Note: Descriptions are shown in the official language in which they were submitted.
33~
BACKGROUNV OF TE~E INVENTION
We have pre~iously discovered a new strain of antino-
mycetes, Streptomyces tenjimariensis SS-939 which was
deposited in -the Japanese public depository "Fermentation
Research Ins-titute" under the deposit number FERM~P 4932 and
in the American Type Culture Collection under ATCC Number
31603, and produced by cultivation of said strain several
different new aminoglycosidic antibiotic substances, ista-
mycin A, istamycin B, istamycin A~ and istamycin Bo (refer to
our published Japanese Patent KOKAI No. 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-N6 , 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
ofconverting istamycin B (which has a higher antibacterial
activity than istamycin A) into the 3-O-demethyl derivative
thereof, and have now succeeded in synthesizing 3-O-demethyl-
istamycin B starting from istamycin BQ, and have found that
3-O-demethylistamycin B is effective against not only
Pseudomonas aeruginosa but also a variety of resistant
_ .
bacteria. In this series of studies, we have also synthesi.zed
3-O-demethylistamycin Bo and 3-O-demethyl--2"-N-formimidoyl-
istamycin B, the former being useful as an intermediate to be
converted into 3-O-demethylistamycin B 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.
3~
--3--
The 3-O-demethylistamycin Bo ? 3-~demethylis'carnycin B
and 3-O~demethyl-2n-N formimidoylis~amycirl B described and
claimed herelII have been dlsclosed by us in J. Anti}:)iotics, 33,
1577-1580 ~December 1980), along with details of their prepara-
tion and their antibacterial activity,
W~3 are aware of UrS~ Pa~en. 4 j25~,421 which discloses
c~rtain O-demethylaminoglycosldes and claims those of ~e
f ormula
Rl
61 f~ NH~2 NH2
~ O ~
NH2 -(:H3
wherein R L and R2 are ~e same or different and are hydxogen
or methyl ~ R3 is hydrogen or an urlsu~stituted or substituted
amino~c yl group having 2 to 4 carbon atoms in the acyl moiety,
the substituent ~eing selected from hydroxyt formyl and
~arbamoyl; prosrided ~at, when Rl~ R2 and R3 are each hydrogen
atoms ~ the methyl~mino group at 'che 4-position i5 not oriented
tran~ ~o the hydroxyl group~ a~ the 3~ and 5-positionsO
Alt~ough the aminocyclitol r~ ngs are nl3mbered
in. the opposite directiorls in U ~ S ., 4, 255, 421 and in the co~n-
pounds di~closed and claim~d herein (compound~ named as 3-o-
deme thyl derivatives herein are named as 5-O-demethyl
derivatives ther~in~, it may be seen ~at t:he claims of T~.S~
4,255,~21 literally i~clude within their scope two of the ~ree
compotmds disclosea and claimed herein, i~ e. 3-Q-demethyl
sta~mycin Bo arld 3~0~demethylistamycirl B. However" U,.5.
4 0 255, 421 dc~es not exemplify ~ither 9f these two compounds
and, fur the:r ~ neit:hex discloses the starting material necessary
for th~3ir preparation nor teaches how the n~cessary starting
material may be prepared~ Thus, 3 O-demethylistamycin Bo and
3-O~demethyllstamycin B con~ain a 6'-N-methyl group ~nd a C-l
amino group which is cis to the sugar moiety, and the starting
material for the demethylat~on reaction ~istamycin Bo herein)
must contaln ~hese same groups and configurations. U.Sc
4,255,421, on the o~her hand, discloses as starting materials
only antibio~ic compl~x KA~6Ç06 [~he sporarioins; see J.
Antibio~ics~ 32, 187 (197~], at leas~ some of whioh contain
a C-l amino group that is cis ~o he sugar moiety, but all
of which have a 6'-C~methyl group; antibiotic complex KA-7038
[the sannamycins; see JO Antibiotics~ 32, 1066 (1979)], some
of which contain a 69~N-methyl group but all of which have a
C-l amino group which is rans to the ~ugar moiety; and
fortimicins A and B Isee J. ~ntibiotics, 30, 552 (1977)3, both
~f which have a 6' C-amlno group and a C-l amino gxoup which
is rans to the sugar moiety D as well as an additional hydroxyl
group on the C-6 position (numbered C 2 herein) which is not
present in the compounds claimed herein. ~hus, the sporaricin,
sannamycin and fort~micin starting materials dis~losed in U.S.
