Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
3~
FIELD OF I~rV~NTION
This invention relates to some n~J processe~ for the
production of a selectively protected N-acylated der1vative
of an aminoglycosidic antibiotic in which some amino or
alkylamino groups at particular positions of the aminoglyco-
side molecule have selectively been protected or blocked
~,~ith an acyl group. This invention thus relates to new
processes fo~ selectively protecting some amino or alkylamino
groups at particular positions of the aminoglycosidic anti-
biotic. Thls invention finds i-ts main uses in the production
of a selectively protected N-acylated derivat~ve of the amino-
glycosidic antibiotic which comprises a deox~streptamine
moiety having a 3"-aminoglycosyl group linked with 6-hydroxyl
group of thedeoxystreptamine moiety in the aminoglycos1de
molecule. The aminoglycosidic antibiotic to which this
invelltion is applicable may be defined more specifically as
an aminoglycosidic antibiotic consisting of a 6-0-(3"-amino
or 3"-alkylamino-3'l-deoxyglycosyl)-2-deoxystreptamine which
may optionally have a 4-0-(6'-aminoglycosyl~ sub~tituent,
and typical examples are kanamycins7 gentamicins, sisomicin,
netllmicin and verdamicin. This invention further includes
an appllcation of these new processes to the produc-tion of
a l-N-(a-hydroxy-~-aminoalkanoyl~-amino-glycosidic anti-
biotics which are kno~m as useful semi-synthetic antibacte-
rial agent active against drug-resistant bacteria.
; PRIOR ART
' Am~noglycosidic antibio~ics such as kanamyclns a~e
3~
-- 3 --
the sub~tanc;e containing ~everal amlno ~unctions and hydroxyl
function.~ having relatively highand various clegrees of re-
activity. Many kinds of semi-~ynthetic aminoglycosidic
antibiotics derlved from the parent aminoglycosidic anti-
biotlcs have been synthetized. In the semi-synthesis o~
these derivatives, it is o~ten neces~ary or preferable to
ensure that ~ome amino groups and/or some hydroxyl groups 1n
the staring aminoglycosidic antibiotlc have selectively been
protected or blocked with one or more suitable protective
groups.
For selective protection of amino group~ and/or hydroxyl
groups in the ami~oglycosidic antibiotic9 various, successful
methods have been developed and are available as ~ar a~
selective protection o~ hydroxyl gro~p is concerned with.
Howe~er, ~or ~lective protection of particular some amino
groups amongst the existing ma~y amino groups of the amino-
glycosidic antibiotic, the presently available methods for
this p~rpose are either difficult to operate or require some
complicated operation~. Thi~ is because all the amino groups
in the aminogly¢osidic antibiotic have no grea* dl~ference
in their reactivity. As a demo~strative example is provided
by 6'~amino group of kanamycin A, however9 such an amino or
methylamin~ ~roup which is bound with a certa$n c~rbon atom
which is, in turn, linking to only one carbon atom in the
aminoglyco~de molecule exhibits a higher reactivity than
that of the amino or methylamino group which i3 bounded with
a cert&in oarbon atom which is li~ci~ to two or more carbon
,
: .
. ~ ,
~3~
atoms in thi~ aminoglycoside molecule. ~or this reason the
former type of amino or methylami.no group is able to much
more preferentially reac-t with an acylation reagent hav$ng
an acyl group to be introduced as the amino-protecting group,
as compared to the latter type o~ amino or methylamino group,
where~y the N-prot.ected derivative hqving the ~ormer type
of amino or methylamino group preferentially blocked with
the acyl group may be produced in a higher yield than the
otherwise N-protected derlvatives. Several years ago, some
of the present inventors ~ound tha-t when amino group and
hydroxyl group are neighboring to each other in a pair.in
the steric configuration of the molecule o~ the aminoglycosidic
antibiotic, thesè amino and hydroxyl groups can selectively
be combined with each other in-to the form of a cyclic carbamate
by treatment with sodium hydride, so that the palr o~ amino
and hydroxyl groups can be blocked simultaneously in the
cyclic carbamate without blocking the other amin~ groups
present in the same molecule (see "Journal of Antibiotics",
25, No. 12, 741-742 (1972); U.S. Patent Nos. 3,925,354 and
3,965,089).
In a recent year, Nagabhushan et al hav~ found that
when a salt of a dlvalent transition metal (M++) sel.ected
from the group cons~sting o~ copper (II), nickel (II), cobalt
(II~ and cadmium (II) ~s reacted in a~ inert organic solvent
with an aminoglycosidlc antibiotic which belongs to the class
of 4-0-(aminoglycosyl)-6-0-(aminoglycosyl)-2-deoxystreptamines
repre~ented by kanamycin~, ge~ltamicins and sisomiGin, thi~
3~
-- 5 --
divalent tran~ltion metal cation is complexed with a pair
o~ amino and hydroxyl groups which ex~st at the partlcular
positions of "vicinal" relationship in the aminoglyco~ide
molecule, ~lereby the am~noglycos~dic antibiotic-transition
metal cation complex i~ ~ormed (see Japanese Patsnt Applica-
tion Pre~publication Sho-52-15~944 and U~S. Patent Application
SN. 697,297 now granted under U.S, Patent No. 4,136,254
issued on January 23, 1979). In this aminoglycosidic anti-
biotlc-transition metal cation complex, the complexed amino
group i~ being blocked with the divalent transition me~al
cation. When this complex is ~ubsequently reacted with an
acyl~tion reagent ha~ing anacyl group, only the non-complexed
amino group~ in the metal complex which are not blocked by
the divalent me-tal cation can be acylated mainly, so that
selective N-protection with the acyl group i~ achieved.
This is illustrated below with reference to }canamycin A as
an example. Thu~ when a divalent transition metal cation
(M++) chosen ~rom cupper (II), nickel (II), cobalt (II) and
cadmium (II) cations is reactd with kanamycin A, complexing
reaction of the divalent metal catlon ~M+~) occurr~ between
l-amino group and 2"-hydroxy group and between ~"-amino ~roup
and 4"-hydroxyl group o~ kanamycin A molecule~ shown by the
~ormula (I) below.
.,
:, .
~3~
-- 6 --
6~
H2NC~2 NH2
4~ o
HO OH
HOCH ~'
_ O / M~
~, OH' (I)
~[++
In the abo~e complexin~ reaction, there~ore9 it is
seen that at least 2 mol o~ the transition metal salt is
required for 1 mol o~ kanamycin A. In the resultant metal
complex, l-amino and 3"-a~ino groups are blocked at the same
time. l~hen this complex o~ the formula (I) is treated with
an acylatlon reagent having an acyl ~roup which is avallable
as an amino-protecting group k~o~n in the con~entional syn-.
thesis of polypeptides, the non-complexed 3-amino and 6'~
amino groups only are acylated mainly to give 3,6'-di-N-
acylated dsri~ati~e (see "Journal of American Chemical
Socisty'l 100~ 5Z5~-5254 (1978)~.
We have recognized the above fact as reported, but
w~ ~till have made our ~urther researches on the interaction
.
''~: ' " ' ,
r~
of another5 various metal catlons with aminoglyco~idic anci-
biotics ~uch as kanamycin A and ka~amycin B as well a~ semi-
synthetic derivatives of the aminoglycosidic antibiotic~.
As a result, we have now found that although divalent zinc
cation has behaviour~ signif.icantly dif~erent from those of
the above-mentiond divalent, nickel, cobalt, copper and
cadmium cations, the zinc cation is ultimately able to
strongly complex with and block both l-amino (or l-alkylamino)
group and ~"-amino (or 3"-all~ylamino) group of an amino-
glyco~lde compound (such as kanamycin At B or C) whichcomprises a deoxy~treptamine moiety ha~ing a 3"-aminoglycosyl
or 3"-alkylaminoglycosyl group linked to the 6-hydroxyl group
of said deoxystreptamine moiety.
~ccording to Nagabhushan et al, it might be expected
that ~Jhen divalent nickel, cobalt, copper or cadmium cation
would be reacted with kanam~cin B, for example, there should
be formed a kanamycin B-met~l salt complex of the following
form~la (II): - -
6' 3
H2NCH2 NH2
4 ~ ~ '
J~ O
(II)
This expectation is supportable by the Nagabhushan
et al~s disclosure of the aforesaid "Journal of American
Chemical Society" according to which vicinal amino-hydroxyl
group pairs should form reversible complexes with the divalent
transition metal cations, in view of the fact that kanamycin
B contains three pairs o~ vicinal amino-hydroxyl groups
between l- and 2"-positions, between 2'- and 3'-positions
and between 2"- and 3"-position~ of the kanamycin B molecule.
However, it has now been found that when kanamycin B is
reacted with a divalent metal cation, zinc cation, the
kanamycin B-zinc salt complex actually ~ormed contains ~ree
2'-amino and 3'-hydroxyl groups which are not being blocked
by the zinc cation, as be contrary to the Nagabhushan's
proposal~ Even if complexing reaction o~ zinc cation with
.
1 ~ 3~
the 2'-amino and 3'-hydroxyl group occurs, the force of com-
plexing ls very low, so that substantially 2'-amino and 3'-
hydroxyl groups are not being blocked in practlce. Therefore,
when the kanamycin B-zinc cation complex is then acylated by
reacting eg. with N-benzyloxycarbonyloxysuccinimido to intro-
duce benzylo*ycarbonyl group as the am~.no protecting acyl
group, tri-3,2',6'-N-acylated derivative in which three, 3-,
2'- and 6'-amino groups have been acylated is produced, in
fac~, in a higher yield than the otherwise N-acylated deriva-
tives, but then the 3,6'-di-N-acylated derivative actually
cannot be obtained (refer to Example l9 given hereinafter).
Thi~ experimental ~act sug~ests that zinc cation shows a be-
haviour different from those o~ the aforesaid four transition
metal cations particularly in that zinc cation does not com
plex with the vicinal 2'-amino and 3'-hydroxyl group pair.
As a further example, when kanamycin A is reacted
with zinc cation followed by acylation with benzyloxycarbonyl
group (refer to the formula (I) hereinbefore), the fact is
observed that 396'-dl-N-benzyloxycarbonylkanamycin A i~
.20 formed as the main acylation product in case zinc cation
is provided just in an amount of slightly more than l mol.
per mol. of kanamycin A~ In this case9 it must be noticed
that thi~ acylation reaction gives formation of 1,3,6',3"~
tetra-N~benzyloxycarbonyl derivative of kanamycin A and
formation of non-acylated, initial kanamycin A simulta-
neously to some extent but actually brings about formation
of tri-N-benzyloxycarbonyl derivative of ~anamycin A only
~3
- io -
in a low yield, ln splte o~ that the Nagabhushan et alls
elucldation of the reaction mechanism might expect that the
tri-N-benzyloxycarbonyl derlvative ~rould be formed in a
hlgher yield than the other N-acylated derivatives (refer
to Example 7 given hereinafter). In the ~pecification and
particularly clalm 4 of U.S. Patent No. 4,136,254, Nagabhushan
et ~1 have stated to the effect that a salt of a divalent
transition metal such as copper (II), nickel (II), cobalt
(II) etc., is necessary to be employed in a total quantity
of at lea t 2 mol. per mol. of kanamycin A for the formation
of kanamycin A-transition metal salt complex, as will be
seen ~rom the formul~ (I) glven hereinbefore. Our experiment
has revealed tha~,in contrast to the four transition metal
cations, zinc cation is able to achieve the effect of block-
ing l-amino and 3"~amino group of kanamycin A when zinc
cation is employed in a total ~uantlty of at least 1 mol per
mol o~ kanamycin A. According to our test, it has been
found that when a nickel salt is reacted in a quantity of
slightly more than 1 mol ~or 1 mol of kanamycin A followed
by acylation of the resulting kanamycin A-nickel salt complex
with benzyloxycarbonyl group, there is obtained only in a
very much low yleld ~,6'-dl-N-benzyloxycarbonylkanamycin A
which would be obtainable in a ~igni~icant yield when
kanamycin A-zinc salt complex wa~ acylated (see Example 7
hereinafter). In view of the above-mentioned facts, it is
Oo~cluded that zinc (II) cation exhiblts a mechanism of
complexing with an aminoglycoslde whlch is dif~erent from
~L~3~
the complexin~ mechani~m of nickel (Ir), cobalt (II), copper
(II) ~nd cadmium (II), cation, and that the aminoglycoside-
zinc cation complex has a complexing s-tab~lity which is
different from that of the complex of the aminoglycoside
with nlckel (II), cobalt (II), copper (II) or cadmium (II)
cation. For the complexing o~ zinc c~tion with the amino-
glycosidic antibiotic, zinc cation may be provided in the
form o~ a zinc salt which is advantageously inexpensive
and unlikely to be a source of polluting the environment.
DETAILED DESCRIPTION OF INVENTION
In consequence, we,the present inventors, have found
that when zinc cation is reacted in ~n inert organic solvent
with an aminoglycosidic antibiotic which contains a deoxy-
streptamine moiety having a 3-aminoglycosyl or 3-alkylamino~
glycosyl group li~ked to 6-hydroxyl group of the deoxy-
- streptamine moiety and possi~ly having an aminoglycosyl
~roup linked to 4-hydroxyl group of the deoxystre~tamine --
moiety, zinc cation is complexed withand block amino-hydroxyl
pairs locating ~t particular positions which may vary
depending on the nature of the aminoglycosidic antib.lotic;
and that when the aminoglycosidic antibiotic-zinc cation
complex so ~ormed is reacted with an acylation rea~ent
havin~ an acyl group which i~ used conventionally for
introduction of an amino-protecting group in the synthesi~
o~ polypeptides, thl~ acylati.on reagent acylates at least
. one of such amino groups in the ami.noglycosldic antibiotic
which are not complexed wi.th and hence not blocked ~y zinc
" ' , '
,
: ' ', :,
3~
- 12 -
cation, ~o that the amino group so acylated is protected;and also that when the resulting acylation product (ie.,
the aminoglycosidic sntibiotic-zinc cation complex having
the acylated amino group(s)) is treated with such a suitable
reagent which will remove zinc cation from said acylation
product, the ~inc complex is destroyed, affording a s~lec-
tively protected N-acylated derlvative of the aminoglycosidic
antibiotic of which the initially zinc-non-complexed amino
group(s) ha~ or have selectively been protected with the
acyl group~ .
According to a first aspect of this invention,there-
-~ore, there i5 provided a process for the produc-tion of a
selectively acylated N-protected derivative of an amino-
glycosldic antibiotic~ this aminoglycosidic antibiotic
comprising a deoxystreptamine moiety having a 3-aminoglycosyl
or 3-alkylaminoglycosyl group linked to 6-hydroxyl group o~
the deoxystreptamine moiety9 and the selectively acylated
N-protected derivative having some amino groups thereof
selectively protected with an acyl group~ which compri~es
the steps of:-
- (a) reacting an acylation reagent having an acyl
group to be introduced as the amino-protecting group, with
an amlnoglycos.idic antibiotic-zinc cation complex which has
been formed by reaction of the aminoglycosidic antibiotio
wit~ a zinc salt in an inert organic solvent, to produce
a complex o~ zinc cations with the ~electively N-acylated
derivative of the aminog~ycosidic antibiotic having the
~3~L~2~3
- :L3 --
initially non~complexed amino group~ acylated,
(b) and reacting the complex of zinc cations with
the selectively N-acylated derivative of the aminoglycosidic
antibioticl with a reagent which removes zinc cations from said
complex, to produce the desired selectively acylated N-
protected derivative of the aminoglycosidic antibiotic.
The process according to this first aspect of the
invention is useful to prepare such a selectively acylated
N-protected derivative of an aminoglycosidic antibiotic by
acylating some aminv groups other than 1- and 3"-amino ~roups
of the starting aminoglycosidic antibiotic, and such selec-
tively N-protected derivative i~ useful in the chemical
synthesis of 1 N~aminoacylated derivatives of aminoglyco~
sidic antibiotics such as kanamycins~ including ami~acin
("Journal of ~ntibiotics" ~, 695-708 (1972)) ~-hich is proved
in the recent years to be an effective antibacterial drug.
These l-N aminoacylated derivati~es of the aminoglycosidic
antibiotics include those derived from a wide range of
aminoglycosides such as kanamycin A~ kanamycin B9 kanamycin C,
gentamicins, si~omicin and other~ as well as ~arious deoxy-
derivative~ thereof, but all of them are common in that
their l-amino group is acylated with an a-hydroxy-~ -amino-
alkanoyl group (see U.S. Patent Nos. 3,781,26~; 3,939,143;
~,9409382; and 4,001~208). By this l-N~aminoacylation, the
aminoglycosid~c antibiotics are imparted with an antibacterial
activity against such resistant bacterial to which the
parent aminoglycosidic antiblotics are not active, and also
.
,,' "
~3~
the aminoglyco~idic antibiotlcs are imparted with an im-
proved antibacterial activity against a wider var~ety of
strains o~ bacteria, as compared to the parent aminoglyco-
sidic antibiotics.
We de~cribe below more fully how to work the process
of the ~ir~t a~pect o~ the in~ention.
The aminoglycosidic antibiotic ~hich is to be reacted
with zinc cation to ~orm the zinc complex (which may also
be termed as a zinc complex salt) according to this invention
includes: ~uch aminoglycosidic antibiotics comprising a
deoxy-streptamine moiety of which 6~hydroxyl group is ~ub-
stituted by a ~-aminoglycosyl or ~-alkylaminoglycosyl group
and of wh~ch 4-hydroxyl group may occasionally be substituted
by an aminoglycosyl group. More particularly, the amino-
glycosidic antibiotic available in thi~ invention for forma-
tion of the zinc cation complex may be defined as such one
comprising 6-0-(3"-amino- or 3"-alkylamino-3"-deoxyglycosyl)-
2-deoxystreptamine optionally ha~ing a 4-0-(amino-glycosyl)
group. Moreo~er~ the aminoglycosidic antibiotic may be a
l-N-alkylaminoglycoside, like netilmicin. Examples of the
aminoglycosidic antibiotics of the class available in this
invention, there may be mentioned kanamycin A-group anti-
- biotics including kanamycin A itself, 6'-N-alkylkanamycin A,
particularly 6l-N-methylkanamycin A, 3'-deoxykanamycin A,
6'-N-methyl-3t-deoxykanamycin A, 4'-deoxykanamycin A, 6'-
N-methyl~4'-deoxykanamycin A, ~t ,4'-dideo~ykanamycin A
(se0 Japane~e Patent Applicati-on No. 11402/79) and 6"-deoxy- `
~13~Z~
~5
or 4",6"-dideo~yka~lmycin A (see Japanese Patent Application
~o. 547~/79); kanamycin B-group antibiotics includine
kanamycin B ltself, 3'-deoxylcanamycin B (le., tobramycin),
4'-deoxykanamycin B, 3',4'~dideoxykanamycin B (ie., dibekac~n),
3',4'-dideoxy-3'-eno-kanamycin B, 6l-N-methyl-~',4'-dideoxy-
kanamycin B; kanamycin C~group antibiotics including kana~ycin
C itself, 3'-deoxykanamycin C, 3',4'-dideoxykanamycin C;
gentamicins A, B and C; verdamicin; sisomicin and netilm~cin
(ie, l-N-et.hylslsomicin) as well as the other known amino-
glycosides r Of course, the process of the ~irst aspect ofthe invention is applicable not only to such a new amino
glycosldic antibiotic which is not yet ~unown a-t present and
will be discovered in ~uture, but also to new semi-synthetic
aminoglycoEidic antibiotic derivative3 which wlll be produced
i~ future by chemical trans~ormati.on of kno~m am.inoglycosidic
antiblotics.
Typical examples of the aminoglycosidic antibiotics
to which the pre~ent invention is applicable inolude kanamycin
A, kanamycin B, kanamycin C; and deoxy-derivatives of these
kanamycins a~ well as 6'-N-alkyl derivatives thereof which
are all represented by the followlng general ~ormula (III):
-- 16 --
6:
R4 C~ NH2
4' ~ 2
,
./
HOCH O
~ ~ .
(III)
. 3ll ~
wherein Rl is hydroxyl group or amino group, R~ and R3 are
each hydrogen atom or hydroxyl group, and R4 is hydroxyl
group or amino group or an alkylamino group containing an
alkyl of 1-4 carbon atoms, particularly methylamino group~
In order to form the aminogl~cosidic antibiotic-zinc
cation complex by reaction o~ the aminoglycosidic antibiotic
with zinc cation in accordance with the invention~ a par-
` ticular aminoglycosidio antibiotic, either in the ~orm of
the free base or in the form of an acid-addition salt thereof,
may ~e dissolved or ~uspended in an appropriate organic
sol~ent or a~ueous organic solvent, and to the resulting
solution or suspension is added ~ suitable zinc salt in a
quantity o~ at least 1 mol per mol o~ the aminoglycosidic
.~,
.
.. ~ .
- 17
antlbiotlc employed. Any ordi.nary org~nic ~olvent may be
employed for this purpose, a~ ~ar a.~ the zinc complex formed
after the addition of the zinc salt is at leas~ partially
soluble in it. However, use of a large volume of a polar
organic solvent and par-ticularly of greater volume of water
should preferably be avoided, because the presen.ce of polar
organic solvent and water is likely to reduce the stability
of theresulting aminoglycosidic antibiotic-zinc cation
complex ~or~ed9 so that the subsequent acylation reaction
for introduction of the amino-protecting group is likely to
give unsatisfactory result~
Thus, it is desirable to use an organic solvent of
high solvent power such as dimethyl-sulfoxide for the solvent
in which the zinc complex is to be formed, but it is feasible
to employ aqueous dimethylsulfoxide, dimethylformamide,
a~ueous dimethylformamide, a mixture of dimethylsulfoxide
and dimethylfo~mamide, tetrahydro~uran, a~ueous tetrahydro-
furan, and even a lower alkanol such as methanol, ethanol
and aqueous methanol.
2Q Zinc cation may be supplied in the form of a zinc
salt to the reaction system where the zinc complex is formed.
