CA1188309A - 3-0-demethyl derivatives of the istamycin b series of compounds and their preparation - Google Patents

Abstract

COMPOUNDS AND THEIR PREPARATION

ABSTRACT OF THE DISCLOSURE

New derivatives of the istamycin B series of compounds are provided, which are 3-0-demethylistamycin B0, 3-0-demethyl-istamycin B and 3-0-demethyl-2"-N-formimidoylistamycin B repre-sented generally by Formula I or specifically by Formulae Ia, Ib and Ic, respectively. Compound Ia is useful as an intermediate for the preparation of Compounds Ib and Ic and the latter two compounds exhibit a high antibacterial activity against a wide variety of Gram-positive and Gram-negative bacteria and are use-ful antibiotics. Also provided are processes for the prepara-tion of the new derivatives starting from istamycin B0.

Description

3~

BACKGROUND OF THE INVENTION
We have previously discovered a new strain of antino-mycetes, Streptomyces tenjimariensis SS-939 which was deposited in the Japanese public depository "Fermentation S Research Institute" under the de~osit number FERM~P4932 and in the American Type Culture Collection under ATCC Number 31603, and produced ~y cultivation of said strain several di~ferent new aminoglycosidic antibiotic substances, ista-mycin A, istamycin B, istamycin Ao and istamycin Bo (refer to our published Japanese Patent KOKAI NoO 145697/80 and No.
43295/81; our published U.K. Patent Application GB 2,048,855A;
and our U.S. Patent No~ 4,296,106. Subsequently, we totally synthesized di-~6 , 03-demethylistamycin A and -found its antibacterial activity against Pseudomonas aeruginosa to be significantly higher than that of istamycin A. We then con-tinued our studies on istamycin antibiotics with the intention ofconvertiny istamycin B (which has a higher antibacterial activity than istamycin A) into the 3-O-deme-thyl derivative thereof, and have now succeeded in synthesizing 3-O-demethyl-istamycin B starting from istamycin B~, and have found that 3-O-demethylistamycin B is effective against not only Pseudomonas aeruginosa but also a variety of resistant bacteria. ~n this series of studies, we have also synthesized 3-O-demethylistamycin Bo and 3-O-demethyl-2l'-N-formimidoyl-istamycin ~, the former being useful as an intermediate to be converted into 3-O-demethylistamycin s by glycylating the 4-methylamino group thereof, and the latter being a 3-O-demethylistamycin B derivative useful as an antibiotic similarly to formimidoylistamycin B and formimidoylistamycin A (see U.S. Patent No. 4,382,926) both of which were synthesized by us.

.~

The 3~0-demethylîstamycirl Bo, 3-~demethyli~tamycirl B
and 3-0 deme~hyl 2~N-forrnimidoylistamycin B described and claimed herein have been disclosed by us in J. Antibiotics, 33, 1577-1~80 ~Dec~Trber 1~80), along witll details of their prepar~-tion ~nd ~eir antib~cterial activ~ ty.

We are aware of U.S. Patent 4,255,421 whic:h discloses certain 0 demethylamirloglycosides and claims tho5e s:~f the f on~lul~
Rl 6~ NE~ R2 ~0 N~2 ~CH3 wherein Rl and R2 are ~e same or dif f el-ent and are hydrogen or me'chyi, R3 is hydrogen or an unsubstituted or substituted aminoacyl group having 2 t~ 4 carlbo~ atoms in the acyl moiety the substituent being selected from hydroxy, formyl and carbamoyl; provided that, when R1J R2 ~nd R3 are each hydrogen atoms, the me~h~lamino group a~ ~he 4-position is not ~riented trans to the hydroxyl groups at the 3- and 5 posikions.

Although ~he aminocyclitol rings are numbered in ~he opposite directions in U.S0 4,255,421 and in ~he com-pounds disclo~d and claimed herein ~eompounds named a~ 3-o~
d~me~hyl derivatives herein axe named a~ 5-0-demethyl derivatiYes therei~0 it may ~e seen th~t ~he claims of V~S.
40255,421 literally include wikhin thelr scope two of the ~hree compounds di~closed hnd claimed herein, iue8 3-0-demethyl-istamycin Bo and 3~0d~me~hyl~stam~cin B. However, U.S.
4,255,421 does not exemp~ify ei~her of these two compound~
a~d, ~urther d neither discloses the starting material necessary or their preparation nor teaGhes how th2 ~ecessary starting material may be prepared. Thu~; 3-~-demethylistamycin Bo and ~.