4,255,421 are not suitahle starting material~ for the prepara-
tion of any of the three compounds disclosed and claimed herein,
since they have the wrong 6'-substitution and~or the wrong
stereo configuxation of the l-amino group, and in ~ome instances
have an additional hydroxyl group on the C-6 position~ The
starting materials disclosed in U . S . 4, 255 " 421 are products
oî fermentation and not synthe'cic products t and there is no
disclosure o~ how ~o ob~ain the necess2lry starting material~
for the pxeparation of he compourld disclosed and claimed
herein. mus, U.S. 4,255,421 does not contain an enabling dis
closure for the prepaxation of the here~n olaimed 3-O-deme~hyl-
istamycin Bo~ 3-O-demethylistamycin B or 3 O-demethyl-2"-~-
formimidoylistamycin B~
3 1~ !
--5--
DETAIL~ DESCRIPTION OF THE INVENTION
_
Accordlng to a first aspect o~ this inventlon,
there~ore~ there are provided as new c~mpounds 3-O-demethyl
derivatives of istamycln B serles compounds of formula I:
CH3N ~
41~2 Y \
~11 ~1
3t NH2¦ ~ ~ OH (I)
4 ~ OH
CH3N~
whereln R represents a hydrogen atom, a glycyl group or an
N-formimidoylglycyl group, and acid addition salts thereof.
Concretely~ the following three compound~ and ~heir a~id
addition ~alts ar~ provided:
3-O~Demethylistamycin Bo (R is HJ
3-O~Demethylistamycin B (R i5 H~NCH2CO ~ )
3~0-Demethyl-21' N-formimiavyli~tamycin B
2~ ln
t1R ;5 HN=~HNHCH2CO ) .
Physico-chemical properties of the new compounds
according to thi5 inYenti~n ~re shown in Table 1.
3~C~
, i~
o
o U o
. ~
. ~ o C)~
o
~> h ~`I ~d
i:: ~ ~
~ ~ ~a _ ~ -
z; ~ ~ h Ei h P' u~ -
- ~ S a) ~ o ^ ~ ~ ,~ _
J~ ~ ~:J O r I~, Z h
I u7 h 3 6~ o
~1 ~ ~ O c~ J
~ ~ ~ ~ S o -- ~ ~ 0C~ ~0 ~ I ~
s ~a) J~ ~0 ~ . . . . ~ ~'~ O
o ~ ~ ~ o * ~ ~ ~ ~ O
a~ ~a td tQ 3 ~ ~L~ ~ ~ tn C~ ~
E~ ~ ~ ~ Sl ~Q
h c~ 3 ~ ~ ~ O O
I ~ ~ O o ~d O ~ a ~
O ~ u~ ~ O h p r~ . . . . . P~
IO ~ 1 0 3 ~ D N0~ O Ul -
~ a ~ ~ ~i o ~
_ ~ ~ O
U~ ~ ~
h h ~ o 0 u~
hr I ~ J~ ~ '~ ~) h
R:~m ~ ~ 0 ~ a~ + ~o o ~ rd QJ
J~ ~ C~ O N ~ ~: ,~ r-l 3 h
J~ ~ O U~ O O* 3~ ~ `-- r1 10 0
t~J a~rl D v~ 3 ~1 3 ~1
c~~ h a~
r~~ ~ ~ ~ ~1 ~ ~_
E~ c~ h L~ ~ ~ #
o I ~ a ~ u~
rO ~ N r-l (~J ~1 I ~ -
C)I tO ~ O OD 2~1 3t-- ~ ~
l~ . I (r~ ~ 3 r~
O ~
N O O
S ~ ,_~ ~ O
,1 ~1 o 3 ~ N N
Q~ s a:l d) p~ ) t~a ~
c:l ~ ~: ~ u~ o o~ N _ ~rl ~ Ifl
.~ ~ ~ U~~D N ~ O O
E-~ E t) O Q) N ~1 ~ ~r
~ ~P ~ 4 Z Z
a ~ ~ h Lr~ ~ o ~ o r~l
I 1~ t~ O I t~l ~ 0 3 \~ L.~ 1~ ~1
O ~ C~ ~ ~ ~ . . . I ~ . 1: :C
I U~ ~ O ~ N ~ e
f~ ~ 'OC~ 3 r~1 ~
_ _. _ _ ~
O ~ ~ O O
. ~ 0
O ~ ~- OS ~ U~
rl O ~ h~ 1~ q)
h -- ~ I o
rJ r~ 3
td ~ ~ ~ ~O S~ C~
hQ. ~ 1~ U:C æ u~
3 h.l::
a~ o ~ p,
c> ~ a) ~ d
a ~
C~ V
.--1 N
1:
~ .
'~ 3~D
~7--
The antibacterial spectra of 3-0-demethylistamycin B~
~esquicarbonate and 3-0-deme~hyl-2"-N-formimidoylistamycin B~
disulphate~rihydrate are shown in Table 2, along with that of
istamycln B for comparison purposes.
