Note: Descriptions are shown in the official language in which they were submitted.
1133~75
3. Detailed explanation of invention:
This invention relates to a novel 3"-acylated macrolide
antibiotics. More particularly this invention relates to a
compound of the formula
C~5 rC~o ~ N(C~ Z OR3
RzO ~ ~ ~ ~ O ~ R'
o
wherein Rl is a hydrogen atom or a (C2-C3) alkanoyl group, R2
is a hydrogen atom or a (C2-C4) alkanoyl group, in which at
least one of Rl and R2 is a hydrogen atom, and one of R' and
R" is an R3 group and the other is an R4 group, in which R3
is a (C2-C6) alkanoyl group and R4 is a (C2-C5) alkanoyl
group, or a salt thereof.
The salt hereinabove means a physiologically acceptable
salt. Preferred examples of the salt are inorganic salts
such as hydrochloride, sulfate or phosphate, or organic
salts such as acetate, propionate, tartrate, citrate, succinate,
malate, aspartate or glutamate. Other non-toxic salts can be
included.
The novel compound [l] has enhanced antibacterial
activities against susceptible or resistant strains as com-
pared with prior known 16-membered macrolide antibiotics such
as leucomycin group antibiotics including josamycin, SF-83
1133~75
group antibiotics, YL-704 group antibiotics and espinomycin
group antibiotics. Especially the said novel compound is
effective against the resistant strains for the other macro-
lide antibiotics such as oleandomycin, erythromycin, carbo-
mycin and spiramycin. Moreover deacylation at the 4" position,an one of the reasons for inactivation of 16-membered macro-
lide antibiotics, can not easily take place and hence the
sustained blood level is increased. Furthermore, the strong
continuous bitter taste generally characteristic of macrolide
antibiotics is decreased, and hence syrups for infants, who
cannot be administered tablets or capsules, is preferably
prepared. The antibiotics [1] of the present invention will
be expected to show excellent clinical infectious therapeutic
effects.
In the nomenclature of the compound [1] of the present
invention, it is influenced by a substituent at position 3 or
substituents at positions 3" and 4" of the formula [1].
Therefore when the position 3" is acylated and if the original
acyl group at the 4" position is not rearranged to position
3" as acyl rearrangement, i.e. in the case of a compound of
the formula [1']
c~5 rC~o O~ ~(C~ Z OR3
RZa ~ /7~ 11 ]
~ C}I3 \
"~,,,~ ~0~
-- 3 --
1133~75
wherein R3 i-s a (C2-C6) alkanoyl group, R4 is a (C2-C5)
alkanoyl group, and Rl and R2 have the same meanings as
hereinbefore, the nomenclature is based on the starting
material known antibiotic of the formula hereinbelow ~2].
When the original acyl group at position 4" is rearranged
to position 3" as an acyl rearrangement, i.e. in the case of a
compound of the formula [1"]
R~O~ \o~ ~ 11"~
\
~o /~
wherein Rl, R2, R3 and R4 have the same meanings as herein-
before, the nomenclature is based on leucomycin U, an anti-
biotic of the formula [2] hereinbelow wherein Rl is acetyl
and R4 is hydrogen atom, and leucomycin V, an antibiotic of
the formula [2] hereinbelow wherein Rl and R4 are hydrogen
atoms. [Japanese Patent Publication No. 48-4555 and "Progress
in Antimicrobial and Anticancer Chemotherapy, Vol. II, 1043-
1049 (1970)].
The said known antibiotic is expressed by the formula
12]
11339~7';
c~5 rC~O D~ N(C~ z ~
E10 ~\~ 0 ~ O ~
~/ ~ OR 1
C~5 ~
~0~o
wherein Rl and R4 have the same meanings are hereinbefore,
and includes the following antibiotics.
Antibiotics Rl R4
Leucomycin Al H COCH2CH(CH3)2
" A5 - H COCH2CF12CH3
7 ~ COCH2CH3
" A9 H COCH3
Leucomycin A3 COCH3 COCH2CH(CH3)2
Josamycin (=Leucomycin A3) " "
YL-704 A4 " "
Leucomycin A4 " COCH2CH2CH3
" A6 " COCH2CH3
YL-704 B3 " COCH2CH3
Leucomycin A8 " COCH3
YL-704 Al COC2H5 COCH2CH(CH3)2
SF-837 A2 COCH2CH2CH3
Espinomycin A2 " COCH(CH3)2
SF-837 COCH2CH3
SF-837 A
YL-704 B
Espinomycin A
^'~;'.^.'~
1133~7'~
YL-704 C2 COC2H5 COCH3
Espinomycin A3 " "
The antibiotic of the formula [2] wherein Rl is a
hydrogen atom, has 4 hydroxyl groups at position 3, 9, 2'
and 3", and in the case where Rl is an acetyl or propionyl
group, the said antibiotic has 3 hydroxyl groups at positiOns
9, 2' and 3". In these groups, the hydroxyl groups at
positions 3, 9, 2' are easily acylated and hence many
acylated derivatives thereof have been reported. However,
the hydroxyl group at position 3" has been reported to be
inactive.
Recently, the acylated derivatives of a hydroxyl
group at position 3" were reported. [Japanese Patent Open.
No. 49-124087 and No. 51-26887]. These compounds thus
reported have originally a propionyl group at position 3 and
an acyl group at position 9. In order to produce the acyl
derivatives at position 3" (hereinafter called as 3"-acyl
derivatives, etc.) having at least one hydroxyl group at
position 3 and 9, especially introducing the acyl group to
the position 3" alone of the known antibiotics hereinabove
has practically been impossible by known acylation methods due
to the highly reactive hydroxyl groups being present at the
positions other than 3".
We have found that the hydroxyl group at the position
other than 3", especially the hydroxyl group at position 3
and/or 9, can be protected by a protective group which can
easily removed without 3"-deacylation occurring after prior
acylation of the hydroxyl group at position 3".
4 '` ' .
~ 6 -
.
1133475
An object of the present invention is to provide a
novel antibiotic expressed by the formula [1] hereinbefore.
Another object of the present invention is to provide
novel antibiotics having higher blood level when administered
and having higher activities against susceptible and resistant
strains.
A further object of the present invention is to provide
novel antibiotics having a less bitter taste.
The compound [1] of the present invention can be pro-
duced by the following processes.Process [A]: A compound [1'] wherein Rl is a (C2-C3)
alkanoyl group and R2 is a hydrogen atom; i.e. the compound
of the formula [la]
C~5 rC~o o~ N(C~5)2 R~,s
~O ~ O ~ ~ O ~ R~
3 5 ~ la~
OR l 1
~ C~5 \
20 ~ O ~
wherein Rll is a (C2-C3~ alkanoyl group, and R3 and R4 have
the same meanings as hereinbefore:
The above compound [la] is produced in the following
ways.