Any zinc salt which is ~ormed by reaction of zinc cation
with an ordinary inorganic or organic acid may be used for
the purpose o~ the present invention. In general, however,
it is desirable to employ a zinc salt of a weak acid, ~uch
a~ zinc acetate, as it is usual that amongst the metal com-
plexes containing amino group~ a complex of non-quaternary
amino ~roup with a metal ~alt is more 3table than-a comple
of an ammoni~-type amine wi~h a metal salt, an~ that the
u~e of the zinc salt of a weak acid normally does not lead
to formation of the relatively in~table metal complex con-
taining the ammonium-type amlne. When the zinc salt of a
strong acid, for example, zinc chloride is employed, the
zinc complex as desired may be formed, too,but it is pref-
erable to add a weakly alkaline salt such as sodium acetate,
in addition to the zinc salt, for neutralization o~ the
medium. Similarly, it is desirable to add an amount of
sodium acetate or sodium hydroxide as a neutralizing agent
when the starting aminoglycosidic antibiotic is used in the
form of its acid-addition salt with a strong acid such as
hydrochloric acid. In this case, however, care should be
taken to avoid using unnecessarily excessive am~unt of the
neutralizing agent, as otherwise zinc hydroxide would pre-
cipitate to disturb the formation of the complex. For
instance, when an aminoglycosidic antibiotic tetra-hydro-
chloride is used for the complexing, 4 mol of sodium hydroxide
should preferably be added to neutralize the reaction mixture.
As long as the total molar quantity of zinc salt used is
at least equal to the molar quantity of the aminoglycosidic
antibiotic, the complexing reaction may proceed. However,
it i8 preferable to use the zinc slat in a quantity of sub-
~tantially more than 1 mol per mol of the aminoglycosidicantibiotic, ~o tha~ the equilibrium of the complexing reaction
i- ~hifted ln favor of the formatlon of the complex.
FavorabIe yield of the zinc complex may be obtained ~Jhen
uslng the zlnc salt in a quantity of about ~ 6 mol per
mol of the aminoglycoside a but ln practice it is most pre-
~erable to use the zinc salt in a quantit~ o~ 4-5 mol per
mol of the aminoglyco~side. Time required for complete com-
plexing reaction after the addition of the zinc salt may
vary dependlng on the nature of the organic solvent used,
and it may be in the range o~ "instantaneously" (when using
aqueous organic solvent) to 20 hours. The complexing reac~
tion normally may proceed at ambient tempera~ure9 but heating
or cooling may be done.
In this way, a ~olution or suspension con-taining the
zinc complex o~ the aminoglycosidic a~tibiotic is prepar~d,
to which is then added an acylation reagent having an acyl
group to be introduced as the amino-protecting group.
The acylation reagent employed according to this
invention may be a usual amino-protecting reagent, and this
is used to ensure that the free~ non-complexed amino groups
~n the resultant aminoglycosidic antlbiotic-zinc cation
complex are acylated by and blocked with the acyl group of
the acylation reagent. The acyl group may be an alkanoyl
group, an aroyl group7 ar alkoxycarbonyl group, an aralkyloxy-
carbonyl group, an aryloxycarbonyl groupy an alkylsulfonyl
~roup, an aralkylsulfonyl group or an arylsulfonyl group
which are all the conven~ional amino-protecting group.
The acylation reagen~ available for this purpose may eitller
~e a carboxylic acid o~ the ~ollowin~ ~eneral formula (IVa~:
- 20 -
R5CoOM (IVa)
wherein R5 is hydrogen, an alkyl group, particularly an
alkyl group of 1-6 carbon atoms, an aryl group, particularly
phenyl, or an aralkyl group, especially benzyl, and these
groups being occasionall.y further sub~tituted, or an acid
halide, acld anhydrlde or active ester of said carboxylic
acld ~IVa); or a chloro~ormate of the ~ollowing general
formula (IVb):
R50-C0-Cl (IVb)
or a p-nitrophenyl carbonate of the following general formula
(IVc):
R50_co-o-C6H5-P-N2 (IVc)
or active N-hydroxysuccinimide ester of the ~ollowing formula
(IVd):
R50-Co-o-N ~ (IVd)
or an azidoformate of the ~ollowing-formula (IYe):
R50-Co-N3 (IVe)
where R5 is as defined a~ove, or a sulfonic acid of the
following general Lormula (IV.f):
R6S03H (IVf)
wherein R~ is a hydrogen, an alkyl group; especially an
alkyl group of 1-6 carbon atoms, an aryl group, particularly
phenyl, or an aralkyl group, especially a phenylalkyl group
Ruch a~ benzyl, and the~e group~ being occasionally further
substituted, or an acl~ halide, acid ~nhydride or active
~ ~.3
- 2~ -
e~ter of ~aid sulfonic acld. Accordingly, it is evident
~hat the acylation ~eact~on for protection of amLno groups
according to this invention is an acylation of a broad mean-
ing, including, for example, formylation, acetylation,
propionylation, trifluoroacetyla-tion, benzyloxycarbonylation,
p-methoxybenzyloxycarborlylation, t-butoxycarbonylation,
phenoxycarbonylation, tosylation, mesylation and other
equivalent ones.
Particular examples of the available acyla-tion reagent
include acetoxyformyl, p-nitrophenyl formate, acetic anhy-
dride, acetyl chloride, propionic anhydride, p-nitrophenol
ester of tri~luoroacetic acid, trifluoroacetic acid ester,
N-benzyloxycarbonyloxysuccinimide (a representive active
- ester), N-benzyloxycarbonyloxyphthal~mide, benzyloxycarbonyl
chloride, p-methoxybenzyloxycarbonyloxy-p-nitrophenyl, t-
butoxycarbonylazide, phenoxycarbonyl chloride, tosyl chloride,
mesyl chloride and others.
The acylation reagent, either as such or as a solution
in a solvent such as tetrahydrofuran and dimethylsulfoxide
or in a mixture of these solve~ts, may be added to the
solution or suspension containing the aminoglycosidic a~ti-
biotic-zinc complex. The molar quantity of the acylation
reagent added may usually be e~ual to or a little excessive
than the number of the non-complexed amino groups with which
the acylation reagent is to react. In so.me cases,however,
the molar quantity of the acylation reagent added may be up
to a molar quantity o~ about 3 times higher than the number .
.
.
~ ~ 3
- 22 -
of the non-complexed amino groups. The acylation reagent
may be added ei~her at once or in porti.ons .510wly over a
duratlon of 2-3 hours, though it may usually be added over
a time of 30 minutes to 1 hour. The acylation may be con-
ducted at a temperature of -20C to 100C but may normally
be e~fected at a temperature ranging from 0C to ambient
temperature. In some ca~es, the reaction tem~erature may
be kept low at the time of addition of the acylation reagent
and be -then elevated gradually as the acylat.ion proceeds.
Normally, the acylation reaction may be effected ln situ
in the organic solvent .in which the aminoglycosidic anti
biotic-zinc cation complex was formed. Thi~ acylation of
the zinc complex produces the N-acylated zinc complex, that
is, the complex of zinc cations with the selectively N-
acylated aminoglycosidic antibiotic derivative.
According to the proce~s o~ the first a~pect of theinvention, the step of the acylation of the aminoglycosidic
antibiotic-zinc cation complex is followed by the step of
removing ælnc cation ~rom the N-acylated zinc complex (namely,
destroying of the zinc complex) to yield the selectively
protected N-acylated derivative of the aminoglycosidic
antiblotic which is free ~rom zinc catlons.
~ or removal of ~inc cation from the N-acylated zlnc
complex, it i~ necessarv to treat the N~acylated zinc complex
with a ~uitable reagent which removes zinc cation ~rom said
N~acylated zinc complex. For this purpose, there are ~any
available method~. The ~ir~t method is to react a zinc-
2, -
precipitating agent, which is capable of converting zinc
cation into a water-insoluble zinc compound ~llch as zinc
sulfide, zinc hyroxide or zinc carbonate while the N-acylated
zinc complex is still reMaining dissolved in the acylation
reaction mixture where the amino~lycosidic antibiotic-zinc
cation complex has been acylated,or a~ter it is trans~erred
into a new solution in a fresh volume o~ an organic solvent
from said acylation reaction mixture.
The zinc-precipita-ting ~gent available in the first
method include hydrogen ~ulfide, an alkali me-tal sulfide
such as sodium sulfide, ammonium sulfi.de; an alkaline earth
metal sulfide such as calcium sulfide and an alkali metal
carbonate such as sodium carbonate or ammonium hydroxideO
In some cases, the removal of zi~c cat.ions from the N-
acylated ~tnc comple~ may be effected merely by addition o~water. According to this ~irst method, addition of the
; ~inc-precipitatlng agent to the solution of the N-acylated
zinc complex brings about a comparatively rapid precipitation
of insoluble zinc compound ~ormed ~rom zinc cations, and the
- 20 precipitate may be removed out by ~iltration. The N acylated
aminoglycosidic antibiotic derivative which then remain i~
the filtrate solution may be recovered by concentratlon of
the solution or by extraction ~rom the solu-tion~ and i~
nece~sary, may be purifled subsequently. For purification,
for example, column chromatography with silica gel $s u~efulO
A second method is (i) ~o concen~ra~e or concentrate to
dryne~s by evaporation of the solvent or (ii) *o dilute wi~h
.
- 2~ -
a liquid diluent the afore~aid acylation reaction mixture
or the new solution oP the N-acylated zinc complex transferred
into the fresh ~olume of the organic solvent so as to give
an oily or solid deposit,concentrate or residue, followed
by recovering the desired N-acylated aminoglycosidic anti-
biotic derivative from said deposit, concentra~e or residue
in any way. The li~uid diluent available in this second
method is water or a such an organic liquid in which the
N-acylated zinc complex as the whole or the N-acylated amino-
glycosidic antibiotic derivative moiety of said N-acylated
zinc complex has no or little soluhility.
According to the aforesaid second method, at first,
the acylation reaction mixture containing the N-acylated
zinc complex (or the new solution of the N-acylated zinc
complex trans~errred into an organic sol~ent) is concentrated
or concentrated to dryness to gi~e the oily or solid-deposit
or residue. When a hardly vaporisable organic solvent such
as demethylsulfoxide etc.~ was employed as the reaction
medium for the N-acylation of the zinc complex, it is possible
that the acylation reaction mixture containing the N-acylated
zinc complex ~s admixed with ~ diluent organic liquid such
as ethylether so that the hardly v~porisa~le organic solvent
medium is dissolved in (or diluted with) the diluent, whereby
a solid or an oil comprising the N-acylated zlnc complex is
deposited therefrom. In these ways, an oily or solid deposit
or residue is obtained, which is normally a mixture composed
o~ (i) the N-acylated zinc co~plex, that is, the complex of
- 25 -
zinc ca-tlonq with the N-acylated amlnoglycosldlc an~ibi¢tlc
derivative, (ii) the N-acylated aminoglycosidic antibiotic
derivative liberated by destroying of the complexing asso~
ciation in a portion of the N-acylated zinc complex due to
the substantial absence of the organic solvent mediu~, (iii)
an amount of the inorganic zinc salt formed by the destroying
of the complexing association in the portion of the N-
acylated zinc complex, (iv) an amount of the zinc salt which
was added initially as an excess and remaining unreacted in
the complexing reaction, and possibly (v) some residual
amount of the organic solvent employed in the preceding
operations.
The above oily or solid deposit or residue (the afore-
said mixture) may subsequently betreated by either of the
procedures (a), (b) and (c) stated hereinunder.
.(a) The oily or solid deposit or residue (the afore-
said mixture) is admixed with water or ~uch a kind of a polar
organic solvent, an aqueous polar organic solvent or mixed
polar organic solven*s which is polar organic liquid(s)
having the effect of destroying the complexing association
of zinc cations in the N-acylated zinc complex present in
said deposit or residue and in which amounts of the zinc
salt liberated and initially present unreacted are soluble
but the desired N-acylated aminoglycosidic antibiotic
derivative Ls insoluble. In this way9 the N-acylated zinc
complex is destroyed to liberate the zinc cations therefrom,
to allow the zinc cat~ons to be dis~olved in and extracted
26 -
out as -the zinc salt with the water or (aqueous) organic
solvent(s) and to leave the desired N-acylated aminoglyco-
~idic antibiotic derivative as an insoluble residue to be
recovered. This resi.due may optionally be purified by
re-dissolution in an organic solvent. The polar organic
solvent available in this procedure (a) includes, for
example~ methanol, ethanol, li~l~id ammonia, ethylamine and
triethylamine. These polar organic solvents and water
serve as the zinc cation-removing reagent, accordingly.
(b) Alternatively, the oily or solid deposit or
residue (the aforesaid mixture) is admixed ~ith such another
kind of polar organic solvent, either anhydrous or aqueous,
which has the effect o~ destroying the complexing associ- -
ation of zinc cations in the N-acylated zinc complex present
in said deposit or residue and in which the liberated
zinc salt is not soluble but the desired N-acylated amino-
glycosidic antibiotic derivative is soluble, whereby the
N-acylated zinc comple~ is destroyed to liberate the
N-acylated aminoglycosidic antibiotic derivative therefrom
and to allow the latter to be dis~solved in and e~tracted
out with said polar organic solvent and hence to be
separated from the ~inc salt which is liberated but re-
maining not dissolved in said polar organic solvent. In
this way, the solution of the desired N-acylated amino-
glycosidic antibiotic deriv~tive in the polar organicsolvent is recovered and, if de~ired, may be purified eg.
chromatographically, ~ollowed by conoentration o~ the
~ 27 ~
purifled solution for isolation of the desired N-acylated
product.
(c) Further alternatlvely, the oily or sollcl deposit
or residue (the aforesaid mixture) a~ obtained in the above-
mentioned seconcl method may be again dissolved as the wholein a suitable organic solvent containing a proportion o~
water, if the whole deposit or residue is soluble or sub-
~tantially soluble in water. The solution so obtalned
may then be sub~ected to a chromatographic procedure during
which the liberated zinc salt and the llberated N-acy'ated
aminoglycosidic antibiotic derivative can be recovered
separately from the solution. We have fou~d that for this
chromato~raphic procedure are useful various kinds of cation-
exchange resins, anion--exchange resin~, ch%late-exchange
resln and water-insoluble high-polymers containing functional
groups capable of combining with a metal, ~uch as chitin
or chitosan. The a~ailable grades of cation-exchange resin
for this purpose include ones containing carboxyl groups
~-COOH~ as the exchange functions, ~nd ones containing
sulfonyl groups (-S03H) as the exchange functions. l~hen
using an cation-excha~ge resin containing carboxylic ~unction~
for the above-men-tloned chromatographic procedure, the
aforesaid oily or solid deposit or residue (the aforesaid
mixture) i5 dissolved in a suitable aqueous organic solvent~
for ex.ample, a mixture of water and me-thanol containing
optio~ally 10% to 90% by ~olume of water or a mlxture of
water and dioxane containing optionally 10% to 90% by ~olume
.
~ ~ 3
- 28
of water, and the re~-~ulting solutlon is charged lnto a
column of said cation~exchange resin. The column is then
wa.qhed well with a further amount of the above-mentioned
aqueous organic solvent, followed by the development using
as the eluent an amoun-t of the above-mentined aqueou~
organic solven-t containing fur-ther a quantity of an acid
or a base. A3 this acid may be used a weak organic acid
~uch as acetic acid, or a diluted inorganic acid such as
dilute hydrochloric acid. As the base may be used ammonium
hydroxide ~or almost cases. The concentration of the acid
or base in the developing solvent (the eluent) may suitably
be 0.01% to 5~ by weight of the developing solvent. The
desired N-acylated aminoglycosidic antibiotic derivative
can be separated from the complexing zinc cations during
the process o~ the development, because the cation-exchange
resin used exerts different adsorptive affinities against
the desired N-acylated aminoglycoside and the zinc cations
so that the ~orce of the former to be associated with the
resin is different from the force of the latter to be
associated with the resin. In thi~ way, the elute can be
collected in fractions so as to give fractions containing
the desired N-acylated aminoglycoside free ~rom the zinc
~alt, which m~y then be concentrated to af~ord the desired
aminoglycosidic antibiotic N-acylated derivative.
When using a cation-exchange resin containing sul~onyl
~unctions for the above chromatographic procedure, the
separation and recovery of the desired N-acylated amino-
.
_ ~9 ~
glyco~ldlc antlbio~ic derivative may be achieved in the same
~ray a~ in the above case, because entirely the same
mechanism i~ involved in the separation of the N-acylated
aminoglycoside ~rom the complexing zinc cations. On the
other hand, when using a weakly or strongly basic anion-
exchange resin for the chromatographic procedure, the
portion of the N-acylated aminoglycoside in the N-acyla-ted
zinc complex which is containing one or more non-acylated
amino group(s) therein is normally not be adsorbed by the
weakly or strongly basic anion-exchange resin owing to the
ionic repellence between them, so that the development of
the anion-exchange resin column with a suitable aqueous
organic solvent permits the N-acylated aminoglycosidic
antibiotic derivative to be elu-ted from the column while
the zinc cations remain in the column.
~ hen the chromatographic procedure is conducted
using a chelate-exchange resin which is able to combine with
zinc cations by the metal-chelating ability of this resin,
a solution o~ the aforesaid oily or solid deposit or residue
(the a~oresaid mixture) in an a~ueous organic solvent is
charged in a column o~ chelate-exchange resin, which i.5 then
developed with a suitable development solvent to allow the
~esired N-acylated aminoglycoside to be eluted preferentially
out of the column, while the zinc cations remain bounded in
the chelate-exchange resin. The water-insoluble high-polymer
containing the functions capable of combining with a metal,
~or example, chitin and chitosan, may be employed in the
~ 30 -
~ame manner a~ when using ~he chelate-exchange resin.
(~) Moreover, a third method is possible, in which
the aforesaid acylation reaction mixt~e in which the acyla-
tion of the zinc complex for pro-tecti.on of the amino groups
wa~ conducted i9 directly charged into a column of a cation-
or anion-exchange resin, chelate-exchange resin, or a water-
insoluble high-polymer con-taining the metal-combining func-
tions, so that the N-acylated zinc complex is adsorbed by
the resin or high-polymer. The column may then be washed
with an aqueous organic solvent~ i~ necessary, and may
subsequently developed with an aqueous organic solvent
containing or no-t containing an acid or a base as mentioned
in the above procedure (c), followed by similar operations
to those of the procedure (c), whereby the removal of zinc
cations from the N-acylated zinc complex as well as the
recovery of the desired N-acylated aminoglycosidic antibiotic
derivative are achieved.
(e) Furthermore, a fourth method is also possible
~or the recovery of the desired N-acylated aminoglycosidic
antibiotic dsrivatiYe, in which method the aforesaid acyla-
tion reaction mixture containing the N-acylated zinc complex
is treated immediately with water by admixing with water,
in case the desired N acylated aminoglycosidic antibiotic
derivati~e itself i~ insoluble or substantially insoluble
in water. ~t2',6'-Tri-N-benzyloxycarbonyldibekacin may be
mentioned as an examples o~ the N acylated amino~lycos1dic
antibiotic deri~ative ~hich is substantially insoluble in
~13~8
waterO In thi~ case; when ~he acyla'cion reaction mixture
containing the N-acylated zinc complex comprislng a ~ub-
stantially water-insoluble N-acylated a~inoglycoside
; derivative is immediately admixed with water, the zinc-
complexing association in the N~acylated zinc complex is
broken to allow the water-insoluble N-acylated aminoglyco-
side derivative to be precipitated as a solid, w~.ile the
zinc sal-t formed from the liberated zinc cations remains
in solutlon, whereby the desired N-acylated aminoglycosidic
antibiotic derivative as a substantially pure product can
be recovered separately frorn the zinc salt.
As be stated in the above, the N-acylationp namely
the amino-protecting reaction is conducted with the zi~c
complex of the aminoglyco.sidic antibiotic in accordance
with the process of the first aspect i~vention, and the
complex of zinc cations with the mono-,-di-, tri- or poly-
N-acylated aminoglycoside deri.vative so formed is such one
in which the zinc cations used are complex-associated with
the structure of the N-acyla-ted aminoglycoside derivative.
There~ore, when the desired N-acylated aminoglycoside
derivative is insoluble or sparingly soluble in water, a
~imple operation of admixing water with the acylation reac-
tion mixture containing the N acylated zinc complex causes
the water-insoluble N~acylatsd aminoglycoside derivative
to be precipita~ed a~ a solid while the liberated zinc
cations are removed -therefrom by disso:Lution in the water
(as ~n the case of the ~ourth method described in the
.
~2
preceding paragraph (e)). The water-lnsoluble precipitate
80 obtained may immediately be employed as an initial
material for subsequent reactions for semi-syn-thetic pro-
duction of a desired final product. More generically~
however, the N-acylated aminoglycosidic antibiotic deriva-
tive itself is often soluble in water or partly soluble in
water, and hence the de~ired N-acylated aminoglycoside
derivative can be recovered only in a considerably lowered
yield if the simple treatment of admixing water immediately
with the acylation reaction mixture is adopted. For these
reasons, better re~ult may rather be obtained when applying
either one of the above-mentioned procedures (b) and (c) in
whi~h the N~acylated zinc complex (that is, the complex of
zinc cations with the N-acylated aminoglycosidic antibiotic
deri~ative formed from the N-acylation reaction) is at first
separated from the acylation reaction mixture~ the N-acylated
zinc complex so separated is then dissolved in water or an
aqueous organic solvent and the resulting solution is further
processed for removal of zinc cations therfrom. Meanwhile,
one of the simple methods of removing zinc cations which
are generally obvious is such ~one in whic~l hydrogen sulfide
or an alkali sulfide is reacted as a precipitating agent
with zinc cations to precipit~te the latter as zinc sulfide
~as one mode of the first method set forth in the above
paragraph (~)). However, zinc sulfide of~en precipitates
a~ colloidal deposit wh~ch is difficult to be filtered
out, and beside~,hydrogen sul~ide and an alkali ~ulfide
.