3~
,1 m
O 00 0~ ~ O ~ O O ~D ) ~ ~D ~O
O O O O ~D O O O O ~ O O ~ ~1
C~ J~ V V V V
~n
~ a~
o
* ~
~d I C)
h Z
_
C~
I u~ o o ao 0 0 s~ O O O ~o ~ O ~D
O ~ --1
C~ S ~O O O O O O O O O I--I O O l~~ r~
J~ O
h ~ ~ v v v v v v v
t~ O ~ ~
a~ J~ n ~
Q ~r1 I E~
0 ,D O h
~ C I ~
td
rl ~ ~U
h * I
~ ~ ~ ~ o~ o oo 0 ~ a~ o o o ~D ~D O ~D ~
a) ~ ~ ~ ~ t-- ~1 ~ ~ ~ ~ ~ u~ ~ u~ ,~
~ ~ ~ C~ . ~ . . . . . . . . . . .
J~ ~ Q~ O ~ O O O O ~1 ~I C~
~ a ~
~: ~ ~ V V V V V
o, ~
;~1 ~ ~
a) . r-
~ o
D
~ o~
E~ o ~1
.C U~ C~
~,q Lr~ ~ O E~
~~ O ~ ~ o~ ~ O ~1 ~:
CQ
~ ~ ~ O ~ ~
rlU~ h1~ 0 H 1~ ~ ~ ~ t~l
a~ ~ o c> ~ ~ td
P~ Z C~
bSh _ ~ ~rl u~ ~ E-~ O b~ æ
h 5 I:L :~ Hr3 ~ CC . Q~
o td Q~ ~> ~ t~ ~ ~i rl
O td P~ ~d ~1 ~q ~ ~q ~1
F~ D~ In r-l h _~ : ~ ::S O
::~ ~ ~d ~:: ~ a~ ~1 E~ c)
~r! ~ ~ a) ~ ,Q h h :~
E~ o C~ ~ ~ ~ ~ O ~ ~1 c~
~ O ~ :~ n~ ~q c~ ~ ~ rl
O _ - O C~ ~ Lg ~ ~ ~ ~ S
a) ,~ ~ o
E~ ~ :~ C) ~ ~ ~ ~1 a~ ~d ~
S: S O ~ r~ ~1 = ,1 S: R Q1
P C4 h c~ q_l rl ~rl ~ O ,5::
~d ~ c~ ~1 c~ c~ ~ ~ O C)
O :~ ~q
u~ u~ ~: u~ s~ a: a~ c~ ~: W
33~
~1 ~ ~I N o ~
N 1~ 1 t\l~1 ~ 15~ ~1 N L~ r I N ~1 N
N P ~
a~ o ~ o ~ ~I ~) o ~1
~I N ~~I -i O O O C7 0 .~
0 ~ ~I r-~ r~l r~ t~l N N t-- O H ~: r-l ~1
~ ~ ~ ~ ~ ~ ~ ¢ CC ~ 3 ~ ~) N ~1 r~l
N N N N N ~ ~ ~ t~l N t~
N ~d ~ m
S
X ~ ~ ~ El h h
ol = ~ = 1~ ~:
S~ a
3~
--10--
O~1 0 ~ D O O ~ D O O O LS~ O ~7
rl O L~ O Lr~ N L~ O
r~ r-l H
t~ Lr\ (rS 1~ ~I r~ N L~
O ~ O ~U ~ t~l O ~ N ~ J N 1~ D O
--l 0 N r l r l r 1 N O
~J 1~ r~ N ~ U~
~1 ~1 N ~1 ~\1 H H ~I N H H C~J
N ~) L
~I H rl
c~ rro o~ r~ H æ o~
~D
U~ r~
r ~ a ~~ lO
,, I ~ ~ æ :z ~a ,I c~ ~
O ~ ~ ~ ~ ~ ~ ~ - = = = =
0 ~ C)
~rl S J~ h h 0 O ~ p~ p,
C: :~ ~:: ~d b~ _c~ 0 ~q
~ J ~ a) ~ ~ h ~ ~
O r l ~ ~dP~ t~l ~q
u~ ,_1 ~ :~ h Ei u~ rl rl cd
~d r-l r-l t~l td cd ~: ~: O
~1 a~ ~ u~ 0 ~ ~ ~ ~ a~ ~
H E~ E~ O O _ h al O O O
.C ~ ~ h
u~ u:~ c/~ ~:4 ~ u~ v~u:~ ~t ~ P~
- ~8~3
td
O o o o o o h
o o o o U~ o
,~ ,1 ~ ~ ~
~ A A V
O
~o
U~
OU~ ~ O. ~
O ~ ~ ~ ,, O e
~1 ~
A O
~d ~
. ~ O
L~ ~ S
N . 1~ a
O O O O ~D O
O h h
r~ ~ h
Q :~
S
C) ~ h
~ ~1 ~U t-- O
::~ N O t-- ~i
~1 0 h J:~
O J~
t-- ~1 ~n ~ -d u L:
,~ I p Gq ~ Z
a~ o ~ c~ bO
~, N ~ ~ t~ r\ ~ U:l U~
cq ,J ~ P ~q
~ ~ ~
~ ~ O
~ ~ a) h o o
C~ ~
. d h S: S S
~3 C~
O O
e O ~ ~ H H
~1
~ P~ * * *
-12-
The antibacterial spectra data given above clearly
demonstrate tha~ 3-0-demethylistamycln B and 3-0-demethyl-
2"-N-~ormimidoylistamycln B exhibit a high antibacterial
actlvlty against a wide variety of Gram-negative and Gram-
posltive bacteria. On the other hand, 3-O~demethylistamycin
Bo has a poor antibacterlal activity, but ls use~ul as an
intermediate for the preparation of 3-0-demethylistamycin B.