3 O demethylistamycin B contaill a 6 ' -N-methyl group and a C-l amino yro~p s~hi::h is cis to the sugar moiety, and the starfcing ma~erial for the deme~hylatlon reaction (i~tamycin Bo h rein) must contain these same groups and conf igurations ~. U . 5 .
4,255,421, on the other handt discloses as star~lng materials only antibiotic complex ~ 6606 [the sporaricins; see J.
~ntibio~ics, 32, 187 (1979)~, a~ least some of which contain a C-l amino group i:hat is cis to the sugar moi ety, but all of which have a 6 ~ -C-methyl group; an~ iotic complex KA~7038 tthe sannamycins ; see J. Anti~iotics ~ 32 0 1066 (1979 3 3, some of which contain a ~ ' -N methyl group but all of which have a C-l arnino group which is trans to the sugar moiety; and fortimicins A and B Isee J. Antibiotics, 30r 552 (1977)], bo~.h o~ which have a 6' C-amino group and a 1:-1 amino group which is trans to the sugar moiety, as well as an additional hydroxyl group on the C 6 pOSitiOII (nun~ered C-2 herein~ which is not present, in the compound.s claimed hereinO Thus t the sporaricin, sannamycin and fortimicin startiny materials disclosed in U~S.
4,255,421 are not suitable starting materials for the prepara-tion of any of the three compound~ disclosed arld claimed herein, since they have the wrong 6 ' ~substitution and/or the wrong stereo conf iguration of the l-amino group 9 and in some instances have an additional hydroxyl group on the C-6 position. q~he starting ma~erial~; disclos~d in U.,S. 4,255,421 are products of f~rmentation and not synthetic products, and ~here is no ~isclosure of how to obt~in the necessary starting material~
for th~ preparation of the compounds aisclosea and claimed herein. Thus, U.S. 4,255t421 does not contain an ena~ling dis-closure for the preparation of the herein claimed 3-0-demethyl-ist~mycin Bo~ 3-0-demethylistamycin B or 3-0-demethyl-2~-N-.
formimidoylist~mycin 8, :.
" ' ' ' ? ';' 3~

DETAILED DESCRIPTION OF THE INVENTION
Accordlng to a ~irst aspect of thls inventlon, therefore, there are provided as new compounds 3-O-dismethyl deriYatives o~ istamycin ~ serles compounds o~ ~ormula I:

H4, ~ O
~1' ~1 3- NH2¦ ~ OH (I) 4\~oH

CH3~
R

wherein R represents a hydrogen atom9 a glycyl group or an N formimidoylglycyl group, and acid addition salts thereof.
Concretely~ the following three compounds and their acid additi~n salts are provided~

3-0-Deme~hylistamycin ~O (R i~ H) 3-Q~Deme~hylis amycin B (R is H2NCH2CO-) 3-O-Demethyl-2"-N-formi~idoylict~mycin B
3 n (R iS HN=CE~NHC~32CCI-)o Phy~ico-chemical properties o~ the new compounds acc~rding to ~hi~ in~ention are shown in Table 1.

~ 'r ~ ~ j ~a o --6-- rl ~
O L) O

ri rl o ~ O 0~
m O tn ~3 ~
i~ J~ ~ ~ _~ $ ~ ~
~ ~ ~o O '~ ~ ~-rl I C~ ~ ~ ~ ~U ~ . s:
z; ~ 5 h e h r~ ~ Il') - ta ~ a~ ~ o ~ ~o u~ r1 (`~ \-rl ~ 01~ l Z ~I a I u~ h ~ 5~ 0 ~ a) a ~1 rl ~ O C~ l ~1 ~ ~ ~L S O ~-- ~J t-- O!) OC~ bl~ r~ I ~
S ~ tl~ J~ ~0 1~ ~ ~ ~1 0 J~ O ~ U~ 1 0 *r~ ~1 ~1 0 >
U~ ~2 r-t t~J 1~1 (~) ~1 U~ V
E~ I S r ~ ~ ~ Iti ~ ~ ~ ~ ~ ~ I ~ ~rq a ~ ~ ~ ~ 3 ~ O O
I E~ ~ O o ~d O ~ J ~I S~
O S~ U~ ~ p r_ . . . , . ~1 I O ~1 0 3 b{l ~ O U~ ..
~- ~ 'a c.) N--' ~1 ~ ~1 __ ~ E
O _~ ~ ~ ~
~ ~ ~ u~ n ~ ~ O ~ ~ ~I
h F~ a) UJ--a-f~ ~ ~ + ~ ~ S~ a o I ~ 3 ~ ~ ,~
~~ ~ O
Q:"m ~ ~ _~ , ~ ~ + ~
S ~ ~ o ~ ~: ~ ,~ ~ 3 s:: o u~ o o* J ~ ~ _, ~ t~ O
,1 Q tn 3 ~ a ~ u~
c~E3 C) ~ a~ o~
~a ~ ~ h ~ + ~ r~ ~
a~I t~ o SO ~ ~ ~ ~ r~
I U~ ~ O ~0 ~53 t-- 3 ~ O
l~ ~ ~ c~
O _ _ c) ,_ u~ ~ ,_ S h X o O
~ r l ~ g :~:
r~r~ O 3 ~ ~ ~D r1 r-l 5:

