropanal, 3-amino-propanal, 4-uroinobutanal, 2-amino-3 -wethylbutanal, 8-amino-ocfcr.nr.l and mono-N-alkyl derivatives thci'eof; and amino and hydroxy disubstitui-ed straight and branched chain •w 40437 alkyl aldehydes such as 2 -hydroxy-5 -aminopentanal, 3-hydroxy-3-methyl-4-aminobutanal, 2-hydroxy-4-aminobutanal, 2-hydroxy-3 -aminopropanal, 2-hydroxy-2methyl -3 -aminopropanal, 2-amino-3-hydroxyoctanal, and mono-N-alkyl derivatives thereof.

In this process, if the aldehyde possesses a chiral center* one can use each enantiotner separately or together as a racemate and there will be obtained the respective diastereoisomers or a mixture thereof, respectively.

The aldehyde reagents useful in the process arc either known compounds or are easily prepared from known compounds utilizing procedures well known in the art. Thus, for example, alkylaldehydes substituted by both hydroxyl and amino functions (e.g. 2-hydroxy-5-araino-pentanal) may be prepared from an aminoaldehyde acetal (e.g. 4-aminobutanal diethylacetal) by protecting the amino function therein as an acetamido or phthalimido group utilizing known procedures followed by removal of the acetal function by acid hydrolysis thereby obtaining an N-protected aminoaldehyde (e.g. by converting 4-aminobutanal diethylacetal to the corresponding N-phthalimido derivative which upon ncid hydrolysis yields 4-phthnlimido- - 32 - butanal). Treatment o?. the M-protoctcd nminoaldchyde with hydrocyanic acid yields the corrcnpiMiding N -prot-noted-nminonlkyl hytl»*oxyn.itr i In (e.g. 2-hydroxy-S-phthnIiinido-vnloronitril'o) which upon catalytic reduction (e.g. hydrogen in the prcncnco of palladium) or by hydride reduction (e.g. with di-isobutylaluminum hydride) yields an N-protected amino-hydroxy aldehyde (e.g. 2-hydroxy-5-phthalimido-pentnnal) which is an aldehyde reagentused in this process.

When carrying out the process whereby a 1-N-um;ubotitutcd-4,G-di-(aminoglycosyl) -1,3-diaminocyclitol is treated v/ith a hydride donor arid an aldehyde, to obtain the corresponding l-*-substitutcd derivative of a 4,6- (aminoglycosyl)-1,3-diaminocyclitol, in order to minimize competing side reactions when an aminoaldehyde is used as a reagent, it is preferable to protect tho amino function in the aldehyde, e.g. with an acyl blocking group such as acetamido, phthalimido, or the like, prior to carrying out tho process, and then removing the N-protecting group in tho compound thereby produced. It may also be advantageous to pi-otect the hydroxy! grouu in hydroxy 1 -containing aldehydes when carrying out the pruc^sc; however, it is not generally n'.cessory. 40437 i It ia also possible to use the acetal or herai-.acetal of the aldehyde reagent in acidic medium which gives rise to in, situ formation of the required aldehyde.

A convenient method of carrying out the process comprises preparing a solution of a l-^unsubstituted-4#6-d£-(aminoglycosyl) -1,3-diaminocyclitol antibacterial agent (e.g. eisctnicin or verdamicin) in a protic solvent, (preferably water), and' adjusting the pH of the solution to from about pH 2 to about pH 5 with an acid {usually dilute sulfuric acid) thereby preparing the requisite acid addition salt of the starting compound. When the pH of the solution is at about pH 5, the acid addition salt thereby produced usually contains about one equivalent of acid for each amino function in the 4,6-di-(aminoglycosyl) -1,3-diaminocyclitol (e.g. per mole of sisaraicin there is present 2.5 moles of sulfuric acid). After the acid addition salt solution is prepared, there is added at least a molar equivalent, and preferably a large molar excess of the desired aldehyde (e.g. acetaldchyde, propanal or butanal) followed within a short time (usually in about 5 minutes) by the addition of about a molar equivalent (based upon the starting 4,6-di-(aminoglycosyl)-1,3-diaminocyclitol) of a hydride donor-reducing reagent, preferably an alkali metal cyanoborohydride, usually sodium 4043? cyanoborohydride. The reaction is frequently completed in loos than 30 minutes as determined by thin layer chromatography and there is obtained the corresponding 1-N-substituted derivative of a 4,6-di-(aminoglycosyl)-1,3-dinminocyclitol (e.g. 1-N-ethylsisom.icin or 1-N-ethyl-verdamicin). Isolation and purification of the derivative thereby produced is effected utilizing known techniques such its precipitation, extraction and, preferably, chromatographic techniques.

The process thus provides a novel, convenient, one-vessel proccss whereby an aminoglycoside is reacted in situ with an aldehyde (preferably in excess quantities) and with a hydride donor-reducing agent to produce as the major product, a mono-N-substituted derivative (e.g. 1-N-othylsisoinicin), in which process the 1-amino group attached to a secondary carbon atom is usually alkylated preferentially over other amino groups attached to primary and other secondary carbon atoms in the 4,6-di-(aminoglycosyl) -1,3-diaminocyclitol starting material.

It is also possible to use parc.ially N-i>rot The requisite starting compounds wherein amino groups are protected may be prepared according to procedurco - 36 - 40437 similar or identical to those set forth hereinbelow in Preparation 3. An used herein, the terms "blocking group" or "protoctiwj uroup" defers to group:: which render the blocked or protected amino groups inert to subsequent chemical roanipuljition, but vhich con easily be removed after the chcmioal manipulation is carried out. Exemplary of ouch amino protecting groups are benzyl, 4-nitrobenzyl, triphf-nylmcthyl, 2,4-dinitrophenyl; acylgroups such as acetyl, propionyl and benzoyl} alkoxycarbonyl groups such as methoxycarbonyl, ethoxycarbonyl, 2,2,2-trichloroethoxy-carbonyl, t>-butoxycarbonyl and 2-iodocthoxycarbonylj and arylalkoxycnrbonyl groups 3uch as carbobenzyloxy and 4-methoieybenzyloxycarbonyl groups.

In the blocking process, the protecting group is usually employed in tho form of an acid imidazole derivative, an acid azido or as active esters such as ethylthioltrifluoroacctai.f, N-benzyloxycarbonyloxy suc-cinimide or i>-nitrophenyltrAchloroethyl carbonate. Thus, blocking groups may be described as being derived from a cctnpound BgLg, wherein Bg bcroraos tho blocking group such an the; ucid portion of an nctivc cntrer, and T/j in a leaving group r.uch ns imidazole.

Alternatively, tho above cr.T.cribed procc.«:s m.~>y be carried out in a manner allowing •'.mc £>».•n i^Lion of a 1-K-Schi f Ei. banc derivative ir.u'l rcckic:' :g the - 37 - 40437 so-obtained Schiffs base. The process for the preparation of the 1-^.-substituted derivatives of the 4,6-di-(amino-glycosyl)-l#3-diaminocyclitols listed above wherein the subatituent is -CHjX and X is as defined above comprises reducing the N,C-double bond in a 1 -N=CHX■ -substituted derivative of one of the above named 4,6-di- (aminoglycosyl) -1,3-diaminocyclitols, wherein all NH2-groups are protected and NHCH^ groups may be protected with X' being as defined above, removing all protecting groups present in the molecule and isolating the desired derivative as such or as a pharmaceutically acceptable acid addition salt.

Those 4,6-di-(aminoglycosyl)-l,3-diaminocylitol3 wherein the 6-aminoglycosyl radical is garosaminyl usually possess a 3M-N-4"-0-protecting group which is identical with the l-W-substitucnt of the starting material because the oxazolidine ring is forced simultaneously with the 1-N-Schiffs base group. Thus, fcr example, 2',3-di-N-trifluoroacetylgentamicin upon reaction with an aldehyde (e.g. benzaldehyde, phenylacetaldehyde or acetaldehyde) is converted to the corresponding 3",4"-oxazolidine-l-ylidcne Schiff base starting material of this proccss (e.g. l-N-3"-N-4"-0-di-bcnzylidcno-2',3-di-N-trifluoro-acctylgentamicin C^, 1-N-3"-N-4"-O-di-phenethylidcnc- - 30 - r 40437 2,,3-di-i*-trifluoroacotylgontaraicin and l-N-3"-N- 4" -O-diothylicJono-2 •, 3 -di-JJ-trif luoroncetylgentnmicin + • C^) vhich, upon reduction with sodium borohydride and methanolic oodium methoxide yields tho corresponding 1-N-CIl2X-3",4"- (e.g. l-W-bonzyl-3"-N-4"-0-benzyli- dencgentamicin C^, 1 -N-phenethyl -3 " -N-4 " -O-phenethy1idene -gentamicin and l-^-othyl-3"-N-4"-0-ethylidenegcntamicin C^, respectively) which upon treatment with acid yields a l-N-CHgX compound of the invention, (e.g. 1-N-benzylgenta-micin C^, 1-N-phene thy lgentamic in and 1-N-ethylgonta-micin C^, respectively)« The novel 1-N-substituted derivatives of the 4,6-di-(aminoglycosyl) -1,3-diaminocyclitols such as defdlned by formulae I, II, III, IV and V may also be prepared by a process which comprises treating a 1-N-substituted derivative of one of the above named 4,6-di-(aminoglycosyl) -1,3-diaminocyclitols, wherein one or more amino-groups may be protected and the 1-N-cubctituent is -C-X" with X" being hydrogen, alkyl, alkenyl, cycloalkyl, cyclo-alkylalkyl, hydro::yalkyl, aminoalkyl, ll-alkylaminoalkyl, aminohydroxynlkyl, N-alkylaminohydroxyalkyl, phenyl, benzyl, tolyl or hydrocarbyloxy, said aliphatic radicals having up to seven carbon atoms, and if substituted by amino and hydro?:y, bearing tho substitucnts on different carbon atomr., and wherein any amino or hydro::y group present - 39 - 40497 may be protected, with an aroido-reducing hydrida reagent and, if required# removing all protecting groups proaent in the moleculei the last process step being followed by isolating tho desired derivative as such or as a pharmaceutically acceptable acid addition salt.

The process is usually carried out in a non-reactive organic solvent which is contemplated to be a solvent in which the starting compounds and the amide-reducing reagent sure soluble and which will not react with the reagent so there is produced a minimum of competing side reactions. Non-reactive organic solvents which are most useful in the reduction process are ethers such as dioxane, tetrahydrofuran, diethyleneglycol dimethyl ether and the like.

Preferred amide-reducing hydride reagents are aluminum hydrides and borohydrides including lithium aluminum hydride, lithium trimethoxy aluminum hydride, aluminum hydride, diboranc, di-isoamylborane, and 9-borabicyclo [3.3.1] nonanc.

In general, it is preferred to use dibornne as the amide-reducing agent cxcept when the starting compound possesses a double bond, e.g. as in 1-N-acyl-sicomicin, .l-N-acylverdamicin, 1-N-acyl-Antibiotic 66—40B, - 40 - 410437 1-N-ncyl-Antiblot 1 c 66-40D, and 1-^-acyl-Antibiotic G-52, which compounds arc conveniently reduced by means of lithium aluminum hydride.

When X" represents 1tydrocar by lorry, such as t~ butoxy, and the amide is subjected to reduction, the corresponding 1-N-methyl compound is formed. glycosyl)-1,3-diaminocyclitol is reduced to the corresponding l-N-CE^X derivative of the invention, if the acyl side chain of the 1-N-acyl-intermediate possesses a ehiral center, one can use each stereoisomer separately or a mixture thereof, and there will be obtained the corresponding diastcreoisotncrs or a mixture thereof, respectively. preparation of 1-N-substituted derivatives of the 4,6-di-(aminoglycosyl)-1,3-diaminocyclitols sisonicin and mutamicin 2, wherein the substituent is with X'" being hydrogen, methyl, ethyl or propyl, and of tho phannn.ceutically acceptable acid addition salts thereof comprises cultivating a mutant strain of tho species Micrcsnonosooto inyoennio. nnjnoly Micnmonoriiorn in"»>onnis strain 15f>0 '-.1.0 or a strain of tlv saine* spccies concr;-pondinj thc/roto in its capab.i lity ol profVicing one of In this process whereby a 1-N -X "-4,6-di-(amino- Another process of this invention, for the - 41 - 40437 the above defined antibiotics* in a nutrient medium comprising a compound of the formula OH Wherein V5 is hydroxy or hydrogen* and X'" is as defined above* until substantial antibiotic activity is imparted .to the medium and isolating the so-obtained compound as such or in the foxm of a pharmaceutically acceptable acid addition salt.

The microorganism Micromonospora invoensis strain 1550F-1G is deposited with U.S. Department of Agriculture* Northern Utilization Research and Development Division* Peoria* Illinois under the numerical designation NRRL 5742) several taxonomical* biochemical* and morphological properties thereof being listed hereinabove.

Some of the requisite 1-N-alkyl-2-dcoxy-D-streptamine aminocyclitols are known, e.g. 1-N-methyl-2-deoxy-D- streptamine (also identified as (-)-hyosamine) which* when cultivated with Micromonospora invocnsis strain 1550F-1G in the manner described in Example 16 4043? herejtibclow produces l-N-mothylsiscraicin.

Other requisite starting aminocyclitols (e.g. 1-N-ethyl and 1-N-propyl-2-deoxy-D-s treptamine are prepared from known aminocyclitols (e.g. (IS.) -l-acetamido-3-amino-1.3-didcoxv-mvo-lno3itol) or from known pseudodi-sacchnridon (e.g. garaminc) via chcmical transformations known in the art as exemplified in Preparations 7-9 hore-inbelow. Cultivation of each of 1 -N-ethyl-2-deoxy-D-streptanine and 1 -N-propyl-2-deoxy-D-streptomine with Micromonosnora invoongis utrain 1550F-1G leadg to 1-N-ethyl-oioomicin and 1-N-propylsisomicin, respectively.

Other aminocyclitol starting compounds (e.g. (-)-mono-N-methyl-2,5-dideoxystreptamine) are prepared 3 5 from ci3-4,8-dioxatricyclo (5.1.0.0 ' ]octane by treatment thereof with a mono-N-lower alkyl hydrazine (e.g. N-methyl-hydrnzine) and hydrogenolizing the thereby produced (—)-mono-N-lower alkyl-6,7-diaza—2,4-dihydroxybicyclo-[3..?.,I] octane. The (i) -mono-N-lower alkyl-2,5-dideo::y-streptaminen thereby produced may separated by optical resolution procedures known in the art to obtain 1-N-lower alkyl-^.S-didoorty-D-stn^itnnino and 3-N-lower alkyl-2,5-didcoxy-D-ctroplnminc, respectively. Alternatively, the racciuate of. the precursor, i.e. (-) -wono-N;-lower alkyl- - 43 - 40437 6,7-diaza-2,4-dihydroxybicyclo (3.2.1Joctane may be separated by optical resolution procedures to obtain 6-N-lower alkyl-G,7-diazn-2,4-dihydroxybicyc.lo [3.2.1]octane and 7-iJ-lower alkyl-6,7-diaza-2,4-dihydroxybicyclo [3.2.1]octane. Hydrogenolysia of the aforementioned 6-IJ-lower alkyl derivative leads to 1-N-lower aIky1-2,5-dideoxy-D-strepta-mine while hydrogenolysia of the 7-N-lower alkyl derivative leads to 3 -N-lower alkyl -2,5 -dideoxy-D|-streptajnine.

• Siailarly, ■ono-H-alkyl-2-d>oa^y»tr>pt«aln» prvp&rad v fraa (i)-1 ,3-^-o«rbo^n*ylai3^,5(£-i»opropyli- dene-2-deoxystreptamine, according to procedures described in Preparation 9, may be separated by optical resolution procedures to obtain 1-N-lower alkyl-2-deoxy-D-atreptamine and 3-N-lower aIky1-2-deoxy-D-streptamine.

To obtain a 1-N-alkyl-4,6-di-(aminoglycosyl)-1,3-diaminocyclitol of this invention, a 1-N-lower alkyl-2-deoxy-D-streptamine (e.g. l-N-tnethyl-2-deoxy-D-strepta-mine and l-N-methyl-2,5-dideoxy-D-streptamine) is cultivated v/ith Micromonospora invoensis strain 1550P-1G, to obtain a 1-N-lower alkylsisomicin derivative (e.g. 1-N-methylsisomicin and l-N-mcthyl-5-deoxy-sisomicin).

Optionally, the racemic mixture of a mono-N-alkyl-strepta-mine, (e.g. (-) -mono-N-methy1-2-deoxyctreptamine and - 44 - - 40437 (-)-mono-N-tnctliyl-2,5-ditU!o:tyotroj»lnm.ino) may be ciiltivatod with mcromow';:pnr.n inyornai n strain 1550F-1G to produce a mixture of l-(nnd 3-) -N-all:ylsinwaicino (e.g. a mixture of 1 -N-roethylsi oonicin and 3-N-raethylsisomicin) and a mixture of l-H-methyl-5-deoxysisomicin and 3-N-methyl-5-dcoxysisomicin which may bo separated via chromatographic techniques to obtain 1-N-alkylaicomicin of this invention, e.g. 1-N-mcthylsisomicin and l-N-methyl-5-deoxysiscmicin.

Fermentation is effected in the following manner. A lyophilized culture or cells from a slant culture of Micromonospora invoensis strain 15SOF-1G NRRL 5742 is transferred to a sterile inoculum medium. The medium is an aqueous one containing assimilable sourccs of nitrogen, carbohydrates and the usual compliment of trace metals. The inoculated medium is permitted to incubate under aerobic conditions at from about 24° to about 49°C., preferably about 35°C for from about 2 to about 5, preferably about 3 days. The pH is maintained in the range of from about G.O to about 8.0, preferably from about 6.8 to about 7.4.

The co-produced inoculum is ancm:i i.ally transferred to a fomentation modium which modi dm may bp tho samo as or different from the inoculum medium. An r.mino-cyclito! may bo ;.ddod to tho fermentation i.~.,*• diurn bofore sterilization, at the time of inoculation or up to 48 hours after inoculation. The aminocyclitol is usually dissolved in water, sterile filtered and added to the fermentation medium. In general, the concentration of the compound is from about lOO to about 1500 mcg/ml. of fomentation broth. .The fermentation is conducted -under aerobic conditions, and under about the same conditions of temperature and pH as in the inoculum. Peak antibiotic production is determined by the assay used for sisoroicin [sec J. Antibiotics (Japan) Vol. XXIII]. The sisomicin derivatives thereby produced are isolated from the fermentation and from co-produced minor components having antibacterial activity by methods generally used din the art for aminoglycoside antibiotics.

Another process for the preparation of 1-N-substituted derivatives of the 4,6-di-(aminoglycosyl) -1,3-diaminocyclitols listed above wherein the substituent is straight chain alkyl having up to five carbon atoms and of the pharmaccutically acceptable acid addition salts thereof comprises reacting one of these 4,6-di-(aminoglycosyl) —1,3-diaminocyclitols, which possesses amino-protecting groups at any position other than position 1, and wherein the 1-amino group may be activated, with an alkylating agent containing the straight chain alkyl group having up to five carbon atoms and a leaving group, removing - 46 - J«j :i 7 tlu? pro lut. ling groups And, il rmiuired, tin- aillvitling group or groups present In the nolecule, and Isolating the derivative as such or as a pharmaceutically acceptable acid addition salt.

Examples of alkylating agents advantageously used In this process are alkyllodlde, alkylbronide, dialkylsulfate* alkylfluoro-sulphonate and alkyl p-toluenesulfonate wherein the alkyl group Is the required straight chain alkyl group having up to five carbon atoms.

Other alkylating agents, wherein the alkyl group preferably has one or two tarbon atoms, are trlalkylanlllnlun hydroxide, trialkyloxonium fluoroborate, trialkylsulfoniun fluoroborate, or trlalkylsulfoxonium fluoroborate. All of these alkylating agents contain a good leaving group, such as Br", i~, OSt^F', di-alkylanilin or dialkylether.

The amino group in position 1 of the 4,6-di-(aminoglycosyl )-l>3-diaminocyclitol can be free or activated. An example of an activating group is trifluoromethylsulfonyl. These activating groups may be introduced into the molecule by reacting a 4,6-di-(amiRog1ycosyl)-l,3-diaminocyc1itol which possesses amino-protecting groups at any position other than position 1, e.g. 3"-N-4"-0-carbonyl-2*,3,6'-tri-N-t-butoxycarbonyl-s1somicin, with a compound providing the activating group, such as trifluoromethylsulfonyl chloride.

The 1-amino group can also be alkylated by way of the corresponding di-(2-cyanoethyl)-derivative which is derived by treatment with acrylonitrile of the 4,6-di-(aminoglycosyl)-l,3-diaminocyclitol which possesses amino protecting groups at any position other than position 1. The l-N-di-(2-cyanoethyl)-derivative thus prepared is then alkylated with one of the above listed alkylating agents followed by removal of the cyanoethyl groups. - 47 - The process of the invention is carried out under conditions similar to those employed In the well-known direct alkylatlon procedures of amines.

Yet other processes for the preparation of 1-N-substltuted derivatives of the 4,6-d1-(am1noglycosyl)-l ,3-diarainocyclltols listed above, wherein the substituent is methyl, and of the pharmaceutically acceptable acid addition salts thereof comprise reacting one of these 4,b-d1-(aminoglycosyl)-l,3-diaminocycl1to1s which possesses amino-protectlng groups at any position other than position 1, either with formaldehyde and a cyclic imide, preferably succinimide, and treating the so-obtained compound with a hydride-donor reducing agent, preferably sodium borohydride, or with formaldehyde In the presence of formic acid, removing all protecting groups present in the molecule and isolating the derivative as such or as a pharmaceutically acceptable acid addition salt. The formation of the 1-N-methyl substituent with formaldehyde and formic - 48 - 4 0 4 A procoso for the preparation of l-ij-aubstituted derivatives of tho 4,6-di-(aminoglycosyl) -1#3-diamino-cyclitols listed abovo, wherein the substituent1 is 2-hydroxyethyl, and of tho phanaaceutically acceptable acid addition salts thereof comprises reacting ono of these 4,6-di-(aminoglycosyl)-1,3-diaminocyclitols which possesses amino protecting groups at any position other than position 1, with ethylene oxide, removing all protecting groups present in the molecule and isolating the derivatives as such or as a pharmaceutically acceptable acid addition salt.

In another of its process aspects the invention relates to the preparation of the amide compounds which arc useful as intermediates in the preparation of tho l-N-CHjX^, 6-di -(aminoglycosyl) -1,3-diaminocyclitols and which possess broad spcctrum antibacterial activity.

The process for the preparation of l-N-substituted derivatives of the 4,6-di-(aminoglycosyl)-1,3-diaminocyclitols gcntamicin A, gentamicin B, gentamicin B^, gentamicin C^, gcntamicin c^a# gentamicin C2» gentamicin ^a' gcntamicin C2h' gentamicin X^, sisomicin, verdnroicin, tobramycin. Antibiotic G-418, Antibiotic 66-40B, Antibiotic 06-4OJ>, Antibiotic J1-20A, Antibiotic JI-20B, - 49 - 40437 Antibiotic G-52, mutnroicin I, mutamicin 2, mutnrnicin 4, mutamicin 5 and mufcanticin l>, wherein tho aubatltuont is -K-Z with Z being hydrogen* alley 1, alkenyl, cycloalkyl, cycloalkylnlkyl, hydroxysIky1, aminoalkyl, N-alkylamino-alkyl, aminohydroxyalkyl, N-alkylaminohydroxyalkyl, phenyl* benzyl or tolyl, said aliphatic radicals having up to seven carbon atoms and, if substituted by amino and hydroxy, bearing the substituents on different carbon atoms, with the proviso that Z together with the carbonyl group to which it is attached is other than S.-3 -amino-2 -hydroxy -prppionyl or S.-4-amino-2-hydroxybutyryl, and, in case of tobramycin, also other than S.-5-amino-2 -hydroxyvaleryl, and of the pharmaceutically acceptable acid addition salts thereof, comprises treating one of the above named 4,6-di-(aminoglycosyl)-1,3-diaminocyclitols which may have amino protecting groups at any position other than position 1, with an acid of the formula 0 II OH-C-Z' with Z' being a group as defined for Z above, wherein any amino or hydroxy group present may be protected, in the presence of a carbodiicdde nuch as dicydohexylcarbodiiaiide or with a reactive derivative of above acid, and, if required, removing all protecting groupu present in tho moleculo, the last process step being followed by isolating the derivative ac such or as a pharmaceutically acceptable acid addition salt. - 50 - - 4043? The 4,6-di-(aminoglycooyl)-l#3-diaminocyclitol {starting compounds of thio process mny have free amino groups or prntcvs.od .imi.no groups. If amino gro»:j«:i are protected in the 4,6-di-(r.mino«|l yf?r»»:y1.) "1 » 3-di nminoeyi-litolr; genl.mnin.in H, gcntamicin c^a« Antibiotic JI-20A, Antibiotic 66-40B, Antibiotic 66-40D, oiscmicin, mutamicin 1, mutnmicin 2, mutamicin 4, mutcunicin 5 and mutamicin 6, it i.c usually tho 6*-amino group being protected. Gentamicin may be protected at positions 2' and 3. The 4, 6-di-(aminoglycosyl) -1,3-diaminocyclitol starting compounds may bo used as a free nitrogen-baso (with or without N-protecting groups) or, in an inventive modification of the process, as a compound wherein 1 to n amino groups of the 4,6-di-(aminoglycosyl)-1,3-diaminocyclitol are neutralized by formation of an acid addition salt whereby n is the number of amino groups in tho molecule. The acid addition salt may also contain N-protecting groups. In a preferred feature of the inventive modification of the acylating process (n-1) amino groups aro neutralized by formation of an acid addition salt. For example, one equivalent of gentamicin having five amino groups (n=>5) requires five equivalent:;; of acid to form tho "per" acid addition salt. In the preferred fenLure, an acid addition salt of gentwnicin Cj i.s used having (n-1), i.e. four, amino groups which are pro:;onnfced. The term "acid nddi.i-.ion :;alfc" cr.ibraocr; .such .sol:.:; rs may be formed 40437 between the basic antibiotic and an acid without regard » . 1 to Whether the acid nay be tensed inorganic or organic. Exemplary of acids embraced by the term are sulfuric, hydro chloric, phosphoric, nitric, acetic, propionic, succinic, oxalic, cyclopropylcarboxylic, trimethylacetic, cialeic, benzoic, phenylacetic, trifluoroacetic or the like.