3-0-Demethylistamycln Bo (dicarbonate) 3 3-0-demethyl
istamycin B (sesquicarbonate) and 3-0-demethyl-2"-N-
formimidoylistamycin B (disulphate trihydrate) are further
characterized by their low toxicity. Thus, when the acute
toxicity o~ these three compounds was estimated by intra-
venous in~ection in mice 3 it was found that all the mice
tested tolerated a dose of 160 mg/kg of each compound.
3~0-Demethylistamycin ~0~ 3-O~demethylistamycin B
and 3-O~demethyl-2"-N-~ormimidoylistamycin B according to
this invention may be obtained ln the form of the free base,
a hydrate or a carbonate thereo~, and more pref~rably in
view of their stability they can be converted into a
pharmaceutically acceptable acid additlon salt thereof by
reacting with a pharmaceutically acceptable acid in a usual
mannerO Examples of the pharmaceutically acceptable acids
are inorganic acids such as hydrochloricg hydrobromic~
sul~uric, phosphoric and nitric acids and organic acid~
such as acetic~ malic, c~tric~ ascorblc and methanesul~onic
acids. The compounds may also exist as salt-hydrates~ e.g.
3-0-demethyl-2"~N-formimidoylistamycin B~disulfat2 trihydxateO
.
83~
-13-
3 O-Demethylistamycin Bo of formula Ia:
CH3N--~6'
4' ~
\ 2' \ NH2
O ~i
(Ia)
4 ~oH
CH3~
H
may be prepared according to this invention by demethylat-
ing at the 3-methoxy group of lstamycin Bo of formula II:
3H ~ 5,
4' ~ '\
2 \ NH2
O ~
(II)
4 ~ 3
OCH3
CH3N
in a manne.- known per se. There are several process well-
known in the art for demethyla~ion of the methoxy group~
s~ch as ~reatment with hydriodic acid or hydrobromlc acld,
treatment wlth a Lewis acid, typlcally a metal halide such
.
-14-
as aluminium trichloride, aluminium trlbromlde, boron
trlchloride, boron ~ribromide, boron trifluoride, zinc
dichloride~ zlnc dii~dide or iron trichloride, or treat-
ment with an alkali metal such as sodlum ~r lithium (see
"The Chemlstry o~ the Ether Linkage", edited by S. Patai,
page 21, publlshed by Interscience Publishers Inc. in
1967). A pre~erred process ~or the demethylation reaction
to be used here is one wherein istamycln Bo is heated with
48% hydrobromic acid at 90 - 100C in a sealed tube, which
can achieve ef~ective demethylation reaction in a short
reaction time as described in published U.K. Application
GB 2 037 743.
3-0-~emethylistamycin B of formula Ib:
3H ~ 5,
4' ~ \
\ 2~ \NH2
O ~ ~ OH
(Ib)
4\~oH
CH3N
COCH2NH2
may be prepared according to thls invention by acylating
the 4-methylamino ~roup of 3-0-demethylistamycin Bo o~
~ormula Ia with glycine. In practlce, this is attalned
by previously protecting the 1- and 2'-amino groups and
-15-
the 6'-met~ylamino group of 3-O-demethyli5tamy~in Bo with a
conventional monovalen~ amino-protecting group to give a 1-,
2'- and 6Z-N-protected derivative of 3-0-demethylistamycin Bo
of Fonnula III,
CH3N ~ '
I O ~ ~ OH
R' ~ (III)
\ ~ 3
4 ~ OH
CH3N
I
H
wherein R' represents a conventional mon~valent amino~protecting
group, reacting the eompound of Formula tIII) with a convention-
ally N-protected glycine whose amino-protecting group is the same
as or different from those on the 1-, 2 9~' and 6'-positlons of
the compound of Formula ~III), or with a reactive derivative
~h~reof, in an inert sol~ent, at a temperatur~ of from about 0C
to about lOO~C, to acylate the 4-me~hyl~nino group, thuS forming
a compound of Formula IV:
CH3NI ~
Rt4 ~ ~ \ NH
9' r~ , ~ OH (IV)
5\
~ ~ 3
4 ~ OH
CH3l /A
COC~2N ~B
~ ~ .