~:~,m ~ ~ _, S ~ ~D + ~0 N '7 r~~~ ~ ~:J O r l o~ ~ u'~
tda~ ~ c ~ O O
c) O ~
JJ ~ ~ r-l r-l + r~ Z Z
e ~ h U~ t-- O Lr~ l C~
I ~ ~ O I ~ C~ o 3 O ~ r~
i u~ O ~ ~ ~ ~ O ~ u~
'CJ C:) ~1 1_ 3 rl . ~ ~ V
O h l O O
. C ~ ~ ~
O ~ --` O S ~
c~ ~ r l e~ a~ h ~ r~l r-l S~ Q) CO rl * al 111 ~I O rl ~ ~ Q, O c) C-~ :~
h Q~ C v~ z C/~ C/lr~
Q) Eo~ r~ 1 h S
R, c) ~ ~ ~ ~
~ O O C~ ~1 C t~ ~ h lil fl5 ~: a ~ cn ~ ~d ~V r-l bO
. _ ~ ~ ~
~1 t~
~1 33~

The antibacterial spectra of 3-0-demethylistamycin s~
sesquicarbonate and 3-0-demethyl-2"-N-formimidoylistamycin ~-dlsulphateotrihydrate are shown in Table 2, along with that of istamyc~n B for comparison purposes.

3~
--8~

m 0~ O ~ U~ O O O O

~1C~ ~1 N t-- ~ N t~J N ~J N U~
O OC::\ O ~D O ~ O O ~1 0 0 r-i r t b~~) C~~ V V V V
~tO
_.~ H
U~
~ m *
~dI c~
hZ;
- ~d n~~ ~ O O 00 ~ O O o C~ o ~ ~ o OH rl N N 1~ t`- ~J N N N N ~ t-- N Ir~
O~ :~ O O O ~ O O O O O ~i 0 0 ~I r-i td ~ O v a~d v v v Y v v E~
c~ o ~ e I ~
DO h 5: I ~o ., ~3~ m ~ O ~ co ~ o~ O o o ~ ~D O ~D

rl~ C~ ~ ~ l N N N 15~ ~ N U~ ~1 Q) ::~, O O O O t~ O O O O ~ i O r~
I ~ V V V V V
O
I U~
rl . O
cOr~ Q o;) C~
r 1 tl~ U~ N O E-l o ~ ~ o~ I o ~ ct:
~3 u~ ~ ~ ~ o h ¦ ---- ~ o ~ o ~, ~

h ~ 1 H _I ~ c~ ~ ~

V . 4~ td S , ~ _ ~ o E~ j v¦ C ~¦ j 0¦

r~ U~ r~ r~ r~
r; ~I r; ~ ~ r; rr ,~ ~ ~1 ~ ~ O ,;

r~ r~ ~ r~ ~ ~ r~ r~ r~ O ~ ~ O ~ rr~

CC) U~ ~D J U~
P: !r; ~; r~ C
~ ~ o ~ n o r~ ~ ~ r~ r~ O O O O O r~ r~
~0 r~ 3 O~ ~ O
O H ~ r-l r-l ,~ r~ ri rl r~ td m ~ X X ~ X ~ ~ g ~ ~

s r~
~ , ,~ ~d S _ D _ ~. _ _ _ _ _ _ _ ~ ~D

3~)~

--1t~1~t~) Lr~ N t"J ~U t~J N
~ . . . .. . .
O ~ ~D~D O O~ D O O O ~ O t~
~ O ~ O

t~lt O~Dt~ t.-) tr) 1~ U:) L~ L
~ ~ t~ t~l t~l o N t~t~ o t~
A

U~ ~ ~ t~O ~ t t~ o t ~ t ~ t t~ t Lr~ ~ ~ Lr~

~ t~7 t~ t~" o t~ ~I H Z t~
Z X ~C ~ C:l 3 tn ~ ~D td r- ~ ~ c~
~ ~ ~ 3 ~D r~l O
~~ ~ ~ æ ~ ,~
g ~ O ~ C~ ~ S:4 ~u ~ = - =
tO ~ C~ ~
5~a~ ~ h u~ O h ~: S~, ~: S~ ~ _ u~
O ~ a~ b~ ~ h R. P. rd ~ ~8 U~ 0 ~ ~ ~ ~ ~ ~ ~
c c ~ - `,1 F~ ,1 P C ~
h h a) h h ~