If it is desired to use as a starting material an acid addition salt, wherein (n-1) amino groups are protonated, this compound is advantageously produced in situ thereby reacting a "per" acid addition salt with an equivalent of strong base, e.g. triethylamine.

In general, the use of reactive derivatives of the acid OH-C-Z* as acylating agents is preferred.

Reactive derivatives of the acid comprise esters, azides, imidazole derivatives or anhydrides. In those instances wherein Z' is unsubstituted, one of the preferred reactive derivatives is the anhydride of the requisite acid. In other instances if may be preferable to use the N-hydroxy-succinimidyl ester of the acid.

When carrying out the process whereby a reactive derivative of an acid containing an amino function is used, it is preferable to protect the amino function 40437 prior to carry ing out. tin* procnut ami Mien retnoviuq Iho N-p.vc»t«*ctj»Kj group in th It is obvious to those skilled in the art that tho ainidc compounds excluded from the scopc of the present invention, i.e. those compounds wherein Z together with the carbonyl group to which it is attached is S.-3-amino-2-hydroxy-propionyl or S.-4-amino-2-hydroxy-butyryl and, in cane of tobramycin, also S_-5-omino-2-hydroxy-valeryl, can bo prepared in a manner similar to that described above for the preparation of the ainidc compounds of this invention. Furthermore, tho preparation of some of these compounds is set forth heroinbelov; in Preparation The following Preparations exemplify tho manufacture of a multiplicity of requisite starting materials and the following Examples illustrate the invention. - 53 - u 40437 PREPARATION 1 Qentomloln C2a Separation of Oentarolcln C^a from Co-produood Antibiotics 5 Dissolve 96 ems. of gentamlcin base (prepared from the sulfate salt obtained by the procedure of Example 4 of U.S. Patent 3*091*572) in 400 ml. of the upper phase which results when methanol* chloroform and 17% ammonium hydroxide are mixed In the volume ratio of 1:2:1. Add one tenth of the 10 Solution to cach of the first ten tubes In a 500 x 80 ml. tube counter current extractor. Pill all of the tubes Including the first ten to capacity with the lower phase of the above-described solvent mixture. Set the solvent reservoir to deliver 40 ml. of upper phase to tube one (1) 15 for each transfer. Set the apparatus for 500 transfers.

When the transfers are complete, sample every eighth tube for chromatography (in duplicate) on Schleicher and Schuell paper No. 589 using the lower phase of the above-described solvent mixture. Permit the chromatograms to develop for 20 about 16 hours and then dry the papers. Plate one paper on an agar plate seeded with Staphylococcus aureus (A.T.C.C. 6558P), spray the duplicate with the conventional ninhydrin solution and heat to develop. Incubate the agar plate at yi° C overnight and combine the solution from tubes containing - 54 - 40437 the.* material that migrates lll?« gcntnmicin (l.o. tubes J290 - J60).

Replace tubes ?90 - 360 with fresh tubca containing. '10 ml. ol' upper phar.e and *10 ml. lower phase. He-set the appar.itua for nn additional 2tt00 tninr.f'rrc and repent the chromatographic procedure porrormett above. Combine tubes 1 -16 and concentrate in vacuo to obtain l.jj cms. of gentamicin C2a, having the folJowlnr, proportion: (a) a molecular weight of 16^ as detennined by maus spectrometry which is consistent with an ompirical formula °r c;»oH4lNt>°Y; (b) a specific optica] rotation n:j Measured by the D line of :;odiuni at P.C°C of +J l'»° - [3° ir. water at concentration; and (c) a proton magnetic resonance (pmr) spectrvim as follows: pmr(ppm) (0,0): S 0.99 (j>H, d, J-6.5Hz, CH-CH^); 1.17 Oil, b, C-CJtj); 2.H7 (311, s, N-CIJ^); 2.51 (JIF, d, j»10.!dlz, H-J>"); 5.75 (1H, rj, J-10.5, H-2"); 4.00 (III, «1, J«lPlIas, Jl-l/'cq); 5-0'l (1H, d, J-Jlll'/., H-O"); 5.1} (Hi, d, J*-3.yJz, 11-1').

Irradiation of the secondary uothyl j;roup .it S 0.tv> ppw reveals li-fi1 as a doublet (.1 = O.^lb*.) nV. £ 7l.l 1 ppx. w • 40487 PREPARATION 2 Gentamlcin C2b Separation of Gentamlcin from Co-produced Antibiotics Separate the major gentamlcin C components (C^, Cg and 5 Clft) as described In U.S. Patent 3,651,042, Example 2, and combine those fraotlons containing predominantly overlaps of gentamlclns and Cg free base (500 g. of gentamlcin C mixture gives 53.4 g. of overlaps). Apply 1.5 g* of this gentamicin C^ and Cg mixture to a column containing 50 g. of 10 silica gel made up In a solvent system comprising chloroform) methanol:15# ammonium hydroxide (1:2:1). Elute the oolumn with the same solvent system and monitor the eluted fractions by thin layer chromatography on silica gel plates using the solvent system chloroform:methanol:22% ammonium hydroxide 15 (1:2:1) as developer. Combine those fractions containing a mixture of gentamlclns C^ and Cg together with gentamicin Cgb (Fractions 39 - 57 [410 mg.)). Rechromatograph fractions 39 - 57 over silica gel using a choroform:methanol:f% ammonium hydroxide (1:2:1) solvent system and combine those fractions 20 (98 - 130) containing pure gentamicin Cg^ as determined by thin layer chromatography (yield 45 mg.) having the following 26 o ^ constants: [alp + 165.5 (c=0.3%» HgO); Mass spectrum m/e 463 (M+l)+, 446, 445, 433, 350, 332, 322, 304, 333/ 305, 287, 191, 173* 163, 145, 160, 142, 118, 143; pmr(ppm)(DgO): 25 S 1.25 (3H, s, C-CH3); 2.'>0 (3H, s, N-CH^); 2.55 3H, s, N-CHj); 5.12 (1H, d, J=4Hz, H-l"); 5.22 (IH, d, J.3Hz, H-l'). - 56 40437 Pure gentr.mleln can bo differentiated from gcntamicin Cj nnd C,, by its mobility on thin layer chromatography using silica Gel plates and a chloroformsmothiinol :22jil> oinmonlum hydroxide (1 :?:1) solvent system a s developer. Tho approxi-5 mate 11 r values In this system arc an follows: gcntamicin 0.47 f.cntamlcin Cg 0.47 gentamlcin Cgb 0.35 PREPARATION 3 10 Selectively blocked Dl-(aminof-lycosyl )-l,3-dlamlno- cyclitols A. 2',3> 6'-Tri-N-butoxycarbony1-3"-N-4"-O-carbonylsi somi cIn 1. Pen ta-N-carbobenz oxy r. 1 r.owi e j. n Dissolve 25 g. of sisomicin and 13 g. of sodium carbonate 15 in 625 ml. of distilled water. Add 100 ml. of carbobenzoxy-chloride to the stirred solution at 25°C and stir the mixture for sixteen hours. Filter off the solid, wash thoroughly with water, dry in vacuo, and then wauli with hexane to obtain penta-N-carbobenzoxysisomicin (62 g.) as a colorless amorphous 20 solid. rn.p. r- 165-173°C [a]£6 + 96.2° (CH^OH) IR: V max (CIICl^) 3>I00, 1720, l'^l'j, 1215, 1050, C'J'J, cm"1 NMK: S(CD(M3) 1.03 (3H, broad singlet, W-C-CII^); 3.02 (3H, broad singlet, ^"-NCJl^); !j.02 (3Oil, broad singlet, CHgC^H^); ,'>.2b, 3.30 ppm (2',ill, brocul yinglets, -CHgC^H^). - 51 - 40437 2. Tetra-H-carbQberi7.oxy-3n-N-4n-0-carbonyl-8l80mlcln Dissolve 5 6* of penta-N-carbobenzoxy-slsomloln In 50 ml. of dimethylformamlde, add 250mg. of sod1 un hydride ; to the stirred solution, and stir the reaction mixture under argon at room temperature for two hours. Filter and add glacial acetic acid (2 ml.) to the filtrate which is then concentrated in vacuo. Extraot the residue with chloroform (200 ml., previously passed through basic alumina), wash the extract with water and dry over sodium sulfate." The solution is evaporated to give tetra-N-carbobenzoxy-3w-N-4M-0-carbonyl-sisomicin as an amorphous powder (3.5. g.) m.p. « 210 - 213°C [a]p6 + 68.8 (C = 0.22) IR: V max (Nujol) 3550, 1840, 1760, 1580 cm"1 NMR: g (CDCl-j) 1.34 (3H, singlet, 4"-CH;5)j 2.68 (3H, singlet, 3W-N-Me); 5.04 (8H, broad singlet, -CHgCgH^). "NUJOL" 1s a Registered Trade Mark. 3* 3w-N-4w->0-Carbonyl-nlsomlcln To a solution of l,3»2',6'-tetra-N-benzyloxycarbonyl-3ll-N-4"-0-carbonyl-sisomicin (10.1 g.) in tetrahydrofuran (200 ml.) add 1 litre of liquid ammonia (redistilled from sodium). To the stirred solution add 6 grams of sodium in small pieces. After stirring for 3 hour3 destroy the excess sodium by addition of ammonium chloride. Allow the solvents to evaporate under a stream of nitrogen. Dissolve the residue in water and pass through a medium - 58 - or Ainboi'litc l!!C-:»0 renin (H* fomi) and wash tho resin HuJl willi water then elulo tho produot with 2N ammonium "AHBERUTE" Is a Registered Trade Mark.' hydroxide solution. / Evnpornte the ammonia cluate In vacuo ;to pivc the title product („//'-N-4n-0-carbonyl-sisomicin). Yield ca H cms.

Ills V max (Nujol) 174b cm"1. The product may be used r in subsequent steps without further purification. However a very pure sample nay be obLainud by chromatography of the product over silica /'.el usinr. tin; lower phase of a chl oroform: methanol.: cone. ;i!iiiiioi)iuin hydroxide (1:1:1) solvent system as eluant. ^ y'> 3*6'-Trt-N-t-butox.vcarbony .1.-3"-N-T'-O-carhonyl-sisomlcin Dissolve 3n-N-V,-0-carbonyl-r.isomicin (1.4 c.,3 mmoles)in 10 nil. of aqueous methanol c oil Lai nine; tri ethyl amino (3»r> mmoles). V/ith stirriri/", acid L-butoxycnrbonyl a/.lde (3»l> mmolen)dropwise. Stir the mixture for two days at room temperature. Add 5 ml. of Amberlite JRA-401S (0H~) ion exchange resin alorif; with 5 ml. methanol and stir for 1/2 hours. Remove the rosin by fi ltration and wash with methanol. Concentrate the filtrat-.- and ehromto/u-aph the rer.idue on a column of silica <:el (60-100 mesh, 20.0 r,. ) usiii/; ehloroformrnethanol :ammonium ii.vdx'oxide (30:10:0.*! ) as the .solvent rysteni. Pool the homogeneous fraction:; 40437 containing the title material and remove the solvent by evaporation In vaouo. Dissolve the residue In methanol and prooipitate with excess ethor. Isolate the solid product, by filtration and dry.

B. 2', 3-Dl-N-Trlfluoroaootyl Gentamlcin 1. 2'-N-Trlfluoroacetyl Pentamicin Dissolve 1.7 g. of gentamlcin Cj in 20 ml. of methanol, cool the mixture to 4°C and add 0.46 ml. (O.563 g.) of ethyl thioltrifluoroacetate with stirring. Allow the reaction to continue for 2 hours and concentrate the solution to a residue in vacuo. Chromatograph the product on 80 g. of silica gel G using the lower phase of a mixture of chloroform:methanol:water:ammonium hydroxide in the volume ratio of 10:5:4:1 as eluant. Combine the fractions containing the major component and concentrate to obtain 1.4 g. of the title compound, m.p. 108 - 111°C, 26 o [dip ® +128 (c-0.3^, HgO). Analysis for Cg^H^gN^OgFyHgO requires C=46.69£; H=7.50#; N=11.84£; F=9.63g.

Found: C=46.66#; H=7.6?$; N=11.60#; F=9.24j6. - 60 40437 ?• 2',3-Di-N-Trlf3uoroncotyl Ocntfimlcln Dissolve 0.66 g. of the product of Step 1 in 10 ml. of methanol, cool the mixture to 4°C and add 0.148 ml. (0.1&2 g.) of ethyl thioltrifluoroacutate dissolved in 2 ml. of 5 methanol. Stir the reaction mixture for about 16 hours and concentrate to a rcslduo ljn vacuo. Chromatograph the product on 50 g. of sllluu gel as described in Step 1.

Monitor the column by thin layer chromatography, combine tho appropriate fractions and concentrate to obtain 0.^2 g. 10 of the title compound, m.p. 121 - 129°C Cal^ o 121° (c « O.35S; HgO). Analysis for C25H4iN^°9P6 re(lulres C=44.84#; H=6.3 7N=10.46£. Found: C=44.94$S; H=6.35^; N=10.17#.

C. 6' -N-TrJ f ] uoroacety 1 -r.l sotnl ci n 15 Dissolve 20 g. of slsomlcin in 1.2 liters of anhydrous methanol and add dropwise a solution of 6 ml. of ethyl thioltrifluoroacetate in 60 ml. of methanol over a j> hour period with stirring. Allow the reaction to proceed for 18 hours at room temperature and remove the solvent in 20 vacuo to give a residue of of product of approxi mately 95# purity having the following physicochernical properties: Mass .'Jpectral data: rn/c 'j'O M+, other definitive peaks at in/1; 413, yj'j, j&'j, '(>'■>> W'j and 126. 40437 NMR (60MHz, DgO) $ 5*37 doublet, J-2Hz, H-l')» 5.12 (doublet, JaHllz, H-lw)j 4.9C (broad singlet, H-4*)j 2.57 (singlet, 1.26 (singlet, C-CU^).

D. 6'-N-li-butoxycarbonyl Gentamicin Cla Dissolve 2.69 g. of gentamicin Clft in 60 ml. of methanol: water (1:1), cool to 5°C and add 1.815 ml. of triethyl- amine. Add with stirrinc 1.91 g* of t-butoxycarbonyl azide dropwise. Stir the mixture at 5°C for 18 hours. v* © Add 20 ml. of Amberlite IHA-401S resin ( OH form), stir for 30 minutes, filter and evaporate the filtrate to dryness in vacuo. Chromatograph the crude product over silica gel (350 g.) using the lower phase of a 2:1:1, chloroform:methanol:concentrated ammonium hydroxide solvent system as eluant. Take 3 n»l« fractions and monitor their contents by TLC. Combine fractions containing the major reaction product and evaporate to obtain the title compound of this example (0.42 g., 1}£), [a]^6 + 137O(CH?0H), PMR £ 1.23 OH, s, C-CH3); 1JI5 (9H, s, C-(CRj)3); 2.53 (3H, s, N-CH^); £ 5.08 (PH, overlapping doublets, J^.SHz, H-l* and H-l") PPM. Mass spectrum m/e 550 and m/e 5^9 (M+). 40437 E. 6'-N-t-hntoxycarbony .1 Qrp.tamlcln 13 Dissolve *1 ft. or gcntamicin B in 30 ml. of 50# aqueous methanol, and cool to 5°C. AddO.297 g. of Jb-butoxycarbonyl azido. dropwise with stirring followed by(\l86 ml. of tri-. 5 cthylamlne and stir the resulting solution for 18 hours.

Evaporate the reaction mixture in vacuo to a residue and chroinatograph the residue on 100 g. of silica gel using the lower phuice of a 2:1:1, chloroform:methanol:concentrated ammonium hydroxide solvent system as eluant. Collect 10 2 ml., fractions and monitor the column effluent by TLC.

Combine fractions containing like material (fracts. 180-230) and evaporate to obtain 0.6j50 g. 61 -N-t-butoxycarbonyl gentamicin n having the following physical constants: PMH (60MHz,1>20) S 1.21(311, s, C-C»3); 1.42 (<>H, s, C(CH 15 2.1i3 (3H, s, H-Cn3); 5-2 (311, d, J=4.5Hz, H-l")j 3.2} (HI, d, J=3.0Hz, II«-1') PPM.

PHF.PARAT10N 4 1 -N-(AMINOHYDMOXyACYl,)-4,6-PI-(AMINOOf.ycOSVL)-l. ;i-DIAMINOCYCMTOI.S 20 A. l-N-(.'3-4-Amino-2-hydro:xybutyryl) Gontamicin C. 1. 1-1J- (f»-4 -Bony.yloxyenrbonylariiJ n o-2-hydroxybutyryl)- Gentamicin C, ■ la I)Ji;.v.olvc 2.8 n. (4 mmo3.es) of gcntamicin C, sulfate in i. &L - 63 - 40437 JO ml. of wator and add 15 ml. of methanol. Add 0.56 ml. (4 mraoles) of triethylamlne and stir for 10 minutes. Add a solution containing 4 mmoles of N-(S-4-benzyloxycarbonyl-araino-2-hydroxybutyryloxy)ouccinimide in 20 ml. of dry dimethylformamlde dropwlse*with stirring to the antibiotic solution. Stir the mixture overnight (16 hours) at ambient temperature. Thin layer chromatography of the reaction mixture via TLC on silica gel using the lower phase of a solvent system consisting of chloroform{methanol: ammonium hydroxide (1:1:1), shows the presenoe of a plurality of minor components and one major component. Concentrate the reaction mixture to a residue in vacuo and triturate the residue with methanol to yield 3.2 g. of white solids containing all the components previously observed by chromatography.

Chromatograph 150 mg. of the product on 50 g. of silica gel using the lower phase of a solvent system consisting of chloroform:methanol:ammonium hydroxide (2:1:1). Pool the fractions containing the major component and lyophilize to give 1-N-(S-4-benzyloxycarbonylamino-2-hydroxybutyryl) gentamicin C1&.

NMR (DgO) S 1.15 (3H, s, C-CHj); 2.49 (3«, 3, NCH^); 4.10 (III, dd, J---G.0, 4.0 J I-/., sidechain H-2); 7.36 (DH, m, phenyl). - 64 - 40497 2. J-M-(S-'l-Amlno-2-hydroxybutyryl) Gentamicin Dissolve tho product of Preparation 4A (1) in a mixture consisting of 12 ml. of methanol and 3 ml. of water, add 20 mi;, of 10# palladium on carbon and hydrogenate at 4 5 atmospheres at room temperature. After 3 hours the reaction is essentially complete. Remove the oatalyst by filtration and lyophlllze the filtrate and obtain 46 mr;. of l-N-(S-4-amino-2-hydroxybutyryl) gentamicin Cla» NMH (D20) 8 1.17 (3H, s, C-C«5); 2.48 (3H, s, NCII^)! 10 4.22 (1H, dd, J^9.5 Hz, 4.0 Hz, side chain CHOH); 5.04 (2H, m, H-l' , and H-l").

Mass Spectral data: (M-HgO) m/e 532.

B. 1-N- (S-4-Amino-2-hydroxybutyryl) Gentamicin B 1. 1-N-(S-4-benzyloxycarbonylamino-2-hydroxybutyryl) 15 Gentamlcin B Dissolve 3»59 g« of gentamicin B sulfate in 48.4 ml. of water and dilute with 23.7 ml. of methanol. Add 0.7 ml. of triethylamine dropwlse with stirring. Dissolve 1.67 g. of N- (S-4-benzyloxycarbonylajaino-2-hydroxybutyryloxy ) 20 succlnlmldc in dimethylformamlde and add the solution drop-wise with stirring to the antibiotic solution. Stir the resulting solution at room temperature for 18 hours then conoontrate to a residue in vaouo. Dissolve the residue - 65 - 40437 In water and treat with dilute barium hydroxide solution with stirring until the pH reaches about 8.0. Remove the precipitated barium sulfate by filtration using a filter aid. Wash the precipitate with water, combine the filtrate and washings and concentrate to dryness in vacuo. Chromatograph the residue on a column containing 600 g. of. silica gel using the lower phase of a solvent system consisting of chloroform:methanol:ammoniun> hydroxide (1:1:1) as the eluant. Pool the material which is eluted immediately ahead of gentamlcin D and concentrate the pooled fractions to dryness and obtain thereby l-N-(S-4-benzyloxy- carbonylamino-2-hydroxybutyryl)gentamicin B as an amorphous » solid. Yield = 0.2 g.

NMR (DgO): S 1.27 (311, s, C-CH^); 2.51 (3H, s, NCH^); 5.0& (III, d, J=4 Hz); i>.25 (1H, d, J=3.5 Hz). 2. l-N-(S-4-amino-2~hydroxybutyryl)gentamicin B Dissolve the product of Preparation 4b (1) in a mixture consisting of 20 ml. of water and 8 ml. of methanol. Hydro-genate the product in the presence of 60 mg. of 5# palladium-on-carbon at 3.5 atmospheres and room temperature for 3 hours. Remove the catalyst by filtration through a filter aid. Wash the filter pad with water and combine the filtrate and washings. Concentrate the combined filtrate and v.'ashings to dryness in vacuo. Chromatograph the - r,(, - V 40437 residue on a silica gel column containing 30 g. of oillca gel using a solution consisting of chloroform:methanol: ammonium hydroxide (1:2:1) as the eluant. Fractions containing the most polar component are pooled, concentrated 5 and lyophlllzed to givo l-N-(S-4-amlno-2-hydroxybutyryl) gcntamicin B.

C. 1-N-(S-^-Amino-2-hydroxybutyryl)-verdamicln 1. 1-N- (S-;»-Phthalimido-2-hydroxybutyryl )verdainicin Dissolve 5.00 g. of verdamicln sulphate in 50 ml. of water 10 and add 25 ml. of methanol. Add 0.50 ml. of triethylamine and stir for 10 minutes. Add a solution containing 2.5 g. °f N-(S-4-phthalimldo-2-hydroxybutyryloxy)succinimide in 10 ml. of dimethylformamide dropwise with stirring. Stir the mixture overnight at ambient temperature then concentrate 15 to a residue in vacuo. Chromatocraph the residue over 160 g. of silica gel, eluting with the lower phase of a chloroform:methanol:conccntrated ammonium hydroxide (1:1:1) solvent mixture. Combine and evaporate fractions containing the major component of the reaction (determined by TLC on 20 silica gel plates) and obtain thereby the compound of this example as a white amorphous solid. - f.7 - 40437 2. ]-M-(a-*l-Amino-;,.iiy«iro.xylmtyryl)vcrdamicin Dissolve the product of Preparation 4c (1) In 40 ml. of ethanol and add 0.2 g. of hydrazine hydrate. Reflux the solution for 3 hours, then evaporate to dryness in vacuo. Chromatograph the residue over 160 g. or silica gel, elutlng with the lower phase of a chloroform:methanol:concentrated ammonium hydroxide (1:1:1) solvent mixture. Combine and evaporate fractions containing the major component of the reaction (determined by TLC on silica gel plates) and obtain thereby the compound of this example as a white amorphous solid.

D. In the procedures of Preparations 4A to 4C, the sulfate salts of other 4,6-di-(aminoglycosyl)-l,3-diaminocyclitols may be substituted for gentamicin C^a» gentamicin B and verdamicln and other N-(S-benzyloxycarbonylaminohydroxy-acyloxy)-succinimides may be substituted for N-(S-4-benzyloxycarbonylamino-2-hydroxybutyryloxy)succinimide or N-(S-4-phthalimido-2-hydroxybutyryloxy )succinimide and there will be obtained the corresponding l-N-(aminohydroxyacyl )-4,6-di-(aminoglycosyl)-1,2-diaminocyclitola. - 68 - PUKPAHrtTION 5 PHKPAHATTON OK A1J>K1IV1)1?. TNTKIiK*J ATKfl A. ?->-Ac«jtnml.do-^-liytlroxyocta»al Protcct the amino function In the 2-amino-3-hydroxyoctanoic acid by conversion thereof to on acetamido function, then esterify the resultant 2-acetamido-3-hydroxyootanoic acid with methanol; reduce the resultant 2-acetamido-3-hydroxy-octanoic acid methyl ester with di-isobutylaluminium hydride according to known procedures to obtain 2-acetamido-j5-hydroxyoctanal.