_, .. . . . . _ .
-16
wherein Ri ha~ the same meanlng as defined above, A represents
a hydr~gen atom and B repxesen~s a conventional monovalent
~mino~protecting group or A a~d B toge ~er form a conventional
divalent amino-protecting gro~p, and then removing all the amino-
protecting ~roups on the compound of Formula IV by conventional
m~ans to give 3~0-deme~hylistamyeln B of Fonmula Ib~
~ n the ami~o-protection step~ the 1- and 2~-amino
groups and th~ 6i methylamino group of 3-0-demethylistamycin Bo
of Formula Ia may be simul~ar~eously protected with a monovalent
amino-protecting group wi~hout affecting th~ 4-methylamino
group thexeof. As an example of a sui~able, conventional mono-
valent amino-protecting group, there may be mentioned an
alkoxycarbonyl group, particularly having 2-7 carbon atoms, such
as tert-butoxycarbonyl a~d ~er~-amyloxy~arbonyl; a ~ycloalkyloxy-
carbonyl group, particulaxly 4-7 carbon atoms such as
cyclohexyloxycarbonyl; an aralkyloxycarbonyl group
such as benzyloxycarbonyl; and an acyl group, partlcularly
an alkanoyl group having 2- 7 carbon atoms such as tri-
rluoroacetyl and o-nitrophenoxyacetyl. The introduction
of such an amino-protecting ~roup may be carried out ln a
manner known in the syntheses of peptides, e.g. by using
a known amino-protecting group-lntroducing reagent ln the
~orm of an acid halide, an acld azide~ an active ester,
an acid anhydrlde, etc. By using such an amino~protecting
group-introduclng reagent ln an amount Or 2.5 - 4.0 moles
per mole o~ 3-V-demethyll~tamycin Bo~ ~t ~s possible to
preferentially ~orm 1,2~ 9 6~trl-N protected 3 0-demethyl-
istamycin Bo o~ formula III above due to the dlrference in
reactivlty of the respective amlno and methylamlno groups
o~ 3-0-demethylistamycln ~0~ Alternatlvely9 132~ ,6'~trl
N-protected 3-0 demethylistamycin ~0 o~' ~ormula III may be
obtained in a hlgher yleld by reacting 3 0 demethylistamycin
7~' ! ~` i
33~1~
-17-
Bo with one molar equlvalent of a divalenk catlon such as
those Or divalent transitlon metals such as copper, nickel
and cobalt and of zinc (II) to form a metal complex~ and
reacting the complex with 3 - 5 moles of an amino-protecting
group-introducing reagent 3 followed by removal of the metal
cation from the reaction product (see, for example, published
U.K. Patent Application No. 2,036,020).
The subsequent glycylation (i.e. acylatlon with
glycine) of the 4-methylamino group of the 1,2',6'-tri-N-
protected 3-0-demethylistamycin Bo of rormula III may be
effected by reacting the compound with glycine or a
reactive derivatlve thereof in accordance wlth any of the
known N-acylation processes ~or peptide-syntheses such as
the dicyclohexylcarbodiimide process, mixed acid anhydride
process, azide process, ac~ive ester process, etc. The
reaction may be conducted over a temperature range of from
about 0C to about 100C in a 501vent such as methanol,
dioxane~ acetonitrile or methylene chloride. It i5
preferable for the glycine reagent to have the amino group
protected~ and the amlno-protecting group for thls purpose
may be the same as or dl~ferent ~rom those on the 1- and
2'-amino groups and on the 6'-methylamino group of 3-0-
demethylistamycin Bo and which ls easily removable. Thusg
the amino-protecting group ~or protecting khe amino group
in the glycine reagen~ may be selected ~rom the above
mentloned ~mlno~protectlng groups and some dlvalent amlno-
protecting groups such as those of a Schiff base type. The
acylation reactlon wlth a glycine reagent is preferably
carr~ed out according to ~n act~ve ester process ln an
~383~
-18-
organlc ~olvent such as dioxane under heatlng to a
temperature of 40 ~ 60C, thus giving a compound of
~ormula IV above.
The removal of the amino-protecting groups on the
amino and methylamlno groups o~ the compound of ~ormula IV
may be effected in a known manner, for example, by hydro-
genolysis in the presence of palladium9 platinum oxlde>
etc. as catalyst for the removal o~ an aralkyloxycarbonyl
group or by hydrolysis in an aqueous solution of trifluoro-
acetic acid~ acetic acid~ etc. or a dlluted aqueous acld
solution such as a diluted hydrochloric acid for the
removal o~ other amino-protecting groups.