The compound of the formula [3]
B - 7 -
li3347S
C ~ a~CH O ~(C ~s) 2~H
~ocO~ ~o~ ~o ~ 3
<y ~ OR
~> C~I5 ~
V\o~=
wherein R5 is a halogenated acetyl, p-nitrobenzoyl or silyl
group, R6 is a hydrogen atom or an R61 group, in which R
is a (C2-C4) alkanoyl group, and Rl and R4 have the same
meanings as hereinbefore, is acylated by a (C2-C6) aliphatic
carboxylic acid halide, under heating in the presence of an
inert organic solvent and a tertiary organic amine to obtain
the mixture of the compound of the formula 14]
~0 ~ 0 ~ o ~ OR~
\ C~I3 C~15 ~4
~ORll
~0~
wherein R is an R61 or R3 group and Rll~ R3, R4, R5 and R
have the same meanings as hereinbefore, and the compound of
the formula 14']
" . I
. -
,~. ,,.,~
11 33~7L;
17 18
C~s FC~OR5 OR8 N(C~ 2 Rs
RsO ~ ~ 2' ~ O ~ ~ ~,-OR~ [4,]
~ ~ ORl~ C~s C~3
~ C~15~
V\o--
wherein Rll, R3, R4, R5 and R8 have the same meanings as here-
inbefore. The said mixture is treated; in the case where R5
is a halogenated acetyl or p-nitrobenzoyl group, by ammonia
in methanol or ethanol to remove the protective group at
position 9, then treated by heating in methanol to remove the
acyl group at position 2'. In the case where R5 is a silyl
group, the said mixture is treated, to remove the silyl group
and acyl groups at positions 18 and 2', by the processes of:
(a) removing the silyl group and acyl group at positions 18
and 2' by heating in aqueous lower alkanol which can contain
a base;
(b) removing the silyl group and acyl group at position 18
by treating with aqueous lower alkanol at room temperature in
the presence of the base, thereafter removing the acyl group
at position 2' by heating in methanol which can contain water;
tc) removing the acyl group at position 18 by treating with
methanol containing ammonia and further removing the silyl
group and acyl group at position 2' by heating in aqueous
lower alcohol; or
(d) removing the acyl group at position 18 by treating with
methanol containing ammonia, thereafter removing the acyl
, . .;;
1133~7S
group at position 2' by heating with methanol and removing
the silyl group by treating with aqueous acid.
The compound [3~ hereinbefore is a compound wherein
the preferred protective group is introduced at position 9 of
the antibiotic of the formula [2] in order to prevent the
acylation of the hydroxyl group at position 9 in the step
of 3"-acylation reaction. The said protective group is a
- group, which can selectively be removed by well known methods
without destroying the chemical structure after 3"-acylation,
for example a halogenated acetyl group such as a chloro-
acetyl, dichloroacetyl, trichloroacetyl or trifluoroacetyl
group, a p-nitrobenzoyl group, or a silyl group. Among
these protective groups, introducing the chlorinated acetyl
group such as a chloroacetyl or dichloroacetyl group were
disclosed in Japanese Patent Open. No. 50-96584. Introduc-
tion of the other protective groups can be made in accordance
with the methods of the said patent specification, i.e.
reacting with a carboxylic acid halide, preferably a carbox-
ylic acid chloride, in the presence of a tertiary organic
amine in an inert organic solvent.
Introduction of a silyl group can be made by silylating
the antibiotic of formula [2] or its 2'-acyl derivative with
a suitable silylation reagent such as a tri-substituted
halogeno silane or hexa-substituted silazane. An example of
a tri-substituted halogenosilane is a tri-lower alkylhalogeno
silane such as tri-methylchlorosilane. An example of a
hexa-substituted silazane is a hexa-lower alkyl-silazane such
-- 10 --
~133at7'~
as hexamethylsilazane. Any silylation reagent can be used
if the introduced silyl group cannot be removed by water.
The above silylation reaction is usually carried out
in an inert organic solvent such as dichloromethane, chloro-
form or methyl isobutyl ketone, at room temperature or below.The amount of silylation reagent can be about 1 - 1.8 molar
equivalent. In the silylation reaction using a trisubstituted
halogeno silane, a tertiary organic amine is preferably used
as the dehydrohalogenation reagent. As examples, pyridine,
quinoline, N-methylmorpholine or dimethylaniline can be
mentioned.
The above silylated product of the formula [3] can be
obtained by pouring the reaction mixture into water and
extracting with a suitable water immiscible organic solvent.
The silyl group of the compound [3~ is stable with respect
to water and cannot be removed in the~presence of water.
As hereinabove explained, in the protection of thQ
hydroxyl group at position 9 of the antibiotic [2], the
hydroxyl group at position 2'-may optionally be protected by
a protective group. An example of the said protective
group is a (C2-C4) alkanoyl group, preferably an acetyl
group. 2'-acetylation can be made by the process described
in Japanese Patent Publication No. 53-7434.
The above compound [3] is 3"-acylated by an aliphatic
carboxylic acid halide. The reaction is carried out in the
presence of a tertiary organic amine in an inert organic
solvent by heating. Examples of an inert organic solvent are
B 11
1133475
acetone, methyl ethyl ketone, ethyl acetate, dimethoxyethane,
tetrahydrofuran, dioxane, benzene or toluene. Examples of
a tertiary organic amine are pyridine, picoline or collidine.
Other known organic amines such as triethylamine, dimethyl-
aniline, tribenzylamine, N-methylpiperidine, N-methylmorpho-
line, quinoline or isoquinoline can be used. Examples of
aliphatic carboxylic acid halides are (C2-C6) aliphatic car-
boxylic acid halides such as acetylchloride, propionyl-
chloride, butyrylchloride, isobutyrylchloride, isovaleryl-
chloride or caproylchloride. The heating temperature maybe 50 - 120C. Reaction time can be varied depending on
the reaction temperature, and since the progress can be
checked by silica gel thin layer chromatography, the
termination can be determined within the range of 1 to 150
hours.
In the above acylation reaction, the hydroxyl group
17 18
at position 3" is acylated and the compound having [-CH=CHOR3]
' 17 18
at positions 17 and 18 instead of [-CH2-CHO] is produced as
a by-product. In the case of a hydroxyl group at position
3 and a previously non-protected hydroxyl group at position
2', these hydroxyl groups are also acylated. Therefore the
amount of aliphatic carboxylic acid halide should be altered
depending on the number of hydroxyl groups to be acylated.
In the reaction in which compound [3] wherein Rl is
a hydrogen atom, is used and a different acyl group is
introduced at positions 3 and 3" thereof, in other words in
order to obtain the compound [4] wherein Rll and R3 are
- 12 -
~ '
1133~75
different acyl groups, an acyl group such as an acetyl group
or propionyl group is previously introduced in the hydroxyl
group at position 3 of the above compound [3], and thereafter
acylated at position 3".
The thus obtained mixture of the compounds [4] and
[4"] can be isolated so that, when the reaction mixture is
adjusted by alkali at pH 8 - 10 in water to precipitate and
filter, and when the reaction solvent is a water immiscible
organic solvent, the reaction mixture is poured into water,
the pH is adjusted to 8 - 10, then extraction with a water
immiscible organic solvent is carried out. Further purifica-
tion can be made by chromatography using silica gel, active
alumina or an adsorption resin-with elution using benzene -
acetone.
Removal of protective groups of positions 9, 18 and
2" of the mixture of the compound [4] and compound [4']
depends on the protective group at position 9. In the
case where R5 is a halogenated acetyl or p-nitrobenzoyl
group, the protective group at position 9 is removed by
treating with ammonia in methanol or ethanol at room
temperature. Progress of reaction can be checked by
silica gel thin layer chromatography and the reaction can
be terminated when the spots of compounds [4] and [4'] have
17 18
not been detected. By this reaction, the group [-CH=CHOR3]
of the compound ~4'] is changed to original [-CH2CHO].
Ammonia and alcohol are distilled off from the reaction
mixture to yield the compound wherein the protective groups
at positions 9 and 19 are removed. The said compound is
- 13 -
,
11334~5
heated in methanol which can contain water to remove the acyl
group at position 2'. Heating is effected by refluxing with
methanol. Reaction can be checked by silica gel thin layer
chromatography.