B. 2-Acetoxy-4-(N-methylacetamldo)butanal Treat the diethylacetal of 2-hydroxy-^-aminobutanal with acetic anhydride in pyridine followed by treatment of the resulting diethylacetal of 2-acetoxy-4-acetamidobutanal with sodium hydride and methyl iodide to obtain the diethylacetal of 2-acetoxy-4-(N-methylacctamido)butanal. Remove the acetal protecting croup by means of acid to obtain 2-acetoxy-4- (N-mcthylacctamido Jbutanal.. - 60 - 40437 PREPARATION 6 (t )-M0N0-N-METHYL-2,3-DIDEOXYSTREPTAMINE Prepared from CIS-4,8-DIOXATRICYCLQ- r5.1.0»0^*5] OCTANE >5". a* (1) -2 ,4 -Dlhydroxy-mono-N-methy1 -6,7-d.t azablcyo lo-[3*2.1.1 octano 1. Mix 2 gm. of cls-4,8-dloxatrlcyclo[5« 1.0.0^* **3-octane, 0.8 gm. of monomethylhydrazine, and 1.4 gm. of anhydrous magnesium sulfate with 200 ml. of 2-methoxyethanol. 10 Heat the mixture at reflux temperature under an inert atmosphere, e.g. argon, for about 40 hours. Remove the solvent In vacuo and extract the resultant solid residue with 100 ml. of hot methanol, filter, and evaporate the filtrate in vacuo to a residue of about 20 ml. and cool. 15 Filter the resultant precipitate comprising ( — )—2,4 — dihydroxy-mono-N-methyl-6,7-diazabicyclo[3.2.1.loctane (yield = 2.1 gm.); m.p. = 177 - 178°C. 2. In the above procedure, by substituting for mono- methylhydrazine equivalent amounts of other monoalkyl- 20 hydrazines, e.g. monoethylhydrazine and monopropylhydrazine, there is obtained the corresponding monoalkyl derivative, e.g. (-)-2,4-dihydroxy-mono-N-ethyl-6,7-diazabicyclo[3.2.1] octane or (-)-2,4-dihydroxy-mono-N-propyl-6,7-diazabicyclo -octane [3.2.1]; respectively. - 70 - 40437 3. Separate (-)-2,-dihydroxy-mono-N-inethyl-6,7-dia7.ablcyclo[3.2.1]octane by optical resolution procedures known In the art to obtain 2,4-dlhydroxy-6-N-methyl-6,7-dlaxnblcyclo[3.2.1]octane and 2,4-dihydroxy-7-N-methyl-5 6,7-dlazablcyclo[3#2.1]octane, respectively.

In similar manner, the (-)-mono-N-ethyl and (-)-mono-N-propyl derivatives, prepared as described in Preparation 6a (2), are separated by optical resolution procedures to obtain 2,4-dihydroxy-6-N-ethyl-6,7-diazabicyclo[3.2.1]-10 octane, 2,4-dihydroxy-7-N-ethyl-6,7-diazabicyclo[3.2.1]-ootane, 2,4-dlhydroxy-6-n-propyl-6,7-diazabicyclo[3.2.1]-octane and 2,4-dihydroxy-7-N-propyl-6,7-diazabicyclo~ [3.2.1]octane, respectively.

B. (-)-Mono-N-methyl-2t5-didcoxystrcptarnine 15 1» Mix 2.1 gm. of (i)-2,4-dihydroxy-mono-N-methyl- 6,7-diazabicyclo[3.2.1]octane and 0.5 em. of Raney nickel in 90 ml. of v/ater and hydiogenate at room temperature with an initial hydrocen pressure of 4 atmospheres, until hydroccnatiori uptake ceases (36 hours). Remove the 20 catalyst by filtration and lyophllize the filtrate to obtain (-)-niono-N-methyl-2,5-dideoxystreptamine ar, a white hydroscopic solid (yield = 2.12 ems.); m.p. 156 - 157°C. - 71 - 40437 2. In similar'manner, treat each of (-)-2,4-dihydroxy-mpno-N-ethyl-6,7-dlazablcyclo[3.2.1]octano and (i)-2,4-dihydroxy-roono-N-propylr6,7-diazablcyclo[3.2.1]octane with hydrogen in the presence of Raney nickel. Isolate and 5 purify each of the resultant products in a manner similar to that described to obtain, respectively* (-)-mono-N-ethyl-2,5-dideoxystreptamine and (-)-mono-N-propyl-2,5-dldeoxystreptamine.

C. l-N-Methyl-2,5-dldeoxy-D-streptamlne 10 1. In the procedure of Preparation 6B (1), by using 2,4-dihydroxy-6-N-methyl-6,7-diazabicyclo[5.2.l]octane as starting compound in place of the racemic mixture of 2,4-dihydroxy-mono-N-methyl-6,7-diazabicyclo[3.2.1]octane, there is obtained l-N-methyl-2,5-dideoxy-D-streptamine. 15 2. Similarly, by hydrocenating each of 2,4-dihydroxy- 6-N-ethyl-6,7-diazabicyclo[2.2.1]octane and 2,4-dihydroxy-6-N-propyl-6,7-diazabicyclof3.2.1]octane in the manner of Preparation 6B (l) and isolating each of the resulting products in a manner similar to that described, there is 20 obtained, respectively, l-N-ethyl-2,5-dideoxy-D-streptamine and 1 -N-propyl -2, fi-dideoxy -D-streptamine. - 72 - 40437 PREPARATION 7 1 -H-ALKYL-g-nTOXY-D-STREPTAMTN K Prepared from (lS)-l-Acetamlrio-3-amlno-'3,3-dldeoxy-ni.vo-Anofiltol A. (lS)-l-Amino-3- C ethy 1 amino) -1,3-dideoxy-miyo-lnosl tol To a solution of 10 gm. of (lS)-l-acetamido-3-araino-l,3-dldcojcy-myo-lnosltol In 100 ml. of ethanol add 6 gm. of acctaldehyde. Allow the solution to starnl at room temperature for 16 hours, then to the reaction mixture containing the (lS)-l-acetomido-3-N-ethylldene derivative thereby produced, cautiously add 5 gm. of sodium borohydrlde.

Allow the mixture to stand for a further 24 hours, then evaporate to a small volume in vacuo. Add 6 N hydrochloric acid and reflux for 6 hours to hydrolyze the 1-acetamido group. Cool, evaporate lr» vacuo,dissolve the resultant residue in water, bring the aqueous solution to neutrality with 1 N ammonium hydroxide, then add the solution to a column of Amberlite IRC-i>0 resin (NH^ ®cyele). Wash the column with water to remove inoreanic salts, then olute the column with 2 N ammonium hydroxide. Concentrate the eluate In vacuo and chromatograph the resultant residue over silica gel (3^0 G">s.) elutinn with a mixture of chloroform:methanol: 7#aiui:onium hydroxide (1:2:1). Combine like fractions as determined by thin layer chromatography and evaporate the coinl;inod fractions to a rcr.idm; comprisinr; (15)-l-ai:utio-3-ethy 1 ami no-1,3-dlucoxy-myo-lnor.l 1;o.l . 40437 B. l-N-Ethyl-2-dooxy-D-strcptamlne To 1 gm. of (IS)-l-amlno-3-(othylamlno)-1,3-dldcoxy-myo-inositol which has been carefully dried and finely powdered, add 10 ml* of acetyl bromide and 10 ml* of acetic anhydride. Seal the mixture In a glass pressure bottle and heat at 150°C for 6 hours. Cool, pour Into 100 ml. of ethanol, evaporate, to dryness, then treat the resultant residue with 10 ml. of acetic anhydride and 20 ml. of pyridine at room i temperature overnight. Evaporate the reaction mixture to dryness and chromatograph the resultant residue over silica gel elutlng with chloroform containing 1*5# methanol.

Combine the fractions containing the major products of the reaction as determined by thin layer chromatography, evaporate the combined eluates to a residue, dissolve the foregoing residue in 100 ml. of 50% aqueous ethanol, add Raney nickel T-4 and Amberlite IR-401S resin (OH" form) and hydrogenate for 2 days at 3*5 atmospheres hydrogen pressure. Filter the solution, evaporate, dissolve the resultant residue in 6 N hydrochloric acid and heat at reflux temperature for 6 hours. Cool, evaporate the solution in vacuo, redissolve the residue in water, neutralize the aqueous solution with ammonium hydroxide and apply the solution to an excess of Amberlite IRC-50 resin (NH^®form). Wash the column with v/ater, then elute the product with 2 N ammonium hydroxide. Combine the like - 74 - I 40437 eluotcs as determined by thin layer chron.atography and evaporate the combined oluatco in vacuo to a residue com- l * prising l-N-cthyl-2-dcoxy-D-ntrcptarainc. Purify by chromatography over silica gel elutlng with the solvent system chloroform:mothanol:7& ammonium hydroxide(l:2:1). Combine tho like fractions as determined by thin layer chromatography and evaporate to a residue of l-N-ethyl-2-deojcy-D-streptomino as a white amorphous r.olld ta]j^ -40° (water)* mass spectrum m/e 191 (M+l)+.

C. Other l-N-Alkyl-2-dcoxy-D-streptamlnor: By substituting propionaldehydc for acetaldchyde in the procedure of Preparation 7A and by treating the corresponding resulting (IS)-l-amino-3-alkyl-amino-l,3-dideoxy-rpyo-lnosltol thereby formed according to the procedure described in Preparation 7B, there is obtained the corresponding 1-N-alkyl-2-dcoxy-D-strcptamine, e.g. l-N-propyl-2-deoxy-D-streptamine. - 75 - 40437 PREPARATION 8 1-N-ALKYL-2-DK0XY-D-STREPTAMINE3 Prepared from OARAMINE A. l-N-Carbobonzyloxy-3,4;3* <41-dl-N,O-carbonylgaramlne To a solution of 1 gm. of 1,3,3'-tri-N-carbobenzyloxy-garamine In 50 ml. of dry dimethylformamlde add 200 mg. of sodium hydride. Stir vigorously under a nitrogen atmosphere for 24 hours then dilute the solution cautiously with water. Add Amberlite IRC-50 resin (H © form) to the solution until neutral, filter off the resin, and wash the resin well with ethanol. Evaporate the combined filtrate and washings in vacuo to a residue to obtain the bls-oxazolldlnone derivative, i.e. l-N-carbobenzyloxy-3,4;3,,4*-di-N,0-carbonylgaramine.

B. l-N-Propyl-2-deoxy-D-streptamine Dissolve the bis-oxazolidlnone prepared in Preparation 8a in 30 ml. of ethanol and hydrogenate for 16 hours at 3.5 atmospheres over a 10% palladium-on-carbon catalyst. Remove the catalyst by filtration and to the ethanol solution of the 3>4;3',41-di-N,0-carbonylgaramine thereby formed add 0.5 gms. of propionaldehydc. Allow the solution to stand at room temperature for 6 hours, then add 0.5 gms. of sodium borohydride. Allow the foregoing reaction mixture to stand - 70 - 40437 for a further 2 hour? then evaporate in vacuo to a residue comprising l-N-propyl-J, '-di-N, 0-carbony lgaramine.

To this residue add 75 ml. of 6 N hydrochloric aeld and heat the solution at reflux temperature for 6 hours. Evap-5 orate In vacuo and redlssolve the resultant residue in water.

Neutralize with ammonium hydroxide and apply the solution to a column of Amberlite IRC-50 resin (NH^® form). Wash the column with water to remove the inorganic salts, then elute with 2 N ammonium hydroxide. Evaporate the ammonium 10 hydroxide cluate in vacuo to a residue comprisinn 1-N-propyl-2-deoxy-g-streptamine. Purify by chromatography over slllcu gel in a manner similar to that described for the purification of 1-N-ethy1-2-dcoxy-D-strepfamine in Preparation 7B to obtain l-N-propyl-2-deoxy-D-streptamine as a 15 white amorphous solid.

C. Other 1-N-A3 kyl-2-deoxy-D-streptamlnes In the procedure of Preparation 8B by substituting acetal-dchyde for propionaldehyde and by isolating and purifying each of the resultant products, there is obtained the c.orres-20 ponding l-N-alkyl-2-deoxystreptamine, e.g. l-N-ethyl-2-deoxy-D-streptamine. - 77 - . 40437 V PREPARATION 9 (-)-M0N0-N-ALKYL-2-DE0XYSTREPTAMINE FROM * • (*)4.3-DI-N-CARB0PENZYL0XY-4.5-0-IS0PB0PYLIDENE-2-DE0XY- STRKFfAMINE 5 A. -1 -N-Carbobcnzy 1 oxy-3, 4 -N. 0-carbony 1 -2-deoxystreptamlne To a solution of 4.7 gniG.of carofullydried/1 "3-di-N- carbobenzyloxy-4,5-0-isopropylidene-2-deoxystreptamine In 75 ml. of dry dimethylformamlde add 0.5 gms. of sodium hydride and stir at room temperature under a dry nitrogen 10 atmosphere for 24 hours. Evaporate the reaction mixture to a small volume, cautiously add aqueous ethanol to destroy any excess sodium hydride then Immediately dilute the reaction product with a mixture comprising 30 ml. of ethanol, 50 ml. of acetic acid and 20 ml. of water. Allow the 15 solution to stand at room temperature for 4 hours, then evaporate in vacuo and extract the resultant residue with hot ethanol. Concentrate the combined ethanol extracts and dilute the resultant residue with a small amount of water. Cool the solution and separate the resultant (i). 20 precipitate by filtration to obtain/L-N-carbobenzyloxy- 3>4-N,0-carbonyl-2-deoxystreptamine as a white crystalline solid.

B. (-)-l-N-Ethyl-3»4-N,0-Carbonyl-g-dcoxystreptamine 1. Dissolve 2 gms. of/l-N-carbobenzyloxy-3,4-N,0-25 carbonyl-2-deoxystreptamino in 60 ml. of acetic acid and - 70 - 40437 hydror.cnate at 3*5 atmospheres hydrocen pressure for 16 i' hours over a 10j» palladium-on-carbon catalyst. Remove the catalyst by filtration* wash the oatalyst with acetic aold then evaporate the oomblned filtrate and washings in vacuo 5 to a residue comprising an oxazolldlnone derivative* I.e. (-)- 3*4-N,0-carbony1-2-deoxystreptamlne. 2. Dissolve the foregoing oxazolldlnone derivative In 20 ml. of ethanol* add 0.5 nil. of acetaldehyde and allow the mixture to stand at 0°C for 4 hours. Add about an equi-10 molar quantity of sodium cyanoborohydride relative to the starting oxzolidinone derivative and allow the mixture to stand for 24 hours at room temperature. Evaporate to a residue comprisingl-N-ethyl-3,4-N,0-carbonyl-2-deoxy-strcptamlnc. 15 C. (-)-Mono-N-ethyl-2-deoxystreptamlne Dissolve theA-N-ethyl-3,4-N,0-carbonyl-2-deoxystreptamine obtained in Preparation 9B in 50 ml. of 6 N hydrochloric acid and heat at reflux temperature for 6 hours. Evaporate In vacuo, redissolve the resultant residue in water and 20 neutralize with ammonium hydroxide. Apply the solution thereby obtained to a column of Amberlite IRC-50 resin (NHj® cycle) and wash the column with water to remove inorganic salts, then elule with 2 N ammonium hydroxide. - 79 - V 40437 Evaporate the eluate In vacuo to a residue comprising (ij-mono-N-ethyl-2-deoxystreptamine. Purify by chromatography over 150 gms. sllloa gel elutlng with ohloroformt methanol:7# ammonium hydroxide (It1.5>1) solvent system.

D. Other (*)-Mono-N-alkyl-2-deoxystreptamlnes In the procedure of Preparation 9B by substituting other 9 aldehydes, e.g. proplonaldehyde and formaldehyde for aoetal-dehyde and Isolating and purifying the resultant product in a manner described, there is obtained the corresponding (i)-mono-N-alkyl-2-deoxystreptamine, e.g. (-)-mono-N-propy1 -2-deoxystreptamlne and (i)-mono-N-methyl-2-deoxystreptamine, respectively.

E. l-N-Alkyl-2-deoxy-D-streptamlne Separate (i)-mono-N-ethyl-2-deoxystreotamine by optical resolution procedures known in the art to obtain 1-N-ethyl- 2-deoxy-D-streptamine and jJ-H-ethyl-2-deoxy-D-streptamine respectively. In similar manner separate each of (-)-mono-N-propyl-2-deoxystreptamine and (i)-mono-N-methyl-2-deoxy-streptamlne by optical resolution procedures known in the art to obtain l-N-propyl-2-deoxy-D-streptamine and 3-N-propyl-2-deoxy-D-streptamine, find 1-N-methy 1-2-deoxy-D-streptamine and 3-N-methyl-2-deoxy-D-streptamlne, respectively. - 80 - 404^7 PREPARATION 10 PRKPARATION OP MUTAMICINS A* Fermentation of Mlcromonospora Inyoensls strain 1550F-10 NRRL 5742 5 Inoculum Preparation Germination Stage l: Under aseptic conditions, add a lyophlllzed culture (or cells obtained from a slant culturc) of inyoensls strain 1550F-1Q to a 300 ml. shake flask containing 100 ml. 10 of the following sterile medium: Calcium carbonate ... 2 g. 1000 ml Incubate the flask and its contents for 2-5 days at 35°C on a rotary shaker (280 r.p.m., 2 inch stroke). 20 Germination Stage 2: Aseptically transfer 25 ml. of the fermentation medium of nomination stage 1. to a two-liter shake flask containing 500 ml. of the aforedescribed sterile germination medium. Incubate the flask and its contents for three days at 28°C 25 on a rotary shaker (280 r.p.m., 2 inch stroke). - 81 - 40437 Fermentation Stage» Aseptloally transfer 500 ml. of the inoculum obtained from germination stage 2 to a 14 1. fermentation tank containing 9*5 1* of the following sterile medium* 5 Dextrin 50 g.

Dextrose 5 g.

Soybean meal 35 g* Calcium carbonate ... 7 g.

Cobalt chloride 10"^ molar 10 Tap water 1000 ml.

Antlfoam (GE 60) .... 10 ml.

Prior to sterilizing the aforedescrlbed medium* adjust the pH to 8 and add an aqueous solution containing 8.0 gms. of streptamine. Ferment the contents under aerobic conditions 15 for 48 - 240 houra, with stirring at 250 r.p.m., with air input at 4.5 liters per 1./minute and at 2.5 atmospheres The pH of the fermentation medium changes slightly during the antibiotic production, varying in the range of about 6.8 to about 7.3. 20 Monitor the antibiotic production using the assay procedure described for slsomlcln and when peak production is attained, harvest the product (the fermentation is usually complete In about 7 days).

Peak production for mutamicins 1, 2, 4, 5 and 6 is in the 25 order of magnitude of from about 5 to about 50 mcg/ml. - 82 - 40d37 B. Isolation of Mutamicin 1 Add 7*0 g. of oxalic add to the whole broth from Preparation A with stirring. Aoldlfy the broth to pH 2.0 using 6 N sulfuric aold. Stir the mixture for about 15 minutes and filter using a suitable filter aid. Neutralize (pH 7.0) the filtrate with 6 N ammonium hydroxide. Pass the filtrate through a 1.0 liter cation exchange resin column in the ammonium form (e.g., Amberlite IRC-50, Rohm and Haas, Philadelphia, Pa.). Discard the spent broth and eluate the column with 2N ammonium hydroxide collecting fractions of about 100 ml. Monitor the column eluate by disc testing each fraction against Staphylococcus aureus ATCC 6538P. Combine the active fractions and evaporate to about 100 ml. In vacuo and lyophllize to obtain a solid product. Triturate the product several times with warm methanol, filter and evaporate the filtrate to a residue. Chromatograph the product on silica gel (25 g.) using the lower phase of a chloroform:methanol:concentrated ammonium dioxide (1:1:1) system as the eluant. Combine and evaporate the fractions containing antibiotic activity to obtain thereby mutamicin 1.

Mass Spectrum: (M+l)+ m/e 464 also m/e 118, 142, 160, 127, 161, 179* 1B9> 207, 315, 233, 320, 338, 348, 366, 378, 446. 40437 C. In a similar manner# by substituting an equivalent quantity of 2,5-dldeoxystreptamlne, 2-epl-streptamlne. 2, 5-dldeoxy-5-amlnostreptamlne or 5-ef>l-2-deoxystreptamlne for streptamine, and toy following the procedure 5 of Preparations A and B, mutamlclns 2, 4, 5 and 6, respectively, nay be produced.

Mass Spectral Data: Mutamicin 2: (M+l)+ m/e 432 also m/e 127, 118, 142, 160, 129, 147, 157, 175, 255, 274, 283, 301, 288, 506, 316, 336, 10 346, 414, 431.

Mutamloln 4: (Mfl)+ m/e 464 also m/e 127, 118, 142, 160, 161, 179, 189, 207, 315, 333, 320, 338, 348, 366, 378, 446.

Mutamicin 5: (M+l)+ m/e 447 also m/e 127, 118, 142, 160, 144, 162, 190, 270, 288, 316, 303, 321, 331, 349, 446. 15 Kutamlcln 6: (M+l)+ m/e 448 also m/e 145, 163, 173, 191, 127, 118, 142, 160, 271, 289, 299, 317, 304, 322, 332, 350, 447. - 04 - 40437 example 1 l-N--Mubr.tlfctitcd-ni 3ccnici n A. l-N-Ethvlsi r.omlcin To a solution of 5 gm. of sisomicin in 250 ml. 5 of water add 1 N sulfuric acid until the pH of the solution is adjusted to about 5. To the solution of sisomicin sulfuric acid addition salt thereby formed* add 2 ml. of acetaldehyde, stir for 10 minute3, then add 0.85 gm. of sodium cyanoborohydride. Continue stirring at room temperature for IO 15 minutes, then concentrate solution in. vacuo to a volume of about lOO ml., treat the solution with a basic ion exchange resin (e.g. Amberlite IRA 401S (0H~)), then lyophi-lize to a residue comprising l-N-ethylsisomicin.

Purify by chrcroatographing on 200 gm. of silica 15 gel, eluting with lower phase of a chloroform-methanol- 7% aqueous ammonium hydroxide (2:1:1) system. Combine the like eluates as determined by thin layer chromatography and concentrate the combined eluates of the major component in vacuo to a residue comprising 1-11-ethyl sisomicin (yield 20 1.25 gi.i.). Further purify by again chromaLographiny on 100 gm. of silico gel eluting with a chloroform-mcthanol-3.5% ammonium hydroxide (1:2:1) system. Pass the combined, like eluates (as determined by thin ];«.ye*r chromatography) through a column of basic ion exchange resin and lyophilj;:e 25 the cluate to ofc( ;un 1-W-othylc.' nonvc • n (y iel d 0.54 gm.) ; - 05 - 40437 (ajp6 + 164° (0.3*, H20)» pmr (ppo) (D20)» S 1.05 (3H, t, J«7Hz, -CHjCH^i 1.19 (3B, 8, -C-Cg3)) 2.5 (3H, 8, N-C£3)> 4.85 (IB, m, «CH-)| 4.95 (IB, d, J-4BS, B1H)|'5.33 (IB, d, J-2.5 Ha, Bj^1), 5 Haas Spectrum* (M + 1)+ m/e 476 also m/e 127, 154, 160, 173, 191, 201, 219, 256, 299, 317, 332, 345, 350, 360, 378, 390, 400.

B. In the procedure of above Example 1A, instead of adding 1 B sulfuric acid to the solution of sisomicin in water until it reaches a pH of about 5, other acids may be used, such as acetic acid, trifluoroacetic acid, j>-toluenesulfonic acid, hydrochloric acid, phosphoric acid, or nitric acid. The acidified aqueous solution is then treated with acetaldehyde and sodium cyanoborohydride and the resultant product is purified as described above, whereby is obtained l-N-ethylsisoraicin.

C. Alternatively, in the procedure of Example 1A replace sodium cyanoborohydride with an equivalent quantity of other 20 hydride donor-reducing agents. For example, with one of morpholinoborano, tetrabutylammonium cyanoborohydride or sodium borohydride, and there is obtained l-N-ethylsisomicin.

D. 1-N-Methvlsisomicin To a solution of 4.64 gms. of sisomicin in 10 15 - 06 - 40437 100 ml. of water ndd 1 N sulfuric acid until the solution lo nt n ptl of nbonl-. y. Adrt 1,2 ml. of 3'A' nquooun foimnl-deliyilf, utir for lo minuter. LIkmi mid 4»*>t> my. or nodi tint cyanohorohydrido. Pass the reaction solution through a column of a basic ion exchange resin (e.g. Amberlite IRA 40IS (0H~ form)) and lyophilize. Chromatograph the resultant residue on silica gel in the lower phase of a chloroform-methanol-7% aqueous ammonium hydroxide (2:ltl) solvent mixture. Combine the like eluates containing substantially 1-N-mothylsiscmicin as determined by thin layer chromatography. Evaporate in. vacuo to a residue of l-N-methylsisomicin» Ial^ + *53° (0.396, H20^ * Mass Spectrum: (M + 1)+ n*/e 462 also m/e 127, 140, 159, 160, 177, 187, 205, 256, 285, 303, 318, 331, 336(w), 346 376, 386.