3-0-Demethyl-2"-N-formimidoyllstamycin B of
~ ormula Ic:
C~3N ~ -
4, ~ .0\
OH
(Ic)
~ ~ H
CH3l
COCH2NHCH=NH
may be prepared according to this invent~on by formimidoy-
lating the 2"~amino group o~ 3~0-demethylistamycin B o~
~ormula Ib. Thus, for thls purpose~ a 1- 3 2'- and 6'-tri-
N-protected derlYatlve of 3~0-demethylistamycin Bo o~
formula III above ls provlded as startlng materlal. The
.
31~D
--19--
compound of Formula III as reacted wi~h a conventionally
N~protected glycine whose amino-protecting group is differ-
ent from those on the 1-, 2'- and 6 t -positions of th~
compound of Formula III, or w.ith a reac~ive d~rivative
thereof~ ~o acylate ~he 4-methylamino group, thu5 forming
a compound of Formula V:
CH3N ~ ' R'
1 4' ~ O
\ 2' \ NH
~b~ ~ O~ (V)
5 ~ ~ 3
4 OH
CH3~
I /A'
COCH2N ~B'
wherein R' represents a conventiona:L monovalent amino-
protecting group, Al xepresents a hydrogen atom and B~
represents a conven~iDnal mon~valent amin~-protecting group
which is differ~nt from R', or A' and B' together form a
co~ventional divalent amino-protecting group. Then, the
amino-protecting group on the 2"-amino group in the glycine
moiety of ~he compound of Formula V is selectively
removed and ~he resulting ¢ompound is reacted with an
iminoether to convert the 2;'~amino group into an amldine
group, thus ~ormlng a compound o~ ~ormula VI:
3~
-20
CH3N ~
R' ~ o \ 7
\ 2' \ NH
~1' ~1
3t IH \o/6~ ~ OH
R~ ~ (VI)
4~oH
CH3l
COCH2NHCH=NH
wherein R' has the same meaning as defined above. Finally,
the remaining amino-protecting groups on the 1-l 2'- and
6'-positions of the compound of` formula VI are removed to
yield 3-0-demethyl-2"-N~formimidoylistamycin B of formula
Ic.
In one preferred example for ~orming an N-protected
intermediate compound of ~ormula V whereln R' is benzyloxy-
carbonyl ~roup, A' is hydrogen and B' is tert-butoxycarbonyl
~roup, 1,2'96'-trl-N benæyloxycarbonyl-3-O-demethylistamycin
Bo is acylated on the 4-methylamino group with the N~hydroxy-
succinimide ester of N-~ert-butoxycarbonylglycine.
According to another prererred embodiment, 1~2' J6' tri-N-
tert-butoxycarbonyl-3-0 demethylistamycin Bo is acylated on
the 4-methylamlno group with the N~hydroxysuccinimide ester of
N-benzyloxycarbonylglyclne~ thus forming an N-protected
intermed1ate compound of formula V ln which R' is tert
butoxycarbonyl3 Al is hydrogen and B' is benzyloxycarbonyl
group.
~311~)
In the ~ormimldoylatlon step~ the iminoether reagent
to be used may be one ha~ing the general formula:
R"OCH=NH
whereln R" represents a lower alkyl group or an aralkyl
group such as benzyl r or an acid addition salt thereo~ such
as the hydrochloride or sulfate. The use of an iminoether
hydrochloride such as ethylformimidate hydrochloride or
benzylformimidate hydrochloride ls preferred. The reactlon
may be conducted in an organic solvent such as dioxane or
methanol or in an aqueous solutlon at a temperature of
below about 30C in a known manner. The resulting compound o
~ormula VI, 1,2',6'-tri~N-protected-3-O-demethyl-2"-N-
formimidoylistamycin 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 o~ rormula ~I may be removed by a knowm method
as above mentioned~ thus to yield the desired 3-0-demethyl-
2"-N-rormimidoylistamycin B of ~ormula Ic.
3-O-Demethylistamycin B and 3-O-demethyl-2t'-N- -
~ormimidoylistamycin B of this invention have a high
antibacterial activity and are of a low toxicity to animals.