3~

~ .
h O o o o o o bO
o o o o In o ~
~ ~ ~:
:~

U~
O U~ Lr O ~ O, J~
O ~I N 11~ r-l O

~ F~
. ~ O
a~ S
. ~q a t--O O O O ~D O ~ ~ ~
o h ~ h A ~ 0 5 ~d ~ a.) S
c~ ~d 5 U~ o ~1 ~ ~ O
~r ~ o r-- ~ rn ,~ a) ~ ~ ,1 o h ~>
Lr~ ~ ~1 O~ O J~
~I tn ~1 I E~ ~ :4 Z
V~ O I ~ bO
~q ~ S~ ~ b ~ ~ C) ~ ~0 0 ~ ~ O
h r~ ~ O &~ h E3 ~ h ~ ~
. ~ h S .C S
~o o E _ _ = -- O ~ ~ H H

cq cq r~
~4 ~ * * ~

30g ~12-The antibacterlal spectra data given above clearly demonstrate that 3-0-demethyllstamycln B and 3-0 demethyl-

2'l-N-formimidoylistamycln B exhlbit a high antlbacterlal actlvity against a wlde variety of Gram~negative and Gram-positlve bacteria. On the other hand, 3-0-demethylistamycln Bo has a poor antlbacterlal activity, but is use~ul as an intermediate for the preparation of 3-0-demethylistamycln B.

3~0-Demethyllstamycln Bo (dicarbonate), 3-0-demethyl-istamycin B (sesquicarbonate) and 3-O~demethyl-2"~N-formimidoyl.istamycin B (disulphate~trlhydrate) are further characterizecl by their low toxici~y. Thus~ when the acute toxlclty of these three compounds was e~timated by intra-venous in~ection in mice~ it was found that all the mice tested tolerated a dos~ of 160 mg/k.g of each compound.
3-O Demethyllstamycin Bo~ 3-0-demethyllstamycin B
and 3-0-demethyl-2"-N-formimidoyllstamycin B according to this invention may be obtained in the form of the free base, a hydrate or a carbonate thereof, and more preferably in view of their stability they can be con~erted into a pharmaceutically acceptable acid additlon salt thereof by reactin~ with a pharmaceutlcally acceptable acid in a usual manner. ~xamples of the pharmaceutlcally acceptable acids are inorganic acids such as hydrochloric3 hydrobromic, sulruric, phosphoric and n~tric acids and organic acids such as acetic, malic, c~trlc J ascorbic and methanesul~snlc acids. The compounds may also exis~ as salt-hydrates~ e.g.
3-0-demethyl-2"-N-formimidoylistamycin ~-disulfate-trihydrate.

3~
-~3-3-O-Demethylistamycin Bo of ~ormula Ia:
CH3N~t

4' ~ ~
\ 2' ~ NH2 O ~
(Ia) 4 ~oH

may be prepared according to this invention by demethylat-inK at the 3-methoxy group of istamycin Bo o~ formula II:

CH3N ~~
41 ~

(II) 4\ ~

in a manner known per se. There are several process well known ln the art ~or demethyla~lon of the methoxy group, s~ch as treatment with hydriodic acid or hydrobromlc acid, treatDIent with a Lewls acld, typically a metal halide such 3~3~

as aluminium trichloride 7 alumlnium tribromide, boron trichloride 3 boron tribromide 9 boron trifluoride, zinc diehloride, zlnc diiodide or iron trichloride, ~r treat-ment wlth an alkali metal such as sodium or lithium (see '9The Chemistry of the Ether Linkage"~ edlted by S. Patai, page 21, published by Interscience Publishers Inc. ln 1967). A preferred pr~cess for the demethylation reaction to be used here is one wherein lstamycln Bo is heated wlth 48% hydrobromic acid at 90 - 100C in a sealed tube~ which can achieve effective demethylatlon reaction in a short reaction time as described in published U.K. Application GB 2 037 743.

3-O-Demethylistamycln B o~ ~ormula Ib:

4' ~
\ 2~ \ N~l2 O ~ ~ OH
(Ib) 4\~;oH

cocH2NH2 may be prepared according to this in~ention by acylating the 4 methylamino ~roup o~ 3-O-demethylistamycin Bo of formula Ia with glycine. In practlce, this is attalned by previously protectlng the 1 r and 2'-amino groups and 8~

the 6'-methylamino group of 3~0 demethylistamycin Bo with a conventional monovalent amino-protecting group ~o give a 1-, 2~ and 6'~oN~pr~tected deriva~ive of 3-O-demethylisthmyoin Bo of Fo~mula III-CH3N--\6 4' ~ ~.

R' ~ (III) \ ~ 3 4~ OH

H

wherein R' represents a conventional monovalent amino-prot~cting group, reacting the compound of Formula (III) with a convention-ally N-protected gly ine w~ose amino protecting group is the same as or different from those on the 1-, 2'- and 6'-position~ of ~he compound o~ Formula ~III), or with a reactive derivative thereof~ in an inert solY~nt, at a temp~rature of from a~out 0C
to about 100C, to acylate the 4-me~hyl~mino groupy thus forming a compound of Formula rv:

CH3N ~' i ~ ~ R' ;' ~ , ~ OH (IV) 4 ~ 0~
CH3l /A
COCH2N~`B

R~

~16-wherein ~I has the same meaning as defined ab~ve, A represents a hydrogen atom and B represen~s a conventional monovalent amino-protecting group or A and 8 togeth r form a conventional di~alent ~mino-protec~in~ gro~p, ~nd then removing ~11 the amino-protec$1ng groups on khe compound of Formula IV by conventional means to give 3 O-demethylistamycin B of Formula Xb.