- 33 ~
ha~e ob~ectionable odor and are not sui-~able for use in com-
mercial working of Jche process. Thus, tYe have made our exten-
sive research in an attempt to provide a practical method of
removing zinc cations from the zinc complex without resorting
on the use of a sulfide, and now we have succeeded in devel-
oping the efficien-t and facile me-tho~s of removing zinc cat-
ions by using the above-mentioned exchange resins or other
polymeric material (as in the case of the procedures (c~ and
(d)). These procedures (c~ and (d) are commercially very
much advantageous and valuable as they are easy to operate,
give high efficiency of the separa-tion of zinc cations and
provide a high yield of the desired N-acylated aminoglyco-
sidic antibiotic derivative.
After all, the above described various methods and
procedures of treating the N-acylated zinc complex with the
zinc cation-removing reagent may be summarized as follow~s:
(i) The complex of zinc cations with the selectively
N-acylated aminoglycosidic antibiotic derivative is once sepa-
rated from the acylation reaction mixture be~ore it is reacted
with a reagent of removing zinc cations from this complex.
(ii) The complex of zinc cations with the selectively
N-acylated aminoglycosidic antibiotic derivative is separated
from the acylation reaction mixture by extraction with an
organic solvent, by evaporating the organic solvent medium
ZS from the acylation reaction mixture or by diluting the
acylation reaction mixture with a diluent organic solvent,
before it i~ reacted with a reagent of remo~ing zinc cations.
~ ~3
- 34 -
(iii) The complex of zinc cations with the selectively
N-acylated amlnoglycosidic antibiotic drivative once separated
is admixed with water or a polar organic solvent, either
anhydrous or aqueous, which ~erves as the zinc ca-tion-
removing reagent. This polar organlc solvent is either such
one in which the zinc salt is soluble bu-t in which the N-
acylated aminoglycosidic antibiotic derivative is insoluble,
or such one in which the zinc salt is insoluble but in which
the N-acylated aminoglycosidic antibiotic derivative i3
soluble.
(iv) The complex of zinc cations with the N-acylated
aminoglycosidic antibiotic derivative once separated is
again dissolved ~holly in anorganic solvent containing a
proportion of water, and the resulting solution is subjected
to a chromatographic procedure using a cation-exchange resin,
an anion-exchange resin, chelate-exchange resin or a water-
insoluble polymer containing functional groups capable of
combinin~ with ametal, which serves as the zinc cation-
removing reagent.
(v) The acylation reaction mixture is direc-tly
passed through a col~unn of a cation-exchange resin, an
anion-exchange re~in, chelate-exchange resin or a water-
insoluble polymer containing the metal-combining functions
for adsorption of the complex of zinc cations with the N-
acylated aminoglycosidic antibiotic derivati~e, and the
column i~ then developed with anaqueous organic solvent
containin~ or not contalning an amount of acid or base,
~ ~ 3
- ~5 -
and the elua~e is collected ln fractlons, followed by
recovery of -the fractions containing the desired selectively
N-acylated amino~lycosldic antibiotic derivative but con-
taining no zinc cations.
(vi) ~hen the desired N-acylated aminoglycosidic
antibiotic derivative is insoluble ~or substantially insoluble
in water, -the acylation reaction mixture is immediately
admixed w~th water, so that said derlvative is precipitated
separately from the zinc salt remaining dlssolved in water.
(vii) The acylation reaction mixture is immediately
treated with hydrogen sulfide, an alkali metal sulfide or
an alkaline earth metal sul~ide which preclpitates zinc
cations as zinc sulfide, or with ammonium hydroxide which
precipitates zinc cations as zinc hydroxide.
In the zinc complex involved in the process of the
first aspect invention, zinc cations are principally complex- -~
ing with l-amino and 3"-amino groups of the aminoglycosidic
antibiotic, and hence the N-acylation of the aminoglycosidic
antibiotic-zinc cation complex *ollowed by the removal of
zinc çations there~rom normally gives the N-acylated amino-
glycosidic antib{otic derivative in which amino and/or
alkylamino groups other than l-amino and ~"-amino groups
are protected by the acyl group. When the N-acylated amino-
glycosidic antibiotic derivative so obtained from the
process of the first aspect invention is then l-N-acylated
with an a-hydroxy~ aminoalkanoic acid in a known manner
as ~et forth ln the a~oresaid U.S. Patent Nos. ~,781,268 and
~!L3~
3,939,143, for instance, followed by removal of the residual
amino-protecting group~ from the resultant l-N-acylated
product 9 there is afforded Q semi-synthetic l-N-acylated
aminoglycosidic antibiotlc which is known as a useful anti-
bacterial agent.
Synthesis of the l-N-acylated aminoglycosidlc anti-
biotics is now described with reference to an illustrative
use of kanamycin A as a starting material. When kanamycin
A is used as the initial material in the process o~ the
first aspect invention, l-amino and 3"-amino groups of
kanamycin A are initially blocked by Gomplexing with zinc
cations upon the ~ormation o~ its zinc complex. Accordingly,
when the kanamycin A-zinc cation complex is a~ylated with
a suitalbe acylation reagent according to the invention or
with another kind of amino-blocking agent, the non-compleY.ed
3-amino and 6'-amino groups of the kanamycin A molecule
can be protected by the acyl group of the acylation reagent
employed or by the other ~ind of amino--blocking group.
After sub~equent removal of the complexing zinc cations from
the N-acylated kanamycin A-zinc cation complex, the resulting
N-acylated kanamycin A derivative is reacted with an acylating
~gent having an acyl group to be introduced into l-amino
group o~ the kanamycin A molecule. Then, this acyl group
reacts only with the unblocked l-amino and 3"-amino groups
of kanamycin A. At this tlme, l-amino group i~ normally
a little more reactiYe than 3"-amino group, so that the
desired l-N-acylated kanamycin A derivative may be obtained
- 37 - -
ln a li-ttle higher yield than the 3"~N-acylated kanamycln A
derivative. Subsequent N-deprotection of the l-N-acylated
kanamycin A derivative so obtained affor(1s the l~M-acylated
kanamycin A as the final desired produc-t. Therefore, when
utilizing the proce~s of the first aspect .tnvention, it will
be obvious that the deslred l-N-acylkanamycin A can be
obtained in a higher yield, a~ compared to when unprotected
kanamycin A or 6'-N-protected kanamycin A is immediately
reacted with an acylating agent for the purpose of l-N-
acylation of kanamycin A. If a kanamycin without any Nprotection is reacted with a l-N-acylating agen-t, it is
found that -there are formed the mixed N-acylated products
containing a very small proportion (usually 1% to a few ~)
of the desired l--N-acylated product.
In ca~ethe process of the first aspect in~ention is
applied to a kanamycin of the aforesaid general formula (III),
some or all of the amino groups other than 1- and 3"-amino
groups o* that kanamycin used are protected to give an N-
acylated kanamycin derivative represented by the following
~ general formula (V):
~3
-- 38 --
CH2-R4a NHR7
R~
HOCH2 0
_O / (V)
H~WV
3" 1H
wherein Rla is hydroxyl group, amino group (-NH2)9 a group
-N~CoR5, or a group -NHCO oR5 or a group -MHS02R6; R4a is
R8
hydroxyl group, a group -NHCoR5 9 a group -N < 5 , a group
R~ gro ~ S~ R6
\Co-oR5 2
-N < 6; R2 and R3 are sach as defined in the general
S2R
formula (III), R7 is a group -CoR5; a group -Co-oR5 or a
group -S02R6; R5 and R6 are as de~ined in the formulae (IVa)
to (IV~); and R8 is alkyl group,especially of 1~4 carbon
atoms.
Thus, in case the process of the first aspect
,
:
_
invention is appl~ed -to a kanamycin, there is usually ob-
talned an N-protected kanamycin derivative of the formula
(V) in which all the amino groups other than the amino and/or
alkylamino groups present at the 1- and 3"-positions of the
lcanamycin molecule are blocked. None-theless, if the acyl
group to be introduced as the amino-blocking group i5 rela-
t1vely large in its steric si~e~ for example~ with t-butoxy-
carbonyl gr~up, or if the molar quantity of the acylation
reagent used is less than the quanti-t~ stoichiometrically
required to acylate all the non-complexed amino groups of
the kana~ycin molecule even though the acyl group of the
acylation reagent employed is of an ordinary size, or if
the acylati.on reaction is stopped at an intermediate stage,
there is obtained such an N~protected kanamycin derivative
in which the ~umber of the acylated amino groups in the
kanamycin molecule is less than in the above case, and then
in particular cases there i~ obtained such a limitedly N-
acylated kanamycin derivative in which 6'-amino or 6'-
alkylamino group is exclusively acylated, owing to tha-t 6~-
amino or 6'-alkylamino group is more reactive than the other
amino groups in the kanamyci~; molecule.
The N-acylated kanamycin derivative of the general
formula (V) is an important intermediate useful in the semi-
synthetic production of various kinds of kanamycin derivatives.
The compound of the ~ormula (V) has an increased value as an
lntermediate material for chemical synthesis, for instance,
particularly when it is involved in a process of producing
.
- 40
ssmi-synthetic 1-N-acylated aminoglycosidic antibiotic~
artlve ~gainst -the kanamycin-resistant bacteria, by acylating
l-amino group of the compound (V) w~th an a-hydroxyl-
~aminoalkanolc acid and then removing the protective groups
from the bloclced amino and/or alkylamino groups of the result-
ing l-N-acylation product.
As an instance, when the intermediate compound (V)
~s to be acylated with an acyl group, eg., with (S)-4-
benzyloxycarbonylamino-2-hydroxybutyryl group, the compound
(V) may be reacted in a suitable organic solvent such as
aqueous tetrahydrofuran with a correspondingly substituted
butyric acid or its equivalent reactive derivative such as
an active ester, for example, N-hydroxysuccinimide ester,
N-hydroxyphthalimide ester or p-nitrophenol ester, whereby
the l-N-acylation product is formed. Subsequently, removal
of the benzyloxycarbonyl group and the protective group (R7)
in the formula (V) from the l N-acylation product may be
effected by a conventinal N-deprotecting technique, eg.,
either by hydroly~i~ with acid or base, or by reduction with
reducing metal, or by catalytlc hydrogenolysi~ with hydrogen,
or by radical reduction with s~dium in liquid ammonia9 to
give a semi-synthetic kanamycin derivative having (S)-4-
- amino-2-hydroxybutyryl group bounded with l-amino group of
kanamycln which i~ active against the resistant bacteria
and i~ represented by the following general gormula (~
: . .
.
- 41 -
R -CH2 NH2
OIH(CH2)2NH2
~ ~ ~ H ~ 1 OH
R3 1
HO-CH2 0
~ \ / (VI)
HO
H
wherein Rl, R2, R3 and R4 each have the s~me meanings as
defined in the formula (III). In the above process, gener-
ally, an N-protected derivative of an a-hydroxyl-~ -amino-
alkanoic acid of the forMula (VII):
HOOCCH( CH2 )nNE12
1H (VII)
wherein n is an integer of 1, 2 or 3 may be employed in
stead of the (S)-4-benzyloxycarbonylamino-2-hydroxybutyric
acid, to give a l-N-((S)-~-hydroxy~ aminoalkanoyl)-
kanamycin derivative.
.
~2 ~
Furthermore, the invention includes further a
process for the production o~ a selectively protected N-
acylated derivative of an aminoglycosidic antibiotic
comprising a 6-0-(3"-amino-or 3"-alkylamino-3"-deoxy-
S glycosyl)-2-deoxystreptamine moiety possibly having a 4-
O-(aminoglycosyl) group in which derivative all the
amino groups (including 3"-amino group) other than 1-
amino group of the aminoglycoside molecule are blocked
or protected by same or different acyl gxoups.
According to the process of the first aspect
invention (hereinafter sometimes called "zinc-complexing"
process), it is feasible to prepare such a selectively
but partially protected N-acylated derivative of the
aminogly~osidic antibiotic in which derivative all the
amino groups other than the two, l-amino and 3"-amino
(or 3"-alkylamino) groups of the aminoglycoside
. molecule are protected by an acyl group and hence 1-
amino and 3"-amino (or 3"-alkylamino) groups are
remaining unpr~tected. Ev n when this partially
2~ protected N-acylated aminoglycosidic antibiotic derivative
is reacted with an ~-hydroxy-~-aminoalkanoic acid or its
.reactive equivalent for the purpose of effecting the l-N-
acylation as mentioned above, it is actual that there
are yielded mixed acylation products comprising (i) ths
l-N-acylated product where only l-amino group of the
aminoglycoside molecule has been acylated with the ~-
hydroxy-~-aminoalkanoic acid, (ii) the 3"-N-acylated
~3~6~8
- ~3 ~
product where only 3"-amino (or 3"-alkylamino) group has
been acylated, (iii) both l-amino and 3"-amino (or 3"-
alkylamino) groups have been acylated, and (iv) the
unreacted material where none of 1- and 3"-ami.no (or
3"-alkylamino) groups have been acylated. In order to
obtain the ultimately desired l-N-acylation product
from the above mixed acylation products, therefore, it
is always necessary to carry out an additional step in
which the l-N-acylation product is isolated therefrom
by chromatography or by any other isolation method. As
the l-amino group is fortunately more reactive than the
3"-amino (or 3"-alkylamino) group, actual yield of the
desired l-N-acylation product usually is about 40~ to
60% and exceeds a theoretically maximum yield of 25
lS which would be calculated with assuming that the
reactivity of 1- and 3"-amlno (or 3"-alkylamino) groups
should be entirely equal to each other. Nonetheless,
even if the reaction conditions for the l-N-acylation
are adjusted to best ones, it is inevitable that~the
undesirably N-acylated products are by-formed, and
always it needs an additional step t`o remove the
undesired N-acylated by-products by subjecting the
mixed acylated products carefully to a column chromato-
graphy.
In order to eliminate this disadvantage, it is
obviously required to prepare such a selec~ively
protected N-acylated derivative of the aminoglycosidic
z~
- ~4 -
antibiotic in which all the amino groups other than 1-
amino group have been protected. In order to meet this
requirement, we have made further research in an
attempt to provide a process which is able to selectively
protect 3"-amino (or 3"-alkylamino) group of the
selectively but partially protected N-acylated amino-
glycosidic antibiotic derivative containing free 1- and
3"-amino groups as obtainded Erom the above-described
"zinc-complexing" process, while l-amino yroup is
remaining unblocked.
As a result, we have now succeeded to find out
that when the partially protected N-acylated amino-
glycosidic antibiotic derivative as obtained from the
"zinc-complexing" process is reacted with an acylating
agent selected from formic acid esters, dihalo- or
trihalo-al]~anoic acid esters, N-formylimida~ole, 3"-
amino or 3"-alkylamino group can preferentially be
acylated for the blocking purpose without acylating 1-
amino group. This selective 3"-N-protecting process may
be combined with the above-described "~inc-complexing"
process ~ie., the process of the first aspect invention)
so that there is produced in a facile and efficiPnt way
such a selectively protected N acylated derivative of
the aminoglycosidic antibiotic comprising a 6-0-(3"-
amino- or 3"-alkylamino-3"-deoxyglycosyl)-2-deoxystrept-
amine moiet~ in which dexivative all the amino groups
other than l-amino sroup of the aminoglycoside molecule
, ~ ' , '
~ .
.,,
- ~5 -
have been protected selectively with same or different acyl
groups. In the combination of the "zinc-complexing"
process with the selective 3"-N-protecting process, an
advantage is obtained that the ultimately desired l-N-
unprotected but other N-fully-protected derivative of
the aminoglycosidic antibiotic can be produced from the
parent aminoglycosidic antibiotic material in an overall
yield of 70% or more. When this l-N-unprotected but
other N-fully-protected derivative is employed for the 1-
N-acylation o the aminoglycosidic antibiotic, there is
provided a further advantage that the undesirably N-
acylated products are substantially not by-formed, so
that recovery and purification of the~ desired l-N-
acylation product is very facilitated.
According to the second aspect of this invention,
therefore, there is provided a process for the production
of a selectivelv protected N-acylated derivative of an
aminoglycosidic antibiotic comprising a 6-0-(3"-amino-
or 3"-alkylamino-3"-deoxyglycosyl)-2-deoxystreptamine
moiety optionally having a 4-0-(aminoglycosyl) group in
which derivative l-amino group of the deoxystreptamine
moiety is unprotected but all the other amino groups in
the aminoglycoside molecule are protected with same or
different acyl groups; the process comprising a step of:-
(a~ reacting an alkanoic acid ester of the
formula (VIII):- .
~3~
- 46 -
R C-~ (VIII)
wherein Ra is a hydrogen atom or a dihaloalkyl or
trihaloalkyl group of 1-6 car~on atoms, and Rb is an
al~yloxy group of 1-6 carbon atoms, an aralkyloxy group,
S especially benzylo~y group, an aryloxy group, especially
phenyloxy group, or an N-formylimidazole as the
acylating agent in an inert organic solvent with a
partially protected N-acylated derivative of-the aminoglycosi
dic antibiotic in which l-amino and 3"-amino or 3" alkyl-
.amino groups are unprotected and all the other amino groups are
protected with an acyl group as the amino-protecting
group, to effect selective acylation of 3"~amino or 3"-
alkylamino group of the partially protected N-acylated
derivative with the acyl group RaCO- of said acylating
agent and thereby give the desired l-N-unprotected and
other N-fully-protected derivative of the aminoglycosidic
antibiotic.
The aminoglycosidic antibiotics which are
available in the process accordin~ to the second aspect
of this invention are the same as these available in the
process of the first aspect invention and mentioned
hereinbefore.
. Embodiments of the process according to the second
aspect of the invention are now described more fully.
The partially protected ~-acylated aminoglycosidic
antibiotic dexivative which is to be reacted with the
" ' "' ' '
..
.
~13~
- 47 -
acylatin~ agent of the formula (VIII) according to the
second aspect of the invention and of which all the
amino c~roups other than l-amino and 3"-amino (or 3"-
alkylamlno) groups i.n the aminoglycoside molecule are
protected may be such one which i.s produced the
af~esa.id "zinc-complexing" process according to the
first aspect invention. ~ccordingly, the acyl group
originally present in the partially protected N-acylated
I am.inoglycosidic antibiotic derivative used in the second
aspect invention is the same as the acyl group(R5CO-,
R5OCo- or R6S02- group in the formula IVa e) of the
acylation reagent employed in the first aspect invention
and generally may be an alkanoyl group, an aroyl group,
an alkoxycarbonyl group, an aralkyloxycarbonyl group, an
aryloxycarbonyl group, an alkylsulfonyl group, an
aralkylsulfonyl group or an arylsulfonyl group known as
the conventional amino-protecting group. Moreover, the
partially protected N-acylated aminoglycosidic antibiotic
derivative employed as the start.ing material may also be
such one which has been prepar~d by the aforesaid
Nagabhushan et al's method according to U.S. patent No.
4,136,254.
In carrying out the process of the second aspect
invention~ the partially protected N-acylated amino-
glycosidic antibiotic derivative having the unprotected1 and 3"-amino (or 3"-alkylamino) groups i5 used as the
starting material and is dissolved or suspended in an
- ~8 ~
,
appropriate inert organic solvent. To the resulting
solution or suspension is added an alkanoic acid ester
of the formula (VIII) or N-formylimidazole as the
acylating agent in an amount which is at least e~uimolar
to the starting material used. The inert organic solvent
may preferably be such one which sllows a high dissolution
power for the starting material, for example, dimethyl-
sulfoxide, dimethylformamide and hexamethylphosphoric
triamide, but it is possible to use tetrahydrofuran,
dioxane, acetonitrile, nitromethane, sulfolane, dimethyl-
acetamide, chloroform,dichloromethane, methanol,
ethanol, n-butanol and t-butanol, as well as aqueous ones
of these solvents. Benzene, toluene~and ethylether may
be used as the reaction medium solvent, though these are
not very suitable as these bring about poorer yield of
the desired product. With the acylating agent of the
formula (VIII), Ra may preferably be a ~ihaloalkyl or
trihaloalkyl group, particularly dichloromethyl,
trifluoromethyl or trichloromethyl,and Rb may preferably
be an alkyloxy group such as methoxy or ethoxy. When Rb
is an aryloxy group, it may be phenoxy. Particular
examples of the acylating agent (VIII) include methyl
formate, ethyl formate, butyl formate, benzyl formate,
phenyl formate, methyl dichloroacetate, methyl
trichloroacetate, phenyl trichloroacetate, methyl
trifluoroacetate, ethyl trifluoroacetate and phenyl
trifluoroacetate. Using this class of the acylatin~
.
.
~3~Z~3
~ ~9 _
agent, 3"-amino group of the startiny material can
preferentially be formylated, dichloroacetylated,
trichloroacetylated or tri1uoroacetylated. Trifluoro-
acetic acid ester, especially ethyl trifluoroacetate is
most preferred. This class of the ac~l group is
advantageous in that it is very easily removable in the
subsequent N-deprotecting step by a conventional
deprotection method. If the alkanoic acid alkyl ester
of the formula (VIII) is not employed as the acylating
agent but in stead thereof a corresponding alkanoic acid
anhydride or an active ester thereof such as the N-
hydroxysuccinimide ester is employed for the acylation
process tnot in accordance with the second aspect
invention), the selective acylation of 3"-amlno group
cannot be achieved but there is involved by-formation of
l-N-acylated product and/or formation of mixed acylation
products mainly comprising the l-N-acylated product. It
is worthy of attention that the aimed selective acylation
of 3"-amino group cannot be then achieved when using an
acid anhydride or active ester of the same alkanoic acid
for the acylating agentO
The acylating agents of the formula (VIII)
available in the second aspect invention are different
in reactivity and their reactivity are in a wide range
of from "strong" to "weak". When an acylating agent of
a strong reactivity is employbd, the acylating reaction
may he conducted for a short reaction time under cooling.
.
~.~.3~
While, when an acyla~ing agent of a weak reactivity is
employed, the acylating reaction may be effected either
under heating or for a prolonged reaction time. In
general, however, the reaction temperature may suitably
be in a range of -30 to -~-120C and the reaction time
may appropriately be in a range of 30 minutes to 24
hours or even to 4~ hours.
The desired selectively 3"-N-acylated product so
obtained may be recovered from the reaction mixture in a
known manner, for example, by evaporation of the solvent
or by precipitation with addition of water, if necessary,
followed by further purification of the product.