E. 1-N- (n-jaropyl) sisoraicxn In a manner similar to that dcccribed in Example 1A treat the sulfuric acid addition salt of sisomicin in water with propanal and sodium cyanoborohydride. Isolate and purify the resultant product in a manner similar to pg that dcscribcd to obtain 1-N-tn-propyl) sisomicin; fc)D +140° (0.3%, H20); pmr (ppm) (DjO?: P. l-N-(n-butvl)sisomicin To a solution of 3 gms. of sisomicin in 200 ml. of water add 1 N sulfuric acid until the solution is at a pH of about 3.5. Add 1.5 ml. of ^-butanal, stir for 10 minutes then add 450 mg. of sodium cyanoborohydride. Continuo stirring for one hour then concentrate the solution in vacuo to a volume of about 100 ml. Pass this solution through a column of a basic ion exchange resin (e.g. Amberlite IRA 401S (0H~)) and lyophilize. Chromatograph the resultant residue on 140 gm. of silica gel in the lower phase of a chloroform-methanol -7% aqueous ammonium hydroxide (2:1:1) solvent mixture. Combine the like fractions containing 1-N-(n-butyl)sisomicin as determined by thin layer chromatography and evaporate the combined eluates in vacuo to a residue comprising 1-N-(n-butyl) sisomicin»[a]£6+ 129° (0.3%, HjO) , pmr(ppm) (D20) £ 0.82 (3H, t, J=7Hz, -CH2CH3), 1.15 (3H, s, C-CH.3) > 2.46 (3H, s, -N-CH3)j 4.82 (lh, m, =CH-) » 4.92 (1H, d, J=4Hz, Hj^); 5.29 (1H, d, J=2Hz, Hj').

Mass Spectrum: (M + 1)+ m/e 504» also m/e 127, 160, 182, 201, - 08 - 40437 219, 229, 247, 256, 327, 345, 360, 373, 3H0, 410, 420.

G, Other l-H-.Mkvl. l-M-Alkonvl ; In each case carry out the reaction in a manner similar to that described in Example 1A and isolate and purify each of tho resultant products in a manner similar 20 to that described to obtain, respectively. 1. 1 -N- (fl-mcthylpropyl) sisomicin. 2. 1 -N- (n-pentyl)sisomicin. 3. 1 -N- (T-nothylbutyl)sisomicin. 4. 1- -K- (fl-mcthylbutyl)sisomicin, 5. 1- N- (0 ,^-dimethylpropyl) Riycwicin - ft'j - 40437 6. l-&-(0'-otl;ylbutyl) sisomicin [aj*6 + 121° (c=0-.4%, H20)» pmr (PPm) (D20)*£o.75 (6H, t, 6.5Hz, CH2-CH3)» 2.4 (3H, 8, N-CH3)| 4.78 (1H, in, cCH-) \ 4.88 (1H, d, 4.0Hz, 5 Hj^i 5.22 (1H, d, 2.0Hz, H^)* Mass Spectrum* (M + 1)+ m/e 532 also m/e 127, 160, 210, 229, 256, 275, 355, 373, 388, 401, 4o6, 416, 446, 456, IO 7. 1-g- (n-octyl) sisomicin, 8. 1-N-O-propenyl) sisomicin 9. 1-N- (/3-ethylH3-hexenyl) sisomicin, 10. 1-N-benzylsisomicin, and 11. 1-N-phenylethylsisomicin. 15 H. 1-N-(Hvdroxvalkvl)siaomicin3 In the procedure of Example 1A, instead of acetaldehyde, substitute equivalent amounts of each of the following aldehydes: 1. 5-hydroxypentanal, 20 2. 2-hydroxypropanal, 3. 2-hydroxy-3-methylbutanal, 4. 2 -hydroxy-2 -me thylpropanal 5. 4-hydroxybu tana1, 6. 8-hydroxyoctanal, and 25 7. 2-hydroxy-4-pentena1. - 90 - 40437 In cnch ease carry out the reaction in a manner similar to that described in Example 1A, and .isolate and purify each of tho resultant products in a manner similar to that dcccribcd to obtain respectively, 5 1, 1-N- (6-hydroxypcntyl) s isoraic in, 2. 1-N-(Q-hydroxypropyl)sisomicin, 3. 1-N- (0 -hydroxy-T'Hnethylbutyl) sisomicin, 4. 1-N- (0 —hydroxy-#* -jnethylpropy 1) sisomicin, 5. 1-N—(£-hydroxybutyl)sisomicin, IO 6. 1-N-((J-hydroxyoctyl) sisomicin, 7. l-N-(fl-hydroxy-£-pontenyl) sisomicin.

I. l-N-(/-Aminobutyl)sisomicin Add 1 N sulfuric acid dropwise to a solution of 3 gm. of sisomicin in 120 ml. of water until the pH 15 of the solution is adjusted to about 5. To the aqueous solution of the sulfuric acid addition salt of sisomicin thereby formed, add 60 ml." of dimethylformamlde followed by a solution of 2 gm. of 4-phthalimidobutanal in 10 ml. of dimethylformamide. Continue stirring for IO minutes P.O then add 420 mg. of sodium cyanoborohydride. After about 20 minutes add the reaction solution to 1 liter of anhydrous methanol with stirring and collect by filtration the resultant precipitate comprising the sulfuric acid addition salt of 1-H- (/-p'nthol.imidobutyl) sowi cin. •'5 Purify by dissolving the precipitato in water - 91 - 40437 and passing the aqueous solution over a basic ion exchange resin. Evaporate jji vacuo to a residue, chromatograph the residue over silica gel eluting with tho lower phase of a chloroform-methanol-7% aqueous ammonium hydroxide (2slsl) 5 solvent mixture, and evaporate the combined, like eluates to a residue comprising 1-N- (^-phthalimidobutyl) -sisomicin.

To 0.5 gm. of l-tf-(£-phthalimidobutyl) sisomicin, add 5 ml .of 5* ethanolic hydrazine hydrate and heat under reflux for 3 hours. Pour the reaction solution into a large 10 volume of tetrahydrofuran and collect by filtration the re sulting precipitate comprising l-W-(S-aminobutyl) sisomicin.

Alternatively, the compound of this example is prepared as follows: (1) 4-Acetamidobutvraldehvde 15 Dissolve 5 gms. of 4-acetamidobutyraldehyde diethyl acetal in 75 ml. of distilled water and 5 ml. of 1 N sulfuric acid. Allow the solution to stand at room temperature until the hydrolysis is complete as determined by thin layer chromatography. Neutralize the solution with 20 sodium bicarbonate then saturate the solution with sodium chloride and extract with chloroform. Distill the combined chloroform extracts to a residue comprising 4-acetamidobutyraldehyde, which can be used without further purification in the following procedure. 25 (2) l-N-($-Acetamidobutvl)sisomicin To 3 gms. of sisomicin in 120 ml. of distilled - 92 - 40437 water ndrl 0.1 H sulfuric acid unti.l tho solubion is at about pll 5. Add 6 gms. of S-acetam.iciobutyraldehyde prepared as described in the preceding procedure followed, after 10 mi nuteo, with GOO gms. of solid sodium cyanoborohydride. After 2 hours, concentrate the solution to a small volume and pour into methanol. Collect the resultant precipitate by filtration# dissolve in water and pass the aqueous solution through a column of Amberlite IRA 401-S (OH**) ion exchange resin. Evaporate the eluant and chromatograph tho resultant residue on silica gel eluting with the lower phase of a chloroform methanol: 7% ammonium hydroxide solvent mixture. Evaporate the eluant to a residue comprising 1-N- (J -acetamidobutyl)sisomicin. (3) 1-N-(^-aminobutvl)sisomicin Treat 1-N-(inoalkyl) si cornicing nnd l-N-(Hvdroxvnmlno-alltvl) sinomicins t In a manner similar to that described in Example II, treat the sulfuric acid addition salt of sisosnicin in aqueous dimethylformamide with sodium cyanoborohydride, and with each of the following amino"substituted aldehydes: 1. 3-phthalimidopropanal, 2. 5-phthalimidopentanal, 3. 2-phthalimidopropanal, 4. 2-hydroxy-5-phthaliraidopentanal, 5. 3 -methyl -3 -hydroxy-4-phthalimidobutanal, 6. 2 -hydroxy-4 -phthalimidobut anal, 7. 2-phthalimido-3 -methylbutanal, 8. ?. -hydroxy-3 -phthalimidopropanal, 9. 2 -hydroxy-2 -methyl-3 -phthalimidopropanal, and 10. 8-phthalimidooctanal.

In each case carry out the reaction in a manner similar to that described in Example 1A, and isolate and purify each of the resultant products in a manner similar to that described to obtain, respectively, 1. 1-N-(V-phthalinidopropyl) sisomicin, 2. lN-(f-phthalimidopentyl) sisomicin, 3. 1-N-(fl-phthaiimidopropyl) sisomicin, 4. 1 -N-(fl-hydror.y-^-phtha 1 imidopotityl) ninomi.cin, 5. 1-N- (Y-mclhyl.->-hydroxy-,/"-phthalirnidobutyl) -sieomicin. 40437 6. l-N-(ft-hydrc>::y-tJ-phtli;vliinidobutyl) sisomicin, 7. l-W-(n-phtlinljniido-r-mcthylbutyl) sisomicin, 0. l-N-(/5-hydro:;y-V-phthal.imidopropyl) sisomicin, 9. 1-N~ (fl-hydroxy-0 -methyl -phthalimidopropyl)- 5 sisomicin, and 10. 1-N- {i-'-phtha 1 iraidooc t y 1) sisomicin.

Treat each of the foregoing N-phthalimidoalXyl sisomicin derivatives with ethanolic hydrazine hydrate as described in Example II to obtain, respectively, IO 1. 1-N-(7-aminopropyl) sisomicin, 2. 1-N- (£-aminopcntyl)sisomicin, 3.. l-N-(/J-aminopropyl) sisomicin, 4. 1-N- (O-hydroxy-^-aminopontyl) sisomicin, 5. 1-N- (T-mcthy1 -Y"-hydroxy-/-aminobu tyl) sisomicin, 15 6. 1-N- (/3-hydroxy—/-aminobutyl) sisomicin, 7. 1-N— (^-amino-Y-methylbutyl) sisomicin, 8 . 1 -N-(ft -hydroxy-Y-aminopropyl) sisomicin, 9. 1 -N- (3-hydroxy-methyl-T^-cuninopropyl) sisomicin, and 20 IO. l-N-(/l/-aminooc tyl) sisomicin.

K. AUcvlaininoalkvl.q.icomi.cTnr. and Other Hvdroxvaminoalkyl- sisoin.tcins (1) 1-N (H-wothvl rwinoethvl) r.i.somlcin In n nu>nu«.u" similar to that described in Example 25 IA treat oii.oiniein in water with 1 N sulfuric acid and - 'J'i - 40437 2-(N-methy1acetam1do)acetaldehyde followed by sodium cyanoborohydride.

Isolate the resultant product in a manner similar to that described to obtain l-N-( 6 -(N-methylacetamido)-ethyl)sisumicin.

Treat the foregoing N-acetylated intermediate with 10% 5 aqueous sodium hydroxide for 3 hours at 100°C, pour the foregoing reaction solution onto Amberlite IRC 50 Ion exchange resin, elute with 2 M ammonium hydroxide, concentrate the combined eluates 1n vacuo to a volume of about 100 ml., then lyophllize to a residue comprising 1-N-( 0 -methylaminoethyl)sisomic1n. 10 (2) In the procedure described in above Example IK (1) substitute for 2-(N-methylacetamido)acetaldehyde other aldehydes such as 2-acetamido-3-hydroxyoctanal or 2-acetamido-4-pentenal as aldehyde reagents to obtain the corresponding l-N-(aminoalkylJsisomicin, e.g. 1-N-{ B -amino- y -hydroxyoctyl)-sisomicin and l-H-( 0 -amino- 6 -pentenyl) 15 sisomicin, respectively.

L. 1-N-( b -Aminoethyl)sisomicin and l-N-( y -Aminopropyl)sisomicin In a manner similar to that described in the alternate procedure of Example 1-1, treat an aqueous solution of sisomicin to which 0.1 N sulfuric acid has been added until the solution is at about pH 5, 20 with p -acetamidoacetaldehyde followed by solid sodium cyanoborohydride.

Isolate and purify the resultant product in a manner similar to that described to obtain l-N-( 0 -acetamidoethyl)sisomicin. Hydrolyze the l-N-( e -acetamidoethyl)sisomicin with 10% aqueous potassium hydroxide and isolate and purify the resultant product in a manner similar to that 25 described in Paragraph (3) of the alternate procedure of Example 1-1 to obtain l-N-( e -aminoethyl)sisomicin. - 96 - 40437 Mass Spectrum: (M ♦ 1) ♦ m/e 491, also 160. 169, 187, 206, 216, 234, 256, 283, 314, 325, 334, 347, 360, 370, 375, 405, 415.

In the above procedure, by substituting y -acetamldo-5 propa rial for 0 -acetamldoacetaldehyde, there Is formed l-N-( y acetamldopropyl)slsomlcln, which when hydrolyzed with 10X aqueous potassium hydroxide and Isolated and purified 1n the described manner yields 1-N-( y -amlnipropyl)sisomicin.

EXAMPLE 2 10 1 -N,-subs ti tuted-Gentami ci n-Cj A. 1-N-Ethylgentamicin Cja To 5 gm. of gentamlcin in 125 ml. of water add 1 N sulfuric acid until the pH of the solution is about 5.2. Then add 2 ml. - 97 - 404 37 solution for 5 minutes* then add 1 gm, of sodium cyanoborohydride. Continue stirring at room temperature for 30 minutes, concentrate the solution vacuo to a volume of about 75 ml., pass the solution through a column of a basic ion exchange resin, (e.g. Amberlite IRA 401S (OH**)), then lyophilize to a residue comprising 1 -N-othylgentainicin Cla* Purify by chromatographing on 200 gm of silica i gel eluting with the lower phase of a chloroform-methanol-7% aqueous ammonium hydroxide (2:1:1) system. Combine the like eluates as determined by thin layer chromatography and concentrate the combined eluates of the major component in vacuo to a residue comprising 1-N-ethylgentamicin C^a (yield - 0.95 gm.). Further purify by again chrcxaatographing the 1-N-ethylgentamicin C^a on 100 gm. of silica gel eluting with chloroform-methanol-3.5% ammonium hydroxide (1:2:1) system. Treat the combined, like eluates (as determined by thin layer chromatography) with a basic ion exchange resin and lyophilize the eluate to obtain 1-N-ethylgentamicin C^a (0.42 gm.), 1°]^ + 118° (c=0.3%, H2°^ * pmr(ppm)(D20): Jl.06 (3H, t, J=7IIz, -CII2CH3)j 1.19 (3H, s, -C-CH-j) , 2.51 (3H, s, -N-CH3)j 4.97 (1H. d, J=4Hz, I^")? 5.16 (1H» d, J=3.5Hz, H1").

Mass Spectrum (M + 1)+ m/e 478 cilso m/e 129, 15',, 160, 173, - 98 - 40437 191, .?Ul, .1X9, 2.S0, 301, 317, 319, 329, 332, 347, KSO, 360, 37H .mi] 402.

B. Tn tho procedure of R;:ampXc 2A instead of using 1 N sulfur.! c acid to adjust the pll to atjout 5.2, add other acicln such as acetic acid, jv-toXuenesuXfonic acid, tri-fXuoroacetic acid, hydrochXoric acid, phosphoric acid, or nitric acid. Treat the acidified aqueous solution of gentamicin thereby produced with acetaldehyde and sodium cyanoborohydride in a manner similar to that described in above Example 2A and isolate and purify tho resultant product in a manner similar to obtain 1-N-ethylgentainicin C. In tho procedure of l£::amplc 2A, by substituting for acetaldehyde other aldehyde reagents, e.g. formaXdehydc, n_-px*opnnaX, ii-butanaX, n.-octanal# hydroxyacetaldehyde, 4-hydroxybufcanaJ. and phonylacctaldehyde there is obtained the corresponding X-N-alkylgentamicin c^a» e.g. 1-N- methylgentnmicin 1-N-(n.-propyl)gentamicin 1-N- (n.-but.yl) gentamicin C- , 1-N- (n-octyX)gentamicin C. , 1-N- xa la — (tf-hydroxyethyl)gcntaraici n C^, X-K-(^-hydroxybutyl)gentamicin and 1-N-(plicnylcthyX)gentamicin C^a» respectively. - 99 - 40437 EXAMPLE 3 1-N-Substitufccd-Verdamicin A. 1-N -Bthvlverdamicin To-O.5 gm. of verdamicin in 65 ml. of water add 1 N sulfuric acid until tho pll of the solution is adjusted to about pH 4.9, then add 0.2 ml. of acetaldehyde. Stir the solution for 5 minutes* add 65 mg. of sodium cyanoborohydride, concentrate the solution in. vacuo to a volume of about IO ml. and pour the solution into 50 ml. of methanol with stirring. Collect by filtration the resultant precipitate comprising 1-N-ethylverdamicin. Purify by chromato-graphing on 100 gm. of silica gel eluting with a chloroform-methanol-3.5% ammonium hydroxide (1:2:1) system. Collect the like fractions as determined by thin layer chromatography and evaporate in vacuo the combined fractions containing the major component to a residue comprising 1-N-ethylverdamicin. Further purify by again chromatographing on 7 gm. of silica gel eluting with the lower phase of a chloroform-methanol-7% ammonium hydroxide (2:1:1) system. Combine the like eluates and evaporate in. vacuo to a residue of 1-N-ethylverdiimicin (yield 50 mg.); Mass Spectrum: (tl + 1)+ m/e 490 also m/e 141, 154, 160, 173, 191, 201.. 219, 270, 313, 317 (w) , 331, 332, 341, 350 (w) , 357, 359 360, 378, 390, 414. -100 - 40437 B. 3m tho procedure of above ExaiujxU* 3A substitute for acel.aldohyde otlu-r aldehyde intermediates, e.g. formaldehyde, n-propanal, rv-butanal, n-octanal# liydr.oxyacetaldehyde, 4-liydroxybutanal and phcnylacctaldehvdo.. Isolate and purify cach of the resultant products in a manner similar to that to obtain, respectively, 1-N-ir.ethy 1 verdamicin, l-N-(n-propyl)-verdamioin lal^ +■ 122° (e«0.3%, l^O); pmr (ppra) (D20): pmr(ppm)(D20)j 1.03 (3H, t, 7Hz. -CH2CH3)» 1.03 (311, d, J»6.5IIz, -CH-CH3)j 1.17 (3H, a, C-CII3)» 2.32 (311, 8, 6'N-CH3)» 2.49 (311, s, 3"-NHCH3)» 4.94 (1H, d, J=4Hz, H^") ) 5.13 (1H, d, J=3.5Hz, H^).

Mass Spectrum: (M + 1)+ xa/o 506 also m/c 154, 157, 160, 173, 191, 201, 219, 206, 317, 329(w), 347, 350, 360, 375, 430.

D. In the procedure of above Example 4A in place of acchaldohydc utilize other aldehyde reagents, e.g. formaldehyde, n-propnnnl, n-butanal, n-oetanal, hydroxvncetaldehyde, 4-hydroxybutnnnl and phonylacetaldehyde. Isolate and purify cach of the resultant products in a manner similar to that to obtain, respectively, 1-N-raethylgentamiein C^, 1-N-(n-propyDgcuLuvnicin l-N-fj^-butyDgeiitrunicin O , 1-N-- - 102 - (n-octyDy.^ntiuniniri ^ , 1 -II- (/«-hyrt ro.: y« • I hy ] ) .5Il2, 6'-CII3), 1.10 (311, s, 4"-CI!3), 2.20 (311, o, 6'-NCH3), 2.43 (3H, s, 3M -NCIlj) , 4.00 (1H, d, J=4Hz, H^"), 5.00 (111, d, J=3.5Ilz, H^) and 7.33 (5H, s. -C6H5)i Mass Spectrum (m + 1)+ m/e 502 also m/e 506, 464, 454, 451, 436, 433, 426, 423, 408, 405(w)', 393, 295, 206, 277, 267, 249, 160, 157> Y max (KBr> 3300, 1050, 1030 cm."1, respectively. - 103 - 40437 KXAMPI.T? 5 1-N-Subat.i. tutod-An fcjbiotic G-52 A. 1-N-Ethv.l Antibiotic G-57.

Dissolve 875 mg. of Antibiotic G-52 in 40 ml. of distilled water and add 1 N sulfuric acid until the pH of the solution is adjusted to about 3.5. Add 0.7 ml. of acetaldehyde, stir the reaction mixture for 10 minutes then add 100 mg. of sodium cyanoborohydride. Monitor the reaction solution by thin layer chromatography, and when the starting Antibiotic G-52 appears to have completely reacted (i.e. about 10 minutes) then concentrate the solution in, vacuo at about 35 to 40°C to a volume of about 10 ml. Pass the concentrated solution through a basic ion exchange resin then lyophilize to a residue comprising 1-N-ethyl Antibiotic G-52. Purify by chromatographing on a silica gel column (4' by 1/2" column) eluting with the lower phase of a chloroform-methanol-7% aqueous ammonium hydroxide (2:1:1) system. Combine the like eluates as determined by thin layer chromatography and concentrate the combined eluates of tho major component in, vacuo to a residue comprising 1-N-ethyl Antibiotic G-52 (yield 60 mg.). Further purify the overlap eluates from the foregoing chromatography by chromatographing on silica gel eluting with the lower phase of a chlorofornw.iothanol-7';6 aqueous ammonium hydroxide (1:2:1) r.ystcia to o'utain an additional. 35 wg. of residue comprising l-JJ-o'chyl Antibiotic G-52. Puss tho - 104 - cofliMnrd rc.riiclucs: or 1-M-ethyl Antjl>Lofcie G-52 through a column of basic ion exchange resin (e.g. Amber l.i to IK.\ 401.'!) and lyophili.-.o the elunl-o to obin 1-N-ethyL Antibiotic G-52 (yield 90 icg); [a]^6 > 122.1° (c-0.356, II20), pmr (ppm) (OgO): t/1.06 (351, t, J- 6.5Ifo, lN-CnoCII3) j 1.21 (3)1, n, 4"-C-CH3)» 2.30 (311, s, 3 "-«-CH3) j 2.50 (311, n, f>*-N-CJl3)j 4.94 (1H, m, l^'); 4.97 (in, d, J«4.0IIz H^)! 5.34 (111, d, J=»2.5Ilz, Mnsu Spcctrum: (M + 1)+ m/e 490 also m/e 141, 154, 160, 173, 191, 201, 219, 270, 313, 317(w), 331, 332, 341, 350, 359, 360, 378, 390, 414.

B. In the procedure of above Example 5A in place of acetaldehyde use other aldehyde reagents such as formaldehyde, n-propanal, n-butanal, n-octanal, hydroxyacetaldehyde, 4-hydroxybutanal and phenylacetaldehyde. Isolate and purify each of the resultant products in a manner similar to that described in Example 5A to obtain, respectively, 1-W-methyl-AntibioUe G-52, l-N-(n-propyl) -Antibiotic G-52, 1-N-(libit tyl) -Antibiotic G-52, l-N-(rc--hydro; IO.

Antibiotic G-418, 11.

Antibiotic JI-20A 12.

Antibiotic J1-20B 13. tobramycin, 14..

Antibiotic 66-40B 15.

Antibiotic 66-40D 16. mutamicin 1, 17. mutanicin 2, 18. mutamicin 4, 19. mutamicin 5, 20. mutamicin 6. - 106 - 40437 Isolate and puri£y each of the rosultnnt products in a manner aidliar to that described in Example 1A to obtain the corresponding 1-N-ethyJ. compound, i.e. 1. 1-M-cthylgcntamicin A, 5 2. l-M-ethylgcntiwicin D, 3. 1-K-i/thylguntamicin 4. l-M-ethylgontnmicAn C^, 5. 1-N-ethylgentamicin cja» 6. 1-N-ethylgentamicin C^, IO 7. 1 -N-ethylgentainicin c~a» 8. 1-N-ethylgentaraicin c2b' 9. 1-N-ethylgentamicin X,, IO. 1-N-ethyl Antibiotic fi-418. 11. 1-N-othyl Antibiotic JI-20A 15 12. 1-N-ethyl Antibiotic JI-20B 13. 1-N-ethyl tobramycin, 14. 1-N-ethyl Antibiotic 66-40B 15. 1-N-ethyl Vmtibiotic 6G-40D 16. 1-N-ethylmutamicin 1, 20 17. l-N-ethylr>uitamicin 2, 18. l-N-ethylmutamicin 4, 19. 1-N-ethylmutnmicin 5, 20. 1 -N-e thylmu Lain i c in f*.

H. )'n the prorrtdivrr of E:;;mplo 6A by utilising pro;>anal '■'5 in pl.aoo oC aceLfl«?.i:iiyd;> M'.-.rc is oM-tt.rwd • corrospoiu? iisg - 107 - W 40437 1 -H-(ri-propyl) derivative of cach of the 4,6-diaminoglycosyl-1,3-diaminocyclitols listed therein.