In the amino-pxotection ~tepO the 1- and 2'-amino groups and the 6'-me~hylamino yroup of 3-0-demethylistamycin Bo of Formula Ia may be simultaneously protected with a monovalent amino-protecting group without affeoting the 4-methyl2mino group thereof~ As an example of a suitable, conventional mono-vale~t amin~-protecting group, ~here may be mentioned an alkoxycarbonyl group, particularly hav~ng 2-7 caxbon atoms, such a~ tert-butoxycarbonyl and tert-amyloxycarbonyl; a cycloalkyloxy-carbonyl group, pzrticularly 4-7 carbon atoms such as cyclohexyloxycarbonyl; an aralkyloxycarbonyl group such as benzyloxycarbonyl; and an acyl ~roup j, partlcularly an alkanoyl group having 2- 7 carbon atoms such as trl-fluoroacetyl and o-nitrophenoxyacetyl. The introductlon o~ such an amino-protectlng group may b~e carried out in a manner known in the syntheses of peptldes, e.g. by using a known amino-protecting group-introducing reagent in the form o~ an acid halide, an acid azide, an actlYe es~er, an acld anhydrlde, etc. By using such an amino-protecting group-introduc~ng reagent in an amount of 2.5 - 4.0 moles per mole of 3-0-demethyllstamycln Bo~ ~t i possible to preferenti211y rorm 13 2'96'-tri-~-protected 3-0-demethyl-istamycin Bo o~ ~ormula III above due to the differenGe ln reactlvity b~ the respective amino and methylamlno groups o~ 3-0-demethylistamycin Bo~ AlternatiYely9 I,27 ~6'-~rl-N~protected 3-0-demethyllstamycin Bo of ~ormula III may be obtained in a hl~her yleld by rea ting 3-0-demethyli~tamycin .~ g ~, .

33~)~

Bo with one molar equlvalent of a divalent catlon such as those of dlvalent transitlon metals such as copper~ nlckel and cobalt and of zlnc (II) to ~orm a metal complex, and reacting the complex with 3 - 5 moles of an amino-protectlng group-introducing reagent, followed by removal of the metal cation from the reaction product (see, for example, published U.K. Patent Application NoO 2,036,020)~
The subsequent glycylation (l.e. acylation with glycine) of the 4-methylamino group o~ the i~2',6'-trl-N-protected 3-0-demethylistamycin 90 of formula III may be effected by reacting the compound wlth glycine or a reactive derivative thereof in accordance with any of the known N-acylation processes ror peptide-syntheses such as the dicyclohexylcarbodiimide process, mixed acid anhydride process~ azide process, active e~ter process, etc. The reaction may be conducted over a t~mperature range of from about 0C to about 100 DC in a solvent such as methanol, di~xane, acetonitrile ox methylene chlorideO It is pre~erable for the glycine reagent to have the amino group ~rotected, and the amino-protecting group for this purpose may be the same as or dlfferent ~rom those on the 1- and 2'-amino groups and on the ~'-methylamlno group of 3~0-demethylistamyc~n ~0 and which is easlly removable. Thus, the amlno-protecting group ~or protecting the amino group in the glycine reagent may be selected from the above-mentioned amino-protecting groups and some divalent amino-protectlng groups such as those of a Schiff base type. The acylation reaction with a glycine reagent is preferably carried out according to an active ester process in an organic solvent such as dioxane under heatlng to a temperature of 40 - 60C, thus glving a compound of ~ormula IV above.
The removal of the amino-protectlng groups on the amino and methylamino groups of the compound of ~ormula IV
may be effected ln a known manner, ror example, by hydro genolysis in the presence o~ palladlum, platinum oxide, etc. as catalyst for the removal of an aralkyloxycarbonyl group or by hydrolysis in an aqueous solutlon of tri~luoro-acetlc acid, acetlc acid, ets. or a diluted aqueous acld solution such as a dlluted hydrochloric acid for the removal of other amino-protecting groups.
3-0-Demethyl-2l'-N-formlmidoylistamycin B of rormula Ic:

CH3H~I
4' ~ \

(Ic) 4 ~ H
CH3l COCH2NHCH=NH

may be prepared according to this ~nvention by ~ormimidoy latlng the 2"~amlno group o~ 3-0-demethylistamycin B o~

~ormula Ib. Thus~ ~or this purpose3 a 1-9 2'- and 6'-tri-N-protected derlvatlve Or 3-0 demethylistamycin Bo o~
rormula III above ls provlded as startlng materlal. The .