The reaction mechanism by which the selective 3"-
N-acylation can be achieved according to the process
of the second aspect of the invention is not yet fully
elucidated. A possible interpretation is that the
acylating agent of the formula (VIII) acylates at first
a hydroxyl group of the starting material to form an
ester product intermediately and this O-esterifying acyl
group is then shifted or migrated to an amino group
(corresponding the 3"-amino or 3"-alkylamino group in
the case of the present process) when this amino group
is neighboring to the esterified hydroxyl intermediately
formed, whereby the acylation of said amino group is
resulted in. If this assumption is followed, it is
possible to explain the reason why the l-amino group
which has no neighboring hydroxyl group cannot be
, ' ' :
.
.
~ 3
- 51. ~
acetylated in the process of ~he second aspect invention.
Besides, there is a fact that the intermediate ester
product cannot be obtained ~hen the trifluoroacetylation
or formylation is conduc~ed acco~ding to the process of
the second aspect invention. Reason why the ester
product cannot be recovered upon the trifluoroacetylation
or formylation, is probably that the O-trifluoroacetyl
group or O-formyl group is instable and that an amount of
the instable O-acyl group which has not undergone the
shifting to the amino group (namely, the known O ~ N
acyl-migration) is removed from the acylated hydroxyl
group in the course of recovery and purification of the
3"-N-acylation product so as to restore the free
hydroxyl group. However~ this invention isnot limited
to the above interpretation of the reaction mechanism
involved in the present process. Anyhow, it seems that
amongst the compounds which are available as the
acylating agent of the formula (VIII) according to the
second aspect of this invention, such ones are more
suitable for the purpose of the second aspect invention
if they have an acyl group which is likely to give a more
instable ester product when this acyl group is trans-
formed into an O-acyl group by ~eacting with hydroxyl
group and thus giving the ester product. Meanwhile,
it is very interesting to notice that when the process
of the second aspect invention is carried out using in
stead of the N-ormylimidazol an N-alkanoyl-imidazole
52 ~
such as N-ace~yl-imidazole, N-propionyl-imidazole and N-
butyroyl-imidazole, the 3"-amino or 3"-alkylamino group
of the partially protected N-acylated aminoglycosidic
antibiotic derivative is not acylated but a hydroxyl
group neighboring to said 3"-amino or 3"-alkylamino
group can be esterified by the alkanoyl group of the N-
alkanoyl-imidazole employed to give an intermediate O-
esterification product. When this O-esterification
product or the whole reaction mixture containing this
O-esterification product is subse~uently treated with an
alkaline reagent such as ammonium hydroxide at ambient
temperature, the O-esteriEying alkanoyl group is caused
to shift or migrate to the neighboring 3"-amino or 3"-
alkylamino group, resulting in a selective acylation
and hence protection of the 3"-amino or 3"-alkylamino
group. Thus, the reaction mixture from the reaction of
the partially protected N-acylated aminoglycosidic
antibiotic derivative with an ~-alkanoyl-imidazole is, at
first, not found to contain the desired 3"-N-acylated
product, but from said reaction mixture can be recovered
the desired 3"-N-acylated product only after the
- reaction mixture has been made alkaline by treating with
an alkaline reagent such as aqueous ammonia (see
Example 71 given hereinafter).
As a valuable application of the processes of the
first and second aspects of this invention, it is
possible to provide a high-yield process for the
~31~
- 53 -
production of the l-N-acylated aminoglycosidic antibiotic
which is known semi-synthetic antibacterial agent.
Thus, this invention further includes a process of
producing a l-N-(~-hydroxy-~-aminoalkanoyl) aminoglyco-
sidic antibiotic starting from the parent aminoglycosidicantibiotic, the process comprising a combination of the
step of preparing by the aforesaid "zinc-complexing"
process of the first aspect invention such a partially
proteeted N-acylated aminoglycosidie antibiotie
ln derivative in which l-amino and 3"-amino or 3"-alkylamino
groups are unprotected and all the other amino groups are
protected; the step of preparing the l-N-unprotected and
; other N-fully-protected derivative by the selective 3"-
N-aeylating process of the seeond aspect invention, the
step of acylating l-amino group of the l-N-unprotected
and other N-fully protected derivative obtained from
the preceding 3"-N-acylation step, with an a-hydroxy-~-
aminoalkanoic acid, especially 3-amino-2-hydroxypropionie
: aeid (isoserine) or 4-amino-2-hydroxybutyric acid; and
finally the step of deprotecting from the l-N-acylation
product so obtained.
More particularly, according to the third aspeet
of this invention, khere is provided an improved proeess
of producing a l-N-(~-hydroxy-~-aminoaikanoyl) derivative
of an aminoglycosidie antibiotic eomprising a 6-0-(3"-amino-
or 3"-alkylamino-3"-deoxyglycosyl)-2-deoxystreptamine
moiety having optionally a 4-0-(aminoglycosyl) group,
- 5~ -
the process compri.sing the consecutive steps of:-
(a) reacting zinc cations with the aminoglyco- -
sidic antiblotic in an inert or~anic solvent to produce
the complex of zinc cations with the aminoglycosidic
antibiotic,
(b) reacting an acylation reagent having an acy
group to be introduced as the amino-protecting group,
with the aminoglycosidic antibiotic-zinc cation
complex formed in the above step (a) in situ in the
inert organlc solvent, to produce a complex of zinc
cations with the selectively N-acylated derivative of
the aminoglycosidic antibiotic having the initially
non-complexed amino groups acylated,
(c) reacting the selectively N-acylated amino-
glycosidic antibiotic derivative-zinc cation complex
obtained in the above step (b), with a reagent which
removes zinc cations from the N-acylated zinc complex,
to give a partially and selectively protected N-acylated
aminoglycosidic antibiotic derivative which is free
from zinc cations and in which l-amino and ~"-amino or
3"-alkylamino group are unprotected but all the other
amino groups in the aminoglycoside molecule are
protected by the acyl group,
(d) reacting the partially and selectively
protected N-acylated derivative ~btained in the above
st.ep (c) with an alkanoic aci.d ester of the formula
(VIII): .
- 55 -
RaC~Rb (VIII)
O
wherein Ra is a hydrogen atom or a dihaloalkyl or
trihaloalkyl group o 1-6 carbon atoms and Rb is an
alkyloxy group of l-6 carbon atoms, an aralkyloxy
group of 1-6 carbon atoms, particularly benzyloxy group
or an aryloxy group, particularly phenoxy group, or
N-ormylimidazole as the acylating agent in an inert
organic solvent to selectively acylate the 3"-amino or
3"-alkylamino group with the acyl group RaCO- of said
acylating agent and thereby to give the l-N-unprotected
and other N-fully-acyla~ed-protected derivative of the
aminoglycosidic antibiotic in which àll the amino
groups other than 1-amino group are protected with
: acyl group,
15(e) reacting the l-N-unprotected and other N-
fully-protected derivative obtained in the preceding
step (d) with an ~-hydroxy-~-aminoalkanoic acid of
the formula (IX): .
; HOOC-CH(CH2)mNH2 (IX)
H
wherein m is 1 or 2 or an equivalent reactive derivative
ther o of which the amino group is either unprotected
or protected, to acylate 1-amino group of said l-N-
unprotected derivative,
tf) and t ~ removing the residual amino-
protecting groups fxom ~e l-N-acylation product obtained
- 56 -
in the above step (e) hy a conventional deprotecting
method.
We describe below more fully how to carry out the
process of the third aspect of this invention.
The aminoglycosidic antibiotics which are
available as the initial material in the first step (a)
of the present process are the same as those described
hereinbe~ore in respect of the process of the first
aspect of this invention, and the reaction of complexing
zinc cations with the aminoglycosidic antibiotic is
achieved in the same manner as described hereinbefore,
too. The acylation of the aminoglycosidic antibiotic-
zinc cation complex so obtained in the `first step (a)
may be effected in the second step (b) of the present
process in the same way as described hereinbefore in
respect of the process of the first aspect invention.
The removal of zinc cations from the selectively ~-
acylated aminoglycosidic antibiotic-zînc cation complex
so obtained may be conducted in the third step (c) of
2~ the present process in various ways as described before,
whereby there is obtained a partially and selectively
protected N-acylated aminoglycosidic antibiotic
derivative which is free from zinc cations and in which
l-amino and 3"-amino or 3"-alkylamino groups are
unprotected but all the othex amino groups in the
aminoglycoside molecule are blocked with the acyl
group of the acylation reagent employed in the step (b)
~3~
- 57 -
of the present process. This partially and selcctively
protected N-acylated derivative of the aminoglycosidic
antibiotic is then reacted with an alkanoic acid ester
of the formula (VIII) or N-formylimidazole in the step
S (d) o the present process in the same manner as
described hereinbefore in respect of the process of the
second aspect of this inventlon, to obtain the selective
3"-N-acylation of the partially N-protected aminoylycosi-
dic antibiotic derivative without acylation of 1-amino
group thereof.
In the fifth step (e) of the present process,
the l-N unprotected and other N-fully-protected
dexivative of the aminoglycosidic antlbiotic obtained in
the preceding step (d) is reacted with an ~-hydroxy-~-
~minoalkanoic acid of the formula (X), particularly 3-
amino-2-hydroxypropionic acid (as DL-isoserine, D-
isoserine or L-isoserine) or L-4-amino-2-hydroxybutyric
acid to acylate l-amino group of th~ aminoglycosidic
antibiotic with the 3-amino-2-hydroxypropionyl or 4-
amino-2-hydroxyburyryl group. This l-N-acylation may be
conducted qenerally as described in the specification of
U.K. patent No. 1,426,908 or U.S. patent No, 4,001,208
according to any known method of synthesis of amides by
reacting the protected aminoglycosidic antibiotic
derivative with an isoserine or L-4-amino-2-hydroxybutyric
acid, either in its free acid form or in the form o
its reactive equivalent such as an active ester, eg. the
1~3~:;Z8
_ 5
dicyclohexylcarbod~mide ester, mixed acid anhydride, acid
azide in an inert organic solvent such as dioxane,
dimethoxyethane, dimethylformamide, tetrahydrofuran
or aqueous ones of these solvents. Isoserine and L-4-
amino-2-hydroxybutyric acid may be such ones of which
amino group has been blocked with an amino-pro~ecting
group. Suitable amino-protecting group for this
purpose may be the same as or different from that one
which was used in the l-N-unprotected but other N~
fully-protected aminoglycosidic antibiotic derivative
to be l-N-acylated. t-Butoxycarbonyl yroup is a
preferred amino-protecting group, as it is readily
removable by treating with a dilute acid such as aqueous
~rifluoroacetic acid, aqueous acetic acid and diluted
hydrochloric acid. Benzyloxycarbonyl group which is
removed by conventional catalytic hydrogenolysis over
palladium or platinum oxide catalyst, as well as
phthaloyl group which is easily removed by hydrolysis
with hydrazine are very convenient as the amino-
protecting group to this end.
The acylating reaction in the l-N-acylation step
(e) of the process of the fourth aspect invention may
preferably be conducted in an aqueous organic solvent
using an active ester of the ~-hydroxy-~-aminoalkanoic
acid (X~. The suitable active ester may be N-
hydroxysuccinimide ester of isoserine or L-4-
benzyloxycarbonylamino-2-hydroxybutyric acid, and this
~3~
- 59 -
active ester rnay be employed in a ~uantity of 1 to 2 mol.,
favorably o 1 to 1.5 mol. per mol. of the aminoglycoside
to be l-N-acylated. The water~n~scible organic solvent
for use in the reaction medium may preferably be
dioxane, dimethoxyethane, dimethylforrnarnide, tetra-
hydrofuran.
Subsequently to the above step (e), the N-
deprotection step (f) of the present process is carried
out to remove all the residual amino-protecting groups
from the l-N-acylation product obtained in the above
step (e). The removal of the xesidual amino-protecting
group may be achieved by a conventional N-deprotecting
technique. Such a residual amino-protecting gxoup which
is of an alkoxycarbonyl type may be removed by hydrolysis
with an aqueous solution of trifluoroacetic acid or
acetic acid or with a diluted acid solution such as
dilute hydrochloric acid. Such a residual amino-
protecting group which is of an aralkyloxycarbonyl type,
for example, benzyloxycarbonyl is readily removed by
conventional catalytic hydrogenolysis. When all the
residual amino-protecting groups are removed from the
l-N-acylation product of t'ne step ~e) of the present
process, the desired l-N (2-hydxoxy-3-aminopropionyl)-
or l-N-(2-hydroxy-4-aminobutyryl)-aminoglycosidic
antibiotic is obtained in a high yield.
Examples of the l-N-(a-hydroxy-~ aminoalkanoyl)-
aminoglycosidic antibiotic which is produced by the
~3~
- 60 -
proeess of the fourth aspeet invention are listed below.
(1) 1-N-(L-4-amino-2-hydroxybutyryl)-
kanamycin A
(2) 1-N-(L-4-amino-2-hydroxybutyryl)-3'-
deoxykanamycin A
(3) 1-N-(L-4-amino-2-hydroxybutyryl)-3',4'-di-
deoxykanamyein A
(4) 1-N-(L-4-amino-2-hydroxybutyryl)-tobramycin
(5) 1-N-(L-4-amino-2~hydroxybutyryl)-dibekaein
(6) 1-N-(3-amino-2-hydroxypropionyl)-dibekaein.
Another applieation of the proeesses of the first
and second aspects of this invention is to produee l-N
alkyl aminoglycosidie antibiotie from the all N-
acylated aminoglyeosidie derivatives eontaining
unproteeted l-amino group, and an example of this
applieation is to produce netilmiein or its l-N-alkyl-
analogues from sisomicin by alkylation with a lower
aliphatie aldehyde and eyanoborohydride.
. ' ' ~ .
~3~6~3
Thl~ inventlon i~ further lllustrated but not limited
by the followln~ Examples.
ample 1
Preparation Or ~ ~ 6~-di-N-benzyloxyca~bonyllcanamycin A
(i) 2.0g (4.13 m mole~) of kanamycin A (free base) was
suspended in a mixture of dimethylsulfoxide (50 m~) and
tetrahydrofuran (20 m~) and 4g (18.1 m moles) of zinc (II)
acetate dihydrate wa~ added to the suspen~ion, followed
by agitation at room temperature until the reaction mixture
formed a homogeneous solution. It took about 4-5 hour~ for
a zinc complex of kanamycin A to be formed and dissol~e.
~he resultant solution was then cooled to 0C, to which
was slo-~ly added over about one hour a cooled solution
(at 0C) of 2.37g (9.5 m moles) of N-benzyloxycarbonyloxy-
o
succinimide (C6H5CH20C00-N ! ) dissolved in a mixture
O
(40 mb) of tetrahydrofuran-dimethrlsulfoxide (1 : 1 by
volume). The reaction solution was allowed to stand at
ambient temperature for 4 hour~ during which the zinc
complex of kanamycin A had undergone benzyloxycarbonylation
(the acylation according to the first aspect in~cntion).
A sample taken from the reaction solution thus
obtained was subjected to silica gel thin layer chromatography
using as developlng solvent the lower liquid pha~e of a
mixture of chloroform-methanol-28% aqueous ammonia
(1 t 1 s 1 by ~olume), which g~a a main spot of the desired
.
, ' .. : ' : . ' :,
. ,~ ' ' .
- ~2 ~
,
product at ~r=0~23 and two or three minor ~pots attributted
to by-products at upper points.
~ii) The above reaction solution wa4 poured into 500 m~
of ethyl et}ler ~md the oil separated was washod several
times with further volume~q of ethyl ether to afford 8.8g
of a tllick syrupy material.
(iii) Remov~l of zinc cation from tlle syrupy material
(substantlally comprising the zinc complex) was performed
by either of the following different procedures:
(A) Procedure using a weakly acidic cation-exchange resin
carrying carboxyl group (~COOH) as functional group (com- -
mercially available as "Amberlite"~CG 50 resin (H~ form)
from Rohln & Haas Co~, U.S.A~)
60 me of Amberlite~CG 50 resin (H~ form) was
preliminarily saturated thoroughly with a mixture of water-
dioxane (2 : 1) and then packed in a column. A solution
of lg of the syrupy substance dissol~ed in 20 mQ of water-
dioxane (1 : 1) was passed through the column, which was
then developed with water-dioxane (2 : 1) containing 1~
acetic acid. The eluate was collected in fractions. The
desired 3,6'-di-N-benzyloxycarbonylkanamycin A which was
positive to ninhydrin reaction was first eluted out of the
column~ and zinc acetate which was sensitive to coloration
by diphenylcarbazide was then eluted out. The fractions
containing the desired product were combined together and
concentrated to drynes~ The re~idue was washed with cthyl
ether to give 340 mg (81~) of 3,6~-di-N-bcnzyloxycarbonyl-
" ' ' ' '
. ~ ,
63 - ~3~
~anamycin ~ as colorless so:lid. (aj25+76 (c 1, water-
dimethylrormamide~ 1 : 2).
El~mental ancllysl~
Calcd. for C34TI4~N4V15 2CH3C2 2
C, 51,Z3; H, 6 56; N, 6 29
~ound: C, 51.02; H, 6 71; N~ 6.22~
(B) Procedure u~ing a weak cation-exchange re_in bearing
carboxylat~ group as functional group (commercially available
as "Amberlite"~CG 50 resin (NH4+ form) from Rshm & Haas Co.)
lg o* the syrup-like material obtained in the above
Example 1 ~ii) was dissolved in 20 m~ of water-dioxane (l : 1)
and the solution was passed through a column of 6G m~ of
Amberlite~CG 50 resin (NH4+ form) and subjected to linear
gradient elution with water-dioxane (1 s 1) containing 0 to
0,1 N an~onia. No zinc ca~ion ~zs eluted but the desired
product, 3,6'-di-N-benzyloxycarbonylkanamycin ~ was eluted.
The fraction~ of the eluate containing th~ desired benzyJ-
oxycar~onylation product was concentrated to dryness to give
328 mg(~9~) of the desired product as colorless ~olid.
(a)25=+86 (c 1, water-dimethylformamide, 1 : 2).
~ ' .
Calcd. for C34H48N4~5 2 2 3
C~ 52.87; H, 6.30; N, 7.15
i ~ound: C~ 52~50; H~ 6.59; N, 7.00~
(C) Procedure using a cation-exchang~ rcsin bearing stron~ly
acidic functional group ~SOgH (commerclally ;-vailable a~
~Dowcx"~50W X 2 re~in from Dow Chsmlcal Co.)
': ' ' ' ~ ' ' ':
i. - 6l ~ ~ 3~ ~
.
30 m~ Or Dowcx 50W X 2 resin (~1~ form) which had
been immersed in water-d~oxana (2 ~ as packed in a
; column~ through which was then pas~ed a solution of lg of
the syrup-liko material obtalned in ~xample 1 (ii) in 20 m~
of water-dioxane (2 s 1). The column was washed with water-
dioxane (Z : 1) until the offluent from the colu~m showed
neutral nature~ and then linear gradient elution was made
.with water-dioxane (2 s 1) containing 0 to 1 N ammonia.
The eluate fractions containing the desired 3,6~-di~-
benzyloxycarbonylkanamycin A was concentrated to drynessunder reduced pressure to afford 311 mg (84%) of a white
solid which wa~ identical to that obtained in Example 1 (iii)
(B)-
(D) Alternati~e procedure using Dowex 50W X 2
A solution of lg of the syrup-like material obtained
in Example 1 (ii) in 20 m~ of water-methanol (3 : 1) was
charged into a column of 30 m~ of Dowex 50W X 2 (H+ form)
pre~iously w~tted with water-methanol (3 : 1). The column
was well washed with water-methanol (3 : 1), then gradient
elution was made with water-methanol (3 : 1) containing 0 to
6 N hydrochloric acid. The active fractions containing the
desired 3,6'~di-N-benzyloxycarbonylkanamycin A were coIlected
~nd admixed with a strongly basic anion-exchange resin,
Dowex 1 X 2 resin (OH form) in an amount sufficient to make
; 25 the admixture slightly acidic.
The ~dmixture was.flltered and the filtrate was
concentrated to dryne~s to give 285 mg (72%) of the desired
- 65 -
product in the ~orm of dihydrochlorlde. (a)D5~79~ (c 1 J
water-dimethylformamide, 1 : 2).
(~) Procedure using an anion~oxcllange rcsin carrying
strongly basic functi.onal quaternary atmnonium group (commer
cially available as Dowex 1 X 2 resin from Dow Chemical Co.)
A solution,of lg of the syrup-litce material obtained
in Example 1 (ii) in water-dioxane (1 : 1) was placed in
a column of 30 mR of Dowex 1 X 2 resin (OH form) previously
impregnated with water-dioxane (1 : 1)~ and then the column
was developed with water-dioxane (1 : 1) at a relatively
hi~h speed. The eluate fractions containing the desired
product were collected and concentrated to dryness to give
305 mg (84~) of a colorless soli.d which was identical to
that of Example 1 (iii)(B).
(F) Procedure using an anion-exchange resin bearing
weakly basic functional group (commercially available as
Dowex WG~ resin~ a product of Dow Chemical Co.)
lg of the syrup-lilce material obtained in Example 1 '
(ii) was dissol~ed in 20 mR of water-dioxane (2 : 1) and the
solution ~as passed through a column of 50 m~ of Dowex WGR
resin (base form) previously saturated.with water-dioxane
(2 ~ followed by ~lution with water-dioxane (2 : 1).
The desired 3,6~-di-N-benzyloxycarbonylkanamycin A was
eluted.out in some fractions together with a trace of zlnc
cation cntrained. Thc~e fractlons were comblned together
and concentrated to dryness to afford 450 mg of a color-
le~9 solid. The ~olid could be directly u~ed as starting
- 6G ~ 6~t3
materlal for the production of l-N-((S)-4-amino-2-
hydroxybutyryl) kanamycin A according to the l~N-acylation
method of Example 31 given hereinafter~ whorein the trace
of zinc cation remalning in the soLid starting matorial has
5 no adverse in~luence on the acylat:ion reactlon involved in
~xample 3]. ~ -
(G) Procedure using a chelate-exchange resin carrying
weakly acidic functional group (commercially available as
Dowex A 1 resin, a product of Dow Chemical Co,, U.S.A.)