C. By treating each of the 4,6-diaminoglycosyl-l,3-di-aminocyclitol starting compounds listed in above Examplo 6A in a manner similar to that described in any of Examples 1A through IK there is obtained the corresponding 1-N-alkyl derivative thereof.

EXAMPLE 7 Preparation of l-N-substituted-4.6-di-(Aminoglvcosvl)-1.3- \ di-aminocvclitols via Selectively Blocked Intermediates A. 1-N-Ethvlgentamicin via a 2'.3-di-N-substituted Intermediate Dissolve 240 mg. of 2',3-di-N-trifluoroacetyl-gentamicin in 10 ml. of water-methanol (2:1) and adjust the pH of the solution to about 3.5 by adding 1 N sulfuric acid. Add 0.19 ml. of acetaldehyde, stir for 10 minutes, then add 27 mg. of sodiun cyanoborohydride, and stir the reaction mixture for an additional 10 minutes. Evaporate the reaction mixture in. vncuo to a residue comprising 1-N-ethyl -2 ' ,3-di-W-trifluoiKicoLylgcntnmicin C^. Dissolve tho foregoing residue in 50 inl. of concentrated ammsniur.i hydroxido ;md a 11 h the renultant renidue over oil Jen gel (12 g.) eluting with the low^r |>lint;e of n mixture of chloroform-me Minnol-lO)C anucoun airouonium hydronicl;- (2:1:1). Coubine the liXo 5 fractions (an determined by thin layer chromatography) and evaporate the combined eluates in vacuo to a residue comprising 1-N-ethylgentamicin C^. (Yield » 80 mg.) B. 1 -H-Ethvloisoinicin via a 6'-N-r:uhstituted tntrrn< diate In a manner similar to that described in above IO Kxanple 7A, treat 6'-N-fc-butoxycarbonylsiconiicin (prepared in a manner tiimilar to that of Preparation 3D) In a<]iu.-our; methanol with miliuric acid, then aceluldoliyde and r.odiujn cyanoborohydride. Allow tho reaction mixture to ptanrt at room temperature for 30 minutes then evaporate jji vnew 15 to obkiiin l-N.-ethyl-G'-N-t.-bufc.oxycarbonylsisomicin. Diesoivo I tho foregoing residue in trifluoroacetic acid and allow the solution to stand for 10 minutes. Then add to an excess of anhydrous methanol, filter the resultant precipitate of the trifluoroacetic acid salt of l-N-ethylsisowicin 20 and clvroinafcograph over silica gel using the lower phase of a chlorororni-methanol-anm.onium hydr.o:ride solvent system in a manner ;;*r.:.\J.ar to that dor.ex'ibed in Kxample 7A to obtain 1-N-othylsi some in. - 109 - 40437 C. l-N-Mothvlsioomlnin from 3"-N-4w-0-carbonvl-nisomicin 3 "-N-4"-O-carbony 1-sisomioin (5 gm.) in distilled water (300 ml.) is treated with 1 N sulphuric acid until the pH of the solution is 2.5. Aqueous formaldehyde (37%) (2 ml.) is added and, after ten minutes, a solution of sodium cyanoborohydride (500 mg.) in water (5 ml.) is added dropwise. After 1 hour, the volume of the solution is reduced to one half by evaporation irj. vacuo, the pB of the concentrated solution is adjusted to 11 with addition of 1 N sodium hydroxide solution, and the solution is evaporated to dryness. The residue is extracted with methanol (3 X 100 ml.) and the combined methanol extracts are diluted with an equal volume of chloroform, filtered and evaporated to dryness to give crude 1-N-methyl-3"-N-4"-O-carbonyl-sisomicin.

The crude product is treated with 10% aqueous potassium hydroxide at 100° C for 5 hours. The cooled solution is passed down an Amberlite IRC-50 (H+) ion exchange resin and eluted with 2 N aqueous ammonium hydroxide and the eluant is concentrated and lyophilized to give crude 1-N-methyl-sisomicin.

Chromatography of the crude material on silica gel (300 g.) in chloroform-«u:thanol-7% ammonium hydroxide 26 ft (2:1:1) gives 1-W-methylsisomicin. talD + 153 (0.3%, h2°j - 110 - Ma;.:n Spootmrat CM I- X) ' m/>> 4('»2 a loo nv'c 127, 140, 159, 160, 177, 107, 205, 256 205, 303, 310, 331, 336 (w), 346, 376, 306.

D. 1-H-mothylvprdnmicin via r> 3".4**-N-0-o;:vzolidonc» intcrmndi nto (1) ' To an aqueous solution of 1 g. of verdamicin add sodium carbonate until the pH is in the range of about 8 to 9. Add. a nolution of 5 g» of j>-nitrophenylchlorocarbon«ite in 25 ml. of dimcthylformamido dropwisc with stirring over a period of 3 hours while maintaining the pll of the reaction mixture in the range of about 0 to 9 by adding more sodium carbonate solution. After the addition is complete, continue stirring at pll 8 to 9 for 16 hours. Evaporate the mixture in vacuo, extract the resultant residue several tines with hot chloroform, combine and evaporate the extracts and chromatograph the resultant residue over lOO g. of silica gel eluting with the lower phase of a (2:1:1) chloroform-methanol -conccntrated ajmuonium hydroxide solvent system. Combine and evaporate the like fractions as determined by thin layer chromatography to obtain verdamicin 3",4"-N,0-oxazo-lidone. (2) * In a manner similar to that described in Example 113 tvo;»fc the foregoing oxazolidone derivative in water with r.nlfuric acid, forma 1 dohyclo, and odium cyanoborohydride. Isolate and purify Ulit: rn^iili-.ant product in a - Ill - , 40437 manner similar to that described to obtain l-jj-raethylverda-micin-3",4"-N,0-oxa2olidone. (3) Diasolvo 0.2 g. of l-Jl-n»ethylvordamicin-.V,#4"- N,0-oxyzolidono in 10 ml. of 2 N sodium hydroxide solution. 5 Heat under reflux for 5 hours# neutralise the reaction solution with acetic acid and evaporate to a residue. Chromatograph the rosidue over 10 g. of silica gel eluting with the lower phase of a (2:1:1) mixture of chloroform-. methanol-1555 unmonium hydroxide. Combine and evaporate 10 li):e fractions as determined by thin layer chromatography to a residue to obtain 1-N-raethylverdamicin. - 112 - 40437 EXAMPT.B 8 1-N-Bcnzvlgentamicln via a tri-W-urotectccj-l-N-Schiff baoe Intermediate (X) Dissolve 0.3 g. of 2',3-di-«-trifluoroacetyl- gontamicin in 12 ml. of ethanol and add 0.9 ml. of benzal-dehyde. Stir the reaction mixture; for 3 hours then evaporate in vacuo. Dissolve the resultant residue .in 0.8 ml. of chloroform and add the solution dropwise to 25 ml. of hoxana-ether (3si). Separate the resultant precipitate by filtration and dry in vacuo to obtain l-N-3" -^-4M -O-bi s-benzylidine-2 • ,3-di-N-trifluoroacetylgentamicin C^. (Yield ° 0.38 g.) i m.p. 128-134°C» [a)£6 + 74.6° (c»0.26%, ethanol). (2) Dissolve 1.37 g. of the product obtained in above Example 8(1) in 100 ml. of ethanol and add to a stirred mixture of 1.37 g. sodium methoxide and 1.94 g. of sodium borohydride in 100 ml. of ethanol. Stir for 3 hours, acidify the mixture to a pH of about 3 with hydrochloric acid then stir for an additional 16 hours. Extract the solution with ether, separate and discard the ether layer. Add ammonium hydroxide to the aqueous phase until it is basic, then evaporate in. vacuo to a residue. Extract the residue with 35 ml. of hot ethanol. Combine the extracts and evaporate in, vacuo. Chromatograph the resultant residue over 75 g. of silica gel eluting w.ith the lower phase of a chloroform -me thoriol -ammonium hydro:;! de-water (2:1:0.2:0.8) - 113 - 4043? solvent system. Combine like fractions as determined by thin layer chromatography and evaporato to a residue com-prising 1-N-benzylgentamicin C^, m.p. 83-88 C, t<*]p + 90 (c=0.3%, H20)» pmr (ppm) (DjO): J 1.03 (3H, d, J=7Hz, HC-CH3>» 5 1.16 (3n, s, C-Cn3)» 2.27 (311, s, N-CHj)» 2.50 (3H, s, N-CHg) j 4.7 (D20 + PhCH2N )} 4.92 (in, d, J=4Hz, H-l")» 5.08 (1H, d, J=>3.5Hz, H-l*) } 7.43 (511, s, aromatic H)» Mass Spectrum: (M + 1)+ m/e 568 also m/e 440, 437, 412, 394, 10 379, 281, 263, 253, 235, 160, 157. (3) In the above procedure by substituting other alde hydes, e.g. propionaldehyde or phenylacetaldehyde for benzalde-hyde there is obtained 1-N-propylgentamicin C^ and 1-N-phenyl-ethylgentamicin C^, respectively. 15 EXAMPLE 9 1—N—Subst«-4.6-Diaminocrlvcosvl-l»3 -Diaminocvclitols Prepared bv Hydride Reduction of the Corresponding 1-N-Acvl Derivatives A. 1-N- (S-r/"-am.ino-/3-hvdroxvbutvl) gentamicin C^ 20 (1) Suspend 98 mg. of 1-N— (S>-J*-aiuino-0—hydroxybutyryl) gentamicin C^ in 8 ml. of tetrahydrofuran. Add 14 ml. of IN dibornne in tetrahydrofuran and heat at reflux temperature for 6 hours under an atmosphere of nitrogen. Carefully add 2 ml. - 114 - of water to decompose any excess diborane and evaporate. Dissolve tho resultant residue in hydrazine hydrate and heat at reflux temperature under an atmosphere of nitrogen for 16 hours. Evaporate the solution and extract tho residue with hot aquoou3 ethanol. Rvapora to the combined ethanol extracts and chromatograph the resultant residue oyer 10 ml. of silica gel eluting with the lower phase of a chloroform-methonol-concentrated ammonium hydroxide (2s1:1) solvent ■ system. Combine and evaporate the like fractions as determined by thin layer chromatography to obtain 1 -N- (J3-c/-amino -fl -hydroxybutyl)gentamicin C^ (yield 14.5 mg), m.p. 93-9B°C, [a)p6 t 72.4° (c»0.35%, H20)i pmr (ppm) (D20) % 1.18 (311, d, J»7Hz, CH-CH3 ), 1.24 (3H, 8, C-CH3) j 2.49 13H, s, N-CHg)} 2.54 (3H, s, H-CH3)» 5.07 (1H, d, J=3.5Hz, H-l")» 5.24 (1H, d, J«3.5Hz, H-l*).

Mass Spectrum : (M+l)+ m/e 565 also m/e 528, 516, 490, 437, 434, 410, 397, 270, 250, 232, 160, 157. (2) In the above procedure substitute 1-N-(S-V -amino-0-hydroxypropionyl) gentamicin C^ for l-N-(S.-cf -aminoH3-hydroxy-butyryl)gentamicin C^ to obtain l-N-(S-Y-aminoH3-hydroxypropyl) -gentamicin C^. (3) Treat each of the following l-N-fS-i/1 -amino-hydroxy-butyryl)-4,6-dianinoglycosyl-l,3-diominocyclitols with diborane in tetrahydrofuran in the manner described in Example 9A (1): - 115 - 1. 1 -N -(S -< / - -«imJ no~n-hydroxybutyry 1)gentamicin A, 2. 1-M-(S-J-nmino-^-hydroxybutyryl)gentamicin B, 3. 1-N-(jS-J-nmino-p-hydroxybutyryl) gcntamicin B^, 4. 1-N- (S-J-rjnino~/3--hydro::ybutyryl) gentamicin C^a# 5 . 1 -N- -amino -& -hydroKybutyry 1) gent sonic in C^# 6. l-N-tS.-t/^amino-^-hydroxybutyryDgenteBBicin C2a' 7. 1 -N-{Sj-tf-amino-0-hydroxybutyryl)gentamicin C2I}, 8. 1 -N~ (S.-cAamino-@ -hydr oxybu tyryl) gentamicin X^, 9. 1-N- (S_-^amino -0 -hydroxybutyryl) tobramycin, 10. 1-N-(S_-c/1aminoH3-hydroxybutyryl)Antibiotic G-418, 11. 1-N-(S_-t/-aminoH3-hydroxybutyryl)Antibiotic JI-20A 12. 1-N-(S_-c/-amino-/3-hydroxybutyryl)Antibiotic JI-20B Isolate and purify each of the resultant products in a manner similar to that described in Example 9A(1) to obtain, respectively, 1. 1-N-(S.-J^amino-£-hydroxybutyl)gentamicin A, 2. 1-N- (S.-c£amino-/3-hydroxybutyl) gentamicin B, 3. l-N-(S.-c^-aminoH3-hydroxybutyl)gentamicin B^, 4. 1 -N_— (S_—j/-amino-0-hydroxybutyl)gentamicin C^a' 5. l-W-(S_-y^aminoH3-hydroxybutyl)gentamicin C^, 6. 1 -N~ no-fi -Tiydroxybutyl) gcntamicin C_ , 7. 1-N- (S_-r^-amino-A-hydro:cybutyl) gentamicin C^, 0. 1-N-(5-./-amino-O-hydroxybu tyl) gentamicin X?, 9. 1-N- amino-/3 -hydroxybu ':yl) tobramycin, - 116 - 40437 10. 1-N-(2.-<£amJno-0-hydroxybutyl)Antibiotic G-418, 11. 1-N-(§---Y-amino-fl-hydroxypropyl)derivatives, i.e. 1. l-N-(S_-'y-aminoH3-hydroxypropyl)gcntamicin A, , 2. 1 -N- (Sj-«ya^110 -0 -hydroxypropy1) gent amic in B# 3. l-«-(S,-*Ytaomino-^-hydroxypropyl) gentamicin B^, 4. 1-N- (S.-f-amino-£-hydroxypropyl) gentamicin 5. l-N-(S,-f-amino-0-hydroxypropyl)gentaraic in , 6. 1-N-(S,-7-aminoH5-hydroxyprqpyl) gentamicin C2a» 7. l-N-(S^-7-amino-0-hydroxypropyl)gentamicin C^, 8. 1-N-(S.-T-amino-0-hydroxypropyl) gentamicin , 9. 1-N- (S,-7-amino-0-hydroxypropyl) tobramycin, 10. l-N-(S_-'Y-araino-<3-hydroxypropyl)Antibiotic G-418, 11. 1-N-(lS-'¥-amino-£-hydroxypropyl) Antibiotic JI-20A, 12. 1-N-(S_—Hf-araino-f}—hydr oxypropyl) Antibiotic JI-20B.

B. 1-N-Ethvlcrentamicin (1) In a manner similar to that described in Example 9A(1) treat 1-N-acetylgentamicin with diborane in tetra-hydrofuran. Isolate and purify the resultant products in a manner similar to that described in Example 9A(1) to obtain l-N-othylgentamicin C^. - 117 - 40437 (2) Treat tho following 1-N-«\c« tyl-4,6-di aminogly cosyl-li3-diaminocyclitols'in the manner of above Example 9B(1): 1. 1-N-acetylgentcunicin A, 5 2. 1-N-acetylgentamicin B, 3. 1-N-acetylgentaraicin B^, 4. 1-N-acetylgentamicin Cla, 5. 1-N-acctylgcntamicin C^, 6. 1-N-acetylgontamicin C2a» IO 7. 1-N-acetylgentamicin c2b' 8. 1-N-acetylgentamicin X2> 9. 1-N-acetyltobramycin, 10. 1-N-acetyl Antibiotic G-418, 11. 1-N-acetyl Antibiotic JI-20A, 15 12. 1-N-acetyi Antibiotic JI-20B.

Isolate and purify each of the resultant products in the manner similar to that described to obtain, respectively, 1. 1-N-ethylgentamicin A, 2. 1-N-ethylgcntamicin B, 20 3. 1-N-ethylgentamicin B^, 4. 1-N-ethylgcntnmicin C^a» 5. 1-N-ethylgcntnnicin C 6. l-N-ethylgonl:ninicin , 7. l-N-cthylgant.r-nicin 25 8. l-N-othylgcnl:;inicin , 9. l-M-cthyltobunnycin, - 118 - 40437 10. 1-N-ethyl Antibiotic G-418# 11. l-fl-ethyl Antibiotic JI-20A# 12. 1-N-ethyl Antibiotic J1-20B.

C. 1-N-Ethvl sisomicin (1) Suspend 1 gra. of 1-^-acetylsisoraicin in 100 ml. of tetrahydrofuran. Add lgm of lithium aluminum hydride# then stir the resultant suspension at reflux temperature for 24 hours under an atmosphere of nitrogen. Cool and decompose the excess hydride by careful addition of ethyl acetate. Evaporate tho reaction mixture to a small volume and dilute with water. Separate the insoluble solids by filtration and wash well with acetic acid. Evaporate the combined filtrate and washings and dissolve the resultant residue in water. Adjust the pH of the aqueous solution to about 7 by addition of ammonium hydroxide. Pass the AMBERLITE solution through a column cf/IRC 50 resin in the ammonium cycle and wash the column well with water. Elute with 0.5 H ammonium hydroxide# evaporate the eluate, and chromatograph the resultant residue over 20 gm. of silica gel eluting with the lower phase of a 2:1:1 chloroform-methanol-concentrated ammonium hydroxide solvent system. Combine and evaporate the like fractions as determined by thin layer chromatography to obtain l-N-ethylsisomicin. (2) Treat the following 4,6-dinminoglycosyl-l,3-di-aminocyclitol.a in tho. mnnncr described in the procedure of - 119 - W 40437 Example 9C(1): 1. ■ 1-N-acetylverdamicin, 2. 1-^-acetyl Antibiotic 66-40B, 3. 1-N-acetyl Antibiotic 66-40D, 4. 1-N-acetyl Antibiotic G-52, 5. 1-N-acetylmutamicin 1, 6. 1-N-acetylmutamicin 2, 7. 1-N-acetylmutamicin 4, 8. 1-N-acetylmutamicin 5, 9. 1-N-acetylmutamicin 6.

Isolato and purify each of the resultant products to obtain, respectively. 1. 1 -N-e thy 1 verdamicin, 2. 1-ii-etnyl Antibiotic 66-40B 3. 1-N-ethyl Antibiotic 66-40D 4. l-j[-ethyl Antibiotic G-52, 5. 1-N-ethylmutamicin 1, 6. 1-N-ethylmutamicin 2, 7. 1-N-ethylmutamicin 4, 8. 1-N-ethylmutamicin 5, 9. -ethylrautamicin 6.

D. 1-N-Hethvlnenhamicin (1) Dissolve 1 gm» of 2 ' ,3—di-.N—t3rif luoroacetylgenta— micin in 30 ml. of 50% aqueous methanol. Cool, to 5°C then add 0.25 gm. of t_-buto;:ycarbonyl azi.de dropwiso with - 120 - stirring followed by 0.1S5 ml. of triethyl amine. Stir tho resultant solution for 10 hourn, ovopoi'nto thn reaction mixture irj vacuo to a residue nnd chromatograph the residue on 100 gm. of silica gel eluting with the lower phase of 2tlil# chloroforn-mcthanol-concentrated ammonium hydroxide solvent system. Combine and evaporate the like fractions of tho major product as determined by thin layer chromatography to obtain l-JI-t-butoxycarbonyl-21 ,3-di-N-trifluoro-acetylgentamicin C^. (2) Dissolve the product, of Example 9D(1) in a mixture comprising 30 ml. of methanol and 20 ml. of concentrated ammonium hydroxide. Allow the solution to stand for 3 days at room temperature, then evaporate to a residue comprising l-i£-£.-butoxycarbonylgentamicin C^. (3) Dissolve 100 mg. of 1-N-t-butoxycarbonylgentanicin C^ in 15 ml. of 1 M diborane in tetrahydrofuran. Reflux the resultant solution for 16 hours under an atmosphere of nitrogen. Add 2 ml. of water to decompose any excess diborane and evaporate the mixture to a residue. Dissolve the residue in 10 ml. of hydrazine hydrate and heat at reflux temperature under an atmosphere of nitrogen for 16 hours. Evaporate the solution, extract the resultant residue with hot aqueous ethanol then evaporate the combined extracts and chromatograph the resultant residue over 10 gm. of silica gel eluting with the lower phase of a 2:1»1 - 121 - 40437 of i.»fuvi!i-iiK»l.l»aii«.«l -IV;.', nicaoniusn hydroxide.

Cf Chromatograph tho crude material on silica gel 20 in th<; lower pli.i.*;r: of a chloroform-vu i.linnol-V,' ammonj um hydroxide (2:1:1) solvent nixture to obtain 1-N-methyl-?G o Einoniic.ni. fa)o + JI2°) ' Mas.': ."ip.vetrum: (M (• 1)^ r.i/o 462 also m/e 12 7, I'D, 159, - 122 - 177, 107, 205, 256, 205, 303, 310, 33J, 336 (w), 346, 376, 306.

EXAMPLE 11 l-N-Methvlsiccwicin 21,3,6' -Tri-N-£.-butoxycarbonyl-3"-N-4"-O-carbonyl-sisomicin (5*j.» in ethanol (loo ml.) is treated with 37% aqueous formaldehyde* (1 ml.) and ammonium formate (5 gm.) and the solution is heated under reflux for 24 hours. The solution is diluted with water (200 ml.) and extracted with chloroform (3 X 100 ml.). The combined extracts are reduced to dryness and the residue containing 2,,3,6'-Tri-N-t-butoxycarbonyl-3"-N-4" -O-carbonyl-l-N-methyl-aisomicin io dissolved in trifluoroacetic acid and after five minutes at room temperature, the solvent is removed in vacuo. The residue is treated with 10% potassium hydroxide (25 ml.) at 100° for 5 hours. The cooled solution is passed down a column of Amberlite IRC 50 (H+) ion exchange resin and the crude product is eluted with 2 N aqueous ammonium hydroxide. Tho combined eluant is reduced to dryness in, vacuo and the residue chromatographed on silica gel (200 gm.) in the lower phase of a chloroform-methanol-7% ammonium hydroxide (2:1:1) solvent system to give l-N-msthylsisomicin. [a]y6 + 153° (0.3%, H20)j Mass Spectrum: (M t- 1)+ ta/c 462 Cairo m/c 122. 140, 159, 160, - 123 - 40437 177, 187, 205, 256, 285, 303, 310, 331, 336 (w) , 346, 376, 306.

EXAMPLE 12 1-N-Methvloisomicln Treat 2 * > 3,6 • -Tri-N-t.-butoxycnrbonyl-3 " -N-4" -o-carbonyl -sicomlcin (0.77 gm.)' in THF (25 ml.) with methylamine (101 mg.) and trifluoromethylsulphonio anhydride (290 mg.) at 0° for 3,0 hours. Reduce the solution to dryness and dissolve the residue in DMF (10 ml.) and stir with methyl iodide (300 mg.) and potassium carbonate (130 mg.) for a further 10 hours. Remove the solvent by evaporation and treat the residue with 10% aqueous potassium hydroxide at 100° for 12 hours. Pass the cooled solution through a column of Aiubc.-x.iito IRC 50 (H ) ion exchange resin. The crude product is eluted with 2 N aqueous ammonium hydroxide. The combined eluant is reduced to dryness in vacuo p.nd the residue chromat9graphed on silica gel (200 giw.) in the lovor phiise of a chloroform-methanol-77> ammonium hydroxide (2:1:1) solvent system to give l-N-meihylsisomicin. talp^ + 153° (0.3%, h.,0) j Mass Spectrum: (M + 1)+ m/e 462 also m/e 127, 140, 159, I60, 177, 107, 205, 25C, 205, 303, 3.10, 33.1, 33-3 (w) , 316, 3 76, 3B6. - 124 - EXWIPI.K .1.3 1 -N-^lnblivlfiinnmlci n 2',3,6• -Tri -N-t_-bnt oxycnrbonyl -3"-N-4" -O-carbonyl-sisomicin (0.77 gm.) in THP (20 ml.) If treated with 37% aqueous formaldehyde (3 ml.) and succinimidc (170 mg.) at room tempcraturo for 18 hours. Tho solution is dropped into a mixture of diothylether-hexane (1:1) and the precipitate is collected by filtration. This material is treated with sodium borohydrido (200 mg.) in ethanol (20 ml.) at room temperature for 5 hours. The ethanol is removed in, vacuo and the residue is treated with 10% aqueous potassium hydroxide for 12 hours at 100°C. The cooled solution is passod down a column of Amberlite IRC 50 (H+) ion exchange resin and tho crude product is eluted with 2 N aqueous ammonium hydroxide. The combined eluant is reduced to dryness in vacuo and the residue chrcmatographed on silica gel (200 gm.) in the lower phase of a chloroform-methanol-1% ammonium hydroxide (2:1:1) solvent system to give 1-N-methylsisomicin. la)^6 + 153° (0.3%, H20) s Mass Spectrum: (M + 1)+ m/e 462 also m/e 127, 140, 159, 160, 177, 187, 205, 256, 285, 303, 310, 331, 336 (w), 346, 376, 386. - 125 - 40437 EXAMPLE 14 1-N-Me bhvlsi pomicin ' Diceolve 21 , 3,6 '-/Tri-W-t-butoxycarbony 1-3 "-N-4 "-0-carbony 1-oisomicin (0.77 gm.) in dichloromethane (loo jnl.) with acry.lonitrile (0.25 gm.) and leave at room temperature for 24 hours. . Remove the solvent in vacuo to leave a residue which is dissolved in dimcthylformamide and treat with methyl iodide (100 mg.) at 50°C for 12 hours. Remove the solvent# and treat the residue with 10% aqueous potassium hydroxide at 100° for 8 hours. The cooled solution is passed down a column of Amberlite IRC 50 (II+) ion exchange resin and the crude product is eluted with 2 N aqueous ammonium hydroxide. The combined eluant is reduced to dryness in vacuo and the residue chromatographed on silica gel (200 gm.) in the lower phase of a chloroform-methanol-7% ammonium hydroxide (2:1:1) solvent system to give 1-N-methylsisomicin. [a]£5 + 153° (0.3%, II20), Mass Spectrum: ' (M + 1) + m/e 462 also m/e 127, 140, 159, 160, 177, 107, 205, 256, 285, 303, 318. 331, 336(w), 346, 376, 306.