In the case where R5 is a silyl group, protective groups
at positions 9, 18 and 2' can be removed in the following
manner:
(A) The silyl group and acyl group at positions 18 and 2'
are removed by heating in an aqueous lower alkanol which can
contain the base;
(B) The silyl group and acyl group at position 18 are
removed by treating with aqueous lower-alkanol at room tem-
perature in the presence of base, and the acyl group at
position 2 is removed by heating in methanol which can
contain water;
(C) The acyl group at position 18 is removed by treating
in methanol containing ammonia, and the silyl group and acyl
group at position 2' are removed by heating in an aqueous
lower alcohol; or
(D) The acyl group at position 18 is removed by treating
in methanol containing ammonia, and removal of the 2'-acyl
group by heating in methanol, and removal of the silyl
group by treating with aqueous acid.
In the above, the lower alkanol is preferably
methanol or ethanol. In the method (A) and (B), tertiary
organic amine, alkaline carbonate or basic resin can be
mentioned. Heating can be made by refluxing the organic
solvent. In (B), for example, a non-heatins condition or
~ . .
- 14
1133~S
where heating is not specified the temperature means
room temperature or below. End point of the reaction
can be checked by silica gel thin layer chromatography.
The desired compound [la] can be obtained from the
thus prepared compound by the process hereinbelow explained.
Process [B]: A compound [1'] wherein Rl is a hydrogen atom
and R2 is a (C2-C4) a;kanoyl group; i.e. the compound of
the formula [lb]
~ CH3 C~O o~ N( ~ 2 OR5
R2lo~ o ~ O ~ OR"
~5C0 0
~5 C~3 ~lb]
C~I3 \
~\ o ~
wherein R21 is a (C2-C4) alkanoyl group, and R3 and R4 have
the same meanings as hereinbefore.
The above compound [lb] can be obtained by reacting
the compound of the formula [5]
17 lB
R~O~ o ~ s]
0
/\0~
1133475
wherein R6 is a hydrogen atom or an R61 group, in which R
is a (C2-C4)alkanoyl group, and R2 and R4 have the same
meanings as hereinbefore, with a (C2-C4) aliphatic carboxylic
acid anhydride in the presence of an inorganic base to pre-
pare the compound of the formula [6]
R~O ~ ~ r I ~ ~ OR~
\
~\ O /~
wherein R7 is an R61 or R21 group, and the R21, R4 and R6
groups have the same meanings as hereinbefore;
reacting the compound [6] with a (C2-C6) aliphatic carboxylic
acid halide in the presence of a tertiary organic amine in
an inert organic solvent with heating to prepare the compound
of the formula ~7]
C~s 17 8 N(C~3)2 OR5
R2lG~ o ~ o ~0~,,
~ ~5CO ~ I C~3 C~s ~7
/~ OH
~\oi~
7S
wherein R21, R3, R4 and R7 have the same meanings as herein-
before, and removing the protective group at position 18 by
treating with a methanol solution of ammonia or an ethanol
solution of aqueous alkaline carbonate, then removing the
2' acyl group by heating in methanol.
The above compound [5] means the antibiotic [2] wherein
Rl is a hydrogen atom, such as leucomycin-Al, -A5, -A7, -Ag
or acyl derivatives thereof. The acyl derivative may be 9-
acyl, 2'-acyl or 9,2'-diacyl.
Introduction of protective groups to positions 3 and
18 of the compound [5] hereinabove is made by reacting with
the corresponding carboxylic acid anhydride in the presence
of an inorganic base.
Examples of inorganic bases are, alkaline hydroxides
such as potassium hydroxide or sodium hydroxide, alkaline
carbonates such as potassium carbonate or sodium carbonate,
and alkaline hydrogen carbonate such as sodium bicarbonate.
Preferable examples are alkaline carbonate or alkaline
hydrogen carbonate. The corresponding carboxylic acid
anhydride may be a (C2-C4) carboxylic acid anhydride such
as acetic anhydride, propionic anhydride or lactic anhydride.
The temperature of introducing the protective group is 30 -
100C, and preferably is 40 - 60C.
When the antibiotic [5] wherein position 9 was pre-
viously acylated is used, the spot on a thin layer chromato-
gram of the antibiotic [5] is checked and when the antibiotic
[5] wherein position 9 is a hydroxyl group is used the spot
of acylated derivative at the said hydroxyl group is checked
- 17 -
1~33~75
for termination of the reaction.
By the above reaction, the aldehyde group at position
18 is acylated, and the hydroxyl group at position 3 is
protected by ring closure between the carbon atom at posi-
tion 18 and the oxygen atom at position 3. Also in the casewhere the hydroxyl group at position 9 is not acylated
previously, and/or the hydroxyl group at position 2 is not
acylated previously by an acyl group, preferably an acetyl
group, these hydroxyl groups can be acylated. Since this
protection of the 3 and 18 positions are the most preferred
protective groups for selective reaction and are quite
stable, it is an excellent and convenient protective group
for the hydroxyl group at position 3.
Separation of the product [6] from the reaction mixture
can be made by the same procedure of separation and purifica-
tion of the compound [4] in the process IA] hereinbefore.
The acylation at position 3" of the compound [6] can
be made by the same process of 3"-acylation of the compound
[3] in the process [A] hereinbefore. The thus obtained
compound [7] can be isolated and purified by the same
procedure of the compound [4] in the process [A] herein-
before.
The removal of the protective groups at position 3
and 18 of the compound [7] can be made quantitatively by
allowing to stand at room temperature in methanol solution
containing ammonia or ethanol solution containing aqueous
alkaline carbonate. Reaction can be checked by silica gel
thin layer chromatography whereby disappearance of the
- 18 -
~1'133~75
spot of the compound [7] reveals the termination of the
reaction.
The 2'-acyl group in the 9, 2', 3"-triacyl derivative,
which can be obtained from vacuum concentration of the
reaction mixture, is removed by heating in methanol which
can contain water. Heating can be made by refluxing
methanol. The reaction can be terminated by checking
the silica gel thin layer chromatogram until the spot of
the above 9, 2', 3"-triacyl derivative cannot be detected.
The product obtained by distilling off methanol is
separated and purified as hereinbelow explained to obtain
the compound [lb].
Process [C]: A compound [1'] wherein Rl and R2 are hydrogen
atoms: i.e. the compound [lc] of the formula
C~ ~~ O~ N~C~ 2 ~s
~~~~ [lc]
D C~13 \
~\0 /~
wherein R3 and R4 have the same meanings as hereinbefore.
The above compound [lc] is prepared by reacting the
compound [8] of the formula
,;., ~ -- 19 --
il~3~7C;
R~O~/~ O ~ --~OR,
~ a CD-~ O cx CH3 [8]
~
~0
wherein R51 is a chlorinated acetyl group, R6 is a hydrogen
atom or an R61 group in which R61 is a (C2-C4) alkanoyl
group and R4 has the same meanings as above, with a (C2-C4)
aliphatic carboxylic acid anhydride
in the presence of an in-
organic base to prepare the compound [9] of the formula
C~5 r C~u OR7 N( ~ 2 O~
Rc,O ~ O ~ ~ ~ OA~ [y~
~ ~--O
~ C~s ~
o
~ O
wherein R7 is an R61 group or R21 group, in which R21 is
a (C2-C4) alkanoyl group and R51, R61 and R4 have the same
meanings as hereinbefore; and
acylating the compound [9] at position 3" with a (C2-C6)
aliphatic carboxylic acid halide in the presence of a
tertiary organic amine in an inert organic solvent.