A solution of lg of the syrupy material obtained in
Example 1 (ii) in water-dioxane (1 : 1) was introduced into
a colunn of 50 mR of Dowex A 1 resln which had been saturated
with water-dioxane (1 : 1) containing 1% ammonia, followed
by gradient elution with mixtures of water-dioxane (1 : 1)
containing 0 to 1 N ammonia. The eluate fractions containing
the desired 3~6~-di-N-benzyloxycarbonylkanamycin A which
were eluted only in a later phase as the 0ffluent from the
column9 were combined together and concentrated to dryness
to give 272 mg t74~) of the desirsd product as a white solid.
(H) Procedure using Chitosan (a water-insoluble polymer
containing functional groups capable of combining with a
metal~ commercially a~ailable a~ a product of Toko ~asei
~oyo Co., Ltd. Japan)
100 m~ of Chitosan was thoroughly impregnated with
watér-methanol (3 : 1~ and packed in a column, through
which was then passed a solution of lg Or the syrupy material
obtained in Example 1 (ii) in water methanol 13 ; 1).
67 - ~ ~ 3~
Th~ column WQS ~u~Jccted to development with wat~r-methanol
(3 s 1), ~hen th~ de~ircd 3,6'-di ~-benzyloxycarbonylkanamycin
A was fir.~t eluted and zinc acetate wa~ eluted much later.
The eluate fractions contain;.ng the former were combined and
concentrate~ to dryne~s to leave a re~i~lue, whicll was dis-
solved in water-dioxane (1 : 1) and the solution was placed .in
a column of Amberlite~CG 50 resin (N~ + f`orm) pretreate~l -
with water-dioxane (1 : 1). The column wa~ well wa~hed with
water-dioxane (1 : 1) and therl subjected to gradient elution
with water-dio~ane (1 : 1) conta;.ning 0 to 0.1 N ammonia~
l`ho~e fraction.~ sensitive to ninhydrin reaction were combined
together and c~ncentrated to dryness to give 301 mg (82~) of
a colorless solid which was i.dentical to that obtained in
Example 1 (iii)(B).
(I) A procedure using a high polymer bcaring carboxyl
functional groups (commercially a~ailable as "CM-Sephadex"~
C-25, which is an ion~exchange gel-filtration agent consisting
of a carboxymethyl-substituted dextran gel, a product of
Pharmacia ~ine Chemical Co., Sweden)
A solutio~ of lg of the syrupy material obtained in
Example 1 (ii) in water-dioxane (1 : 1) was passed through
a column of 40 m~ of CM-Sepha~ex~C-25 (~H4~ form) which had
been well qaturated with watar-dioxane (1 : 1), The column
was w~hed wlth 200 m~ of water-dioxane (1 : 1~ and then
3ubjected to gradient alution using water-dioxane (1 : 1)
containing 0 to 0.1 N ammonia, No ~inc cation was eluted
out o~ the column but only the de~ired 396'-dl-N-benzyloxy-
.
- 6~
carbonylkanamycin A ~luted. The eluate was concentrated
to dryne~s to gi~ 303 mg (82%) of a colorless solid identical
to that of Example 1 (iii)(B).
(J) ~ procedure using hydro~en sulficle as zinc-precipitating
agent
lg o~ the syrupy material obtained in Examp]e 1 (ii)
was dissolved in 20 m~ of water-methanol (I : 1~, to which
was then added aqueous ammonia, followed by introduction of
a sufficient amount of hydrogen sulfide. The reaction mixture
containing the zinc sulfide precipitate formed was filtered
on a glass filter which was filled with "Celite"~filter aid,
and the filtrate was concentrated under reduced pressure
to leave a syrupy material~ which ~as well washed with ethyl
ether to give a solid residue. This residue was taken up in
a volume of water-dioxan0 (1 : 1) and tha solu-tion was
chromatographed on a column of 30 mQ of Arnberlite~IRA 900
(OH form~ strongly basic resin, a product of Rohm & Haas Co.)
using water~dioxane (1 : 1) as developing solvent. The
eluate was collected in fractions, and the frac~ions contain-
ing 3,6'~di-N-benzyloxycarbonylkanamycin A were combined
together and concentrated to ~ryness to gi~e 235 mg (64%)
of a colorless solid which was identical to that of E~ample
1 (iii)(B)-
~
500mg (1 03 m mole~) of kanamycin A (fro0 base) wassusp~nded in 15 la~ of dl-cthyliulfoxld-, to whlch wore th~n
'' '.
. . .
- G9
3~
added ll20 m~ (3.09 m llIole~) Or ~inc cI~ioride and 8I,o mg
(6,18 m ~loles) of 80dium ace$ate trihydrate. After stlrring
the mlxture at ambient temperature for 10 hours, to the
mixture containing the kanamycin A-zinc complex formed was
5 slowly added over about ono ilo~Ir a so]ution of G75 mg ( 2 . 27
m moles) of N~benzyloxycarbonyloxyphthalimide
(C6H5-cll20cooN\ J ~ ) di~so/ved ln 10 m~ of dimethyl-
sulfoxide. The resultant mixture was ailo~Jed to qtand at
room temperature for 4 hours.
Subsequently, the reaction mixture was treated in
the same m~lner as described in Example 1 (ii) and (iii)(I)
to yield 598 mg (74~) of 3,6'-di-N-benzyloxycarbonyl-
kanamycin A in the form of colorless solid.
Example, 3
~
600 mg (0.95 m moles) of Icanamycin A tetrahydrochloride
and 150,mg (3.8 m moles) of sodium hydroxide in 15 mQ of
dimethylsul~oxide were agitated for one hour, to which was
then added lg (4.55 m moles~ of zinc acetate dihydrate~
followed by continued agitation for further 5 hours. To the
mixture containing the kanamycin A-zinc complex formed was
added over 30 minutes a ~olution of 545 mg (2.2 m moles) of
N-benzyloxycarbonyloxysuccinimide dissolved in 5 m~ of
dimethylsulfoxide~tetrahydrofuran (1 : l). After agitating
the resultant mixture at ambient tempe3ature overnight~
.
~ ~ 3
- 70 -
ethyl ather wa3 addcd thereto to deposit the N-acylated
zinc complex A8 a preclpltate. The precipitate wa~ then
treated followi~g the ~ame procedure as described in
Example 1 (iii)(H) to give 581 mg (78%) of a colorles~
solid of the titled compound.
xample 4
Preparation of`_~ ~6~-di-N-benzyloxycarbon~lkanamycln A
ti) 500 mg (1.03 m moles) of kanamycin A (free base)
was dissolved in 20 mQ of-a mixture of water-dimethylsulfoxide
(1 : 9), to which were then added lgr (4~55 m moles) of zinc
acetate dihydrate and subsequent]y 590 mg (2.4 m moles) of
N-benzyloxycarbonyloxysuccinimide. After allowing the
mixture to stand at amblent temperature overnight, a great
amount of ethyl ether was added to the mixture, resulting
in separation of a watery ~yrup layer, which was washed
several times with ethyl ether to give a thick syrupy layer.
(ii) The syrupy material thus obtained was dissolved in
water-methanol (3 : 1) and the solution was passed through
a column of 200 m~ of Chitosan. The column was eluted with
water-methanol (3 : 1) and the eluate was collected in
fractions. The fractions positive to ninhydrin reaction
were combined together and concentrated to a small volume.
Tha concentrate was placed into a column of Amberlite CG 50
resin (NH4~ form) and the column was well washed with a
mixture of water-dioxane (l : l) and then subjected to
gradiant alutlon with w&ter-dioxane (1 : 1) containing 0 to
0.1 N ammoniA, The eluate fraction~ containing the clesired
.
.
., , '.
71- 113~
.
product werr~ combinecl togeth~r and concentrated to dr-yne~s
to aff`ord 494 mg (61%) of a colorless solitl which was
identical to t~lat obtained in Example 1 (iii)(B).
~ ation of ~,6_~-di~N-benzy~_xycarbonyllcan.~m~cin ~
500 mg (1.03 m moles) of Ican~mycin ~ (free base) was
dissol~ed in 20 mQ of a mixture of water-tetrahydrofuran
(1 : 3), to which was then added lg (4.55 m moles) of zinc
acetate dihydrate~ followed by addition of 590 mg (2.4 m moles)
of N-benzyloxycarbonyloxysuccinimide. The mixture was
allowed to stand at ambient temperature overnight and the
reaction solution so obtained was concentrated under reduced
preSsure. The residue was pa~sed throu~h a column of 200 m~
of Chitosan and the effluent coming from the column was
subsequently treated in the same way as in Example 4 (ii) to
give 414 mg (51%) of a colorless solid of the titled compound.
Preparation of 3 6~-di-N~benzylo.YycaL9~ o~
(i) 500 mg (1.03 m moles) of kanamycin A (frce base) was
dissolv~d in 15 mQ of a mi~ture of ~ater methanol (1 : 7),
to which was then added 1.5g (Ç.8 m moles) of zinc acetate
dihydrate,followed by addition Or 590 mg (2.4 m moles) of
N-benzyloxycarbonyloxysuccinimide in 7 m~ of tetrahydrofuran.
The mixture was allowed to stand Mt ambient temperature
overnight and the reaction ~olution so obtained was concentrated
under reduced pre~sure. The residue was passed through a
colun~ of 200 m~ of Chitosan and the effluent coming out of
'.
_ 72 - ~ ~3~
tho column was ~ub30querltly trsated in tho same way as in
Examplc 4 (ii) to give 442 mg (55~) of a colorless solid
of the titled compound.
Example 7
Pre~a3;ation o ~ N-ben~y]oxycl ~ ycin A
500 m~ (1.03 m moles) of ]~anamycin A (free base) was
susp~nded in 20 m~ of dimethylsulf`oxide and 272 mg (1.24 m
moles) of zinc acetate dihydrate wa5 added to the suspension.
The mixture was stirred at room temperature for 10 hours
to form a substantially transparent solution, to which was
then added in small portions over about two hours 540 mg
(2.17 m moles) of N-benzyloxycarbonyloxysuccinimide. After
allowing the resultant mixture to stand at ambient temperature
overnight~ a large volume of ethyl ether wa~ added and the
oily material separated was taken off and washed several timQs
with ethyl ether to give a thick SyI~py material.
Silica gel thin layer chromatography of a sample
talcen from the syrupy material using chloroform-methanol-
28% aqueous ammonia (1 : 1 : 1 by volume, lower phase) as
developing solvent indicated the following spots:-
- minor spot at Rf 0.4 of 1,3,6',3"-tetra-N-
benzyloxycarbony1kanamycin A (which~developed a
color by being sprayed with sulfuric acid and
then heatlng);
- ~aint spot at Rf 0.28;
- main spot at Rf 0.23 of the desired product9
3~6~-di-N-benzylo~Tcarbonylkanamycin A;
" :
- ~3 ~
- minor spot at n~ 0.12 of 6~-N-ben~yloxycarbonyl-
kanamycin ~; And
- extremely w~nk spot at ~r of unreacted
kanamycin A.
No ~pot corresponding to tri-N-benzyloxycarbonylkanamycin
A was substantially observed which might appear at Rf 0.28
to 0.4.
The above thiclc syrupy material was dissolved in
water-dioxane (1 : 1) and the solution was passed through a
column of 100 m~ of CM-Sephadex C-25 resin (NHI~ form)
pre~iously wetted with water-dioxane (1 : 1). Subsequently~
the column Wa9 subjected to the elution process in the same
way as described in Example 1 (iii)(I), whereby zinc cation
was removed and the desired product separated from the other
produts to yield 412 mg (51%) of the titled compound as
colorless solid.
By way of comparison, the procedure as mentioned
~ust above was repeated but replacing the zinc acetate
dihydrate by 308 mg (1.24 m moles) of niclcel (II) acetate
Z0 tetrahydrate, with the result that the desired 3,6'-di-N-
benzyloxycarbonyllcanamycin A was obtained as colorless solid
only in a poor yield of 59 mg (7.3%).
~
500 mg (1,03 m moles) of Icanamycin A (free base) was
su~pended in 12 ml of.dimethylsulfoxide and lg (4.55 m moles)
~ ~ .
:; ' ' ' ~
.
~ _ 71~ 3~ ~2 ~
of zinc acet~te dihydrate wa~ added to the sllspension.
The mixture was stirred at room tempcrature until it formed
a homogeneous ~olution, to which was then added over approx,
30 mlnutes a solution of 789 mg (2.6 m moles) of p-
methoxycarboben~,oxy p-nitrophenyl ester
(p-CH30c6H~cH20cooc6~ p-No2) dissolved in 10 m~ of
dimethyl.sulfoxide, The resultant mixture was allowed to
stand overnight at ambient temperature and subsequently
treated in the same manner as in Example 1 (ii) and (iii)(B)
to afford 722 ~g (83%) of` a colorless ~olid of the titled
compound. (a)D5+87 (c 1, water-dimethylformamide, 1 : 2).
Elemental ~
Calcd. for C36H52N417 2 2 3
c, 51.95; H, 6,33; N, 6.64%
Found: C, 51.56; H, 6.41; N9 6.53
Example 9
Preparat.ion of 6s~N-(t-butoxycarbonyl~
kanamy _n A
~ollowing the same procedure as described in Example
8 sxcept that the p-methoxycarbobenzoxy p-nitrophenyl ester
was replacéd by 220 mg (1. 54 m,moles) o~ t-butoxycarbonylazide5
the titled compound was obtained in the form of colorless
solid. Yield 627 mg. (~)D5=~960 (c 1, water-dimet~yl-
formamidet 1 : 2) .
~5 ~ Q
lcanamycin A
, ~.
'" ' , ~ '
- 75 - ~ * 3~
500 In~ 3 m moles) of kanamycin A (free basej ~/as
su~pended in 12 m~ of dinlcthyl.Qulfoxide and lg (4.55 m moles)
of zinc acotate dihydrate wa~ added to the suspen3ion. The
mixture was stirred at room temperature until it formed a
homogeneous solution1 to which wa~ then added a solution of
1,2g (5.1 m mole3) of p~-nitrophenol es-ter of trifluoroacotic
acid dissolved in 10 m~ of dimethylsu~foxide. The resultant
mixture was allowed to stand overnight at ambient temperature
and subsequently treated with ethyl ether as set out in
Example 1 (ii). The ether-in~oluble syrupy material was
further treated in the same way as in Example 1 (iii)(~) to
give 590 mg (70~) of the titled compound in the form of
colorless solid. ~a)25~81 (c 1, water-dimethylformamide,
1 : 2)-
E].emental Analysi~
Calcd, for C~2~I34N413~6 2CH3C2H 2
C9 38.33; H, 5.44; N, 6,88; F, 13.99%
~ound: C~ 38.03; H, 5.48; N, 6,54%
Example.ll
. 20 ~
500 mg (1,03 m moles) of kanamycin A (free base) wasYuspended in a mixture of dimothylsulfoxide (15 m~) and
tetrahydrofuran (5 mb) and lg (4.55 m molss) of zinc acetate
dihydrate was added to the suspension, followed by agitation
at room temperature un-til tho reaction mixture formed a
homogeneous qolution. Tho re~ultant solution was then cooled
to 0C, to which wa~ slo,wly added a covled solution ~at 0C)
.
'
' ,, .
31 ~ 8
~ 76 -
of 400 mg (2.55 m moles~ of phenoxycarbonyl chloride
(C6H50COC~) in 3 mQ of tetrahydrofuran. The reaction solution
wa~ brought to room temperature ovor one hour and then allowed
to stand at tha-t tempe~atllre for 3 hours. Subsequently, the
reaction mixture was treated with ethyl ether as mentioned
in Example 1 (ii) and the ethcr-inso:Luble syrupy material was
further treated by the same procedure as in Example 1 (iii)(A)
~o give 625 ~g (70~) of a colorless solid of the titled com-
pound, (a)D5~73 (c 1, water-dimethylformamide, 1 : 2).
Elemental Anal~sis
Calcd. for C32H44N415 2CH3C2 2
C, 50.11; H, 6.31; N, 6.49
Found: C, 49.77; H, 6.60; N, 6.11
Example 12
Preparation of 3, 6 ' ~di-N-acetylkanamycin A
The reaction mixture obtained by the same procedure
. as in Example 8 except using 260 mg (2.6 m moles) of acetic
anhydride in place of the p-methoxycarbobenzoxy p-nitrophenyl
ester was treated in the same way as described in Example 1
~iii)(A). There was thus prepared 525 mg t72~) of the titled
compound as colorless solid. ~a)D5=+93 (c 1~ water-
dimethylformamide, l : 2).
e~a~
Calcd- for C22H40N413 2CH3C2 2
. C, l~4.19; ~I, 7.13; N~ 7.93%
~ound: C~ 44.20; ~I, 7.07; N, 7,85%
- 7~ -
_eparation of ~6l-cli-N-formylkanamycin Q
500 mg (1.03 m moles) of kanamycin ~ (free base) was
suspendod ln 12 m~ of dirrlethylsulfoxide and lg (4.55 m moleY)
of zinc acetate dihydrate was added to the suspension. The
mixture waR stirr~d at room tempernture until it formed a
homogeneous solution, to which was then added 690 mg (4012 m
moles) of p-~nitrophenylformate (OHCOC6H4-p-N02). The resultant
mixture was allowed to stand overnight at ambient temparature
and subse~uently treated in the same manner as in Example 1
(iii)(H). The fractions positive to ninhydrin reaction were
combined together, bubbled with ~aseous carbon dioxide and
then concentrated to dryness. There was thus obtained 430 mg
(67~) of the titled compound as colo~rless so]id. (a)D5+101
1$ (c 1, water~.
Ana~y~i~
Calcd. for c2oH36N4ol3 H2 3 2
C, 40,64; H, 6~50; N, 9.03%
~ ound: Cl 40.43; H, 6.47; N9 8.83%
Example 14
~. . .
Preparation of 3,6'-di-N-tosylkanamycin A
500 mg (1,03 m moles~ of kanamycin A (free base) was
suspended ln 15 m~ of dimethylsulfoxide and lg (4.55 m moles)
of zinc acetate dihydra$e was added to the suspen~ion. The
mixture was stirred at room temperature until it form0d a
homogeneous Qolution~ to whioh was then added slowly a
solution of 400 mg (2.1 m moles) of tosyl chlorido in 7 m~ of
', ' '- '
:'
113~G~
~ 78 -
tetrahydrofuran. The resultant mixturo was allowed to stand
at amblent temperatllre for one hour, ~ollowed by further
addition of 200 mg of tosyl chlorlde dissolved in 3.5 m~
of tetrahydrofuran. The rcaction mixture was allowed to
stalld for ~`tlrther two 1~OIII`S ~nd then treated by the procedure
identiccll to that described in Example l (ii) and (iii)(A),
affording 270 mg (28~) of a colorles~ solid of the titled
compound. (~)25~680 (c 1, water-dimethylformamide, 1 : 2).
AnalYsi s
C~lcd- for C32T148~415S2 2CH3C 2 2
C~ 46.44; H, 6.28; N, 6.02; S, 6.89
~ound: C~ 46.31; 11~ 5,98; N, 6 31; S, 6.55~
l~here the above reaction procedure was repeated but
omitting zinc acetate, no substantial amount of the colorless
solid was recoveredO
Example 15
methylkanamvcin A
500 mg (1.0 m mole) of 6'-N-methyl-kanamycin A (free
base) was suspended in 12m~ of dimethylsulfoxide and lg
(4.55 m moles~ of zinc acetate~dihydrate was added to the
suspension. The mixture was stirred at room temperature
- until it formed a homogeneous solution, to which was then
added over 30 minutes a solution of 550 mg (2.2 m mole6) of
N-benzyloxycarbonyloxysuccinimide dis~olved in 5 m~ of
dimethylsulfoxide-tetrahydrofuran (1 ; 1), The re~ultant
mixture was allowed to stand o~ornight at ambient temperature
- ,
~ 79 -
and ~ubsequently treated in the ~am~ manner as in ~xampl~ 1
(ii) alld (lii)(A) to afford 720 mg (79%) Or a colorles~
solld of th~ ti.tled compound. (a)25~74 (c 1, water-
dimethylf`orln~de, 1 s 2).
The ~ubsequent treatlllent of the ti.tled compound by
the proceclure si~ilar to that descri~c3d ill Example 31
herelnb~low gave l-N-((S)-4-amlno-2-hydroxybutyryl)-6'-N-
methyllcanalllycln A.
P ~ at~.on of ~,6~-di-N-ben~lox~carbonyl~3'~
dc~ A
.
The titled compound in the form of colorless solld
wa~ obtained in a yield of 765 mg (82~) by repeating the
same procedure as in Example 15 but starting from 500 mg
(1,07 m moles) of 3'-deoxykanamyci.n A (free base) and using
610 mg (2.45 m moles) of N-benzyloxycarbonyloxysuccinimide.
)D5=+76 (c 1, water~dimethylformamide, 1 : 2).
Calcd. for C34I-I48N4014 2CH3C 2 2
20C, 52.16; H, 6.68; N, 6.40%
Found: C~ 51.99; H" 6.75; N, 6.20~
The subsequent treatment of the titled compound by
the procedure similar to that described in Example 31 gave
l-N-((S)-4-amino-2-hydroxybutyryl)-3'-deoxykana~lycin A.
~ le 17
. '
:' " ' :
.
'' . .
:, :,
~3~6~
80 -
The titled compound was obtained in a yield of 737 mg
.(80%) by repeating the san~e procedure as in Example 15 but
starting froln 500 mg (1.04 m moles) of 3'-deoxy-6'-N-
me$hylkan~lnlycin A (free.base) and usin~ 595 mg (2.4 m moles)
of N-ben7yloxycarbonyloxysuccinimide. (a)25~73~ (c 1, water-
dimethylf`ormamide~ 1 : 2).
The subsequent treatmen-t of the titledcolnpound by
the procedure slmilar to that described in Example 31 gave
l-N-((S)-4-amino-2-hydroxybutyryl 3 -3~-deoxy-G~ methyl-
kanamycin A.