EXAMPLE 15 1-N-(Tydroxycthyl) sisomicin Dissolve 2 ', 3, t>1 -Tri-n-fc_-bul.o::ycnrbonyl -3"-_W-4" -0-carbony si.f,or.iicin (1.0gm.) in methanol (30 ml.) in n stainless - 126 - 40437 steel presouro reactor (500 ml. capacity) that is connccted to an ethylene oxide cylinder through a shut-off valve and which io equipped with a heater and a pressure gauge.

Fill with ethylene oxide uutil a pressure inside the reactor of 2 atmospheres is obtained, neat the reactor to 60°C while rocking for 24 hours. Cool the reactor and depressurize.

Remove the solvent to obtain a residue containing 1-4T-(hy droxye thy 1)-2■ ,3,6'-tri-n-t^-butoxycarbonyl-3"y N-4O-[ carbonyl-siccinicin.

Heat the residue with 1GG» aqueous potassium hydroxide (20 ml.) at lOO°Cfor 8 hours and cool. The cooled solution is pasccd down a column of Amberlite IRC 50 (H+) ion exchange resin and the crude product is eluted with 2 N aqueous ammonium hydroxide. The combined eluant is reduced to dryness In vacuo and the residue chromatographed on silica gel (200 gro.) in the lower phase of a chloroform-methanol-1% ammonium hydroxide (2:1:1) solvent system to give 1-N-hydroxyethyl)-sisomicin. - 127 - 40437 EXAMPLE 16 Preparation of l-N-Alkvlainoniein rnd Derivatives thereof bv Cultivation of Mtcrmononpora inyoonnis Strain lr»5Ql'-lo in tho Prcncnco of a 1 -M-A .He v.! -2 -nr o:: v -D-S t r opt ran 1 n o or 5 Derivatives Tl'ercof A. Preparation of Inoculum of Micromonoapora invonenaia Strain 155QF-1G Germination Stage It Under asceptic conditions* add a lyophilized 10 culture (or cells obtained frcn a slant culture) of MlcromonoBPora invonenaia strain 1550F-1G to a 300 ml. shake flask containing 100 ml. of tho following sterile medium: Beef extract 3 gm. 15 Tryptose 5 gm.

Yeast extract 5 gm.

Dextrose 1 gm.

Starch 24 gm: Calcium carbonate 2 g. 20 Tap Water 1000 ml.

Incubate the flask and its contents for 2-5 days at 35°C. on a rotary shaker (280 r.p.m., 2 inch stroke).

Germination Starrc 2: Aseptically transfer 25 ml. of the fermentation 25 medium of germinabion stage 1 to a two-liter shake flask - 128 - 4043? containing 500 ml. of tho ;ifcrcdc«jcribirf/e t Asoptically transfer 500 ml. of tho inoculum obtained from germination stage 2 to a 14 1. fermentation » tank containing S.5 1. of fche folowing sterile medium: Dextrin Dextrose Soybean meal Calcium carbonate Cobalt chlori.dn Tap Water Antifoam (GE 60) 50 gm. 5 gm. 35 gm. 7 gm. _6 10 molar lOOO ml. IO ml.

B. "Preparation of l-N-Hethvlsiscnticin (1) Tho requisite 1 -N-methyl-2-deoxy-D-streptamine (also known as (-)-hyosamine) is prepared by hydrolysis of destomycin A according to the procedure of S. Kondo et. al, J. Antibiotics Ser. A., 18, 192, (1965). (2) Prior to sterilization of the medium described in Example 16 A, adjust the pll to 8 and add an aqueous solution containing 8.0 gms. of 1-N-methyl-2-deoxy-D-strepta-mine. Ferment the contents under aerobic conditions for 48-240 hours, with stirring at 250 r.p.m., with air input at 4.5 liters per minute and at 2.5 atmospheres. The pll - 129 - 40437 of tho fermentation medium changes slightly during the antibiotic production, varying in the range of about 6.8 to about 7*3. (3) Monitor tho antibiotic production using the assay procedure described in J. Antibiotics (Japan) Vol. XXIII, and when peak production is attained, harvest the product (the fermentation is usually complete in about 7 days) • C. Isolation of 1-M-f^ethvlsi.^omicin Add 7.0 gm. of oralic acid to the whole broth from Example 16B with stirring. Acidify the broth to pH 2.0 using 6N sulfuric acid. Stir the mixture for about 15 minutes and filter using a suitable filter aid. Neutralize (pll 7.0) the filtrate with 6 N ammonium hydroxide. Pass the filtrate through a l.O liter cation exchange resin column in the ammonium form fc.g., Amberlite IRC-50, Rohm and Haas, Philadelphia, Pa.). Discard the spent broth and elute the column with 2 N ammonium hydroxide collecting fractions of about 100 ml. Monitor the column eluate by disc testing each fraction against Staphylococcus aureus ATCC 6538P. Combine the active fractions and evaporate to about 100 ml. in. vacuo and lyophilize to obtain a solid product. Triturate the product several times with warm methanol, filter and evaporate the fill-rate to a residue. Chromatograph the product on silica gel (25 gin.) using tho lower phase of a chloroform-mothnr.ol-concentrated ammonia:.* - 130 - hydroxide (Is 1:1) uyaton an tlu* cltuutl. .nut cvnpo- ral:u Llio fmcLJoiiM eontniiiinrj antibiotic activity to obtain thereby 1-N-mcthylsioomiain.

D. l-WWilkvlnifiomicins (1) . . In a manner similar to that described in Example 16B by substituting for 1 -N-tnethyl-2-deoxy-D-atreptamine equivalent quantities of other mono-N-alkyl-2-deoxystrepta-mines* e.g. 1-N-e thyl-2-deoxy-I3-streptamine, l-N-propyl-2-deoxy-D-streptamine , 1-N-methyl-2,5 -dideoxy-J)-streptamine „ there is obtained the corresponding 1-N-alkylsisomicin, e.g. 1-N-ethyloisomicin, 1 -N-pr opy1 -sisomicin, and 1-N-methyl-5-deoxysisomicin.

E. Preparation of Mono-N-diethyl —5-Deoxysisomicin (1) Perment Micromonospora invoensis strain 1550F- 1G in the presence of (-) -mono-N-methyl-2,5-dideoxystreptamine according to the procedure described in Example 16B and isolate the resultant antibiotic thereby formed in a manner similar to that described in Example 16c to obtain an antibacterial substance comprising a mixture of 1-N-methyl-S-deoxysisomicin and 3-N-methyl-5-deoxy-sisomicin.

By susbsituting (i) -mono-N-methyl-2 -deoxystreptamine for (±) -mono-H-methyl-2,5-dideoxystreptamine in tho above procedure thore is obtained a mixture comprising l-^J-mefchylsis-omicin and 3-N-methylsisomicin. - 131 - 40437 (2) Chromatograph tho mono-N-methyl-5-deoxysicomicin mixture prepared in Examplo 16E(1) according to procedures known in tho /art,, combine like fractions as determined by thin layer chromatography and evaporate each of the com-5 bined fractions to a residue to obtain 1-N-methyl-5-de- oxysisotoicin and 3-N-methyl-5-deoxysisomicin, respectively.

Similarly, chromatograph the mono-N-methyl -sisomicin mixture obtained as described in the second para-; graph of Example 16E(1) to obtain 1-N-methylsiscmicin and 10 3-N-methylsisomicin, respectively.

EXAMPLE 17 ACID ADDITION SALTS A. Sulfate Salts (Sulfuric Acid Addition Salts) Dissolve 5.0 gms. of 1-^T-ethylsisamicin in 15 25 ml. of water and adjust the pD of the solution to 4.5 with 1 N sijlfuric acid. Pour into about 300 ml. of methanol with vigorous agitation, continue the agitation for about 10-20 minutes and filter. Yfach tho precipitate with methanol and dry at about 60°C in. vacuo to obtain 1-N-ethylsisomicin 20 sulfate.

In like manner, the sulfate salt of the compounds of Examples 1-16 may also be prepared. - 132 - 40437 B. Hydrochloride Salta Dissolve 5.0 grams of l-N-ethylverdomicin in 25 ml. of water* Acidify with 2 N-hydrochloric acid to pH 5* Lyophilizo to obtain 1-N-ethylverdaroicin hydrochloride.

In li];o manner, the hydrochloride salt of tho compounds of Examples 1-16 may alco be prepared. - 133 - 40437 KXAMIM.KIM .1 -N-Acetvl b i KOtnicin (A) Dissolve 1.25 g of sisomicin sulfate in 2(30 ml. of methanol-water (2:3, v/v) and chill the solution. Add 1.5 ml. of acetic anhydride and after approximately 10 minutes add 0.125 ml of kriethylamine in 10 ml of methanol over a 15 minute interval. Allow the reaction mixture to warm to room temperature over a 2 hour interval then evapor> ate the solvent in vacuo. Dissolve the residue in water and convert the product to the free base by passage of an aqueous solution thereof through Amberlite IRA-401S resin in the hydroxide ion cycle. Lyophilize the column eluate and chromatograph the residue on 50 g of silica gel using the lower phase of (2:1:1) chloroform, methanol, 1% arrcnoniu;.. hydroxide solvent system as eluant. Monitor the fractions via TLC and combine like fractions to obtain thereby the title compound.

Yield - 0.185 g, .u.p. 128°-130°C, fu]2jj 150° (0.3%II20), NMR: (D20)J-1.22 (3H, s. -C-CH^) ; 2.02 (3H,s, NH-C0-CH3) » 2.53 (3H, s. N-CH3) * 4.88 (1H, m, =CH-); 5.08 (111, d, J=4Hz, Hj/*); 5.35 (1H, d, J=2Hz, H^).

Mass spectrum: (M+l)+ m/e 490, M+ m/e 489. - 134 - 40437 (B) In a similar manner, treat an equivalent quantity of tho sulfate salt of tlio following antibiotics to the process of Example I8A1 gentamicin C^» gentamicin gentamicin C2 # gontamicin C2a» tobramycin. Antibiotic G-418, Antibiotic 66-40B, Antibiotic 66-40D, Antibiotic JI-20A, Antibiotic JI-20B, Antibiotic G-52, mutamicin 1, mutamicin 2, mutamicin 4, mutamicin 5, gentamicin X2» gentamicin A, gentamicin C2b, verdami cin, gentamicin B, and gentamicin B^. mutamicin 6, Isolate the respective products in the manner described in Example 18A and obtain thereby the following: 1-N-acetylgentamicin C^, 1-N-acetylgentamjcin - 135 - O 4043? 10 15 20 25 1-H-acetylgentamicin C2, 1-ji-acetylgentamicin C2q, 1-N-acetylgentamicin 1-N-acetylgentamicin C2b, 1-N-acetyl verdamicin, 1-^-acetyl tobramycin, 1-N-acetyl Antibiotic G-410, 1-N-acetyl Antibiotic 66-40B, 1-N-acetyl Antibiotic 66-40D, 1-N-acetyl Antibiotic JI-20A, 1-N-acetyl Antibiotic JI-20B, 1-N-acetyl Antibiotic G-52, 1-N-acetylmutamicin 1, 1-N-acetylmutamicin 2, 1-N-acetylmutamicin 4, 1-N-acetylmutamicin 5, 1-N-acetylmutamicin 6, (C) In the procedure of Example 1BA by substituting other acid anhydrides, e.g. rj-octanoic acid anhydride, phenylacetic acid anhydride, propenoic acid anhydride and trans-O-Phenylacrvlic acid anhydride there is obtained the corresponding 1-N-acyl derivative, e.g. 1-N-(n-octanoyl)-sisomicin, 1-N-phenylacetylsisomicin, 1-N-propenoylsiso-micin and 1-N-(J^rnns-fl-plicnylpropcnoyl) sisomicin, respectively. 1-N-acetylgentamicin A 1-N-acetylgentamicin B, and 1-N-acetylgentamicin B^. - 136 - 40437 EXAMPLE 19 l-N-Propionvlsisomicin A. Dissolve 1.25 g of sisomicin sulfate in 200 ml of methanol water (2*3, v/v) and chill the solution. Add 1.5 ml of propionic anhydride followed by 0.125 ml of tri-bthylamino Jn 10 ml of methanol over a 15 minute interval. Allow the reaction mixture to warm to room temperature over a 2 hour interval then evaporate the solvent in vacuo. Dissolve the residue in water and convcrt the product to the free base by passage of an aqueous solution thereof through Amborlite IRA-401S resin in the hydroxide ion cycle. Lyophilize the column eluate and chromatograph the residue on 50 g of silica gel using the lower phase of (2:1:1) chloroform, methanol, 7% ammonium hydroxide solvent system as eluant. Monitor the fraction via TLC and combine like fractions to obtain thereby 1-N-propionyl-sisomicin.

Yield 0.18 g m.p. 125°-130°C, fa]*6 = +147°(0.3% H20) NMR: (D^JJl.OS (3H, t,J=7.5 Hz, CHj-QLj) , 1.18 (3H, s, C-£H3)» 2.25 (2H, m, CH2CH3)* 2.48 (3H, s.NBCH.^ j 4.87 (111, m, = CH-) j 5.07 (1H, d, J= 4Hz, H^); 5.34 (1H, d,J=2llz, Ilj').

Mass spectrum M+ +1 m/e 504, M+ m/e = 503. - 137 - 40437 B. In a similar manner, treat an equivalent quantity of acid addition salt of those antibiotics set forth in Example 18B to the process of Example 19A, isolate the respective products in the manner described in Example 19A and obtain thereby the following! ( 1-N-propionylgentamicin C^, 1-N-propionylgentamicin 1-N-propionylgentamicin C2, l-N-propionylgentamicin C2a< l-N-propionylgentamicin . 1-N-propionylgentamicin A, 1-N-propionylgentamicin C2b» 1-N-propionylverdamicin, 1-N-propionyltobramycin, 1-N-propionylantibiotic G-418, 1-N-propionylantibiotic 66-40B, 1-N-propionylantibiotic 66-40D, 1-N-propionylantibiotic JI-20A, 1-N-propionylantibiotic JI-20B, 1-N-propionylantibiotic G-52, 1-N-propionylmutamicin 1, 1-N-propionylmutamicin 2, 1-N-propionylmutamicin 4, 1-N-propionylmutamicin 5, 1-N-propionylmutamicin 6, 1-N-propionylgentamicin B, and 1-N-propionylgentamicin B^.

EXAMPLE 2Q 1—N-Acetvlverdamicin A. Dissolve 1.25 g of verdamicin sulfate in a mixture of 10 ml of methanol and 25 ml of water. Add 0.125 ml of triethylamine followed by 1.5 ml of acetic anhydride. Chill the solution to 0°-5°C and allow to react for a 15 minute interval. Allow the reaction mixture to warm to room temperature over a 2 hour interval then evaporate the solvent jLn - 138 - 40437 vacuo. Dissolvo tha rosidue in water and convert the product to the free base by passage of an aqueous solution thereof through a suitable anion exchange resin column in the hydroxide ion cycle e.g. Amberlite IRA-401S. Lyophilize the column cluate and chromatograph the residue on 50 g of silica gel using the lower phase of (2:ltl) chloroform, methanol. 7% ammonium hydroxide solvent system as eluant. Monitor the fraction via TLC and combine like fractions to obtain 1-N-acetylverdamicin.

Yield: 0.241 g.

B. By the process of Example 20, other 1-N-acyl derivatives of vcrdamicin may be prepared, such as those wherein the acyl group is derived from propionic, butyric, phenylacetic, isobutyric or valeric acids or the like. In like manner, 1-N-acyl derivatives of other 4,6-di-(aminoglycosyl) aminocyclitol antibiotics, such as those set forth in Example 18B, may be prepared.

EXAMPLE 21 1-N-Acetvlcientamicin C^ Dissolve 250 mg. of gentamicin C^ sulfate in a mixture of 25 ml of water and 10 ml of methanol. Add 0.065 ml of triethylamine. Allow the solution to stand for ten minutes then add o.5 ml of acetic anhydride.

Let the solution stand for one hour at room temperature then evaporate to a residue in vacuo. Dissolve the residue in water and treat with Amborlite IRA 401S in - 139 - tho hydroxyl. ion form. Filter the suspension and lyophilize the filtrate. Chromatograph the residue over a silica gel column containing 30 g of adsorbent using the lower phase of a chloroform, methanol, 1% ammonium hydroxide (2:1:1) system and obtain thereby the product of this example.

Yield: 50 mg fa]*6 » +124° Mass Spectrum (M+l)+ m/e = 520 NMR (D20); 2.45 (3H, s, 3' N£03)» 5.03 (1H, d, J=4.5 Hz, Hj^) * 5.09 (1H, d, J=3.5 Hz, EXAMPLE 22 A. 1-N-(5-Aminopentanovl)gentamicin C^ (1) 1-N- (5-Phthalimi.dopentanovl)gentamicin C^ Dissolve 2.5 g of gentamicin C^ sulphate in 250 ml of water and add 100 ml of methanol. Add 0.65 ml of triethylamine and stir for IO minutes. Add a solution of 1.2 g N-(5-phthalimidopentanoyl.oxy)succinimide in 20 ml of dry dimethylformamlde dropwise with stirring to the solution of the antibiotic. Stir the mixture at ambient temperature for 16 hours. Concentrate the reaction mixture to a residue in vacuo and triturate tho residue with methanol to yield - 140 - 3.4 9 of white solids. Chromatograph t.he residue on 200 g of silica gel in the lower phase of a chloroform , methanol, 7% ammonium hydroxide (2tlsl) system to give 1-N- (5-phthalimidopentanoyl)gentamicin C^. (2) 1-N-(5-Aminopentanovl)gentamlcin C^ Heat 0.4 g of 1-N- (5-phthalimidopentanoyl)gentamicin C^ in 5 ml of 5% ethanolic hydrazine hydrate under reflux for 4 hrs. Concentrate the solution and add tetrahydrofuran to precipitate 1-N- (5-aminopentanoyl) gentamicin C^ which is collected by filtration.

B. In a similar manner, treat an equivalent quantity of acid addition- salt of the following antibiotics to the process of Example 22A(1) gentamicin Cla» gentamicin C2, gentamicin C2a, gentamicin Xj, gentamicin A, gentamicin C2b, verdamicin, tobramycin. Antibiotic G-418, Antibiotic 66-40B, Antibiotic 66-40D, Antibiotic JI-20A, Antibiotic JI-20B, Antibiotic G-52, mutamicin 1, mutamicin 2, mutasicin 4, mutamicin 5, mutamicin 6, gentamicin B, gentamicin B^, and sisomicin.

Isolate the respective products in the manner described in Example 22A(2) and obtain thereby the following: - 141 - V 40437 lo 15 20 l-M" (5-aminopontanoyl 1—U— (5-aminopontanoyl 1-J&- (5-aminopentanoyl 1-N-(5-aminopontanoyl 1-N-(5-aminopentanoyl l-fi-.(5-aminopentanoyl 1-H-(5-aminopentanoyl 1-N-(5-aminopentanoyl 1-N-(5-aminopentanoyl 1-N- (5-aminopentanoyl 1-N-(5-aminopentanoyl 1-N-(5-aminopentanoyl 1-N-(5-aminopentanoyl 1-N-(5-aminopentanoyl 1-N-(5-aminopentanoyl 1-N-(5-aminopentanoyl 1-N-(5-aminopentanoyl 1-N-(5-aminopentanoyl 1-N-(5-aminopentanoyl 1-N-(5-aminopentanoyl 1-N- (5-aminopentanoyl 1-N-(5-aminopentanoyl gcntamicin C^a# gentamicin Cj» gcntamicin C2a, gcntamicin Xj, gentamicin A, gentamicin C.^# vcrdamicin, tobramycin, antibiotic G-418, antibiotic 66-40B, antibiotic 66-40D, antibiotic JI-20A, antibiotic JI-20B, antibiotic G-52, mutamicin 1, mutamicin 2, mutamicin 4, mutamicin 5, mutamicin 6, gentamicin B, gentamicin B^, and sisomicin. - 142 - 4 0 4 37 A. 1-N- (S-Hvdroxvpentanovllqentamicin Dissolve 2.5 g of gentamicin in 250 ml of water and add 100 utl of methanol. Add 0.65 ml of triethyl-amine and stir for fifteen minutes. Add a solution of 1.0 g o£ N~ (5-acetoxypentanoyloxy)succinimide with stirring to t ho solution of the antibiotic, and stii at ambient temperature for K> lira. Evapornlo tho solution in vacuo to leave a solid residue. Dissolve the residue in 5 ml of 5% ethanolic hydrazine hydrate and heat under reflux for fifteen minutes. Concentrate the solution in vacuo to leave an oily residue and chromatograph it on 200 g silica gel in the lower phase of a solvent system consisting of chloroform, methanol and 7% ammonium hydroxide (2:1:1) to give 1-N-(5-hydroxypentanoyl)gentamicin C^.

D. In a similar manner, treat an equivalent quantity of acid addition salt of those antibiotics set forth in Example 22D to the process of Example 23A, isolate tho respective products in the manner described in Example 23A and obtain thereby the following: 1-N-(5-hydroxypentanoyl)gentamicin C^a< 1-N-(5-hydroxy-pentanoyl)gentamicin C£« 1-N-(fi-hydroxypentanoyl)gentamicin ^2ai 1-N- (5-liydroxypontunoyl)gentamicin X^, 1-N-('j-hydroxypeni anoyl) yentam.i <~rin A, 1-N- (5-hydi.oxypcntanoyl .)- - 143 - 4043? gentamicin C2b, (5-hydroxypentnnoyl)verdamicin, 1-N- (5-hydroxypuntanoyl.) I ol»ramyci it, 1-.N- (5-hydroxypentanoyl) -ant.ihi.oL.ic C--4JH, 1-N- (%• Itydroxypentnnoyl)nnLibiot Iu 06-40D, 1-N- (li-hydioxypcsntanoyl) antibiotic 66-40D, 1-N- (5-hydroxy-pcntanoyl)antibiotic JI-20A, 1-N-(5-hydroxypentanoyl)antibiotic JI-20B, 1-N-(5-hydroxypentanoyl)antibiotic Gt52, 1-N-(5-hydroxypentanoyl)mutamicin 1, 1-N-(5-hydroxypentanoyl)-mutamicin 2, 1-N-(5-hydroxypentanoyl)mutamicin 4, 1-N-(5-hydroxypentanoyl)mutamicin 5, 1-N-(5-hydroxypentanoyl)-mutamicin 6, 1-N-(5-hydroxypentanoyl)gentamicin B, 1-N-(5-hydroxypentanoyl)gentamicin B^, and 1-N-(5-hydroxypentanoyl)-sisomicin.

EXAMPLE 24 A. 1-N-Formvlqentamicin Dissolve 2.5 g of gentamicin sulphate in 250 ml of water and add 100 ml of methanol. Add 0.65 ml of triethyl-amino and stir for ten minutes. Add a solution of 2.0 g of N-formyloxysuccinimide in 20 ml of dry dimethylformamide dropwise with stirring to the solution of the antibiotic.

Stir the mixture at ambient temperature for 16 hours. Concentrate the reaction mixture to a residue in vacuo and triturate the residue with methanol to yield the product of this example as a white amorphous solid which may op tionally be further purified by the chromatographic procedure set forth in the preceding Example. - 144 - B. In a similar manner, treat an equivalent quantity of acid addition salt of those antibiotics set forth 1n Example 22B to the process of Example 24A; Isolate the respective products in the manner described in Example 24A and obtain thereby the following: 1 -H-f orroy 1 gentami ci n Cla* -fortnylgentamicin Cg, 1-N-formylgentamicin C2a* 1-N-formylgentami ci n X^, 1-N-formylgentamicin A, 1-N-formyl-gentamlcin C2b^ -N-formylverdamicln, 1-N-formylobraraycin, 1-N-formyl-antibiotic G-418, 1-N-formylantibiotic 66-40B, 1-N-formylantibiotic 66-40D, 1-N-fonqylantibiotic JI-20A, 1-N-formylantibiotic JI-20B, 1-N-fonnylantibiotic G-52, 1-N-formylmutamicin 1, 1-N-fomylmutamicin 2, 1-N-formylmutamicin 4, 1-N-formylmutamicin 5, 1-N-formylmutamicin 6, 1-N-formylgentamicin B, 1-N-formylgentamicin Bp and 1-N-formylsisomicin.