- 20 -
,
1133~5
Also the compound [lc] is prepared in the following
manner:
The 2'-acyl antibiotic [lO] of the formula
CH5 r c~o o~l N(C~ 2 5~
~ ~ ~ i ~C ~ CR~
3CO-< O \
~o~ C~5 C~3 [10]
,~ C~3 \
10 V\o/~=
wherein R4 and R6l have the same meanings as hereinbefore,
is protected at positions 3 and 9 by chlorinated acetyl
halide in the presence of a tertiary organic amine in an
inert organic solvent to prepare the compound [11] of the
formula
C~s C~O ~ N(C~5)2
~ ~ ~ O ~ ~ -GR~
~ ~sCO ~ C~3 C~3 [11]
~ C~5 \
~ 0~o
wherein R4, R51 and R61 have the same meanings as herein-
before. The said compound [11] is 3"-acylated by a (C2-C6)
aliphatic carboxylic acid halide in the presence of a
tertiary organic amine in an inert organic solvent to obtain
B
1~33475
the compound [12] of the formula
17 18
o _<~/~ o_/ ~ G ~)--OR~ ]
5 CO -~_
CHS ~_
~\o/
wherein R21, R3, R4, R5 and R7 have the same meanings as
hereinbefore, or the compound [13] of the formula
RD,O ~ /~O "~ ) 2 OR~
~ H5CO-<~ C~3 C~5 [13
~a2~U
~ CHS \
~o/~
wherein R3, R4, R51 and R61 have the same meanlngs as
hereinbefore.
The said compound [12] or [13] is treated by ammonia
in methanol or ethanol to obtain the compound [14] of the
formula
- 22 -
~.
1133475
CH3 C~O OF~ N(C ~ Z OR5
~IO~ o -~ O ~/~0~
~CO ~/ o o / L14]
C~3 C~5
C~3 \
~ ~0
wherein R3, R4 and R6 have the same meanings as hereinbefore,
and the said compound [14] is heated in methanol which can
contain water to remove the acyl group at position 2'.
The above compound [8] is the antibiotic [2] in which
there is first introduced a protective group to the hydroxyl
group at position 9 for preventing the acylation of hydroxy
groups at positions 3 and 9 in the forthcoming 3"-acylation
reaction. The said protective group is a group which can
easily be removed without destroying the chemical structure on
post-3"-acylation, and is preferably a chlorinated acetyl
group such as a chloroacetyl, dichloroacetyl or trichloro-
acetyl group.
These 9-protected compounds can, if required, have a
previously or posteriorly protected hydroxyl group at
position 2'. The preferred example is a C2-C4 alkanoyl
group such as an acetyl group.
Then the hydroxyl group at position 3 of the compound
[8] is protected by reacting with the corresponding carboxylic
acid anhydride, preferably acetic anhydride, in the presence
of an inorganic base to protect the positions at 3 and 18.
v, ,
- 23 -
113~1~75
Introduction can be made by the same procedure as described
for process [B] hereinbefore. Disappearance of the spot of
the compound [8] on a silica gel thin layer chromatogram can
reveal the termination of the reaction. The compound [9] can
be obtained by the same procedure as that of the isolation
procedure of the process [A].
In this process, the compound [11] can be used instead
of the compound [4] to obtain the compound [lc].
The compound [11] is a compound in which there is
introduced a protective group for the hydroxyl groups at
positions 3 and 9 for preventing acylation in the forth-
coming 3"-acylation. The hydroxyl group at the 2'-position
should preferably be protected by the other protective
group. A preferred example is the acetyl group. Hydroxyl
lS groups at positions 3 and 9 of the 2'-acylated antibiotic
[10] are protected by suitable protective groups. Preferred
examples are a chlorinated acetyl group such as chloro-
acetyl, dichloroacetyl or trichloroacetyl. Introduction
of the protective group is made with a 2 - 3 times excess
amount of chlorinated acetyl halide.
Isolation of the compound [4] in the process [A] can
be applied for obtaining the compound [12] or [13] by 3"-
acylation of the compound [9] or [11].
Removal of protective groups at positions 9, 3 and 18
of the compound [12] or at position 3 or 9 of the compound
[13] can be made by treating with methanol or ethanol solu-
tion containing ammonia. The reaction can be carried out at
room temperature. Termination of the reaction can be checked
B 24 -
1133~75
by disappearance of the compound [12] or [13] on a silica gel
thin layer chromatogram.
The compound ~14] obtained by distilling off ammonia
and methanol from the reaction mixture is heated in methanol
which can contain water to remove the 2'-acetyl group. Heat-
ing is made by refluxing methanol. The product obtained by
distilling the methanol can be purified to prepare the
compound [lc].
Further, the compound [lc] can be prepared as follows:
The compound [21] of the formula
C~3 r CEO OF~ ~(C~ 2
~ ~ O ~ o ~ OR~
o O
~ 5 c~3 C~ [21]
~ C~s ~
~\oi~
wherein R53 is a silyl group, R4 and R6 have the same
meanings as hereinbefore, is acylated by a (C2-C6) aliphatic
carboxylic acid halide under heating in an inert organic
solvent to prepare the mixture of the compound [22] of the
formula
C~3 r c~o OE~ N(C~ 2 OR5
~ O ~ ~ ~ r~
~ ~OR55
~3 ~o~O
11~3~7S
wherein R8 is an R6 group or R3 group, and R3, R4, R53 and
R61 have the same meanings as hereinbefore, and the compound
~22'] of the formula
C~5 17 18 N(C~S)2 OR3
RS3~ o '7 ~ OR~
5 > aRS ~S ' C~5 [
~o
wherein R3, R4, R53 and R8 have the same meanings as herein-
before. -
The mixture is treated as follows:
(A) removing the silyl group and acyl groups at position 18
and 2' by heating in an aqueous lower alkanol which can
contain base;
(B) removing the silyl group and acyl group at position 18
by treating under non-heating conditions in a aqueous lower
alkanol in the presence of a base, and removing the 2'-acyl
group by heating in methanol which can contain water;
(C) removing the acyl group at position 18 by treating with
methanol containing ammonia, thereafter removing the silyl
group and 2'-acyl group by heating with aqueous lower alcohol;
or
(D) removing the acyl group at the 18 position by treating
with methanol containing ammonia, thereafter removing the
2'-acyl group by heating with methanol, and removing the
... - 26
1133~75
silyl group by treating with aqueous acid.
The compound 121] can be prepared by the same process
of producing the compound [3], wherein R5 is a silyl group,
in the process [A] hereinbefore, and the amount of silylat-
ing reagent is preferably a 2 - 5 molar excess with respect
to the antibiotic 12] or its 2'-acyl derivative.
The compounds [22] and [22'] prepared by 3"-acylation
of the compound [21] can be obtained by the same process
for preparing the mixture of compounds [4] and [4'] by
acylating at position 3" of the compo~nd [3] as described in
the process [A] hereinbefore.
Further removal of the silyl group and the 18- and
2'-acyl groups of the mixture of~the compounds [22~ and [22']
can be made by the same process as in the process [A] herein-
before, wherein the silyl group and the 18- and 2'-acyl
groups of the mixture of the compounds [4] and [4'] in which
R5 is a silyl group are removed.
Process [D]: A compound [1"] wherein Rl is a C2-C3 alkanoyl
group and R2 is a hydrogen atom; i.e. the compound [ld] of
the formula
C~s rC~O O~ N(C~S) OR~
~ ~ ~ O O~ OR3
~ ~3CO ~ O
25~ ~ oRll ~3 C~5 lld]
' ~0>~
B 27 -
1133~L7ri
wherein Rll is a C2-C3 alkanoyl group, and R3 and R4 have the
same meanings as hereinbefore, can be prepared as follows.