Example 18
Preparation of 3 6'-di-N-benz~lox~carbonyl-4'~
deoxykanamycln A
Starting from 500 mg (1.07 m moles) of 4'-deoxykanamycin
A free base (see "Journal of Antibiotics", Vol. 27, pp. 838-
849 (1974); "Bulletin of the Chemical Society of Japan",
Vol. 50, pp. 2362-2368 (1977)), the titled compound in the
form of colorless solid was obtained in a yield of 666 mg
~71%) by the same procedure as in Example 15 except that
580 mg (2.3 m moles) of N-benzyloxycarbonyloxysuccinimide
dissolved in 4 m~ of dimethylsulfoxide.was slo~ly added over
one hour to the homo~eneous solu$ion. (a)D5=~77 (c 1,
water~dimethylformamide, 1 : 2).
Ana lv Y i S'
Calcd- for C3l~ll48N418 2C~3C2 2
C~ ~Z.16; H~ 6068, N, 6,40%
~ound: C, 51.77; H, 6,79; N, 6.31
, - 81 - ~3~
Preparat.iorl of ~,2~,6' tri-N-ben7,~0x~carbollyl-
kan~ll)ycin B
500 In~r (1,03 m moles) of Icanamycin B (free base) was
suspended in a mlxture of 12 mQ of dimethylsulfoxide and
4 m~ of tetrahydrofuran arld 1~ (4,55 m moles) of ~inc acetate
~ihydrate was added to the suspension. The mixture was
stirred at room temperature until it formed a homogeneous
solution, and then cooled to 0C, Into the cooled solution
was slowly added over one hour a cold solution of 825 mg
(3,3 m mole~) of N-benzyloxycarbonyloxy~uccinimide dis~olved
in 10 m~ of tetrahydrofuran~dimethylsulfoxide (1 : 1). The
resultant mixture was allowed to stand at 0C for 2 hours and
then at ambient temperature overnight, whereupon the mixture
was treated in the same ~ay as stated in ~xample 1 (ii) and
(iii)(A) to yield 740 mg (70%) of the titled compound as
colorless solid. (a)D5=~63 (c 1, water-dimethylformamide,
1 s 2).
Analysis
Calcd. for C42H55N$16 2C~3C2 2
C; 53.95; H, 6.40; N~ 6.84%
Found: C~ 53.66; H~ 6,67; N, 6.63%
The subsequent treatment of the titled compound by
the procedure sisnilar to that described in Exarnple 31 gave
1-N-((S)-4~amino-2-hydroxybutyryl.) kanamycin B.
.
., .
.
. .
~z ~ 6~
~x~ 20
~paration of 3,2~6'-trl-N-
benzyloxycarbonyltobramycin
480 mg (1.03 m moles) of tobramycin (free base) was
suspended in 12 m~ of dimethylsulfoxlcle and 1~ (4.55 m mo]es)
of zinc acetate dihydrate was added to ~he susp~nsion. The
mixture was ~tirred at room temperature during one hour to
form a homogeneous solution, to which was then added over
approx. one hour a solutioll of 850 mg (3.4 m moles) of N-
benzyloxycarbonyloxysuccinimide dissolved in 10 m~ of
tetrahydrofuran-dimethylsulfoxide (1 : 1). After allowi~g
the mixture to ~tand at ambient temperature overnight1 the
reaction solution obtalned was treated ~ith a large ~olume
of ethyl ether as mentioned in Example 1 (ii) to give a thick
syrupy material.
The syrupy material wa~ further treated in the same
way as in Example 1 (iii)(A) but using water-dioxane (1 : 2
instead of 2 : 1) to afford 810 mg (78%) of the titled com-
pound as a colorless solid. (a)D5=~65 (c 1, water-
dimethylformamide, 1 : 2).
nalysis
Calcd. for C42~55N5~15 2CH3C2 2
C, 54.81; H~ 6.50; N, 6.95~
Found: C, 54~77; H, 6.71; N, 6,88%
The subsequent treatment of the titled compound by
,
the procedure ~imilàr to that de~cribed in Example 31 ga~e
((S)-4-amino 2~hydroxybutyryl)-tobramycin.
'
- 83 ~ ~ ~3~
Propnration of 3,2',6~-tri-N-ben~,yloxvcarbonYl-
6'-N-methyltobramy~in
The titled compound in the form of colorle~s solid
was obtainec~ ln a yield of 890 mLr (81l~;) by repeating the
same procedure as in Example 20 but starting from 500 mg
(1.04 m moles) of 6~-N-methyltobramycin (fre~ base).
(a)~5=+63 (c 1, water-dimethylformanlide, 1 : 2).
Example 22
Preparation of ~ _
4~-deoxykana~ycin B
Starting from 480 In~ (1.03 m moles) of 4'-deoxy-
kanamycin B free base (see "Bulletin of the Chemical Society
of Japall", VO1D 50~ pp.2362-2368 (1977~), the titled compound
in the form of colorless solid was obtained in a yield of
815 mg (79~) by the same procedure as in Example 20.
(a)D5+63 (c 1, water-dimethylformamide, 1 : 2).
ExamPl e ? ~3
Preparation of ~921 6'-tri-N~
~
600 mg (1.33 m moles) of dibekacin (3 ,4 ?_
dideoxykanamycin B) (free base) was suspended in 15 m~ of
dimethylsulfoxide and the suspension was agitated to form
~ solution, to which was added 1.4g (6.4 m moles) of 2inc
acetate dihydrate,followed by furtller agitation. 'rO the
re~ultant solu~ion was slowly added over about one hour a
solution of l.lg (4.4 m'mole~) of N-benzylnxycarbonyl-
_ 8~ Z8
oxysuccinimide in 12 ml of dimethylsulfoxide, and the mixture
- was allowed to stand at ambient temperature overnight.
Then, a large volume of ethyl etller was admixed with the
reaction solution to separate an oily deposit (mainly comprising
the N-benzyloxycarbonylated dibekacin-zinc complex as
tho desired product and a proportion of dimethylsulfoxide),
which was washed with ethyl ether to give a thick syrupy
material.
This syrupy material was repeatedly washed with water,
whereby the N-acylated zinc complex was destroyed with water
and the liberated zinc cation removed tvgether with the
initially existing excess of zinc acetate. There was thus
obtained l.lg of a water-insoluble solid comprising the
N-acylated dibekacin. The solid was subjected to silica gel
thin layer chromatography Usillg chloroform-ethanol-18~ aqueous
ammonia (1 : 1 : 1, lower phase) as developing solvent to
give a single spot at Rf 0.3, indicating that the solid
consi~ted e~sentially of 3,2~,6~-~ri-N-benzyloxycarbonyl~
dibekacin with a trace of zinc .
~he subsequent treatMent of the titled compound by
the procedure similar to that described in Example 31 gave
l-N-((S~4-amino-2-hydroxybutyryL)-dibekacin.
~or further purification~ the crude product as
obtained above of the titled compound was washed with 3M
ammonia solution to give product without contamination of
zinc lon. (a)D5~71 (c 1~ watcr-dimethylformamide~ 1 : 2).
~. . ..
~L~3~
Pr~pnr~tion of~_~,2~ ! 6~ tri-N-benzyloxvcarbonYl-
500 mg (1.07 m moles) of 6l-N-methyldibelcacin (free
base) and 1.2g (5.1~5 m mol~) of ~inc acetate dihydrate
were di~solv~d in 20 mQ of dimetllylstllfoxldo~ to w~ich was
qlowly added over about 30 minutes ~10 mg (3.6 m moles) of
N-benzyloxycarbonyloxysuccinimi.de. The reaction solution
was allowed to stand at ambient temperature overnight and
subsequently treated in the same way as mentioned in Example
23, affording 910 mg of the tltled compound which was ~ub-
stantially pure.
The subsequent treatment of the titled compound by
the procedure similar to that described in Example 31 gave
1-N-((S)-4-amino-2-hydroxybutyryl)-6'-N-methyldibekacin.
Example 25
Pre~aration of ~ 2'-di-N-benzvloxvcarbonyl-
}canamy~_n C
The titled compound in the form of colored solid was
obtained in a yield of 730 mg (79~) by following the same
procedures as described in Example 1 (i), (ii) and ~iii) A
but starting from 500 mg (1003 m mo~es) of kanamycin C (free
ba~ )25+750 (c 1~ water-dimethylformamide, 1 : 2).
The subsequent treatment of the titled compound by
the procedure similar to that described in Example 31 gave
l-N-((S)-4--amino-2-hydroxybutyryl)-lcanamycin C.
: , '
-
BV 3 ~3,,3~
Preparation of 6'-N-benzYloxYcarbonyllcan~mYcin A
500 m~ (1.03 m moles) of Icannmycin A (free base) was
suspendod in 20 m~ of dimethylsulfo~ide and 0.5g (2.3 m moles)
of zinc acetato dihydrate was adcled to the suspension. The
mlxture was stirred at room temperature until it formed a
homo~eneou~s solution, to which was then aclded 283 Ing ( 1.13
m moles) of N-benzyloxycarbonyloxysuccinimide. The resultant
mixture was allowed to stand overnight at ambient temperature
and subsequently treated in the same manner as in Example 1
(ii) and (iii)(I) to afford 556 mg of the titled compound
as a colorless solid. (a)25=~92 (c 1, water).
Preparation of 6'-N-benzyloxycarbonyldibekacin
Following the procedure as described in Example 25,
382 mg of the titled compound was obtained using 500 mg of
dibekacin (free base), 12 m~ of dimethylsulfoxide, 0.7g of
zinc acetate dihydrate and 305 mg of N-benzyloxycarbonyl~
oxysuccinimide. (a)D5~105 (c 0.5, water)
Example 28
Preparation of ~I?'~6'-tri N-benz~loxycarbonyl-
500 mg ~1.11 m moles) of 3'~4'~dideoxy-3'-enokanamycin
B free base (see "Bulletin of the Chemical Society of Japan",
Vol. 50~ pp. 1580-1583 (1977)) was dissolvcd in 12 m~ of
dimethylsulfoxide, and lg (4.55 m moles) of ~inc acetate
dihydrate was added to the solution, followed by agitation
87~ 28
for one nour. 'rO the resultant ~oluti,on was slowly added
ovcr 30 minutc~ 870 mg (3.49 m moles) of N-benz,yloxycarbonyl-
oxysuccinimid0, After a]lo~ing the mixture to stand at
arllbient telllperature overnight, the reaction so],u-tion obtained
was troated with a large volume of othyl ether as mentioned
in Examplo 1 (ii) to give a thic}c syrupy m-,lterial.
The syrupy material was further treated in the same
way as in Example 1 (iii)(B) but using water-dioxane (1 : 2
instead of 2 : 1) to afford 784 mg of the titled compound
as colorless ~olid. (~)D5+300 (c ls water-dimethylformamide,
1 : 2)-
'Example 29
P paration_of ~,2~ tri-N benzyloxy- -
~ omicin
The titled compound ln the form of colorless solid
was obtained in a yield of 780 mg by following the same
procedure as in Example 28 but starting from 500 mg (1~12
m moles) of sîsomicin (free base). ~)D5=+110 (c 1, water-
dimethylformalllide9 1 : 2).
Example ~
_inS
787 mg of the titled compound was obtained in the
form of colorless solid by following the same procedure as
in Exampl~ 28 but starting from 500 mg of mixed gentamicins
(C, ~la~ C2 0tc.).
.
L3~Z~
EY~ample~ a rcfcrence)
Preparatiorl of ~-N-l(S)-li-amillo-2~ydroxybutvr
kanamy~in ~(amikacirl)
55 I"g(0,062 m moles) of 3,6'-di-N-benzyloxycarbonyl
]canamycin A acotate prepàred as described in Example 1 was
disqolvod in 1.5, mQ of water-tetrahydrofuran (2 : 5)~ to
which were added 13 mg (0.12 m moles) of anhydrous sodium
carbonate and subsequently 23 mg (o.o66 m moles) of N-
hydroxysuccinimide e~ter of (S) 4-benzyloxycarbonylamino-Z-
hydroxybutyric acid. The mixture was allowed to stand atambient temperature for 10 hours. The reaction solution
obtained was concentrated to a small volume and the, concentrate
was taken up in 4 mQ of water-dioxane (1 : 1). A small
amount of acetic acid was added to the solution to render
it weakly acidic~ and the solution was subjected to hydro-
genolysis by passing therethrough hydrogen gas under atrnospheric
pressure for one hour in the presence of palladium black
(for removal of benzyloxycarbonyl group). The re~ultant
reaction solution was filtered and,concentrated and the
concentrate was passed through a column of CM-Sephadex C-25
~NH4~ form) (a product of Pharm~cia ~ine Chemical Co., Sweden).
The colunn was subjected to gradient elution with 0 to 0.5 N
aqueous ammonia. The eluate fractions containing the de~ired
product were combined together and-concentrated to dryness
to give 24 mg (yield 60%) of the titled compound as it~
monocarbonate, who~e physical properti~s and alltibacterial
potency were identical to tho~e of an authentic ~ample.
.
_ f~9 ~
~= .
58 mg (o.o6 m moles) o~' 3,2',G'-tri-N-benzyloxycarbonyl
dibekacin prc~pared as in Examplo 23 was dissolved in 1.5 m~
of wator-tetrahydrofuran (2 : ~), to which were added 13 mg
(0~12 m moles) of anhydrous sodium carbonate and then 21 mg
(o.o63 m lllole~) of N hydroxysuccinilnide ester of N-benzyl-
oxycarbonyl-DL-isoserine~ The mixture was allowed to stand
at room.temperature and subsequently treated by the procedure
as described in Example 31 to afford 21 mg (yield 59~) of
the titled compound as its monocarbonate, who~e physical
.properties and antibacterial potency were identical to those
. of an authentic sample.
_ 90 -
Production of 3,6'-di N-benz,ylo~,ycar
l~trifluoroacetylkanam~cln A
A solution of 50~ mg of 3,6'~di-N-benzyloxycarbonyl-
kanamycin A ~see E~ample 1) in 4 m~ of dimethylsulfoxide
was admixed wlth 220 m~ of ethyl trifluoroaceta-te, and the
admixture obtained was allowed to stand overnigh*, at
ambient temperature. After a small vo]ume of trlfluoro-
acetic acid was added to the reaction mixt~e, the reaction
solution ~las poured into a large volume of ethyl ether and
the resu'ltant oily materlàl deposited was washed well with
ethyl ether to af-ford the solidified material. This
material was dried ~ell to obtain 640 mg of the titled
compound a~ a solid substance, Yield 99~o9 ~a]25 ~ 98
(c 1, pyridine)
Elemental analysis
CalcdO for c36H47~4ol6F3 CF3COOH
C 47,40; H 5.02; N 5.82%
Found: a 47.13; H 5.15; N 5,79~u
E_ample 34
Production of 3,6'-di-~-benzyl~ __n~
N-trifluoroacet~lkana~
A solution o-f 20 mg of 3,6'-di-~~benzyloxycarbonyl-
kanamyc~n A in 0.4 ml of dimethylsulfo~ide was admixed with
~5 6 mg of phenyl trifluoroacetate, and the admixture obtained
was allowed to stand overnight at ambient temperature.
Subseqtiently~ the reaction mixture wa~ processed in the
~ame manner as i,n ~ample 33y affordingj 24.8 mg of the
titled product wl-ich was found identical to that of
Examp:Le 33. Yield 97%.
Examp].e 35
Procluction of ~06'-di-N-ben_yloxYcarbon~1-3"-
N-trifluoroacet,yllcanam~cin A
A ~o].ution o~ 10 mg of 3,6'-di~-benzyloxycarbonyl-
kanamycin A in 0.3 m~ of hexamethylphosphoric triamide was
admixed with 7 mg of ethyl trifluoroacetate, and the
admixture obtained was allowed to stand overnight at
ambient temperature. The reaction soluti.on was admiY~ed
with a small volume oP trifluoroacetic acid and then
poured into a large volume of e-thyl ether. The oily
material deposited was washed well with ethyl ether and
the resultant solid substance dried to give 11.7 mg (yield
91~) of the titled product as its mono~trifluoroacetate in
the form of a solid.
Example 36
~roduction of 3,6'-di-N-benz~lox~car~on~
N-trifluoroacet~lkanam~cin A
A suspension of 10 mg of 3,6'-di-N-benzyloxycarbonyl-
kanamycin A in 0~3 m~ of dimethylformamide wa3 admixed with
7 mg of ethyl trifluoroacetate, and the admixture obtained
was allowed to stand overnight at ambient temperature,
The homogeneous reaction solution thus obtained was admixed
with a small ~olume of trifluoroacetic acid and then poured
into a large volume of ethyl ether. ~he oily material
,
. .
~ 92 ~ 6Z~
depo~ited wa~ ~ashed well with ethyl ether to solidi~y
it and the reaultant 30]id substance dried, affording
11,5 mg (~ield 90~o) of the titled product as it3 mono-
trifluoroacetate ln the form of a solld.
ExamF)le 37
Production of 3,6'-di-N-benæ~lox~carbon~
N-trifluoroacet~ cin A
A suspension of 10 mg of 3,6'-dl-N-benzyloxycarbonyl-
kanamycin A in 0,35 m~ of ~ul~olane was admixed with 7 mg
of ethyl trifluoroacetate, and the admixture was stirred
overnight at ambient temperature, Subsequently, the
reaction mixture was processed in the ~ame manner as in
Example 33~ affording 12,0 mg tYield 94~) of the titled
product as the mono-trifluoroacetate in the form of a
solid substance.
Exam~le 38
Production of 3,6'-di-N-benz~loxycarbon~1-3"-
N-trifluoroacet~lkanam~cin A
.
A suspension of 22 mg of 3,6' di-N-benzyloxycarbonyl-
kanamycin A in 0,8 m~ o~ tetrahydro~uran was admixed with
10 mg of ethyl trifluoroacetate, and the admixture was
stirred for 2 days. The resulting homogeneous reactlon
solution was admixed with 15 mg of ethyl trifluoroacetate
and 8 mg of anhydrous sodium carbonatey stirred overnight
and then allowed to stand ~or 2 days, The resultant
reaction solution was concentrated to a small volume9 and
the concentrate was wa~hed with water and then dried to
- 95 ~ ~L31~iiZ~3
give a 301id material l`he solid material was suspended
~n a s~all volume o~ tetrahydrofuran together with a .~mall
amount of trifluoroacetic acid. The admixt~re so obtained
was stirred followed by addition of ethyl ether. The
precipitated solid ~ta~ filtered off, washed with ether
and dried, giving 21 mg (Yield 74~) of the titled product
mono-trifluoroacetate as a solid sub~tance. [a]D5 ~ 98
(c l, Pyridine)
~ . .
Production of 3,6'-dl N-benæ~lox~carbon~1-3"-
N-tri~luoroacetylkanam~cin A
A solution of lO mg of 3,6'-di-N-benzyloxycarbonyl-
kanamycin A in water-tetrahydrofuran (l:l, 0.3 mR) was
admixed with a solution o~ 5 mg of ethyl trifluoroacetate
in 0.1 m~ of tetrahydrofuran, and -the resultant admixture
was allowed to stand at ambient temperature for one day.
Subsequently, a mixture of ethyl trifluoroacetate (10 mg),
anhydrous sodium carbonate (4 4 mg) and tetrahydrofuran
(0.1 mQ) was added to the resultant solution at 5 hours
intervals (four times in all) to effect the 3"-N-tri
fluoroacetylation. The reaction solution was concentrated
and then treated in the same manner as in Ex~mple 38 to
give 5.5 mg (Yield 4~) of the titled product mono-tri-
fluoroacetate as a solid sub~tance.
- 25
.
N-tr1~luoro~cetvlkanamvoin A
.' .
,
- g4 ~ 6~ ~
~ solution o~ 10 mg of 3,6'-di-N-benzyloxycarbonyl-
kanarnycin A in water-ethanol (2:3, 0.6 m~ as admixed with
a solution of 5 mg of ethyl trifluoroacetate ln 0.1 m~ o~
tetrahydrofuran, and the admix-ture was allowed to stand at
ambient temperature for olle day. The reac-tion solution
wa~ thereafter processed in the same way. as in E~ample 38,
a~fording 2.3 mg (Yield 18r~o) of the titled produc~ mono-
trifluoroacetate as a solld ~ubstance~
Example 41
Production of 336'-di-N-t-buto~carbon~1 3"-
N-tr _luoroacetylk2nam~cin A
(a) Pr~paration o~ 3,6'-di~N-t-but~ ar~
kanamvcin A
~,
500 mg (1.0~ m moles~ of kanamycin A (free base)
was suspended in 12 mR o~ dimethylsulfoxide, and 1 g
(4.55 m moles) o~ zinc acetate dihydrate was added to the
suspension obtained. The mixture was stirred at room tem-
perature until it formed a homogenous solution, to which
was then added 370 mg (2~59 m moles) of t-butoxycarbonyl
azide. ~he resultant mixture was allowed to stand over-
night at room temperature and subsequently treated in the
same manner as described in Exampls 1 (ii) and (lii) ~
to af`ford 590 mg ~80~o~ of a colorles~ ~olid of the titled
compound [a]25 ~ 89- (c 1, watcr~dimethylformamiae, 1;2
(b) ~ ~
1~
~6l-di-N~t-butoxyoarbonyl~{~.namycin A (60 mg) ~as
' ~ .
î~31
- 95 --
di~solved in 0~5 mR of dimethylsulfo~ide9 and the rcsulting
~olution wa~ admixed with 25 mg of ethyl trifluoroacetate,
followed by allowing the admix-ture obta:lned to stand o~er-
night at ambient temperature. Subsequently, the reaction
solution was processed ~n the same manner as described in
Example 33, giving '76.~ mg (Yield 98~) of the titled
compound trifluoroacetate as a solid. [a]25 ~ 72 (c 1,
water-dimethylformamide, 1:2).