3~3~

eompound of Formula III is reacted wi~h a conventionally N-protected glycine whose amino-protecting group ls differ~
ent from those on the 1-, 2~ and 6'-positions o the compound of Formula ~II, or wi~h a reactive derivative thereof, to acylate ~he 4-methylamino group, thu5 forming a compound of Pormula V:

CH31 ~ ' R' 4' ~ 0 \ 2' \ NH
~1 R' ~ (V) 4 ~ 0H

I /A' cOcH2N ~B' wh~rein R' represen~s a conventionaL monovalent amino-p.rotecting group, A' represents a h~ydrogen atom and BD
rep:resents a conventional monovalent amino-protecting group which is different from R', or A and B' toge~her form a conventional divalen amino-protectirlg group., Then, the amino-protecting group on the 2"-amino group in the ylycine moiety of the csmpound of Formula V is selectively removed and the resulting compound ls reacted with an imlnoether to convert the 2"-amino group into an amidine group, 'chu6 ~orming a compound Or rormula VI:

. .

3~3 4~
\ 2t \ NH
~1' ~1 3' ~ ~ OH (VI) 4 ~ 3 CH
COCH2NHCH=NE~

wherein R' has the same meaning as defined above. Finally~
the remaining amino-protecting groups on the 1-, 2'- and 6'-positions of the compound of formula VI are removed to yield 3-O-dernethyl-2~'-N-formimldoyllstamycin B of formula Ic.
In one preferred example for forming an N-protected lntermedlate compound of formula V wherein R' is benzyloxy-carbonyl group, A' is hydrogen and B' is tert-butoxycarbonyl group, 1~2',6'-tri-N-benzyloxycarbonyl-3-O-demethylistamycin Bo ~s acylated on the 4-methylamino group wlth the N-hydroxy-succlnimide ester of N-tert~butoxycarbonylglycine.
According to another preferred embodimentg 1,21,6'~tri-N-tert-butoxycarbonyl-3-O-demethyllstamycin Bo is acylated on the 4-methylamino group wlth the N-hydroxysuccinimide ester of N-benzyloxycarbonylglycine, thus forming an N-protected intermedlate compound of formula V in which R' is tert-butoxycarbonyl, Al i~ hydrogen and B' i5 benzyloxycarbonyl group .

3~3 In the ~ormlmidoylatlon step~ the lmlnoether reagent to be used may be one havlng the general formula:
R"OCH-NH
whereln Rn represents a lower alkyl group or an aralkyl group such as benzyl, or an acid addition salt thereof such as the hydrochloride or sulfate. The use o~ an iminoether hydrochloride such as ethylformimidate hydrochloride or benzylformimidate hydrochloride is preferred. The reaction may be conducted in an organic solvent such as dioxane or methanol or in an aqueous solution at a temperature o~
below about 30C in a known manner. The resulting compound of formula VI, 1,2',6'-tri-N-protected-3-O-demethyl-2"-N-formim~doylistamycin B, or an acid addition salt thereof, may be purified by a column chromatography using a silica gel or the like.
The remaining amino-protecting groups on the 1- and 2'-amino groups and on the 6'-methylamino group of the compound of formula VI may be removed by a knowm method as above-menSioned 9 thus to yield the desired 3-O-demethyl-2~'-N-formimldoylistamycin ~ of ~ormula Ic.
3-O-Demethylistamycin B and 3-O-demethyl-2"-N-formlmidoylistamycin B of this invention have a high antibacterial activity and are of a low toxicity to an~mals.
Accordingly, these compounds are useful similarly to the known antibacterial antiblotics and may be ~ormulated into known pharmaceutical rorms and administered ln the same manner as the known antibacterial antlbiotics. According to a further aspect o~ this invent~on, there is provided a pharmaceutical compos~tion comprlsing an antibacterially ~22-e~ectlve amount Or 3-O-demethyllstamycl~ B or 3-0-demethyl-2~'-N-~ormimidoyllstamyci~ B~ or a pharmaceutically accept~ble acld addition salt thereof, ln combinatlon with a pharma-ceutically acceptable carrier. It will be appreclated that the actual preferred dosages of the ackive new compoundS of thls inventlon used will vary accordlng to the particular compound being used, the particular composition ~ormulated, the mode of application and the particular situs and organlsm belng treated. Many factors that modify the action of the drug will be taken into account by the skilled ln the art, for exampleg age, body weight, sex, diet, time of admlnistration, route of administration, rate of excre-tion, drug combinations, reaction sensitivities and severity of the dlsease. Optimal application rates for a given set of conditions can be ascertained by the skilled in the art using conventional dosage determination ~ests in view of the above guidellnes.
The following Examples further illustrate the pre-paration ~ the compounds according to this invention.
Example 1 Preparat~on of 3-O-demethylistamycin Bo Istamycin Bo monocarbonate (500 mg, 1.27 mmol) was dissolved in 48% hydrobromic acid (50 ml) and the solution was heated ln a sealed tube at 90- 93C ~or 4 hours. The reaction solutlon was concentrated to dryness in vacuo and the resldue was dissolved in water (50 ml~. The solution was ad~usted to pH 8.5 wlth addition o~ 7M aqueous ammonla and passed ~hrough a column (21 x 550 mm~ of 200 ml o~