A 2'-acyl antibiotic [15] of the formula
CH5 rCHO ~l N( ~ 2- OH
O ~ O ~ OR~
~3CO-< o O llS]
ORl C~s
T15 ~
~/~o/
wherein R61 is a (C2-C4) alkanoyl group, and Rl and R4 have
the same meanings as hereinbefo~e, is reacted with p-
nitrobenzoyl halide in the presence of a tertiary organic
amine in an inert organic solvent to prepare the compound
[16] of the formula
c~3 rCHO OR~<,O~d 2
~ ~ s' ~ o ~ ~ OR~
~ 3CO~ C~3 C~3 [16]
~/ ,~ OR 1
~0
wherein R52 is a p-nitrobenzoyl group, and Rl, R4 and R61
have the same meanings as hereinbefore, and the said compound
[16~ is acylated by heating with a (C2-C6) aliphatic
carboxylic acid anhydride in the presence of alkaline
carbonate or a tertiary organic base to prepare the mixture
- 28 -
11339~75
of the compound [17] of the formula
17
C~3 F F3 OR~ N(a~ 2 OR~
5~2 / ~ \ ~ ~ ~ ~\ ~ OR2
~0~o
n Rll, R3, R4, R52 and R8 have the same meanings as
hereinbefore, and the compound [18] of the formula
R~20~ ~ ~OR2
~ ~ O-R~ c~s CHs
~0~
n Rll, R3, R4, R52 and R8 have the same meanings as
hereinbefore. The said mixture is treated with ammonia in
ethanol to remove the protective group at position 9 and
the acyl group at position 18, then heated in methanol, which
can contain water, to remove the 2'-acyl group.
The above 2'-acyl antibiotic ~15] can be prepared by
a known method such as the process disclosed in Japanese
Patent Publication No. 53-7434 and J. Med. Chem., 20 (5),
732 - 736 (1977).
~ - 29 -
11;~34~75
Protection of the hydroxyl group at position 9 of the
2'-acyl antibiotic [15] can be made by reacting with p-
nitrobenzoyl halide, preferably p-nitrobenzoylchloride, in
the presence of a tertiary organic amine in an inert organic
solvent. Examples of inert organic solvents are acetone,
methyl ethyl ketone, dichloromethane, ethyl acetate, di-
methoxyethane, tetrahydrofuran and dioxane. Examples of
tertiary organic amines are pyridine, picoline or collidine,
but other known tertiary organic amines can be used. The
above reaction proceeds under ice-cooling or at room tempera-
ture.
If a chlorinated acetyl group such as monochloroacetyl
or dichloroacetyl group is used-in place of a p-nitrobenzoyl
group as the protective group for position 9, the acyl group
at position 3 will simultaneously be removed at the removal
of the said protective group in the forthcoming process,
therefore a p-nitrobenzoyl group is preferred as the protec-
tive group for position 9.
The thus obtained compound [16] can be obtained by
isolating, in the case where a water miscible organic sol-
vent is used as the reaction medium, the precipitate by
filtration with adjustment of the medium to a pH of 8 - 10.
Or the compound can be obtained by extracting, in the case
where a water- water immiscible organic solvent is used as
the reaction medium, with a water immiscible organic solvent
after pouring the said reaction mixture into water and
adjusting its pH to 8 - 10. Further purification can be
made, if required, by chromatography using such as silica
B - 30 -
1133~75
gel, alumina or an adsorption resin.
For introduction of different acyl groups to posi-
tions 3 and 3" of the compound [15], wherein Rl is a
hydrogen atom, namely in case where there is obtained the
compounds [17] and [18], wherein Rl and R3 are different
acyl groups, the desired acyl group such as an acetyl or
propionyl group is previously introduced into the hydroxyl
group at position 3 of the compound [16].
Further, acylation of the compound [16] with a corre-
sponding aliphatic carboxylic acid anhydride is made byheating in the presence of a base. Examples of suitable
bases are alkaline carbonates such as potassium carbonate
or sodium carbonate, and tertiary organic amines such as
pyridine, picoline or collidine. Known tertiary organic
amines other than those having a pyridine group can be used.
The reaction temperature may usually be 50 - 120C, pre-
ferably 80 - 100C. Reaction time can be varied depending
on the reaction temperature, since the progress of the
reaction can be checked by silica gel thin layer chromato-
graphy, the disappearance of a spot of compound [16] on thechromatogram reveals the termination of the reaction, and
is usually 1 - 100 hours.
As a result of the above reaction, the original 4"-
acyl group is rearranged to position 3", and the (C2-C6)
alkanoyl group, for example, a acetyl or propionyl group,
is introduced to position 4". Further, in the case of the
2'-acyl antibiotic [15], wherein Rl is a hydrogen atom, the
hydroxyl group at position 3 is acylated. Furthermore, the
aldehyde group at position 18 is acylated to a considerable
- 31 -
1133~7S
extent, and hence the compounds 117] and [18] are prepared.
The thus prepared mixture of compounds 117] and 118]
can be separately purified, if required; however the
mixture itself can be used for the following reaction.
Next, removal of the protective group at position 9
of the compounds 117] and 118] can be made by treating with
ammonia in methanol or ethanol at room temperature. By
this reaction the acyl group at the 18 position of the
compound 117] is removed. Termination of the reaction can
be checked by silica gel thin layer chromatography, there-
fore it can be determined by disappearance of the spots
corresponding to the compounds 117] and 118] on a thin layer
chromatogram.
The compound, wherein the protective group of position
9 has been removed, is obtained by distilling of ammonia and
alcohol from the reaction mixture, and the removal of the
2'-acyl group of the said compound is made by heating for
reflux with methanol, which can contain water.
The product lld] can be obtained by distilling off
methanol and purifying.
Process IE]: A compound 11"] wherein Rl is a hydrogen atom
and R2 is a (C2-C4) alkanoyl group; i.e. the compound lle]
of the formula
- 32 -
--- 1133475
c~3 C~O ~ ~(~J 2 OR~
R2l~
~[ ~ C ~ 3 [ le ]
~3>~
wherein R21 is a (C2-C4) alkanoyl group, and R3 and R4 have
the same meanings as hereinbefore, is prepared by reacting
the compound [5] with a (C2-C4) aliphatic carboxylic acid
anhydride in the presence of an inorganic base to prepare
the compound ~6], acylating the compound [6] with a (C2-C6)
aliphatic carboxylic acid anhydride by heating in the pre-
sence of an alkaline carbonate or a tertiary organic amine
to prepare the compound [19] of the formula
17 18
C~3 rC~OR2l OR7 (C~ 2 OR~
" R2l0~ 47 ~ OR~ [19]
~ ~sc~\ I O
~ ~ C~3 C~3
~ C~s \
~\0~
wherein R21, R3, R4 and R7 have the same meanings as here-
inbefore, and removing the protective group at position 18
of the said compound [19] by treatinq with ammonia in
methanol or with aqueous alkaline carbonate in ethanol, then
removinq the 2'-acyl group by heating with methanol.
- 33 -
113~75
The preparation of compounds [5] and [6] hereinabove
are illustrated in the process [B] hereinbefore.
Acylation of the compound [6] by employing the corre-
sponding carboxylic acid anhydride for preparing the compound
[19] can be made by the same process for preparing the
compounds [17] and [18] from the compound [16] illustrated
in the process [D] hereinabove.
Removal of the protective group at positions 3 and 18
of the compound 119] can be made by treating the compound [19]
with methanol containing ammonia or e.thanol containing aqueous
alkaline carbonate at room temperature or with heating if
required. By this reaction, the acyl group at the 18-posi-
tion is removed to form the original aldehyde group. Reac-
tion can be traced by checking the disappearance of a spot
corresponding to the compound Il9] on a silica gel thin
layer chromatogram.