EXAMPLE 25 1-N-Acetylgentamicin B Dissolve 0.582 gms. of 6'-N-_t-butoxycarbonylgentamir.in B in 20 ml. of 3:1 water:methano1. Cool to 5°C in an ice bath, then while stirring, add dropwise 0.143 gms. of 0-acetyl -N-hydroxysuccinimide. Dissolve in 1.5 ml. of dimethylformamide. Stir for two hours, then add an additional 0.56 gms. of O-acetyl-N-hydroxysuccinimide in 0.6 ml. of dimethylformamide. Stir for 18 hours, then evaporate in vacuo to a residue. Chromatograph the residue on 70 gms. of silica gel eluting with the lower phase of a 1:1:1 chloroform:methanol:ammonium hydroxide (28X) solvent system. Take 5 ml. fractions and combine the like fractions as determined by thin layer chromatography. Evaporate the combined fractions (45-49) to a residue comprising 1-N-acetyl-6'-N-t-butoxy-carbonylgentamicin B. Add 1 ml. of trifluoroacetic acid to the foregoing dried 1-N-acetyl-6'-N-jt-butoxycarbonylgentamicin B, and, after three minutes, add 40 ml. of cold ether. Dissolve the precepitate in water and treat with 10 ml. of Amberlite IRA-401S (0H~) ion exchange resin. ' 40437 Dry the compound in vacuo over phosphorous pentoxlde. Wash the resin with water, lyophilize the combined filtrate and water-wash to obtain 1-N-acetylgentamlcln B; « _7^6 ♦ 132 (c ■ 0.4 in MeOH); Mass Spectrum: m/e 233, 205, 187, 215, 160, 162} pmr(ppra) 5 (020); 6 1.24 (3H, s), 2.02 (3H, s), 2.56 (3H, s), 5.11 (1H, H-l", Jj", 2" • 4.5Hz)» 5.40 (1H, H-V, J,*, 2' - 3.0Hz).

All percentages referred to herein, except those relating to "aqueous" organic solvents are expressed on a weight basis. The percentages relating to "aqueous" organic solvents and the ratios of the 10 components of the chromatography solvents are expressed on a volume basis. - 146 - 40437 Tho present Invention includoc wll.hin its Kcope ph.'irmaceu Ileal compositions comprint nr. at least one of Uiu novel. I-N-Gub-utiluted derivatives of Jj,6-di-(aiiiinotf.lyeor.yl)l,3-diajnino-cyolitols defined below together with a pharmaceutically acceptable carrier or coating. Also included within the invention is a method of eliciting an antibacterial response in a warm-blooded animal having a susceptible bacterial infection which comprises administering to said animals a r»on-toxic, antibacterially effective amount of a 1-N-substituted derivative of a 4,6-di-(aminoglycosyl)-l, j5-diaminocyclitol selected from gentamicin A, gentamicin B, gentamicin B^, gentamicin C^, gentamicin C^a» gentamicin C^, gentamicin Cga, gentamicin C2b> gentamicin Xg, sisomicin, verdamicin, tobramycin. Antibiotic G-'Jl8, Antibiotic 66-40B, Antibiotic 66-'»0D, Antibiotic JI-20A, Antibiotic JI-20B, Antibiotic G-52, mutamicin 1, mutamicin 2, mutamicin 4, mutamicin 5 and mutamicin 6 wherein the substituent is -CHgX with X being hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkyl-alkyl, hydroxyalkyl, aminoalliyl, N-alkylaminoalkyl, amino-hydroxyalkyl, N-alkylaminohydroxyalkyl, phenyl, benzyl or tolyl, said aliphatic radicals having up to seven carbon atoms and, if substituted by amino and hydroxy, bearing the substit-uentc on different carbon atoms; I or wherein the substituent is -C-Z with Z being X defined above and with the proviso that Z together with the carbonyl group - 147 - 40437 to which it is attached i.s other than S-3-amlno-2-hydroxy-proplonyl or S-'l-aniino-P-hytlroxybutyryl ani| in the case of tobramycin also other than S-5-amino-2-hydroxyvalerylj or of a pharmaceutically acceptable acid addition salt of any 5 of the 1-N-substituted derivatives.

Thi; compound;; of tills invention are broad spectrum antibacterial agents which, advantageously, exhibit activity against many organisms, particularly gram-negative organisms, which are resistant to their 1-N-unsubstituted precursors. 10 Thus, the compounds of this invention can be used alone or in combination with other antibiotic agents to prevent the growth or reduce the number of bacteria in various environments. They may be used, for example, to disinfect laboratory glassware, dental and medical equipment contamin-15 aled with Staphylococcus aureus or other bacteria which may bo Inhibited by the compounds of this invention. The activity of the compounds of this invention against gram-negative bacteria renders them useful for combating infections caused by gram-negative organisms, e.g. species of 20 Proteus and rseudomonas. The 1-N-substituted derivatives of the 4,6-di-(aminoglycoayl)-l,3-diaminocyclitols, e.g. 1-N-ethylsisoinlcin and 1-N-cthylverdamicin have veterinary application;;, particularly in the treatment of mastitis in cuttle and Srtlmonol 1 a induc-M diarrhea in domestic animals 25 such as the do*: and the cat. - 148 - 40437 10 Tho following tal»l c vetr. forth tlm Minimal .Tnhtl»1 tory Con eon-trntlcii (MTC) oi* cth'^kmuhIo of thlr. Invent.!«»ii. 'Uto |.or>1-*< W Minimal Inhibitory Concentration 1) 1-N-acetylr.cntamicin 2)1-M-proplonylslsomlcln 3) l-N-acetylsieomlcin MK/kn (Mueller-Hlnton Broth pH 7.2) E. Coll 1 2 JL 0.3 7.5 0.3 3.0 7.5 3.0 7.5 17.5 3.0 3.0 7.5 3.0 3.0 7.5 3.0 3.0 3.0 3.0 3.0 3.0 0.75 17.5 >25 3.0 >25 >25 7.5 7.5 17.5 3.0 3.0 3-0 .075 17.5 17.5 3.0 >25 >25 3.0 >25 >25 3.0 W 677/R55 * JR 66 * St. M. 589 15 Baker 2 F-14-HK LA 290/R55 * ATCC 10556 Pseudomonas Aeruginosa 20 St. M. 762 1395 MRRL 3223 Stone 20 Stone 39 25 Stone 130 * Stone 138 * Klebsiella pneumoniae Adler 17 3.0 3.0 0.75 Adler 18 3.0 3.0 0.75 30 Georgetown 369'! * 17.5 17.5 0.3 Georgetown 3020 * 3.0 7.5 0.30 Staphylococcus aurcu3 2°9 P 3.0 17.5 0.75 W00^ 3.0 7.5 0.3 35 Streptococcus pyogenes C . >25 >25 3-0 A. Alvarez 3.0 7.5 7.5 Bacillus subtills 6623 .075 0.3 0.03 40 * gentamicin resistant - 149 - 40437 In general, the donate administered of the compounds of this Invention will be dependent upon the age and weight of the animal species beirsr, treated, the mode of administration, and the type and severity of bacterial infection 5 being prevented or reduced. In general, the dosage of the derivatives of the 6-di-(aminoglycosyl)-1,3-diaminocyclitols employed to combat a given bacterial Infection will be similar to the dosage requirements of the corresponding 1-M-unsubstituted-'l,6-di-(aminoglycosyl)-l,3-10 diamlnocyclitols.

The compounds of this invention may be administered orally. They may also be applied topically in the form of ointments, both hydrophilic and hydrophobic, in the form of lotions which may be aqueous, non-aqueous or of the emulsion type 15 or in the form of creams. Pharmaceutical carriers useful in the preparation of such formulations will include, for example, such substances as water, oils, greases, polyesters, polyols and the like.

For oral administration the compounds of this invention 20 may be compounded in tho form of tablets, capsules, elixirs or the like or may even be admixed with animal feed. It is in these dosage forms that the antibacterlals are most effective for treating bat:U;rlal infection:; of tho gastro-intcr:t j nal trnrt. which 1 n(\i on:: ili aiThi.-a. - 150 - 4043? In general, tho topical preparations will contain from about 0.1 to about 3.0 cms. of the compounds of the Invention per 100 gros. of ointment, creams or lotion. The topical preparations are usually applied gently to lesions from about 2 to about 5 times a day.

The antibacterlale of this Invention may be utilized in liquid form such as solutions, suspensions and the like for otic and optic use and may also be administered parenterally via Intramuscular injection. Ohe injectable solution or suspension.will usually be administered at from about 1 mg. to about 15 mgs. of antibacterial per kllrgram of body weight per day divided into about 2 to about 4 doses. The precise dose depends on the stage and severity of the infection, the susceptibility of the infecting organism to tho antibacterial and tho individual characteristics of the animal species being treated.

The following formulations are to exemplify some of tho dosage forms in which the antibacterial agents of this invention may be employed: - 151 - 40437 Formulation 1 Tablet 10 ir.i% Tab. 25 m«.

Tab. 100 mp,.

Tab 1-N-ethylslsomlcln 10.50* mg. 26.25* mg. 105.00* mg Lactose, impalpablo powder 197.50 mg. 171.25 mg. 126.00 mg Corn Starch 25.00 mg. 25.00 mg. 35.00 mg, Polyvinylpyrrolidone 7.50 mg. 7-50 mg. 7.50 mg, Magnesium Stearate 2.50 mg. 2.50 mg. 3.50 mg, * 5# excess Procedure Prepare a slurry consisting of the 1-N-ethylsisomlcln, lactose and polyvinylpyrrolidone. Spray dry the slurry. Add the corn starch and magnesium stearate. Mix and compress into tablets.

Formulation 2 Ointment 1-N-ethylverdamicin 1.0 gm.

Methyl paraben U.S.P. 0.5 gm.

Propyl paraben U.S.P. 0.1 gm.

Petrolatum to 1000 gm.

Procedure (1) Melt the petrolatum (2) Mix the 1-N-ethylverdamicin, methyl paraben and propyl paraben with about 10# of the molten petrolatum. (j5) Pass the mixture through a colloid mill. (4) Add the remainder of the; petrolatum with acitation arid - 152 - 40437 cool tho mixture until it becomeu semi-solid. At this singe « the product may be put Into suitable containers.

Ointments of other l-N-substltutcd derivatives of the 4,6-di-(aminoglycosyl)-l,3-diaminocyclltols and of the acid addition salts thereof are prepared by substituting an equivalent quantity of another compound of this invention for 1-N-ethylverdamicin in the foregoing example and by following substantially the procedure of the example. 10 15 Injectable Solution 1-N-ethylslsomlcln sulfate Formulation 3 Per 2.0 ml. vial Methyl paraben, U.S.P.

Propyl paraben, U.S.P.

Sodium bisulfite, U.S.P.

Dlcodium Ethylenediamine tetraacetate dihydratc, R.C.

Water, U.S.P. q.s. 84.0 ings. * 3.6 mp;s. 0.4 mgs. 6.4 Rigs. 0.2 mgs. 2.0 ml.

Per 50 Liters 2100.0 #gms. 90.0 gms. 10.0 gms. 160.0 gms. 5.0 gms. 50.0 liters * Includes a 5jb manufacturing overcharge. 20 Procedure; For a r)0.0 liter batch Charge approximately 35 liters of water for injection to a suitable stainless steel Jacketed vej;sel and heat to about 70°C. Charge the methylparaben and propylparaben to the heutcd water for injection and dissolve with agitation. When - 153 - w f 40437 the parabcns are completely dissolved, cool the contents of the tank to 25 - 30°C by circulating cold, water through the tank jacket. Sparge the solution with nitrogen gas for at least 10 minutes and keep covered with nitrogen gas 5 for at least 10 minutes and keep covered with nitrogen during subsequent processing. Charge and dissolve the dlsodlum EDTA and sodium bisulfite. Charge and dissolve the 1-N-cthylslsomlcln sulfate. Bring the batch volume up to 50.0 liters with water for injection and agitate 10 until homogenous.

Under sterile conditions, filter the solution through a suitable bacteria retentive filter collecting the filtrate in a filling tank.

Pill the filtrate aseptlcally into sterile pyrogen-free 15 multiple dose vials, stopper and seal.

In like manner, injectable solutions of 1-N-propylsisomicin, 1-N-ethylverdamicin and 1-N-propylverdamicin and especially acid addition salts of such antibacterial agents, may be prepared by substituting an equivalent.quantity of such 20 compounds for 1-N-cthylsisomicin sulfate and by following the procedure set forth above. - 154 -

Claims (109)

40437 ctjvims

1. Process Tor the preparation of 1-N-aubstituted derivatives of tlie 4,G-di-(ii»jinog.tycosyl)-l,3-di»in»ino-cyclitols gentamicin A, gcntamicin B, gentamicin B^# 5 gcntamicin C^, gcntamicin Cla, gentamicin C2» gentamicin C2a» gontnmicin gcntamicin X2, sisomicin, verdamicin, tobramycin. Antibiotic G-410, Antibiotic 66-40B, Antibiotic 66-40D, Antibiotic JI-20A, Antibiotic JI-20B, Antibiotic G-52, mutoraicin 1, mutamicin 2, mutamicin 4, 10 mutamicin 5 and mutamicin 6, wherein the substituent is -ch2x with X being hydrogen, alkyl, alkenyl, cycloalkyl, cyclo-aIky 1 alkyl, hydroxyaIky1, arainoalkyl, N-allcylaminoalky 1, aiainohydroxyalkyl # N-alkyl-15 aminohydroxyalkyl, phenyl, benzyl, or tolyl, said aliphatic radicals having up to seven carbon atoms and, if substituted by amino and hydroxy, bearing the substituents on different carbon atoms, 20 and of the pharmaccutically acceptable acid addition salts thereof, which process comprises treating one of the above named 4,6-di-(aminoglycosyl) -1,3-diaminocyclitols which may have amino-protecting groups at any poni tion other than position l,with an alcohyde of the formula - 155 - 40437 X'-CHO with X* being a group as defined for X above wherein any amino or hydroxy group present may bo protected, in the presence of a hydride donor-reducing agent# and, if required, removing all protecting groups present in the molecule tho last process step being followed by isolating the derivative no such or as a pharmaceutically acccptable acid addition salt.

2. Process as claimed in claim 1, wherein the reaction is carried out at a pH in the range of from 1 to 11.

3. Process as claimed in claim 1 or 2, wherein the reaction is carried out at a pH in the range of from 2 to 5.

4. Process as claimed in any of claims 1 to 3, wherein the reaction is carried out at a pH in the range of from 2.5 to 3.5. - 156 - - 40437

5. Process as clnimod in any of claims 1 to 4, Wherein tho reaction is carricd out in on inqrt solvent.

6. Proccss as claimed in any of claims 1 to 5, wherein the reaction is carried out in an inert, protic solvent.

7. Process as claimed in any of claims 1 to 6, wherein the 4,6-di-(aminoglycosyl)-1,3-diaminocyclitol is treated with at least one equivalent of aldehyde in the presence of one equivalent of hydride-donor reducing agent.

8. Process as claimed in any of claims 1 to 7, wherein the hydride-donor reducing agent is a dialkyl-aminoborane, a tetraalkylammonium cyanoborohydride, an alkali metal cyanoborohydride or an alkali metal borohydride.

9. Process as claimed in any of claims 1 to 8, wherein a hemi-acetal or acetal of the aldehyde is used in acidic medium and the required aldehyde is formed in. situ.

10. Trocess as claimed in any of claims 1 to 9, wherein as 4,6-di-(aminoglycosyl)-1,3-diaminocyclitol sisomicin or verdamicin is used, the ciaino groupr. of which are in the free form or protected j.n position 6' or in positions 2 ■ and 6 • or in positions 2 ■, 3 # and C'# - 157 - 40437

11. Process as claimed in any of claims 1 to 10, Wherein in the aldehyde of the formula X'-CIIO, X* represents methyl, ethyl, or propyl. i

12. Process for the preparation of lHN-substituted derivatives of tho 4,6-di-(nminoglycosyl)-1,3-diamino-cyclitols gentamicin A, gcntamicin B, gentamicin B^, gentamicin C^, gentamicin gentamicin C^, gentamicin i C2n, gentamicin C^, gentamicin X^, sisomicin, verdamicin, tobramycin. Antibiotic G-418, Antibiotic 66-40B, Antibiotic 66-40D, Antibiotic JI-20A, Antibiotic JI-20D, Antibiotic G-52, mutamicin 1, mutamicin 2, mutamicin 4, mutamicin 5 find mutamicin 6, wherein the substituent is "C^X w*-th X being hydrogen, alkyl, alkenyl, cycloalkyl, cyclo- alkylalkyl, hydroxyalky1, aminoalkyl, N-alkyl-aminoalkyl, aminohydroxyalkyl, N-alkylamino-hydroxyalkyl, phenyl, benzyl or tolyl, said aliphatic radicals having up to seven carbon atoms, and, if substituted by amino and hydroxy, bearing the substituents on different carbon atoms, and of the phnrmaceutically acceptable acid addition salts thereof, which process comprises reducing the tie- double bond in a 1-N=CHX'-substituted derivative of one of the above named 4,6-di-(«uninoglycosyl) -1,3-diaminocyclitols, wherein - 158 - 40437 all NH2 groups aro protected and NHCH^ groups nay be protected} with X* being a group as defined for X above, wherein any amino or hydroxy group present may 5 be protected, removing all protecting groups present in the molecule and isolating the desired derivative as such or as a pharmaceutically acceptable acid addition salt.

13. Process as claimed in claim 12, wherein the 10 N,C-double bond is reduced in a 1-N-substituted deriva-Antlbietio 66-^OD or at tive of/one of the 4-aminoglycosyl-6-garosamiRyl-1,3-diaminocyclitols gentamicin B, gentamicin B^, gentamicin C1# gentamicin Cla# gentamicin C2, gentamicin C2a, gentamicin C2b' gentamicin X^t sisomicin, verdamicin, 15 Antibiotic JI-20A, Antibiotic JI-20B, Antibiotic G-52, Antibiotic G-418, mutamicin 1, mutamicin 2, mutamicin 4, mutamicin 5, and mutamicin 6 having a 3H-N-4"-o-protecting group of the formula ^rCHX* 20 where X' iB as defined in claim 12 and is identical with X' in the 1-N=CHX* moiety. - 159 - 4043?

14. • Process as claimed in claim 12 or 13, wherein tho groups of the 4,6-di-(nminoglycosyl) -1,3-diamino- Z \ cyclitol btarting materials are protected with triphenylmethyl, 2,4-dinitrophenyl, acetyl, trifluoroacetyl, propionyl, benzoyl, methoxycarbonyl, ethoxycarbonyl, 2,2,2 -trichloroethoxycarbonyl, t-butoxy-carbonyl, 2-iodoethoxycarbonyl, carbobcnzyloxy or 4-methoxy benzyloxycarbonyl.

15. Process as claimed in any of claims 12 to 14, wherein the reducing agent is a dialkylaminoborane, a tetra alkyl ammonium cyanoborohydride, an alkali metal cyanoborohydride or an alkali metal borohydride.

16. Process as claimed in any of claims 12 to 15, wherein reduction is effected with sodium borohydrido in the presence of sodium methoxide.

17. Process for tho preparation of l-N-substituted derivatives of the 4,6-di-(aminoglycosyl)-1,3-diamino-cyclitols gentamicin A, gontnmicin B, gentamicin B^, - 160 - 40437 gcntamicin C^, gcntamicin c^a« gentamicin C2» gcntamicin C2a» gentamicin gontnmicin X2, siscanicin, verdamicin, tobramycin, Antibiotic 0-410, Antibiotic 66-40B, Antibiotic 66-40D, Antibiotic JI-20A, Antibiotic J1-20B, /mtibiotic G-52, mutamicin 1, mutamioin 2, mutamicin 4, mutamicin 5 and mutamicin 6, wherein the substituent is with X being hydrogen, alkyl, alkenyl, cycloalkyl, cyclo-alkylalkyl, hydroxyalkyl, amino- alkyl, N-alkylaminoalkyl, aminohydroxyalkyl, N-alkylaminohydroxyalkyl, phenyl, benzyl or tolyl, aaid aliphatic radicals having up to seven carbon atoms, and, if substituted by amino and hydroxy, bearing the substituents ✓ on different carbon atoms, and of the pharmaceutically acceptable acid addition salts thereof, which process comprises treating a 1 -^.-substituted derivative of one of the above named 4,6-di-(aminoglycosyl)-1,3-diaminocyclitols, wherein one or more amino-groups may be protected and the 1-^j-substituent is -C-X" with X'being hydrogen, alkyl, alkenyl, cycloalkyl, cyclo-alkylalkyl, hydroxyalkyl, aminoalkyl, N-alkylaminoalkyl, aminohydroxyalkyl, N-alkyl-aminohydroxyalkyl, phenyl, benzyl, tolyl or hydrocnrbyloxy, said aliphatic radicals having up to seven carbon atoms, and if sub- - 161 - 40437 ntitutod by sunlno and .hydroxy# bearing \ the subaitutcnts on different carbon atoms, and wherein any amino or hydroxy group present nay be protected. with an amide-reducing hydride reagent and, if required, removing all protecting groups present in the raoleculcj the last process step being followed by isolating the desired derivative as. such or as a pharmaceutically acceptable acid addition salt. ♦

18. Proccss as claimed in claim 17, wherein the amidc-reducing hydride reagent is an aluminum hydride or a borohydride.

19. Process as claimed in claim 17 or 18, wherein the amide-reducing hydride reagent is lithium aluminum hydride or diborane.

20. Proccss as claimed in any of claims 17 to 19, wherein the 4,6-di-(aminoglycosyl)-1,3-diaminocyclitol is sisomicin or verdamicin, X" in the 1-N-substituent is methyl, ethyl or propyl and the reducing agent is lithium aluminum hydride.

21. Procer.s for the preparation of 1-N-substituted derivutives of the 4,6-di-(aminoglycosyl) -1,3-diam.inocyclitols - 162 - 40437 sisomicin and mutamicin 2, wherein the substituent io -CII2X'" with X"* being hydrogen, methyl, ethyl or propyl, and of tho pharmaceutically acceptable acid addition ealts 5 thereof, vhich process comprises cultivating a mutant strain of the species Micromonospora invoensis. namely Micromonospora invoensis strain 1550F-1G or a strain of the same species corresponding thereto in its capability • of producing one of the above defined antibiotics, in 10 a nutrient medium comprising a compound of the formula OH wherein Vg is hydroxy or hydrogen, and X,M is as defined above, until substantial antibiotic activity is imparted to the medium and isolating the so-obtained compound as 15 such or in the form of a pharmaceutically acceptable acid addition salt.

22. Process as claimed in claim 21, wherein fer mentation i3 conducted at a pll in the range of from 6.0 to 8.0 and at from 24° to 49° C. - 163 - 40437

23. Proccso for tho preparation of 1-^-substituted derivatives of the 4,6-<3i-(aminoglycosyl)-l#3-dinrainocy-clitols gentamicin A, gentamicin B, gentamicin B^, gentamicin C^, gentamicin gentamicin Cj, gentamicin C2a, gentamicin C2b» gentamicin sisomicin, verdamicin, tobramycin. Antibiotic G-418, Antibiotic 66-40B, Antibiotic 66-40D, Antibiotic JI-20A, Antibiotic JI-20B, Antibiotic G-52, mutamicin 1, mutamicin 2, mutamicin 4, mutamicin 5, and mutamicin 6, wherein the substituent is straight chain alkyl having up to five carbon atoms and of tho pharmaceutically accepteible acid addition salts thereof which process comprises reacting one of the above named 4,6-di-(aminoglycosyl)-1,3-diaminocyclitols, which possesses amino-protecting groups at any position other than position 1, and wherein the 1-amino group may be activated, with an alkylating agent containing the straight chain alkyl group having up to five carbon atosns and a leaving group, removing the protecting groups and, if required, the activating group or groups present in the molecule, and isolating the derivative as such or as a pharmaceutically acceptable acid addition salt.

24. Process as claiinod in claim 23, wherein the free 1-amino group of tho 4, G-di—(aminoglycosyl)-1,3-diaminocyclito.l is alkylated. - 164 - 40437

25. Procesn as clnimocl in claira 23, whoroin the 1-amino group of tlio 4,6-tli-(t»minoglycoityl) -1,3-rtinmino-cyclitol is alkylated in the Tom of tho trifluoromothyl-oulphonyl derivative.

26. Proccss as claimcd in claim 23, wherein the 1-amino group of the 4,6-di-(aminoglycosyl)-1,3-diaminocyclitol is alkylated in the form of the Di-(2-cyanoethyl)-derivative.

27. Process as claimed in any of claims 23 to 26, wherein the alkylating agent is an alkylfluorosulphonatc, a dialkylsulfate or an alkyliodide.