~3-CM-Sephadex C-25 (NH4-rorm, a product o~ Pharmacia Co., Sweden). The ~olumn was eluted gradiently wlth 0.15M
aqueous ammonla (1120 ml) and 0.70M aqueous a~monla (1120 ml), The eluate was collected in 16 ml-~ractions. The rrac~ions Nos. 85 to 102 were combined together and concentrated to dryness in vacuo to afford 275 mg of a colorless powder of 3~0-demethyllstamycln Bo dicarbonate. Yield 49%.
Ex~
Pre aration o~ 3-0-demeth listamycin B
P Y
3-0-Demethylistamycln Bo-dlcarbonate (150 m~, 0.34 mmol) obtained in Example 1 was dissolved ln methanol (12 ml) and to the solution N-benzyloxycarbonyloxysuc-cinimide (329 mg, 1.34 mmol) was added over 2 hours with stirring and under cooling to -10C, and the mixture was stlrred for further two hours. The reaction solution was concentrated in vacuoto form a syrup, which was then dissol~ed in chloroform (25 ml). The solution was washed with water (8 ml x 2) and the chloroform layer was dehydrated over anhydrous sodium sulfate and concentrated to dryness in ~acuo to yleld a crude powder o~ 1~2',6'-trl-N-benzyloxycarbonyl-3-O-demethylistamycin Bo. This powder was dlssolved in dioxane (9 ml) and to the solution were added trlethylamine (0.5 ml~ and N-(N-benzyloxycarbonyl-glycyloxy)succinimide (250 mg, oO82 mmol)~ and the mixture was heated to 55C for 2 hours under stirring. The reaction solutlon was concentrated ln vacuo and the residue was dissolved in chloro~orm (25 ml~ and washed with water (8 ml x 2). The chloroform layer was dehydrated over anhydrous sodium sul~ate and concentrated to dryness in vacuo to give * trade mark.

a crude powder (389 mg). This powder was purifled by column chromatography on silica gel (30 g of Wako gel C-200~ a product o~ Wako Pure Chemical Industries, 1td., Japan; the column size: 14 x 350 ~m) de~eloped wlth a mixture of ethyl acetate-toluene (5:2 by volume) ~o afford a colorless powder (83 mg) of l,2',6',2"-tetra~N-benzyloxy-carbonyl-3 0-demethylistamycin B. This powder was dissolved in a mlxture of methanol (5 ml) 3 water (l ml) and acetic acid (0.5 ml) and the solution was sub~ected to hydro-genolysis under a hydrogen stream in the presence of 5%
pallad1um-carbon (15 mg; a product of Kawaken Fine Chemical Company, 3apan) as catalyst at room temperature for l.5 hours. After the catalyst was removed by flltration, the reaction solution was concentrated to dryness in vacuo to yield a crude powder (60.9 mg). A 60 mg portion of the powder was dissolved in water ~3 ml) and the solution was ad~usted to pH 8 with addition of aqueous ammonia and passed through a column (8 x 95 mm~ o~ 5 ml of Amberlite CG-50 (NH4 ~orm, a product Or Rohm & Haas Co.~ U~SoA~) ~ The column was washed with water ~lO ml) and then eluted wlth 002M a~ueous ammonia t70 ml) and o.8M aqueous ammonia (70 ~l~
in a manner of gradient elution. The eluate was collected in 1.4 ml-fractions. The fractlons Nos. 22- 38 were combined together and concentrated to dryness in vacuo to af~ord a colorless powder ( 35 . 5 m~) o~ 3-0-demethylistamycin B-sesqu~carbonate. Yield: 23%.

* trade mark.

3~

Example 3 Preparation of 3-O demethyl-2"-N formimidoyl~
istamycin B
3-O-Demethyllstamycin Bo~dicarbonate (260 mg9 0.59 mmol) obtained in Example 1 was dlssolved in methanol (24 ml) and to the solution were added ~riethyl amine (0.32 ml) and 2-(t _ -butoxycarbonyloxyimino) 2-phenyl-acetonitrile ~433 mg, 1.76 mmol) (BOC-ON, a product of Aldxich Co.~ U.S.A.) under stirring and cooling (0-8C), and the mixture uas allowed to stand o~ernight. The reaction solutlon was concentrated in vacuo and then dissolved in chloro~orm. The chloroform solution was purified by column chromatography on silica gel (20 g of *