The 9, 2', 3"-triacyl derivative thereof, which can
be obtained by distilling off ammonia and methanol or
ethanol from the reaction mixture, is treated by the same
procedure as for the process ID], in which deacylation at
position 2' is performed by heating with methanol (which
can contain water), to remove the 2'-acyl group. After
distilling off methanol, and purifying~the compound Ile]
can be prepared.
Process IF]: A compound [1"] wherein Rl and R2 are hydrogen
atoms; i.e. the compound llf] of the formula
. _~
- 3~ -
1133475
c~5 _C~O o~ ~(C~ 2 OR~
~ ~ O ~ ~ O ~ ~ .OR3 llf)
\ ~3CO ~ o O
~ ~ O~ C~5 C~3
~ O >
wherein R3 and R4 have the same meanings as hereinbefore, is
produced by reacting the compound [8] with a (C2-C4) aliphatic
carboxylic acid anhydride in the presence of an inorganic
base to prepare the compound [9], acylating the said compound
[9] by heating with a (C2-C6) aliphatic carboxylic acid
anhydride in the presence of an alkaline carbonate or a
tertiary organic amine to prepare compound [20] of the formula
. C~a rc~o ~ OR7 N(C~ 2 OR~
R5lO ~ ~ O ~ ~ ~ OR5
~ ~5CO ~ I C~5 C~3
~ o
wherein R51 is a chlorinated acetyl group, R7 is a R61 group
or R21 group, and R21' R6l' R3 and R4 have the same meanings
as hereinbefore, removing the protective groups at position
9 and at positions 3, 18 by treating with ammonia in
methanol, then removing the 2'-acyl group by heating with
methanol.
- 35 -
1133~7S
The production process of the compounds [8] and [9] is
illustrated in the process IC] hereinbefore.
Acylation of the compound [9] by a carboxylic acid
anhydride for preparation of the compound [20] can be made
by the same procedure for preparing the compounds [17] and
[18] from the compounds [16] as in process [D].
Removal of the protective groups at position 9 and at
positions 3, 18 can be made by treating the compound [20] with
ammonia in methanol at room temperature. By this reaction
the acyl group at position 18 is removed and changed to the
original aldehyde group. The reaction is checked by dis-
appearing of a spot corresponding to the compound [20] on a
silica gel thin layer chromatogram.
The 2', 3"-diacyl derivative, obtained by distilling
off the ammonia and methanol from the reaction mixture, is
heated in methanol (which can contain water) by the same
procedure of 2'-deacylation as in the process [D] herein-
before, to remove the acyl group at position 2'. The product
obtained by distilling off methanol is separated and purified
to obtain the product [lf].
Isolation of the compound [1] from the reaction mixture
is performed in accordance with the known separation and
purification methods for macrolide antibiotics such as con-
centration, extraction, washing, transferred extraction and
recrystallization, and chromatography using silica gel,
active alumina, an adsorption resin or an ion-exchange resin.
Antimicrobial spectra of the compound [1] of the present
invention are shown in the table. These data show that the
'O
_ 36 ~
~33~7X
compound [1] of the present invention exhibits enhanced anti-
microbial activity against not only the susceptible strains
but also resistant strains as compared with the known anti-
biotics.
- 37 -
'~3
11;~39L7~
,~ o o o
,,
~ ~ /\ /\ ,~ U~
a~
D ~ O O O O
. . . . o o o o ~
~1 ~ ~ ~ O
/\ ~ /\ /\ rl~
,, ,~
r~ ~ In ~ ~
o~ O , t~ o
~ ~ _, ~ o o ,, ~9 ~
~ ~ /\ ~0 ~
, ,, U~
l ~n ~D ~O ~ ~ O U~ O CO
a~ I . ~ ~ . o . o .
H ~; ~ ~ l
:E~ .
~ , ~1
l ~ "~ ~ ~
O ~ C) ~ CO ~ D O u~ O ~r ~
.,1 ` a~ I . . . o o ~ ,1 o
~ ~ O ~1 0 ~ 1 0 U~ ~
~a ~ /~ ~ s~ a)
_ N
c) I ~ ,,, tn
~ r-l R
O ~ C) ¢ ~ ~ D O O O ~ O
C~ ` ~ I . . . O U~ O S~_~
~ ~ l O ~ C) g
O R ~0
.~ ~ U~ ~ _
.,1 I ~ ~ CD ~D er a~ o o o oo ~ u~
~ _ ~ I . . O Ln O ~.
H ~ ~ ~ O _I O O~1 0 ~ ~a
I ~
R l ~1
O ~
0~ ~ o Ul o ~ ~o~
X o ~1 0 0 0 O ~
I ~ ~ CO O ~) O et' t~ ~
a~ * . . o . o. ~ u~
O O ~ 1 0 0--1
a.) o
/ .,, a~ . R
o / u~
/ . ~ ~ o_l a
/ ~ a)aJ a)o s~ o
/ ~$-1 C ~ 11S h O ~ S~ ,~ h
Ul I U~ ~ ~ ~ ~
Q / R f~ ~1I~S ~a5 (~)' (~ ~ (~ ~ . ~1 ~
U~ .C ~ ,5 ~ .~ ~ ~0 ~ ~ S ~ ~--I ~ ~--
/ ~ ~ ~ h ~ a~ ~ td O (3 0 S~ ~1 * *
/ Ul ~ ~ ~ ~ .IJ ~ ~ ~ ~ *
/ h U~
~ ,~
-- 38 --
113347S
The following examples illustrate the present invention.
Rf values in the examples are measured, if not specified,
by the following thin layer chromatography.
Carrier: silica gel 60 (Art. 5721, Merck Co.)
Developer:
A: n-hexane - benzene - acetone - ethyl acetate -
methanol (90 : 80 : 25 : 60 : 30)
B: benzene - acetone (3 : 1)
; C: benzene - acetone (5 : 1)
Example 1.
3,3"-diacetylleucomycin A5:
To 9-dichloroacetyl leucomycin A5 (RfA = 0.55,
RfB = 0.11) (lO g) dissolved in~ dry acetone (250
ml) was added dry pyridine (11.5 ml), acetyl
chloride t9.5 ml) was added, and reacted at 50C for 18 hours.
The reaction mixture was poured into ice-water (250 ml),
adjusted to pH 9.5 by adding aqueous ammonia and extracted
twice with chloroform (250 ml). The chloroform layer was
dehydrated by anhydrous sodium sulfate and dried in vacuo to
obtain a brownish colored powder (9.82 g) of 3,2',3"-triacetyl-
9-dichloroacetyl-leucomycin A5 (RfB = 0.62, RfC = 0 35) as
a main component.
This powder was dissolved in ammonia saturated methanol
solution (300 ml), allowed to stand for 1 hour at room tempera-
ture and dried in vacuo to obtain a powder of 3,2',3"-tri-
acetyl leucomycin A5 (RfA = 0.67, RfB = 0.27, RfC = 0 09)
as a main component.
- 39 -
B
1133~75
The powder was dissolved in methanol (300 ml), refluxed
for 20 hours and concentrated in vacuo. The residue was chroma-
tographed on a silica gel column by eluting with benzene -
acetone (7 : 1). Fractions showing RfA = 0.58 were collected
and concentrated in vacuo to obtain the product (875 mg).
RfA = 0.58, RfB = 0-15
Mass (m/e) : 855 (M ), 796 (M -59), 768 (M -87).
NMR (CDC13, 100 MHz): 1.43 (3"-position: CH3), 2.02
(3"-position: OAc), 2.29 (3-posi-
tion: OAc), 9.79 (CHO) ppm.
The above 9-dichloroacetyl leucomycin A5 was prepared
by the process described in Japanese Patent Open. No. 50-96584.
Example 2.