28. Process as claimed in any of claims 23 to 27, wherein the 4,6-di-(aminoglycosyl)-1,3-diaminocyclitol, which possesses amino protecting groups at any position other than position 1, is 3"-N-4"-0-carbony1-2',3,6'-tri-N-t-but oxycarbony1-sisomicin.

29. Process for the preparation of 1-H-substituted derivatives of the 4,6-di-(ominoglycosyl)-1,3-diaminocyclitols gcntamicin A, gentamicin B, gentamicin B^, gentamicin C^, gentamicin C^a, gcntamicin C^, gcntamicin gentamicin °2b' gentamicin sisomicin, verdamicin, - 165 - 40437 tobramycin,. Antibiotic G-418, Antibiotic 66-40B, Antibiotic 66-40)), Antibiotic JI-20A, Antibiotic J1-20B, Antibiotic G-52, mutamicin 1# mutamicin 2, mutamicin 4, mutamicin 5 and mutamicin 6, wherein the substituent is methyl, and of the pharmaceutically acceptable acid addition salts thereof vhich process comprises reacting one of the above named 4,6-di-(aminoglycosyl) -1,3-diaminocyclitols which possesses amino-protecting groups at any position other than position 1, with formaldehyde and a cyclic imide, treating the so-obtained compound with a hydride-donor reducing agent, removing all protecting groups present in the molecule and isolating the derivative as such or as a pharmaceutically accepteible acid addition salt.

30. Process as claimed in claim 29, wherein the cyclic imide is succinimide.

31. Process as claimed in claim 29 or 30, wherein the hydride-donor reducing agent is sodium borohydride.

32. Process for the preparation of 1-^-substituted derivatives of the 4,6-di-(aminoglycosyl)-1,3-diaminocyclitols gentamicin A, gentamicin B, gentamicin B^, gentamicin C^, gentamicin gentamicin C^, gentamicin C2a* gentamicin C2b' gentamicin x2' sisomicin» verdamicin, tobramycin. - 166 - 40437 Antibiotic G-418, Antibiotic 6S-40D, Antibiotic 66-40D, Antibiotic JI-20A, Antibiotic JI-20B, Antibiotic G-52, mutamicin 1# mutamicin 2, mutamicin 4, mutemicin 5, and mutamicin 6, wherein the substituent is methyl, and of tho pharroaccutio.vlly acccptablo acid addition salts thereof which process comprises reacting one of tho above named 4,6-di-(aminoglycosyl)-1,3-diaminocyclitols which possesses amino protecting groups at any position other than position 1, with formaldehyde in the presence of formic acid, removing all protecting groups present in the molecule and isolating the derivative as such or as a pharmaceutically acceptable acid addition salt.

33. Process for the preparation of 1-N-substituted derivatives of the 4,6-di-(aminoglycosyl)-1,3-diaminocyclitols gentamicin A, gentamicin B, gentamicin B^, gcntamicin C^, gentamicin Cla# gentamicin C2, gentamicin C2a« gentamicin C2^» gentamicin' X^, sisomicin, verdamicin, tobramycin. Antibiotic G-418, Antibiotic 66-40B, Antibiotic 66-40D, Antibiotic JI-20A, Antibiotic J1-20B, Antibiotic G-52, mutamicin 1, mutamicin 2, mutamicin 4, mutamicin 5, and mutamicin 6, wherein the substituent is 2-hydroxyethyl, and of the pharmaceutically acceptable acid eiddition salts thereof which process comprises reacting one of the above named 4,6-di-(iwiinoglyconyl) -1,3-diaminocyclitols which - 167 - 40437 » « possessos amino protecting groups at any position other than position 1,-with ethylene oxide, removing all protecting groups present in the moloculo and Isolating tho derivatives as such or as a pharmaceutically acceptable acid addition salt.

34. Process for the preparation of 1-^-substituted derivatives of the 4,6,-di- (aminoglycosyl) -1,3 -diaminocy-t clitols gentamicin A, gentamicin B, gentamicin B^, gentamicin C^, gentamicin Cla* gentamicin C2,"gentamicin C2a» gentamicin C2h' 9enta®icin ' sisomicin, verdamicin, tobramycin, Antibiotic G-418, Antibiotic 66-40B, Antibiotic 66-40D, Antibiotic JI-P.OA, Antibiotic JI-20B, Antibiotic G-52, mutamicin 1, mutamicin 2, mutamicin 4, mutamicin 5 and mutamicin 6, wherein the substituent is J!-Z with Z being hydrogen, alkyl, alkenyl, cycloalkyl, cyclo-alkylalkyl, hydroxyalkyl, aminoalkyl, N-alkylaminoalkyl, aminohydroxyalkyl, N-alkyl-aminohydroxyalkyi, phenyl, benzyl or tolyl, said aliphatic radicals having up to seven carbon atoms and, if substituted by amino and hydroxy, bearing the substituents on different carbon atoms, with the proviso that Z together with the carbonyl group to which it is attached is other than G-3-omino-2- - 168 - 4043? hydroxypropionyl or S.-4-amino-2-hydroxy-butyryl, and, in ease of tobramycin, also other than S_-5-amino-2-hydroxyvaleryl, and of the pharmaceutically acceptable acid addition salts thereof} which process comprises treating one of the above named 4,6-di-(aminoglycosyl)-1,3-diaminocyclitols which may have amino protecting groups at any position other than position 1, with an acid of the formula O II OH-C-Z' with Z' being a group as defined for Z above, wherein any amino or hydroxy group present may be protected, in the preaeaoe of a oaxfcodllalda, or with a reactive derivative of of above said acid, and, if required, removing all protecting groups present in the molecule, the last proccss step being followed by isolating the \ - 169 - 40437 derivative as such or as a pharmaceutically acceptable acid addition salt.

35. Process as claimed in claim 34# wherein the 4,6-di-(aminoglycosyl) -1,3-dinminocyclitol starting compound, is used as a free baso.

36. Process as claimed in claim 34 or 35# wherein one of the 4,6-di-(aminoglycosyl)-1,3-diaminocyclitols tdbr«njoin gcntamicin B, gentamicin Clg, Antibiotic JI-20A, Antibiotic 66-40B, Antibiotic 66-40D, sisomicin, mutamicin 1, mutamicin 2, mutamicin 4, mutamicin 5 and mutamicin 6 having a 6'-amino protecting group is used.

37. Process as claimed in claim 34 oi 35, wherein any of the 4,6-di-(aminoglycosyl)-1,3-diaminocyclitol starting compounds having free amino groups is used.

38. Process as claimed in claim 34, wherein 1 to n amino groups of the 4,6-di-(aminoglycosyl)-1,3-diaminocyclitol starting compound nve neutralized by formation of an acid addition salt and n is the number of amino groups in the molecule.

39. Proccss as claij.ioc' in claim 30, wherein (n-1) amino groups are neutralized by formation of an anid addition salt. - 170 - 40437

40. Prorono no clnimrtl in nny of cluiuis 34 to 3(1, whrrojn a rc?jiel;iv«» i1«tJv.-»1:1 vo of the ;icicl jo unml tin acylating agent.

41. Process as claimed in any of claims 34 to 40, 5 wherein the rcactive derivative of the acid is an ester, an azide, an imidazole derivative or the anhydride.

42. . 42. Process as claimed in any of claims 34 to 41,

43. Wherein the reactive derivative of the acid is the N-hydroxysuccinimidyl ester. 10 43. A process as claimed in any of claims 1 to 42, substantially as herein described.

44. A process as claimed in any of claims 1 to 42, substantially as herein described in any of the Examples. one of

45. A 1-^-substituted derivative of /the 4,6-di-(awino-glycosyl)-1,3-diaminocyclitols gentamicin A, gentamicin D, gcntamicin B^, gentamicin C^, gentamicin Cla» gentamicin C2# gentamicin C?a, gentamicin C2b, gentamicin X2, sisomicii;, vcrdamicin, tobramycin. Antibiotic G-418, Antibiotic 6G-40B, Antibiotic 66-40D, Antibiotic JI-20A, Antibiotic 20 JI-20B, Antibiotic G-52, mutamicin 1, mutamicin 2, muta- - 171 - 40437 micin 4, mutamicin 5 and mutamicin 6, wherein tho ouboti- ♦ tucnt is -CI*2X with X beintj hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, hydroxyalkyl, aminoalkyl, N-ajLkyliuninoalkyl, aminohydroxy-5 alkyl, N-alkylaminohydroxyalkyl, phenyl, benzyl or tolyl, said aliphatic radicals having up to seven carbon atoms and, if substituted by amino and hydroxy, bearing the substituents on different carbon atans. one of 10 46. A 1-N-substituted derivative of/tho 4,6-di-(amino glycosyl) -1,3-diaminocyclitol3 gentamicin A, gentamicin B, gentamicin B^, gentamicin C^, gentamicin Cla» gentamicin C^, gentamicin C2a' gentamicin C2t), gentamicin X2, sisomicin, verdamicin, tobramycin, Antibiotic G-410, Antibiotic 66-40B, 15 Antibiotic 66-40D, Antibiotic JX—20A, Antibiotic JI-20B,

46. Antibiotic G-52, mutamicin 1, mutamicin 2, mutamicin 4, mutamicin 5, and mutamicin 6, wherein the substituent is -CH2X with X being hydrogen, alkyl, alkenyl, hydroxyalkyl, aminoalkyl, N-alkylaminoalkyl, aminohydroxy-2o alkyl, N-alkylaminohydroxyalkyl, phenyl, benzyl or tolyl, said aliphatic radicals having up to seven carbon atoms and, if substituted by amino and hydroxy, bearing the substituents on different carbon atoms, 25 or a pharmaccutically acceptable acid addition salt thereof. - 172 - 40437 a

47. a 1-N-substituted tl"rlvaH\v of/ 4,G-di-(nmJno-glyconyl)-1,3-diarainocycli fc«>l , whoroin said 6-aminoglycosyl is garosaminyl, tho 4-am.?nixjlycosyl-6 -garonaminyl-1,3-di-aminocyclitol being gentamicin B, gentamicin B^# genta- 5 micin C^, gentamicin C^, gentamicin C2, gentamicin C2a# gentamicin C2b* gentamicin X2» sisomicin, verdamicin. Antibiotic J1-20A, Antibiotic J1-20B, Antibiotic G-52, Antibiotic G-418, mutamicin 1, mutamicin 2, mutamicin 4, : mutamicin 5 or mutamicin 6, wherein the substituent is 10 -CHjX and X is as defined in claim 45. a

48. a 1-N-substituted derivative of /l,6-di-(amino-glycosyl)-1,3-diaminocyclitol , wherein said 6-aminoglycosyl is garosaminyl and said 1,3-diaminocyclitol is 2-deoxystreptamine, the 4-aminoglycosyl-6-garosaminyl-2-Uooxy- <15 streptamines being gentamicin B, gentamicin B^, gentamicin C^, gentamicin Cla' gentamicin C2, gentamicin c2a» gentamicin C, gentamicin X2, sisomicin, verdamicin, Antibiotic JI-2QA, Antibiotic JI-20B, Antibiotic G-52, or Antibiotic G-418, wherein the substituent is -CH2X and 20 X is as defined in claim 45. a

49. A 1-N-substituted derivative of/4,6-di-{r.mino~ glycosyl)-1,3-diaminocyclitol , wherein said 6-aminoglycosyl is garosaminyl and said 1,3-diarr.inccyclitol is 2-deoxystreptamine, the 4-aminoglycor-.yl-G-garosaminyl-2- - 173 - 40497 deoxystreptajnino boing gcntamicin B, gcntamicin B^, gcntamicin C^, gen-ar.icin cla» gcntamicin C2, gontamicin X2, sisomicin# verdamicin# Antibiotic JI-20A# Antibiotic /7I—20B# Antibiotic G-52 or Antibiotic G-418# wherein the substituent in -CU^X and X is as defined in claim 45. a

50. A 1-&-substituted derivative of /4#6-di-(aminoglycosyl) -1,3-diaminocyclitol , wherein said 6-aminoglycosyl is garosaminyl and said 1,3-diaminocyclitol is 2-deoxystreptamine, the 4-aminoglycosyl-6-garosaminyl-2 -deoxystreptamine being gentamicin C^, gentamicin C^a# sisomicin, verdamicin or Antibiotic G-52, wherein the substituent is ~CH;>X and X is as defined in claim 45. one of

51. A 1-W-substituted derivative of/the 4,6-di-(amino-glycosyl)-1,3-diaminocyclitols sisomicin, mutamicin 1, mutamicin 2, mutamicin 4, mutamicin 5 and mutamicin 6, wherein the substituent is -CI^X with X being hydrogen or alkyl having up to seven carbon atoms, or a pharmaceutically acceptable acid addition salt thereof.

52. A compound as claimed in claim 51, wherein X is hydrogen or alkyl having up to two carbon atoms. - 174 -

53. A 1-1£-substituted derivative of sisomicin. Wherein the substituont is -CH2X and 31 *s as dQf*ned *n claim 45*

54. A 1-W-substituted derivative of verdamicin, Wherein the substituent is -CH2X and X is as defined in data 45.

55. A 1-N-substituted derivative of Antibiotic 66-40D, Wherein the substituent is -CH^X and X is as defined in claim 45.

56. A compound as claimed in any of claims 45 to 50 and 53 to 55, wherein X is hydrogen, alkyl, hydroxyalkyl, aminoalkyl, aminohydroxyalkyl, phenyl or benzyl, said aliphatic radicals having up to seven carbon atoms, and, if substituted by amino and hydroxy, bearing the substituents on different carbon atoms.

57. A compound as claimed in any of claims 45 to 50 and 53 to 56, wherein X is hydrogen or alkyl having up to seven carbon atoms.

58. A compound as claimed in any of claims 45 to 50 and 53 to 57, wherein X is hydrogen, methyl-, ethyl or propyl. - 175 - 40437

59. A compound as claimed in any of claims 45 to 50 and 53 to 58, whore in X is methyl or ethyl.

60. A compound, as claimed in any of claims 45 to 50 and 53 to 56, wherein X is aminoalkyl having up to 5 seven carbon atoms.

61. A compound as claimed in any of claims 45 to 50 and 53 to 56, wherein X io hydroxyalkyl having up to seven carbon atcms.

62. A compound as claimed in any of claims 45 10 to 50 and 53 to 56, wherein X is aminohydroxyalkyl having up to three carbon atoms and bearing the substituents on different carbon atoms.

63. A phariflaceutically acceptable acid addition salt of any of the compounds of claims 45, 47 to 50, 15 and 53 to 62.

64. 1-N-Ethylsisomicin or a pharmaceutically acceptable acid addition salt thereof.

65. 1-N-Ethylverdamicin or a pharmaceutically acceptable acid addition salt thereof. - 176 - *0497

66. l-N-Rthylgonticnicin C^a or a pharmaceutically aeccptable acid addition salt thereof.

67. 1-N-Ethylgentaraicin or a pharmaccutically acceptable acid addition salt thereof.

68. 1 -N-Ethy 1-Antibiotic g-52 or a pharmaceutically acceptable acid addition salt thereof.

69. 1-N-(n-Propyl)sisomicin : or a pharmaceutically acceptable acid addition salt thereof.

70. 1-N-(n-Propyl)verdamicin or a pharmaceutically. acceptable acid addition salt thereof.

71. 1-N- (/-ftminobutyl) sisomicin or a pharmaceutically acceptable acid addition salts thereof.

72. 1-N-(o*-/\m1nopropyl)s1son1c1n or a pharmaceutically acceptable acid addition salt tfiereof.

73. 1-N— (n.—Butyl) sisomicin or a pharmaceutically acceptable acid addition salt thereof.

74. 1-N-Methylsisamicin or a pharmaceutically acceptable acid addition salt thereof.

75. 1-K-fiothylverdnmicin or a pharmaceutically acceptable acid addition salt thereof.

76. 1-N-Ethylmutam1c1n 6 or a pharmaceutically acceptable acid addition salt thereof. - 177 - ^ 40437

77. 1-N-(j^-Butyl)verdamicin or a pharmaceutically acceptable acid addition cp.lt thereof.

78. 1-N- (!S-2-Hydroxy-4-aniinobutyl) -gentamicin or a phnrmaceutically acceptable acid addition salt thereof.

79. 1-N- (S.-2-Hydroxy-4-arainobutyl) -sisomicin or a pharmaceutically acccptablo acid addition salt thereof

80. l-4J-(S.-2-iiydroxy-4-nminobutyl) -verdamicin or a pharmaccutically acceptable acid addition salt thereof

81. A compound of any of claims 45 to 80 , whenever prepared by the proccss as claimed in any of claims 1 to 33, 43 and 44. one of

82. A 1-N-Substituted derivative of/ the 4,6-di-(amino glycosyl)-1,3-diaminocyclitols gentamicin A, gentamicin B, gentamicin B^, gentamicin C^, gentamicin gentamicin C2 gcntamicin Cj>n» Sontamici" C2b' 9entamic;*-n x2' sisomicin» verdamicin, tobramycin, Antibiotic G-418, Antibiotic 66-40B Antibiotic 6G-40D, Antibiotic JI-20A, Antibiotic JI-20B, Antibiotic G-52, mutamicin 1, mutamicin 2, mutamicin 4, mutamicin 5 and mutamicin 6, wherein the nMbstit 'ont is - 178 - 40437 -11-2 with Z being hydrogen# alkyl# alkenyl, cycloalkyl# cycloalkylalkyl, hydroxyalkyl, aminoalkyl# N-alkylaminoalkyl, aminohydroxyalkyl, N- alkylaminohydroxyalkyl# phenyl# benzyl or tolyl# said aliphatic radicals having up to seven carbon atoms# and# if substituted by amino and hydroxy# bearing the substituents on di££erent carbon atoms# with the proviso that Z together with the carbonyl group to which it is attached is other than S.-3-amino - 2-hydroxypropionyl or £-4-amino-2-hydr oxy- and gentamlcin C2 butyryl# and# in case of tobramycin/# also other than S.-S-amino-2-hydroxyvaleryl, or a pharmaceutically acceptable acid addition salt thereof. one of

83. A 1-N-substituted derivative of /the 4,6-di-(amino glycosyl)-1,3-diaminocyclitols gentamicin A, gentamicin B, gentamicin B^, gentamicin C^, gcntamicin Cla, gentamicin C2, gentamicin c2a» gentamicin C2b' gentamicin X^, sisomicin, verdamicin, tobramycin. Antibiotic G-418, Antibiotic 66-40B, Antibiotic 66-40D, Antibiotic JI-20A, Antibiotic JI-20B, Antibiotic G-52, mutamicin 1, mutamicin 2# mutamicin 4, mutamicin 5 and mutamicin 6, wherein the substituent is -I-Z with 55 being hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, hydroxyalkyl, aminoalkyl. - 179 - 40437 N-n Iky 1 aminoalkyl, aminohydroxyalkyl with • at least 5 carbon atoms, N-alkylnminohydroxy-alkyl, phenyl, benzyl or tolyl, said aliphatic radicals having up to sevon carbon atoms and, if substituted by amino and hydroxy, bearing the substituents on different carbon atoms, or a pharmaceutically acceptable acid addition salt thereof.

84. A compound of claim 82, wherein Z is hydrogen, alkyl, alkenyl, cycloalkyl, cyclo-alkylalkyl, hydroxyalkyl, aminoalkyl, N-alkyl-aminoalkyl, phenyl, benzyl or tolyl, said aliphatic radicals having up to seven carbon atoms. one of

85. A 1-N-substituted derivative of/the 4,6-di(aminoglycosyl) -1,3-diaminocyclitols gentamicin A, gcntamicin B, gentamicin B^, gentamicin C^, gentamicin c^a» gentamicin C2» gentamicin C2a' gentnmicin X2, sisomicin, verdamicin, tobramycin, Antibiotic G-418, Antibiotic 66-40B, Antibiotic 66-40I>, Antibiotic JI-20A, Antibiotic CTI-20B, Antibiotic G-52, mutamicin 1, mutamicin 2, mut?r.iicin 4, mutamicin 5, f? and mutamicin 6, wherein the substituent is -C~7. with Z being as defined in claim 82 , or a phnrmaceutically - 180 - 40437 acccptablo acid addition salt thereof. one of

86. Al-N-subotituted derivative of/the 4,6-di-(aminoglycosyl) -1,3-diajninocyclitols gentamicin A, gentamicin B, gentamicin B^, gcntamicin C^, gentamicin cja» gentamicin C2* gentamicin 9^ntamicin X2» sisomicin, verdamicin, tobramycin. Antibiotic G-418, Antibiotic 66-40B, Antibiotic 66-40D# Antibiotic JX-20A, Antibiotic JI-20B, Antibiotic 6-52, mutamicin 1, mutamicin 2, mutamicin 4, mutamicin 5 and mutamicin 6, wherein the substituent is -1-Z with Z being hydrogen, alkyl, alkenyl, cycloalkyl, cyclo-alkylalkyl, hydroxyalkyl or aminoalkyl, said aliphatic radicals having up to four carbon atoms, or a pharmaccutically acceptable acid addition salt thereof. a

87. A 1-N-substituted derivative of/4,6-di-(aminoglycosyl) -1,3-diaminocyclitol , wherein the 6-aminoglycosyl is garosaminyl and the 1,3-diaminocyclitol is 2-deoxy- - 181 - v-< 40437 streptamine, the *»-aminoe:lycosy]-6-garo8aminyi-2-dcoxy-streptamines being gentamicin D, gentamioln B^, gentamlcin Cj, gentamlcin C^a# gentamlcin Cg, gentamlcin C2a' gentamlcin C2b' gentamlcin Xg, slsomlcln, verdamicln, Antibiotic G-418, Antibiotic JI-20A, Antibiotic JI-20B, or Antibiotic G-52, wherein the substituent ia -I-Z with Z being as defined in any of claims 82 to 84 or a pharmaceutically acceptable aold addition salt thereof. a

88. A 1-N-substituted derivative of/4,6-di-(amino glycosyl )-l,3-diamlnocyc]ltol , wherein the 6-aminoglycosyl is garosaminyl and the 1,3-diaminocyclitol is 2-deoxystreptamlne, the 4-aminoglycosyl-6-p;arosaminyl-2-deoxystreptamines being gentamicin C^, gentamicin C1&, sisomicin, verdamicln or Antibiotic G-52, wherein the substituent is -C-Z with Z being as defined in any of claims 82 to 84 or a pharmaceutically acceptable acid addition salts thereof.

89. A 1-N-substituted derivative of sisomicin,wherein the substituent is -C-Z with Z being as defined in any of claims 82 to 84 or a pharmaceutically acceptable acid addition salts thereof.

90. A 1-N-substituted derivative of verdamicin, whoroin the substituent is -b-Z with Z being as defined in any of claims 82 to 84 or a pharmaceutically accept able acid addition salts thereof. - 182 - 40437

91. A 1-H-substituted derivative oC Antibiotic 66-40D wherein the substituent is -H-Z with Z being as defined in any of claims 82 to 84 or a pharmaceutically accepteible acid addition salts thereof. 5

92. The compounds as claimed in any of claims 82 to 91* wherein Z is methyl* ethyl or propyl.

93. 1-N-Acety lsisomicin or a pharmaceutically acceptable acid addition salt thereof.

94. 1-N-Acety!verdamicin or a pharmaceutically q acceptable acid addition salt thereof.

95. 1-N-Acetylantibiotic G-52 or a pharmaceutically acceptable auid addition salt thereof.

96. 1-N-Acetylgentamicin or a pharmaceutically acceptable acid addition salt thereof. j. gy A compound of nny of claims 82 to 96 , whenever prepared by tho process as claimed, in any of claims 34 to 44. - 183 -

97. Uv 40437

98. a pharmaceutical composition comprising as active ingredient at least one compound a3 claimed in claim 45, together with a pharmaceutical carrier or excipient.

99. A composition as claimed in claim 98 , in the form of dosage units.

100. A composition as claimed in claim 98 or 99, Wherein the active ingredient is a compound claimed in any of claims 64 to 80'. .

101. A p harmaceutical composition comprising at least ono compound as claimed in claim 82 , together with a pharmaceutical carrier or excipient.

102. A composition as claimed in claim 101, in the form of dosage units.

103. A composition as claimed in claim 101 or 102, wherein the active ingredient is a compound claimed in any of claims 93 to 96.

104. A pharmaceutical composition as claimed in claims 98 or 101, substantially as herein described with specific reference to tho Examples. - 184 - 40437

105. Process for preparing a pharmaceutical copposition as claimed in any of claims 98 to 104, wherein the active ingredient is brought into a form suitable for therapeutical administration.

106. Proccss as claimed in claim 105* wherein the active ingredient is admixed with a pharmaceutical carrier or excipient.

107. '107. Pharmaceutical composition whenever obtained by a process as claimed in claim 105 or 106.

108. A method of eliciting an antibacterial response non-human in a warm-blooded /animal having a susceptiblo bacterial infection, which comprises administering to said animal a non-toxic, antibacterially effective amount of a compound as claimed in any of claims 45 to 80*'

109. A method of eliciting an antibacterial response non-human in a warm-blooded/animal having a susceptible bacterial infection, which comprises administering to said animal a non-toxic antibacterially effective amount of a compound as claimed in any of claims 82 to 96. Dated this 1st day of August, 1974. TOMKINS & CO., Applicants' Agents, (Slgned)^^j—> >.-uartmouth Road, DUBLIN. 6. - 185 -