Accordingly, these compounds are useful similarly to the
known ant~bacterlal antibiotics and may be formulated into
known pharmaceutlcal ~orms and administered ln the same
manner as the known antibacterlal antlblotics. According
to a further aspect of this inventlong there is provided a
pharmaceutlcal composition comprising an ant~bacterially
3~
-~2-
e~fectlve amount o~ 3-O-demethylistamycin B or 3-O-demethyl-
2"-N~formimidoylist~mycin B, or a pharmaceutically acceptable
acld addition salt thereof, ln combination with ~ pharma-
ceutically acceptable carrier. It will be appreciated that
the actual preferred dosages of the active new compoundS of
khls invention used wlll vary according to the particular
compound belng used, the particular composition formulated,
the mode of appllcation and the particular situs and
organism belng treated. Many factors that modify the
action of the drug will be taken into account by the skilled
in the art, for example, age3 body weight, sex, diet, time
of administration, route of adminlstration, rate of excre-
tion, drug combinations, reactlon sensitivities and severity
of the disease. Optimal application rates for a given set
o~ conditions can be ascertained by the skilled ln the art
using conventional dosa~e determination tests in view of
the above guidellnes.
The ~ollowing Examples ~urther illustrate the pre-
paration of the compounds according to this invention.
Example 1
Preparation of 3-O-demethylistamycin Bo
, .
Istamycin Bo monocarbonate (500 mg, 1.27 mmol) was
dissolved in 48% hydrobromlc acid (50 ml) and the solution
was heated ln a sealed tube at 90 - 93C for 4 hours. The
reaction solutlon was concentrated to dryness in vacuo and
the resldue was di~solved in water (50 ml)O The solutlon
was ad~usted to pH 8.5 wlth addition o~ 7M aqueous ammonia
and passed through a column (21 x 550 mm) o~ ~OO ml o~
.. . .. _ . , _ _ . _ .. , . _ . . .... . . . . .
3~
-23-
*
CM-Sephadex C-25 (NH4-~orm, a product o~ Pharmacla Co.,
Sweden). The ~olumn was eluted gradlently with 0.15M
aqueous ammonla (1120 ml) and 0.70M aqueous ammonia (1120 ml~,
The eluate was collected ln 16 ml-fractions. The rractiOns
Nos. B5 to 102 were combined together and concentrated to
dryness in vacuo to afford 275 mg o~ a colorless powder of
3-0-demethylistamycin Bo~dicarbonate. Yield ~9%.
Ex~ple ?.
Preparation of 3-O~demeth~listamycin B
3--0-Demethyllstamycln Bo-dicarbonate (150 mg, 0.34
mmol) obtained ln Example 1 was dissolved in rnethanol
(12 ml) and to the solution N-benzyloxycarbonyloxysuc-
cinlmide (329 mgg 1.34 mmol) was added over 2 hours with
stirring and u~der cooling to -10C, and the mixture was
stirred for ~urther two hours. The reactlon solution was
concentrated in vacuoto form a syrup, which was then
dlssolved in chloroform (25 ml). The solutlon was washed
with water (8 ml x 2) and the chloroform layer was
dehydrated over anhydrous sodium sulfate and concentrated
to dryness in vacuo to yield a crude powder of 1,2',6'-tri-
N-benzyloxycarbonyl-3-0-demethylistamycin Bo. This powder
was dissolved ln dloxane (9 ml) and to the solution were
added triethylamine (0.5 ml) and N-(N-benzyloxycarbonyl-
glycyloxy)succinlmide (250 mg, 0.82 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 chloroform ~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.
- 2~ -
a crude powder ~389 mg). Thls powder was purified by
column chromatoKraphy on sllica gel (30 g of Wako gel
C-200, a product of Wako Pure Chemical Industries, Ltd.,
Japan; the column size: 14 x 350 mm) developed with a
mixture Or ethyl aceta~e-toluene (5:2 by volume) ~o afford
a colorless powder (83 mg) of 1,2'~6'~2"-tetra-N-benzyloxy-
carbonyl 3-0-demethylistamycin B. This powder was dissolved
in a mixture o~ me~hanol (5 ml)~ water (1 ml) and acetlc
acid (0.5 ml) and the solutlon was sub~ected to hydro-
genolysis under a hydrogen stream ln the presence of 5%
palladium-carbon (15 mg; a product of Kawaken Flne Chemical
Company, Japan) as catalyst at room temperature for 1.5
hours. After the catalyst was removed by flltratlon, the
reaction solut.ion was concentrated to dryness in vacuo to
yield a crude powder (60.9 mg). A 60 mg portion of the
powder was dis~olved ln water (3 ml) and the solutlon was
adJusted to pH 8 wlth addition of aqueous ammonia and passed
through a column (8 x 95 mm) of 5 ml of Amberlite CG-50
(NH~ form, a product of Rohm & Haas Co., U.S~A.). The
column was washed with water (10 ml) and then eluted with
0.2M aqueous ammonia ~70 ml) and o.8M aqueous ammonia (70 ~)
ln a manner Or gradient elution. The eluate was collected
in 1.4 ml-fractions. The ~ractions Nos. 22- 38 were
combined together and concentrated to dryness in vacuo to
af~ord a colorless powder (35~5 mg) of 3-O-demethylistamycin
B sesquicarbonate. Y~eld: 23~.