3"-acetyl leucomycin A3:
To 2'-acetyl leucomycin A3 (2 g) dissolved in dry di-
chloromethane (10 ml) was added dry pyridine (0.7 ml) and
dichloroacetylchloride (0.7 ml) with stirring under ice-cooling,
for 1 hour at room temperature. To the reaction mixture was
added water (10 ml), then the reaction mixture was adjusted
to pH 2 by adding 1 N-HCl. After separating the aqueous layer
the dichloromethane layer was washed with water and with
saturated sodium bicarbonate in this order. After drying with
anhydrous sodium sulfate, the solution was dried in vacuo to
obtain 2'-acetyl-9-dichloroacetyl leucomycin A3.
This was dissolved in dry acetone (10 ml) and thereto was
added dry pyridine (2 ml). After adding acetylchloride (1.4
ml) under cooling with stirring, stirring was continued for
20 hours at 50C. The reaction mixture was added to ice-water
- 40 -
1133~5
(100 ml) and adjusted to pH 9.5 by adding concentrated aqueous
ammonia to obtain a precipitate by filtration, which was washed
with water and dried completely. The dried material was
chromatographed on a silica gel column by developing with benzene
- acetone (18 : 1). Fractions containing the main component
were dried in vacuo to obtain 2',3"=diacetyl-9-dichloroacetyl
leucomycin A3 (RfB = 0.62, RfC = 0.36) (460 mg).
After dissolving the said product in ammonia saturated
methanol solution (10 ml) and allowed to stand for 2 hours,
the solvent was distilled off under reduced pressure. The
residue was dissolved in methanol (20 ml) and refluxed with
heating for 17 hours. The reaction mixture was dried in vacuo
and the residue was treated with silica gel column chromato-
graphy by eluting with benzene - acetone (6 : 1). Eluted
fractions showing RfA = 0.62 were collected and dried in vacuo
to obtain the product. Yield: 310 mg.
RfA = 0-62, RfB = 0-17
Mass (m/e): 869 (M ), 810 (M -59), 768 (M -101).
The above 2'-acetyl leucomycin A3 was prepared by the
20 process described in Japanese Patent Publ. No. 53-7434.
Example 3.
9,3"-diacetyl leucomycin A5:
To leucomycin A5 (RfA = 0.38, RfB = 0 04~ RfC = 0.01)
(20 g) dissolved in acetic anhydride (40 ml) was added sodium
hydrogen sulfate (17.4 g), and the reaction mixture was stirred
at room temperature for 1 hour and at 60C for 5 hours. The
reaction mixture was then added to ice-water (400 ml) and
- 41 -
.l6~
1133~L7S
adjusted to pH 9.5 by adding aqueous ammonia. The precipitate
was filtered, washed with water and dried to obtain a powder
(22.4 g). This was treated with silica gel column chromato-
graphy by eluting with benzene - acetone (9 : 1). The eluted
fractions showing RfA = 0.76 were collected and dried in vacuo
to obtain 9, 18, 2'-triacetyl-3,18-0-cyclo-leucomycin A5
(15.8 g).
RfA = 0-76, RfB = 0 50~ RfC = 0-22
Mass (m/e): 897 (M ), 810 (M -87), 750 (M -87,-60)
NMR (CDC13, 100 MHz): 2.06 (2'-OAc), 2.10 (18-OAc), 2.20
(9-OAc) ppm.
m.p.: 106 - 111C (no clear melting point)
W: ~EtaxH = 235.2 mu (~ = 2.6 X 10 )
To the above product (5 g) dissolved in dry ethyl acetate
(50 ml) was added dry pyridine (5 ml) and there was added
dropwise acetylchloride (4.0 ml) with stirring under ice-cool-
ing for 10 minutes, then further reacted at 60C for 45 hours.
The reaction mixture was added to ice-water (500 ml), adjusted
to pH 9.5 by adding aqueous ammonia and extracted twice with
chloroform (300 ml). The chloroform layer was washed with
water, dried by anhydrous sodium sulfate and dried in vacuo
to obtain a powder containing 9, 18, 2',3"-tetraacetyl-3,
18-0-cyclo-leucomycin A5 (RfB = 0.71, RfC = 0.46) (5.06 g).
To this powder dissolved in ethanol (150 ml) was added
5% aqueous sodium carbonate (11.5 ml) and the mixture was
stirred at room temperature for 48 hours. Ethanol was
distilled off under reduced pressure and the residue was
- 42 -
~1334~5
dissolved in chloroform. After washing the solution with
water, chloroform was distilled off. The residue was dissolved
in methanol (50 ml), refluxed for 18 hours and the reaction
mixture was dried in vacuo. The thus obtained residue was
chromatographed on a silica gel column by eluting with benzene
- acetone (10 : 1). The eluted fractions showing RfA = 0.67
were dried in vacuo to obtain the product.
Yield: 1.26 g.
RfA = 0-67, RfB = 0-28
Mass (m/e): 855 (M ), 796 (M -59), 768 (M -87).
NMR (CDC13, 100MHz): 1.43 (3"-CH3), 2.03 (3"-OAc), 2-03
(9-OAc), 9.91 (CHO) ppm.
Example 4.
3"-acetyl leucomycin A5:
In Example 3, leucomycin A5 was replaced by 9-dichloro-
acetyl leucomycin A5 to obtain 18, 2'-diacetyl-9-dichloro-
acetyl-3, 18-0-cyclo-leucomycin A5 (14.7 g.)
RfA = 0 79~ RfB = 0-51, RfC = 0-22
NMR (CDC13, 100 MHz): 2.06 (2'-OAc), 2.11 (18-OAc),
6.38 (9-COCHC12) ppm.
To the above compound (1 g) dissolved in dry ethyl ace-
tate (10 ml) was added ~-collidine (1.5 ml) and there was
added dropwise acetylchloride (0.72 ml) with stirring under
ice-cooling. Thereafter, stirring was continued at 60C for
20 hours and at 70C for 24 hours. The reaction product
was dissolved in chloroform (60 ml) and washed with 0.1 N-HCl,
water, saturated aqueous sodium bicarbonate and water, in this
order. The solution was dried by adding anhydrous magnesium
~ ~c
1133475
sulfate and concentrated under reduced pressure. The residue
was chromatographed on a silica gel (20 g) column by eluting
with benzene - acetone (15 : 1). The eluted fractions show-
ing RfB = 0 74 were dried to obtain 18, 2', 3"-triacetyl-3,
18-0-cyclo-9-dichloroacetyl leucomycin A5 (RfB = 0 74' RfC =
0.51) (604 mg).
The said compound was dissolved in ammonia saturated
methanol (10 ml), allowed to stand for 20 hours and dried in
vacuo. The residue was dissolved in methanol (20 ml), refluxed
for 15 hours, then dried in vacuo. The residue thus obtained
was chromatographed on a silica gel (10 g) column by eluting
with benzene - acetone (3 : 1). Fractions showing RfA = 0 45
were concentrated in vacuo to obtain the product (450 mg).
RfA = 0 45~ RfB = 0.10
Mass (m/e): 813 (M ), 754 (M -59), 726 (M -87)
NMR (CDC13, 100 MHz): 1.43 (3"-CH3), 2.02 (3"-OAc),
9.88 (CHO) ppm.
Example 5.
3,4"-diacetyl-3"-butyl leucomycin V:
To 2'-acetyl leucomycin A5 (2g) dissolved in dry di-
chloromethane (20 ml) was added dry pyridine (0.46 ml) and
p-nitrobenzoylchloride (960 mg) to react at room temperature
for 15 hours. The reaction mixture was added to water (10
ml), adjusted to pH 2 by adding 1 N-HCl, the aqueous layer
separated and the dichloromethane layer washed with water
and saturated aqueous sodium bicarbonate, in this order.