E Gl~ _
Calcd. for C30H5lN~ol6F3-c~ COOH:
C 42.95; H 5.86; N 6,26%
Found: C 42.77; H 5.92; N 6.38
_ 42
Production of 3,6'~di~N-(p-methox~benz~lox~carbon~
3"-N-trifluoroacetylkanam~cin A
A solution of 40 mg of 3,6' di-N-(p-methoxyben~yloxy~
carbonyl)kanamycin A (see Example 8 hereinbe~ore) in 0.4 m~
of dimethylsulfoxide was admixed with 18 mg of ethyl tri-
fluoroacetate, and the admixture was allowed to stand over-
night at ambient temperature. Subsequently, the reactionsolution was processed in the same manner as in E~ample 33,
affording 49,3 mg (Yield 98~) of the titled compound as a
solid substance. [a~D5 ~ 78 (c 1, water-dimethylformamide,
1:2)
Elemental analy~is
.
Calcd. for C38H51N418F3 CF3CH
C 46.9i; H 5.i2; N 5.48
_ 96 - ~133~iZ~
Found: C 47.18; H 5,03; N 5~31
Example 43
Production of 3,6'~3"~tri-~-trifluoroacet~=
~ cin A
75 mg o~ 3,6'-dl-N-tri~luoroacetylkanamycin A
~see ~xample lO hereinbefore) and tr1ethylamine (12 mg)
were admixed with 006 m~ of dimethylsulfoxide and then
with 35 mg of ethyl trifluoroacetate, and the admixture
was stlrred oYernight to effect the desired 3"-N-tri-
~luoroacetylation, The reaction solution was then
processed in the same manner as in Example 33, affording
94,2 mg (Yield 96~) of the titled compound as a solid
substance. [a]25 ~ 76~ (c l, water-dimethylformamide,
1:2)
Elemental anal~sis
Calcd. for C24H33N414F9 CF3
C 35,22; H 3.87; N 6,32~
Fo~ld: C 35,09; H 3,99; N 6,07%
Example 44
Production of 3,6'-d -~-phenoxycarbonyl-3"-
A solution of 5~ mg o~ 3,6'-di-N-pheno~ycarbonyl-
kanamycin A (see Example ll) and triethylamine (9 mg) in
0,5 m~ o~ dimethylsulfoxide wa~ admixed with 23 mg o~
methyl trifluoroacetate, and the admixture was then
proce~sed in the same manner as i~ Example 33, a~ording
65 mg (Yield 95~) o~ the titled oompound as a solid material.
.
'
~ 97 ~ 6~ ~
[a]25 + 70 (c 1, water-dimethylformamide, 1:2)
Elemental ansl/fi1s
Calcd. for C34H43N416F3 CF3
C 46.26; H 4.74; N 5 99%
Found: C 45.88; H 4.g6; N 5.77~o
Example 45
Production of 3,6'~3"-tri-N~-~orm~lka~ cin A
A mixture o~ 62 mg o:~ 396'-di~N-formylkanamycin A
(see Example 13), 90 mg of ethyl formate and 1 m~ of
dimethylsulfoxide was heated at 100C for 12 hour~ in a
sealed tube to e~ect the desired 3"~N-formylation. The
reaction solution was admixed with a little amount of formic
acid, then poured into a large volume o~ ethyl ether, and
processed in the same manner a~ in Example 33, affording
69 mg (Yield 98~) of the titled compound as a solid material
positive to ninhydrin. [a]25-t 109~ (c 1, water-dimethyl-
formamide, 1:2)
,
Calcd. ~o 21 36 4 14
C 43 00; H 6.23; N 9.12%
Found: C 42.83; H 6019 N 9~10%
.~
Production of 3,6'~di-N-benzyloxy
meth~l-3"~N-trifluoroacetylkanamycin A
A mixture o~ 68 mg of 3,6'-di-N-benzylox~carbonyl-
6'-N-methylkanamycin A (see Example 15), and triethylamine
(11 mg), 30 mg o~ ethyl trifluoroacetate and 0.7 mQ of
.
- dimethyl~ulfoxide wa~ treated in the same manner as ln
E~ample 33, af~ording 86 mg (Yie~d 99~o) of the titled
compound mono-trifluoroacetate as a solid substance.
[a3D5 ~ 65 (c 1, water-dimethylformamide,1:2)
_a pl~e
~roduction_ f 3,6'-di-N-benz~y]ox~carbonAy~
deoxy-3''-N-tr:Lfluoroac_t~ylkan~mycln A
A solution of 52 mg of 3~6'-di-N-benzyloxycarbonyl-
3'-deoxykanamycin A (see Example 16) and triethylamine
(11 mg) in 0.4 m~ of dimethylsulfoxide was admixed with
21 mg of ethyl trifluoroacetate, and the admixture waæ
allowed to ætand overnight at ambient temperature. Sub
sequently~ the reaction solution was processed in -the ~ame
manner as ir~ Example 33, affording 64.8 mg (Yield 97%) of
,15 the titled compound as a solid material, [a]25 + 70
(c 1, water-dimethylformamide, 1:2)
Elemental anal~
Calcd. for C36H47N415 3 3
C 48~ 21; H 5.11; N 5. 92~o
Found: C 47.94; H 5~35; N 5.77%
~ ' .
Production of 3,6' di-N-benz~lox,ycarbon,yl-3'-
deox~-3"-N-fo ~
A solution of 78 mg of 396'~di-N-benzyloxycar~Donyl-
3'-deoxykanamycin A in 0.7 m~ of dimethylsulfoxide was
admixed with 65 mg of phenyl formate, and the admixture
wa~ heated o~ernight at 50-G for the 3"-N~formylation.
,
.
- 99 ~3~;21~
The reaction ~olution was admixed with a ~mall amount o~
formic acid, and proce~sed in the eame manner a~ in Example
33, giving 83 mg (Yield 97~) of the titled compound mono-
~ormate as a solid sub~tance, [a]25 + 84 (c 1, water-
dimethylformamide, 1:2)
ExamPle 42
Production of 3,6'-di-N-benz~lox~c2rbon~3"-
N-dichloroacet~1~3'-deox~kanamycin A
A solution of 35 mg of 3,6'-di-N-benzyloxycarbonyl-
3'-deoxykanamycin ~ in 0,5 m~ of dimethylsulfoxide was
admixed with 12 mg of methyl dichloroacetate, and the
admixture wa~ allowed to stand overnight at ambient tem-
perature. The reaction solution was admixed with a ~mall
volume of dichloroacetic acid and then treated in the same
manner as in Example 33, giving 44.5 mg (Yield 96~) of the
titled compound as a solid substance. [a]25 + 65~ (c 1,
water-dimethylformamide, 1:2)
Elemental a~
Calcd, f 36 48 4 15 2 2
C 46,73; H 5.16; N 5.74; CQ 14,52
Found: C 46,58; E 5,33; N 5,62; CQ 14,28
~0
.
A solution of 58 mg of 3,6'-di-N-benzyloxyczrbonyl-
3'-deoxykanamycin A in 0~7 mQ of dimethyl~ulfoxide wa~
admlxed with 25 mg cf methyl trichloroacetate, and the
'. ' . ~
.
loo 1~3~
admixture ~ra,s allowed to stand overnight at 50C. The
reaction solution ~ras admixed with a ~mall volume of tri-
chloroacetic acid and then processed in the same manner
a~ in Example 33~ affording 80.5 mg (Yield 98%) of the
titled compound as a solid sub~tance. []25 + 65 (c 1,
water-dimethylformamide, 1:2)
Elemental anal~si~
Calcd, ~or C36H47N415C~3 cc~3c02
C 43.65; H 4.63; N 5.36; C~ 20~34~
Found: C 43.44, H 4.77; N 5.30; C~ 20.19%
Example 51
,Production of 316'-di-N-benz~loxycarbon,yl-3'-
deox~-3"-N-trifluoroacetyl-6'-N-methylkanamycin A
A solution of 72 mg of 3,6'-di-N-benzyloxycarbonyl-
3'-deoxy-6' N-methylkanamycin A in 1 m~ o, dimethylsulfo~ide
~as admixed with 30 mg of ethyl trifluoroacetate, and the
admixture was allowed to stand overnight at ambient tem-
perature. Subsequently, the reaction solution wa~ processed
in the same manner as in Example 33) affording 89.5 mg
(Yield 97%) of the titled compound, mono-trifluoroaoetate
as a soli~ substance. ~a]25 + 70o (~ 1, water-dimethyl
formamide, 1:2)
Examp1e_5 ?
Production of 3,6'-di~N-benzyloxycar_~yl~
A solution of 71 mg o~ 3,6'-di-N-benzyloxycarbonyl-
4'-deoxykanamycin A (see Exa~ple 18 hereinbefore)
.
,
.~ . .
~ lol_ ~1316'~8
trie~hylamine (12 mg) and 30 mg of ethyl trifluoroacetate
in 1 m~ of dimethylsulfoxiae wa~q proces~ed ln the same
manner as in Exc~mple r~3 ~ glving 90 mg (Yield 99~o) of the
titled compound mono-trifluoroacetate a~s a solid substance.
~a]25 ~ 72 tc 1, water-dimethylformamide, 1:2)
Exam~le 53
Production o~ 3,~'-dl~N-benæ~lox~carbon~ 3',4'-
dideox~-3"-N-trifluoroacetylkanamycin A
A solution of 75 mg o~ 3 ~ 6 ~ -di-N-benzyloxycarbonyl-
3',4'-dideo~ykanamycin A and 30 mg of ethyl tri~luoro-
acetate in 1 mQ of dimethylsulfoxide was treated in the
same manner as in Example 33, affording 96 mg (Yield 99~o)
of the titled compound aq a solid substance. [a]D5 ~- 72
(c 1, water-dimethylsulfoxide, 1:2)
Elemental anal~is
Calcdr for C36H47N414~3 3
C 49 03; ~ 5.20; N 6 ~ 02%
~ound: C 48.83; H 5.46; N 5.87~o
ample 54
Production of 3,6'-di~N-benzvloxycarbon~1~3'~4'-
d~deoxy-3"-N-form~lkanamycin A
75 mg of 3,6'~di-N-benzyloxycarbonyl-3',4'-dideoxy-
kanamycin A and 65 m~ of phenyl formate were dis~qolved in
1 m~ of dimethylsulfoxide and the resultant solution was
processed in the same manner a~ in Example 48, affording
80 mg (Yield 97~) o~ the titled compound monoformate as
a solid substance. ~a]D5 ~ 80- (c 1, water-dimethyl~orm-
- 102 - ~ ~ 3
amide9 1:2)
Eæ~l~ple 55
Production of 3,~' dl-~-benzy]..oxycarbo~1-3', _
dideox~3"-N-dichloroacet~ylkcm ~
A solution of 68 mg of 3,6'-di-N-benzyloxycarbonyl-
3',4'-dideoæykanamycin A in 0.9 mR ~;~ dimethylsulfoxide
was admixed with 25 mg of methyl dichloroacetate, and the
admixture was allowed to stand overnight at ambient tem-
perature. The reaction solution was admixed with a small
amount of dichloroacetlc acid and then processed in the
, same manner as in Eæample 33, affording 88 mg (Yield 97%) of the titled compound mono~dichloroacetate as a solid
sub,stance~ [a]25 + 67 (c 1, water dimethylformamide9
1:2)
Exam~le 56
rod~L~L~ benzylox:vcarb~a=~
N-trifluoroacetylkanam~cin B
A solution of 78 mg of 3,2',6'-tri-N-benzylox~-
carbonylkanamycin B ~see Example 19 hereinbefore) a~d
triethylamine (11 mg) in 1 m~ of dimeth~ylsulfoxide ~a~
admixed with 35 mg of ethyl trifluo~oacetate, and the
admiæture was processed i,n the same manner as in Example 33,
affordi.ng 92 mg (~ield 95~) of the titled compound mono~
trifluoroacetate as a solid substance. [a]25 ~ 60 (c 1,
water-dimethylformaMide~ 1:2)
103 - ~ ~ 3~ ~ 8
Example 57
Production o-f 3. ? ', 6 ' -tri-N-benz~lox~carbon~1-3"-
N-form~ltobramycin
A ~olution of 82 mg of 3,2',6'-tri-N-benzyloxy-
carbonyl-tobr~lmycln (see Example 20 hereinbefore)and tri-
ethylamine (12 mg) in 1.2 m~ of dimethylsulfoxide was
admixed with 60 mg of phenyl formate, and the admixture
was processed in the same manner as in Example 48, affording
86 mg (Yield 97%) of the titled compound as a solid sub-
stance~ ~a]25 ~ 71 (c 1, water~dimethylformamide, 1:2)
a~G~9 .
Calcd. for C43X55N516 HC
C 55.98; H 6,09; N 7.42%
~ound: C 55,50 H 6.22; N 7.28
E~ample 58
Production of 3,2',6'-tri-N-benz~loxycarbonyl-6'-
N-meth~1-3"-N-trifluoroacet~ltobramycin
A solution of 80 mg of 3,2',6'-tri-N-benzyloxy-
carbonyl-6'-N methyltobramycin (see Example 21 herein-
before) and triethylamine (12 mg) in 1.2 m~ of dimethylsulfoxide wa~ admixad with 30 m~ o~ ethyl trifluoroacetatag
and the admixture was then processed in the same manner
as in Ezample 33, affording 97 mg (Yield 98~) of the
titled compo~d mono-trifluoroacetate as a solid ~bstance.
~a]25 + 60 (c 1, water-d1methylformamide~ 1:2)
- 104
~roduction o~ 3,2',6'-trl-N~be_
N-trifluoroace~ldibelcacin
A solution o~ 82 m~ of 3,2'~6'-tri-N-benæyloxy-
carbonyl~dibekacin (see Example 23 hereinbefore) in 1 mQ
of dimethylsul~oxide was admixed with 30 mg of ethyl tri~ -
fluoroacetate, and the admixture was processed in the same
manner as in Example 33, affording 100 mg (Yield 98%) o~
the ti~led compound as a solid substancQ. [a]-25 ~ 61
(c 1, water-dimethylformamide, 1:2)
Elemental anal~is
Calcd~ for C44H54~515F3 CF3C
. a 51.93; H 5.21; N 6.58
Found: C 51.84; H 5.38; N~6.47
~
Production of 3.2~.6',3"-tetra-N-trifluoro-
acetyldibekacin
~ mi~ture of 71 mg of 3,2',6'-tri-N-trifluoroacet~1-
dibekacin and 30 mg o~ ethyl trifluoroacetate in 1 m~ of
dimethylsulfo~ide was allo~ed to stand overnight at 40C.
Subsequently, the reaction solution was processed in the
same manner as in Ex~mple 33, a~fording 90 mg (Yield 99%~
o~ the titled compound as a solid substance. ~a]25 ~ 70
(c 1, water-dimethylformamide, 1:2)
Elemental analysis
Calcd. for C26~33N5l2F12 CF3
C ~5.42; II 3.61; N 7.38%
,
. ::
~ ' .
:,
- 105 1~3~z~
Found: C 35.40~ H 3.89; N 7.17
Example 61
Production of 3~ 2'~6'~tri-N-ben~yloxycarbonyl-
3"-N~forra.,;~ldibeka.cln
__
~ mixture of 79 mg of 3~?' ,6'-trl-N-benzyloxy-
carbonyl~dibekacin and 60 mg of phenyl formate in 1.1 m~
of dimethylsulfoxide was processed in the same m~nner a~
in Example 48, affording 84 mg (Yield 98%) of the titled
compound monoformate as a solid substance. [a]25 ~ 70
(c 1, water-dimethylformamide, 1:2)
Example 62
3"~N-di _loroacet~yldibekacin
A solv.tion of 84 mg of 3,2',6'-tri-N benzyloxy--
carbonyl-dibekacin in 1.2 m~ of dimethylsulfogide ~as
reacted with 25 mg of methyl dichloroacetate in the same
manner as in Example 49, affording 104 mg (Yield 97~) of
the titled compound mono dichloroacetate as a solid sub~ .
stance, [a]25 + 59 (c 1~ water-dimethylformamide, 1:2)
~
Production of 3,2',6'-tri~~-benzyloxycarbonyl
A solution of 85 mg of 3,2',6'-tri-N-benzyloxy~
carbonyl-~ r-methyldibekacin (see Example 24) in 1 m~ of
dimethylsulfoxide was admixed with 30 mg of ethyl tri-
~ fluoroacetate, and the admixture was processed in the
same manner a~ in E~ample ~3, affording 103.5 mg (Yield 98%~
.
- :Lo~
.
of the titled compound mono-trifluoroacetate as a ~o].ld
substance [a]~5 ~- 60 (c l, water-dimethylformamide, 1:2)
xample 6~
_oducti.on of 3,2'-di N~ o7:~carbon~1-3"-
N- r ~ cin C
A solution of 81 mg of 3,2'r~i-N-benzylo~.ycarbonyl-
kanamycin C (see ~xample 25) and triethylamine (14 mg) in
1.5 m~ of di~ethylsulfoxide was admixed with 90 mg of ethyl
formate, and the admixture obt~ined was trea'ced in the
same manner as in Example 48, affording 85.5 mg (Yield 96%)
of the titled compound monoformate as a solid substance.
[a~D5 + 81 (c 1, water-dimethylformamide, 1:2)
~3~oi~6~5
P duction o ~ ~loxycarbon~1-3"-
N-trifluoroacet~lsisomicin
A solution of 82 mg of 3,2',6'-tri-N-ben~yloxy-
carbonyl-sisomici.n (see Example 29 hereinbe~ore) in 1.5 m~
of dimethylsulfoxide was admixed with 30 mg o ethyl tri~
fluoroacetate, and the admixture was processed in the
same manner as in Example 33, affording 99 mg (Yield 97~o)
of the titled compound mono-trifluoroacetate as a solid
substance. ~125 + 151~ (c 1~ water-dimethylformami.de,
1:2)
xam~le 66
Production of 3,2',6'-tri--N-benz~loxycarbonyl-3~'-
N-trifluoroacet~lnetilmicin
A solution of 85 mg of 3,2'~.6~-tri-N-benzyloxy-
_ 107 -
carbonyl-netilmicln ln 1 3 mQ of dimethylsulfoxide was
admixed with 30 mg of ethyl trifluoroacetate, and the
admixture was processed in the same manner as in ~xample 33,
affording 103 mg (Yield 9~) of the titled compound mono-
trifluoroacetate as a ~olid substance. [~]25 + 145 (c 1,
water-dimethylfornamide, 1:2)
Example 67
Production of 3,6'-di--N-benz~lox~carbonyl-3'~-
N-trifluoroacet~lgentamicin B
A solution of 72 mg of 3,6'-di N~-benzyloxycarbonyl-
gentamicin B ln 1.2 m~ of dimethylsulfoxide was admixed
with 30 mg of ethyl trifluoroacetate, and the admixture
wa~ processed in the same manner as in Example 33, afford-
ing 91 mg (Yield 99~) of the titled compound mono-tri-
fluoroacetate as a solid substance. [a]25 + 92~ (c 1,
water-dimethylformamide, 1:2)
~xam~le 68
Production of 3~ 6'- o L~ 3C~L~ 9~L~
~-trifluoroacetylgentamicin Cl and Cla mixture
A solution of 84 mg of 3~2',6' tri-N-benzyloxy-
carbonyl-gentamicin Cl and Cla mixture in 1.5 m~ of di-
methylsulfoxide was admixed with 30 mg of ethyl tri-
fluoroacetate, and the resultant admixture was processed
in the same manner as in Example 33, affording 101 mg
of the titled compound mono-trifluoroacetate aæ a solid
æu~stance. [a]D5 + B7 (c 1, water-dimethylformamide,
1:2)
~ .
., ' ' '' ' .
:::
_ ~,o~ 3
ple_~9
oduction of 3J2~,6'--tri-N~,enzyl Y~ bon~l-
3',4' dideox~-3'-eno-3"~N-tri,fluoroacet~lk~.nam2cin B
A mixture of 83 mg of 3,2',6'~tri-N-benzyloxy-
carbony~-3',4'-dideoxy-3'-eno-kanamycln B (see Ex~mple 28
hereinbefore) and 35 mg of ethyl trifluoroace-tate in 1.2 mQ
of dimethylsulfoxide was allowed to stand overnight at
ambient temperature. Subsequently, the reaction solution
was processed in the same manner as in Example 33, afford-
ing 99.5 mg (Yield 96%) of the titled compound mono tri-
fluoroacetate as a solid substance. [a]D5 ~ 26 (c 19
water-dimethylformamide 9 1 2)
Example 70
Production of 3,6'-di-N~benz~lox~carbon~1.-3'-deox~
3~ Q~ylkanam~
A solution of 90 mg of ~,6'-di-N-benzylo~ycarbonyl-
3'-deoxykanamycin A in 0.8 n~ of dimethylsulfoxide was
admixed with 13 mg of N-formylimidazole, and the admixt~e
~as allo~ed to stand at ambient temperature overnightO
The reaction solution was admixea with a little amount of
formic acid and then treated with ethyl ether as in Example
33, affording 94 mg (Yjeld 95%) of the titled compound
monoformate as a solid substance,
Example 71
Producti~ 6' 3"-tri N-acet~lkanamycin A
A mixture of 100 mg o~ ~76'-di-N-acetylkanamycin A
and 20 mg (1.03 molar plopor-tlon for 1 mol of the starting
: :
~ 109 ~
material) of N-acetylimidazole in 1 m~ of dimethylsulfoxide
- wa~ stirred under ice-cooling for 3 hours and then allowed
to stand at ambient temperature overni~ht. The reaction
solution wa~ made alkaline by admixing with 0,3 m~ of 28
aqueous ~lmonia an(l then allowed to stand at ambient tem
perature for 3 days, The re~ultant reaction mixture wa~
treated with ethyl ether to gi~e an ether-in~oluble syrup.