* trade mark.
.
- 25 -
Example 3
Preparation o~ 3~0-demethyl-2"-N-formlmidoyl~
3-O-Demethylistamycin Bo-dicarbonate (260 mg, 0.59
mmol3 obtained in Example 1 was dissolved in methanol
(24 ml) and to the solution were added triethyl amine
(0.32 ml) and 2-(tert-butoxycarbonyloxyimino)-2-phenyl-
acetonitrile t433 mg9 1.76 mmol) (BOC-ON, a product of
Aldrich Co~, U.S.A.) under stirring and cooling (0-8C),
and the mlxture was allowed to stand overnlght. The
reactlon solution was concentrated in vacuo and then
dissolved in chloroform. The chloroform solution was
purified by column chromatography on silica gel (20 g of
Wako gel C-200; the column slze: 14 x 250 mm). The column
was washed with chloroform (80 ml) and developed with a
mixture of chloro~orm-methanol (10:]. by volume), and the
eluate was concentrated to dryness to afford a colorless
powder (255 mg) of 1, 2 ', 6 ' -tri-N-tert-butoxycarbonyl-3-0-
demethylistamycin Bo. Yield: 66%.
This powder (215 mg~ 0.33 mmol) was dissolved in
dloxane (7 ml) and there were added to the solution tri-
ethylamine (0.Q72 ml) and N-(N benzyloxycarbonylglycyloxy)
succlnimide (160 mg, 0.52 ~mol)3 and the mixture ~as
maintalned at 5SC ~or 2 hours to hasten ~he reaction.
The reaction solutlon was concentrated in vacuo and puri~ied
by column chromatography on sillca ~el (26 o~ Wako gel C-200;
* trade mark.
3~3
- 26 -
the column size: 14 x 310 mm) developed with a mlxture of
ethyl acetate-toluene (11:4~ ~o yleld a colorless powder
~197 mg) o~ 2" N-benzyloxycarbonyl-1,2',6'-tri~N-tert-
butoxycarbonyl-3-O-demethylistamycln B. Yield: 75%.
This powder (190 mg, 0.23 mmol~ was dissolved in a
mlxture of methanol (6 ml) J water (1 ml) and acetic acid
(o.05 ml~ and the solution was sub~ected to hydrogenolysis
in a hydrogen stream in the presence of 5% palladlum-carbon
(30 mg) as catalyst at room temperature for 3 hours. After
the catalyst was removed by filtration, the reaction solu-
tion was concentrated to dryne~s in vacuo to afford a
colorless powder (160 mg) o~ 1,2'~6'-tri-N-tert-butoxy-
carbonyl-3-0-demethylistamycin B-monoacetate. Yield: 93%.
This powder (150 mg, 0.20 mmol) was dissolved in a
mixture of methanol (27 ml) and water (4 ml), and to ~he
solution there was added dropwise over 15 minutes a solution
of` benzylformimidate hydrochloride (209 mg~ 1.22 mmol) in
methanol (5 ml) under ice-cooling, while the pH of the
reaction solution was adJusted to 8.0 - 8.5 by addition of
a 0.5N potassium hydroxlde~ The reaction solution was
furthe~ stirred ~or 30 minutes under ice-cooling~ then
adJusted to pH 4.0 by addltion of lN hydrochloric acid and
concentrated in vacuo to give a syrup. The syrup was dis-
solved in butanol (50 ml) and the solutlon was washed with
water (25 ml x 2). The butanol layer separated was
concentrated to dryness in vacuo and th~ residue was
3~
- 27 ~
purlfled by column chromatography on sillca gel (20 g of
Wako gel C-200; the column size: 12 x 370 mm) developed
with a mixture of chloroform-methanol (4:1 by volume) to
yield a colorless powder (63 mg) of 1,2',6'-trl-N-tert-
butoxycarbonyl~3-0-demethyl-2"-N-formimidoyllstamycin B.
Yield: 42%.
This powder (63 mg) was dissolved in a90% tri-
fluoroacetic acid (2.5 ml) and the solution was allowed to
stand for 1.5 hours and then concentrated to dryness in
vacuo. The residue was dissolved ln water (2 ml) and the
solution was passed through a column (8 x lO mm) of 5 ml
of Amberllte IRA-400 (S04 form; a product of Rohm & Haas
Co., U.S.A.) to e~fect a salt exchange. The effluent
(6 ml) was concentrated to dryness in vacuo to obtain a
colorless powder (53 mg) of 3-O-demethyl-2"-N-formimidoyl-
istamycln B-disulphate-trihydrate. Yield: 95%.