Wako gel C-200; the column size: 14 x 250 ~n). The column was washed wlth chloroform (80 ml) and developed with a mixture of chloroform-methanol (10:1 by volume), and the eluate was concentrated to dryness to afford a colorless powder (255 mg) o~ 1,2'~6l-tri-N-~ert~bu~oxycarbonyl~3-O-demethylistamycin B0. Yield: 66%.
Thls powder (215 mg, 0.33 mmol) was dissolved in dioxane (7 ml) and there were added to the solution tri-ethylamine (0,072 ml) and N-(N-benzyloxycarbonylglycyloxy) succinimide (160 mg, 0.52 ~mol~, and the mixture was maintained at 55C ~or ~ hours to ha~ten the reaction.
The reaction solution was concentrated in vacuo and purlfied by column chromatography on sllica gel t26 of Wako gel C-200;

* trade mark.

the colu~n size: 14 x 310 mm) developed with a mlxture of ethyl acetate~toluene (11:~) to yield a colorless powder (197 mg) o~ 2"-N-benzyloxycarbonyl-1,2' ,6'-tri-N-tert-butoxycarbonyl-3-0-demethylistamycln B. Yleld: 75%.
This powder (190 mg, 0.23 mmol) was dissolved in a mlxture of methanol (6 ml), water (1 ml) and acetlc acid (0.05 ml) and ~he solution was sub~ected to hydrogenolysis in a hydrogen stream in the presence o~ 5% palladlum-carbon (30 mg) as catalyst at room temperature for 3 hours. After the catalyst was removed by filtration, the reactlon solu-tion was concentrated to dryness in vacuo to afford a colorless powder (160 mg) o~ 1,2',6'-tri-N-tert-butoxy-carbonyl-3-0-demethylistamyc~n B-monoacetate. Yield: 93%.
Thls powder (150 mg~ 0.20 mmol) was dissolved in a mixture of methanol (27 ml) and wat~r (4 ml), and to the solution there wa~ added dropwise over 15 minutes a solution Or benzyl~ormimidate hydrochloride (209 mg, 1022 mmol) in methanol (5 ml) under ice-co~ling, while the pH of the reaction solution was ad~usted to 8.o - 8.5 by addltion o~
a 0.5N potassium hydroxide. The reactlon solution was ~urther stirred for 30 minutes under ice-cooling, then a~justed to pH 4.0 by additlon of lN hydrochloric acid and concentrated in vacuo to give a syrup. The syrup was dis-solved in butanol (50 ml) and the solution was washed with water (25 ml x 2) The butanol layer separated was concentrated to dryness ln vacuo and the residue was purifled by column chromatography on silica gel (20 g of Wako gel C 200; the column size: 12 x 370 mm) developed wlth a mlxture of chloroform-methanol (4:1 by volume) to yleld a colorless powder (63 mg) of 1,2',6'-tri-N-tert-butoxycarbonyl-3~0-demethyl-27'-N-formlmidoylistamycin B.
Yield: 42%.
This powder (63 mg) was dissolved ln a 90% tri-fluoroacetic acid (2.5 ml) and the solution was allowed to stand for 1.5 hours and then concentrated to dryness ln vacuo. ~he resldue was dissolved in water (2 ml) and the solution was passed through a column (8 x 10 mm) of 5 ml of Amberlite IRA-400 (S04 form; a product of Rohm & Haas CO~ ~ U~S~Ao ) to effect a salt exchange. The effluent (6 ml) was concentrated to dryness in vacuo to obtain a colorless powder (53 mg) of 3-0-demethyl-2"-N-formimidoyl-istamycin B-disulphate-trihydrate. Yield: 95%.

Claims (2)

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

1. A process for the preparation of 3-0-demethyl-istamycin B of Formula Ib:
(Ib) or an acid addition salt or salt-hydrate thereof, which comprises reacting a 1-, 2'- and 6'-N-protected derivative of 3-0-demethylistamycin Bo of Formula III:

(III) wherein R' represents a conventional monovalent amino-protecting group, with a conventionally N-protected glycine whose amino-protecting group is the same as or different from those on the 1-, 2'- and 6' positions of the compound of Formula (III) or with a reactive derivative thereof, in an inert solvent, at a tempera-ture of from about 0°C to about 100°C, to acylate the 4-methylamino group, thus forming a compound of Formula IV:
(IV) wherein R' has the same meaning as defined above, A
represents a hydrogen atom and B represents a convention-al monovalent amino-protecting group or A and B together form a conventional divalent amino-protecting group, and then removing all the amino-protecting groups on the compound of Formula IV to give 3-0-demethyl-istamycin B of formula Ib.

2. The compound 3-0-demethylistamycin B, in a pharma-ceutically acceptable acid addition salt or salt-hydrate thereof, whenever prepared or produced by the process of claim 1 or an obvious chemical equivalent thereof.