The syrup was taken up into water and then passed through
a column of CM-Sephadex C-25 (NH4+ -form) (a product of
Pharmacia Eine Chemicals Co., Sweden), The resin column
was developed with 0,05N aqueous ammonia. The fractions
containing the desired product eluted out were combined
together and concentrated to dryness. The concentrate was
taken up into water, and the aqueous solution was neutralized
with acetic acid and again concentrated to dryness, afford- -
ing 109 mg (Yield 90~o) of the titled compound as a solid
product. ~a~25 ~ 98 (c 17 water-dimethylformamide, 1:2)
Elemental anal~si~
~alcdO for C24~42N414 CH3C00 H2
C 45,~4; H 7.02; N 8.14%
Found: C 45.22; H 7.20; N 8.11
(a) Preparation of 3,6'-di-N-benzyloxycarbonyl)~
kanamycin A
(i) A ~uspension of 2~0 g (4.13 millimol) of
- 110 ~ 2 ~
kanamycln A (free base) in a mixed solvent of dimethyl-
~ulfoxide (50 m~) and tetrahydrofuran (20 m~) was admixed
with 4 g (18.1 millimol) of zinc (II) acetate dihydrate,
and the resulti.ng admixture was stirred at ambient tem-
perature until the reaction admixture formed a homoge~neous solution. It took about 4-5 hours by the suspended
kanamycin A dissolved with ~orming a kanamycin A-zinc cation
complex. The resulting solution was then cooled to 0C,
and.to this solution was dropwise added over about one
hour a cold solution (at 0C) of 2.37 g (9.5 millimol) of
N-benzyloxycarbonyloxysuccinimide in 40 m~ of a mixed
solvent of tetrahydrofuran-dimethylsulfoxide (1:1 by volume)0
Then the reaction solution was allowed to stand for 4 hours
at ambient temperature. During the period of this time,
the zinc complex o~ kanamycin A was subjected to benzyloxy-
carbonylation, The resulting reaction solution was sub-
jected to silica gel thin-layer chromatography using a
lower layer of chloro~orm-methanol-28~ aqueous ammonia
(1:1:1 by volume) as the development solvent? and it was
then observed that the silica gel plate showed a main spot
at Rf 0,2~ and two or three slightly perceptible minor
spots which are above the main spot and attributable to
other byproducts,
. (ii) The reaction solution containing the N~ben~yloxy-
carbonylated kanamycin A-zinc cation complex obtained in the
above stage (i) was poured intc 500 m~ of ethyl ether, and
the precipitated oily product was then washed with ethyl
' ~:
~ ~.3~
-
ether ~everal times to ~ive 8.8 ~ of a thick syrup-lik~
product comprising the N-benzyloYycarbonylated complex.
(iii) Removal of the zinc cation from the-syrupy
complex product was made in the undermentioned way using a
weakly acidic cation-exchange resin containing carboxylic
functions (-COOH) [Amberlite CG-50 resin (H~-form), a product
of Ro~m and Haas Co., U.S.A.].
60 m~ of Amberlite CG-50 (H+-form) resin was previ-
ously saturated well ~ith water-dioxane (2:1 by volume). A
column ~as filled with this resin, and then a solution of 1 g
of the syrupy complex product in water-dioxane (1:1 by volume)
wa~ passed through the column, which was then developed with
water-dioxane (2:1 by volume) containing 1% acetic acid. ~he
eluate fractions containing the desired product, 3,6'-di-N-
benzyloxycarbonyl-kanamycin A positive to ninhydrin firstly
run out, and then the fractions containing zinc acetate posi-
tive to colorization with diphenylcarbazide were collectedD
The former fractions containing the desired product were com-
blned together and concentrated to dryness9 and the concen-
trate was washed with ethyl ether to give 340 mg (Yield 81%)
of 3,6'-di-N-benzylo~ycarbonyl-kanamycin A in the form of a
colorless solid. [a]25+76(c 1, water-dimethylformamide,1:2)
Elemental anal~sis
Calcd. for C34H48N415 2CH3C2~ 2
~ 51.23; H 6.56; N 6029
~ound: C 51.02; H 6.71; N 6.22~
(b) Preparation of 396'-di-N-benzylo~ycarbonyl-3"-N-
trifluoroacetylkan~mycin A trifluoroacet~te ~-
" ~
31~i~Z8
'~he product o~tained in the abo~e procedure (a) waa
proce3s in tl~e same way ag in Example ~ but with addition
of 1.5 molar equivalent of triethylami.ne,affording the
titled compound.
(c) Preparation of l N~ 4-amino-2-hydroxybutyryl)-
kanamycln A
A soluti.on of 60 mg of 3,6'-di-N-benzyloxycarbonyl-
3"-N-trifluoroacetylka~amycin A trifluoroacetate obtained
in the above procedure (b) in 1.5 m~ of water~tetrahydro~
furan (1:1 by volume) was admixed with 7 mg of anhydrous
sodium carbonate followed by addition of 2~ mg of N-hydroxy-
.succinimide ester of ~4-benzyloxycarbonylamino-2-hydro~y-
butyric acid, and the admixture was allowed to stand at
ambient temperature for 10 hours~
The reaction solution th.us obtained was concentrated
to a small volume and admixed ~ith water, giving a solid
precipitateO The solid was taken up into 3 mR of 2N aqueous
ammonia~tetrahydrofuran (5:3 by volume) and the solution was
allowed to stand overnight at ambient temperature to effect
the removal of the 3"-N-trifluoroacetyI group. The reaction
mixture was concentrated to dryness to give a solid residue.
This solid residue was dissolved in 4 mR of water~dioxane
(1:1), a.nd the solution was made weakly acidic by addition
of a very small volume of acetic acid and subjected to cataly~
. 25 tic hydrogenolysis with hydrogen at atmospheric pressure for
one hour in the pres.ence of pallad.ium bla.ck catalyst to
ef~ect the rsmo~al of the benzylo~ycarbonyl groups, Ths
,
- '
- 113 ~
r0sultant reaction solution was filtered and concentrated,
and the concentrate was passed through a column of CM-
Sephadex C-25 (NH4~~-form) (a product of ~harmacia Fine
Chemicals Co., Sweden), which was then gradient~developed
with 0-~0 5 N aqueou~ ammonia ~he fractions containing
the desired product were combined together and con-
centrated to dryness to give 36 mg ~Yield 8g~) o:f the
monocarbonate of the titled compound. The physicochemical
properties and the antibacterial activities of this product
were found to be perfectly identical to those of an authentic
sample.
~E~ 3
-
Synthesis of l-N~ 4-amino-2-hydrox~butyr 1]-
3'-deo~kana~y~
(a) Preparation of 3,6'-di-N-benzyloxycarbonyl 3'-
deoxykanamycin A
A suspension of 500 mg (1.07 millimol) of 3'-deoxy-
kanamycin A (free base) in 12 m~ of dimethylsul-foxide wa3
admixed with 1 g ~4055 millimol) of zinc acetate dihydrate,
and the resulting admixture was stirred until it formed a
homogeneous solutionO To this solution was added a solutio~
of 610 mg (2 45 millimol) of N-benzyloxycarbonyloxy-
succinimide in 5 m~ of dimethylsulfoxide-tetrahydrofuran
(1:1 by vol~me), and the reaction solution was then allowed
to stand at ambient temperature overnight. Subsequently7
the reaction solution was processed in substantially the
same manner as in Example 72 (a) (iii), affording 765 mg
- 114 ~
(Yield ~) of the above titled compound in the form of a
- colorle~s solid. [a]25 -~ 76~ (c 1J water-dimethylform-
amide, 1:2)
Elemental ana].~si~
aalcd ~ f or C34H48N414 2~H3 2 2
C 52.16; H 6.68; N~ 6.40,~
Found: C 51.99; H 6.75; N 6.20~o
(b) Preparation of 3, 61 -di-N~benzyloxycarbonyl-3'-
deoxy-3"-N-trifluoroacetylkænamycin A trifluoroacetate
The product obtained in the above procedure (a)
was processed as in Example 47 to give the titled compound.
(c) Preparation of l-N~ 4~amino-2-hydroxybutyryl3-
3'-deoxykanamycin A
A solution of 50 mg of 3, 6 1 -di`-N benzyloxycarbonyl-
3'-deoxy-3"-N-trifluoroacetylkanamycin A trifluoroacetate
obtained in the above procedure (b) in 1.5 m~ of water-
tetrahydrofuran (1:2 by volume) was admixed with 6 mg of
anhydrous sodium carbonate~ followed by addition of 20 mg
of N-hydroxysucci~imide ester of ~-4-benæyloxycarbonyl-
amino 2~hydroxybutyric acid.~ The admixture was allowed to
- stand at ambient temperature for 8 hours~ The reaction
solution was concentrated to a small volume and admixed
with water, giving a solid precipitate.
The solid was admixed with 3 m~ of 2N-aqueous
ammonia-tetrahydrofuran (1:1 by volume) and the admixture
was allo~led to stand overnig~t at ambient temperature
to effect the removal of the ~"-N-trifluoroacetyl group.
: .
.
- 115 ~
The reaction ~olution wa~ cor,centrated to dryness to give
a sol~d residue, and this residue was admixed with 4 m~ of
water~dioxane (1:1 by volume). The so]ution was made weakly
acidic by addition of a very small volume Or acetic acid and
subJected -to hydrogenolysis with hydrogen under atmospheric
pressure for one hour over palladium black catalyst to
effect the removal of benzyloxycarbonyl groups. Sub-
sequently, the hydrogenolysis reaction solution was
processed in the same manner as in Example 72 (c)~ a4ford-
ing 30 mg (Yield 87'~o) of the titled compound as its mono-
carbonate monohydrate, ~a]D5 + 89 (c 1, water)
xam~le 74
S~nthesis of l-N-(I-4-amino-2~h,~droxybut~r~
3',4'-dideox~kanamycin A
A solu-tion of 70 mg of 3,6'-di-N-benzyloxycarbonyl-
3',4'-dideoxy-3"-N-trifluoroacetylkanamycin A trifluoro~
acetate obtained in ~xample 52 in 2 m~ of water-tetra-
hydrofuran (1:2) was admixed with 9 mg of anhydrous sodium
carbonate 7 followed by addition of 28 mg of N-hydroxy-
succinimide ester of ~-4-benzyloxycarbonylamino-2-hydroxy-
butyric acid. The admixture was allowed to stand at
ambient temperature for 10 hours. The reaction was con-
centrated to a small volume and admixed with water to give
a solid precipitate,
The solid was admixed with 4 mQ of a mixed solvent
o~ 3N aqueous ammonia-tetrahydrofuran (1:2), and the
admixture was allowed to stand at ambient temperature
~ 8 '
- 116 -
overnigh-t, The reaction solution was concentrated to dryne~
to ~ive a solid residue, The residue was admixed with 6 m~ o
water-dioxane (1:3), and the solution was made ~eakly acidic
by addition of a very small volume of acetic acid and sub-
~ected to hydrogenolysis with hydrogen at atmosphericpres~ure for 1.5 hours over palladium black catalys-t added.
Subsequently, the reaction solution ~as proc'essed in the
~ame manner as in Example 72 (c), affording 42 mg (Yield
91%) of the titled compound as its monocarbonate.
[a]25 ~ 91 (c 1, water)
,Example 7,5
Synthesis of 1-N-(L-4~amino-2-h~droxAybutyr,y~
in
(a) Preparation of 3,2',6'-tri-~ benzyloxycarbonyl-
tobramycin
A suspension of 480 mg (1.03 millimol) of tobramycin(free base) in 12 m~ of dimethylsulfoxide was admixed with
1 g (~.55 millimol) of zinc acetate dihydrate, and the
admixtura was s-tirred for one hour. To the reaction solution
containing the tobramycin-zinc cation complex was dropwise
added over about one hour a solution of 850 mg (~.4 millimol)
of N-benzyloxycarbonylo~ysuccinimide in 10 m~ of tetra-
hydrofuran~dimethylsulfoxide (1:1 by ~rolume), and the
reaction-mixture so fol~ed was allowed to stand at ambient
temperature overnight. The resulting'reaction solution
was treated with a large'volume of ethyl ether in the same
manner a~ ample 72 ~a) ~ii), affording a thick syrupy
,
: ., ' ' .
. .
- 117 -
product comprising the N-benzyloxycarbonylated tobramycin-
zinc complex. Subsequently, the syrupy complex product
was processed in the same manner as in ~xample 72 (a) (iii)
with exceptin~ that the rntio of water-dloxane (2:1) was
changed in1;o 1:2 by volume. 810 mg (Yield 78%) of the
titled compound in the form of a colorless solid was
afforded. [~25 ~ 65 (c 1, water-dimethylformamide,
1:2)
E ental anal~sis
Calcd. for C42H55~5015-2CH3C02H H2
C 54.81; H 6.50; N 6.95~
Found: C 54.77; H 6.71; N 6.88%
(b) Preparation of 3,2',6'-tri-N-benzylo~ycarbonyl-
3" N-formyltobramycin monoformate
The product obtained in the above procedure (a) ~as
processed in the same manner as in Example 57 to give above
the titled compound.
(c) Preparation of l-N~ 4-amino-2-hydroxybutyryl)-
tobrc~mycin
A solution of 100 mg of 3,2',6'-tri-N-benzyloxy-
carbonyl~3"-N-formyltobramycin monoformate obtained in the
above procedure (b) in 3 m~ of water-tetrahydrofuran (1:~)
- was admixed with 12 mg of anhydrous sodium carbonate,
followed by addition of 40 mg of ~-hydroxysuccinimide
ester of (~)-4-benzyloxycarbonylamino-2-hydroxybutyric
acid. The admixture wa~ allowed to stand at ambien-t tem-
`perature for 10 hour~ he reaction ~olution ~o formed
was concentrated to a 3mall volume and admixed with water
to depo~it a ~olid precipitate~
The solid was ~uspended in 2 m~ o~ 10~ aqueous
hydrogen peroxide, and the suspension was stirred vigorously
at 60C for 3 hours and then ~iltered to a:eford a solid
residue comprising the de-N-~ormyl derivative, ~he solid
residue was taken up into 8 m~ of water-dioxane (1:3), and
the solution was made weakly acidic by addition of a ~ery
small volume of acetic acid and subjected to hydrogenolysis
at atmospheric pressure for 1,5 hours over pallaclium black
catalyst, Subsequently, the reaction solution was processed
in -the same manner as in Example 72 (c) and passed through
the CM-Sephadex C-25 column, which was then gradient-
developed with O~lN aqueous ammonia. The fractions con-
taining the desired product were combined together and
concentrated to dryness to give 67 mg (Yield 87~o) of the
above captioned compound as its dicarbonate dihydrate.
[a]25 + 78 (c 1, water), This product was coincident
with an authentic product.
Example 76
S~nthe~is ~ ~
dibekacin
(a) Preparation of 3~2',6'~tri N-benzyloxycabonyl-
dibekacin
600 mg (1.33 millimol) of dibekacin (free base)
was admixed with 15 m~ of dimeth~lsulfoxide under stirring~
The solution was admixed with 1.4 g (6,4 millimol) of zinc
. : .
: . , .
.
.. . .
~13~8
-- llg --
acetate dihydrate under stirring, ~o the solutlon was
dropwise added o~er about one hour a solution of 1.1 g
(4,4 millimol) of N-benzyloxycarbonyloxysuccinimide in
12 m~ of dimethyl~.ulfoxide, and the admix-ture was allowed
to stand at room temperature overnl~r,ht. The resultant
reaction solution was further admixed with a large vol~ne
o~ ethyl ether to give an oily deposit comprising mainly
the desired product and a proportion of dimethylsulfo~ide.
The resultant oily deposit was separated -from the upper
liquid phase and further washed with ethyl ether to afford
a thick syrupy product,
`? ~he syrupy product was washed repeatedly with water,
With this water treatment, the initially existing excess
o~ zinc acetate was removed and also the N~benzyloxy-
carbonylated zinc complex was destroyed, affording 1.1 gof a water~insoluble solid residue. This solid gave a
single spot at R~ 0,1~ in a silica gel thin layer chromato-
graphy developed with the lower phase of chloroform-methanol-
18~ aqueous ammonia (1:1:1 by volume) as the development
solvent a~d was comprising almost pure 3~2',6'-tri-N-
benzyloxycarbonyl~dibekacin together with a trace of zinc
incorporated therein, [a3D5 + 71 (c 1, water-dimethyl-
~ormamide, 1 2)o If, however, the solid was ~rashed with
3M aqueous ammonia solution, pure product without con-
tamination of zinc cation was obtainedO
(b~ Prepara~ion of 3;2',6'-trl~N-benzyloxycarbonyl-
3"-N-tri~luoroacetyldibokacin trifluoroacetate
- 120 ~
'rhe product of the above procedure (a) was processed
as in ~xample 59 to afford the ti-tled compound.
(c) Preparation of l-N~ 4-amino-2-hydroxybutyryl)-
dibekacin
A solution of 170 m~ of 3,2',6'~tri-N-benzyloxy--
carbonyl-3"-N-trifluoroacetyldibekacin trifluoroacetate
obtained in ~he abo~e state (b) in 5 m~ of ~ater-tetra-
hydrofuran (1:3) wa~ admixed with 18 m~ of anhydrous sodium
carbonate, followed by addition of 60 mg of N-hydroxy-
succinimide ester of ($)~4-benzyloxycarbonylamino-2-
hydrox~butyric acid, and the admixture was allowed to -
stand at ambient temperature ~or 9 hours. The reaction
solution was concentrated to a small volume and admixed
with water to deposit a solid precipitate.
The solid was admixed with 12 m~ of 4N aqueous
'ammonia-tetrahydrofuran (1:3) and the admixture was allowed
to stznd overnight at ambient temperature~ The reaction
solution was then concentrated to dr~Tness to give a solid
residue. The resultant solid was dissolved in 12 m~ of
water-dioxane (1:~), and the solution was made weakly
acidic by addition of a very s~all amount of acetic acid
and ~ubjected to hydrogenolysis at atmospheric pressure
'for 105 hours over palladium black. Sub~equently, the
' reaction solution was processed in the same manner as in
Example 75 (c), affording 96 mg (Yield 895') of the titled
compound as its dicarbonate~ ~a]25 ~ 86 ~c 1, water)0
It was observed that the physicochemical properties and
121 -
the antibacterial activities of this product was coincident
~1ith those of an authent~c sample [Journ~l of Antibiotic3
Vol, 26, p 412 (1973)~.
ExamPle 71
dibekacin, i~e. 1-N-D~-isoseryldibekacin
A ~olution of 150 mg o~ 3,2',6'-tri-N-benzyloxy-
carbonyl-3"-N~trifluoroacetyldibekacin trifluoroacetate
of Example 59 in 5 m~ of water-tetrahydrofuran (1:3) was
admixed with 16 mg of anhydrous sodium carbona-te, follo~ed
by addition of 51 mg oD N-hydroxysuccinimide ester of D~-
3-benzyloxycarbonylamino-2~hydroxypropionic acid (i.e.
D~-3-benz~loxycarbonylisoserine). ~he admixture was
allowed to stand at ambient temperature for 10 hours.
Subsequently, the reaction solution was processed in the
same manner as in Example 76 (c), affording 82 mg (Yield
88~) of the titled compound as its dicarbon&te. [a~25 + 82
(c 0.32, water)
The physicochemical properties and the antibacterial
activities of this product were found to be identical to
those of an au-thentic sample.
~ .
dibekacin
(a~ Preparation of 3,2',6'-tri-N-p-methoxybenzyloxy-
carbonyldibekacin
500 mg (1.11 m moles) of dibekacin ~free base) was
~ 2
- 122 ~
suspended in 15 m~ of dimethylsul~oxide and the suspension
was stirrcd to form a ~olu-tion, to which was added.1.2 g
(5.5 m moles) of zinc acetatc dihydrate under stirring.
To the re~ultant solution wa~ dropwise added over about
30 minutes a solution of 1.17 g (3.86 m mo~.es) of p-methoxy-
carbobenzoxy p-nitrophenyl ester dissolved in 10 m~ of
dimethylsulfoxide, and the mix-ture was allowed to stand
overnight at room temperature The resultant solution was
then processed in the same manner as described in Example
76(a) to give 893 mg (Yield 85%) of the titled compound.
[a~25 ~ 69 (c 1, water-dimethylformamide, 1:2)
(b) Rreparation of 3,2',6'-tri-N-p-methoxybenzyloxy-
carbonyl-3"-N-trifluoroacetyldibekacin trifluoroacetate
A solution of 160 mg of 3,2',6'-tri-N-p-methoxy-
benzyloxycarbonyldibekacin in 2 mR of dimethylsulfoxide was
admixed with 48 mg of ethyl trifluoroacetate, and the mixture
was processed in the same manner as described in Example 33,
affording 188 mg (Yield 96%) of the titled compound as a
solid substance. [a]25 + 58~ (c 1, water-dimethylformamide,
1:2)
(c) Preparation of l-N-(L-4-amino-2-hydroxybutyryl)-
.dibekacin
A solution of 150 mg o~ 3,2',6'-tri-N p-methoxy-
benzyloxycarbonyl-3"-N-trifluoroacetyldibekacin trifluoro-
acetate obtained in the above s-tage (b) dissolved in 5 m~
of water-tetrahydrofuran ~ ) was admixed with 14 mg of
anhydrous sodium carbonate~ followed by addition of 54 mg
~ ]23 ~ 1 1 31 ~2 ~
N-hy~ro~y3uccinimidc ester of ~$)-4-(p-mctho~yben~yloxy-
carbonyl)~rnino 2~hydro~ybutyric acid, and the mi~ture wa~
allo~led to ~tand at room temperature for 8 hours. The
reaction solution ~as concentr~ted to ~ small volume and
~las admi7.ed with ~rater to dcposit a sol~d precipitate.
To the ~olid was added a so:Lution of lN-iIC~ in
aqueous me-thanol (1:3, 6 m~), and ~he miY~ture ~as heated
at 60~C for 4 hours for removal of the p-methoxybenzyloxy-
carbonyl group. The solution was concentrated to a small
volume, to which was added 5N aqueous ammonia until the
~olution showed pH 10. The solution was allowed to stand
at room temperature overnight and the solution was con-
centrated to give a residue. The residue was dissolved in
water and the solution was charged on a column of CM-
Sephadex C-25 (NH4t ~orm), which was washed with water
thoroughly and then gradiently developed with 0-~lN aqueous
ammonia~ The fractions containing the desired product
were combined together and concentrated to dryness to
give 77 mg (Yield 87'~o) of the titled compound as its
dicarbonate. ~a]25 + 85 (c 1, water3
This is a division of copending Canadian application
Serial Mo. 339, 531, filed Movember 9, 1979.
.