After drying the solution with anhydrous sodium sulfate, the
reaction mixture was dried in vacuo to obtain almost
- 44 -
~13347S
quantitatively 2'-acetyl-9-p-nitrobenzoyl leucomycin A5. To
this compound dissolved in dry pyridine (20 ml) was added
acetic anhydride (2.5 ml) and reaction was continued for 3
days at 100C. The reaction mixture was concentrated under
reduced pressure and the residue was dissolved in chloroform
(20 ml). Water (lO ml) was added thereto, the reaction
mixture was adjusted to pH 2 by addition of l N-HCl and the
separated chloroform layer was washed with water and saturated
aqueous sodium bicarbonate solution, in this order, then
dried by adding anhydrous sodium sulfate and dried in vacuo.
To the residue was added ammonia saturated methanol solution
(50 ml), allowed to stand for overnight and concentrated in
vacuo. The residue was dissolved in methanol (50 ml), refluxed
for overnight and dried in vacuo. The thus formed residue
was chromatographed on a silica gel column by developing with
benzene - acetone (6 : 1). The eluate showing RfA = 0.58 was
concentrated in vacuo to obtain the product. Yield: 225 mg.
RfA = 0-58, RfB = 0-17
Mass (m~e): 855 (M ), 796 (M -59), 768 (M -87)
The above 2'-acetyl leucomycin A5 was prepared by the
process described in Japanese Patent Publ. No. 53-7434.
Example 6.
4"-acetyl-3"-isovaleryl leucomycin U:
To 2'-acetyl leucomycin A3 (a g) dissolved in dry di-
25 chloromethane (20 ml) was added dry pyridine (0.43 ml) and p-
nitrobenzoylchloride (896 mg) and reacted for 3 days at room
temperature. To the reaction mixture was added water (10 ml),
the reaction mixture was adjusted to pH 2 by adding lN HCl,
B - 45 -
1133475
the aqueous layer was separated and the dichloromethane layer
was washed with water, saturated aqueous sodium bicarbonate
and water, in this order. After drying with sodium sulfate,
the solution was dried up to obtain 2'-acetyl-9-p-nitrobenzoyl
leucomycin A3. This was dissolved in dry pyridine (20 ml),
acetic anhydride (2.5 ml) was added thereto and reacted at
100C for 3 days. The reaction mixture was concentrated in
vacuo, chloroform (20 ml) and water (20 ml) were added the
aqueous layer was adjusted to pH 2 and the aqueous layer
separated. The chloroform layer was washed with water and
saturated aqueous sodium bicarbonate, and dried up in vacuo.
The residue was dissolved in ammonia saturated methanol (50
ml), allowed to stand for 3 days-at room temperature then
concentrated in vacuo. The thus obtained residue was dissolved
in methanol (50 ml), refluxed by heating for 20 hours and
concentrated in vacuo. The residue was chromatographed on a
silica gel column by developing with benzene - acetone (7 : 1)
and the eluted fractions showing RfA = 0.60 were collected
and dried in vacuo to obtain the product. Yield: 190 mg.
RfA = 0-60, RfB = 0-16
Mass (m/e): 896 (M ), 810 (M -59) 768 (M -101)
Example 7.
9,4"-diacetyl-3"-butyryl leucomycin V:
To 9, 18, 2'-triacetyl-3, 18-0-cyclo-leucomycin A5
(5.0 g), obtained in Example 3, dissolved in dry pyridine
(30 ml) was added acetic anhydride (15 ml) and reacted at
100C for 39 hours. The reaction mixture was added to
water (500 ml) containing aqueous ammonia (pH 9.5) and
- 46 -
~133475
extracted twice with chloroform (300 ml). The extract was
washed with water (500 ml), dried by adding anhydrous sodium
sulfate and dried up in vacuo to obtain a powder containing
9, 18, 2', 4"-tetraacetyl-3"-butyryl-3, 18-0-cyclo-leucomycin
V as a main component (5.05 g).
RfB = 0 73~ RfC = 0 50
Mass (m/e): 939 (M ), 880 (M -59), 852 (M -87).
To this powder dissolved in ethanol (150 ml) was added
5~ aqueous sodium carbonate (11.5 ml), then the mixture was
allowed to stand at room temperature for 45 hours, and at 70C
for 12 hours. Ethanol was distilled off under reduced pressure
and the residue was dissolved in chloroform (150 ml), which
was washed twice with water (100-ml), dehydrated by adding
anhydrous sodium sulfate and dried up in vacuo to obtain the
powder (4.73 g). The powder was dissolved in methanol (50 ml),
refluxed for 17 hours, then concentrated in vacuo. The residue
was chromatographed on a silica gel column by developing with
benzene - acetone (10 : 1), and the eluted fractions showing
RfA = 0.66 were collected, dried up in vacuo to obtain the
product. Yield: 2.12 g.
RfA = 0.66, RfB = 0-28
Mass (m/e): 855 (M ), 796 (M -59), 768 (M -87)
NMR (CDC13, 100 MHz): 1.44 (3"-CH3), 2.02 (9-OAc),
2.15 (4"-OAc), 9.90 (CHO) ppm.
Example 8.
4"-acetyl-3"-butyryl leucomycin V:
In example 3, leucomycin A3 was replaced by 9-chloro-
acetyl leucomycin A5 to obtain 18, 2'-diacetyl-9-chloroacetyl-
3, 18-0-cyclo-leucomycin A5.
' ~'.i
- 47 -
113~4~'5
Yield: 74%.
RfA = 0.78, RfB = 0 50' RfC = 0.22
NMR (CDC13, 100 MHz): 2.07 (3H, 2'-OAc), 2.12 (3H,
18-OAc), 4.31 (2H, 9-COCH2Cl)
ppm.
To the above substance (5 g) dissolved in acetic anhy-
dride (15 ml) was added potassium carbonate (3.5g), reacted
at 90C for 26 hours and at 100C for 6 hours. The reaction
mixture was poured into the water (200 ml), adjusted to pH
9.5 by adding aqueous ammonia and extracted twice with chloro-
form (200 ml). The extract was washed with water, dehydrated
by anhydrous sodium sulfate and dried up in vacuo to obtain
the powder (5.07 g). The powder was chromatographed on a
silica gel column by eluting with benzene - acetone (16 : 1).
Eluates showing RfB = 0.72 were collected and concentrated
in vacuo to obtain 18, 2', 4"-triacetyl-3"-butyryl-9-chloro-
ace~yl-3, 18-0-cyclo-leucomycin V (RfB = 0.72, RfC = 0 47)
(2.47 g), which was dissolved in ammonia saturated methanol
solution (60 ml), allowed to stand at room temperature for
17 hours and dried up in vacuo. The residue was dissolved
in methanol (60 ml) and refluxed for 20 hours. The reaction
mixture was dried up in vacuo and chromatographed on a silica
gel column by developing with benzene - acetone (4 : 1).
Eluted fractions showing RfA = 0 45 were collected and dried
up to obtain the product. Yield: 1.72 g.
RfA = 0 45~ RfB = 0-10
Mass (m/e): 813 (M ), 754 (M -59), 726 (M -87)
NMR (CDC13, 100 MHz): 1.44 (3"-CH3), 2.16 (4"-OAc),
9.93 (CHO) ppm.
B
- 48 -
1133475
Example 9.
3"-acetyl-SF-837:
To SF-837 substance (4.0 g) dissolved in acetone (40 ml)
was added acetic anhydride (2.5 ml) and stirred for 3 hours at
room temperature. To the reaction mixture was added ice-water
(400 ml), the pH was adjusted to 8.5 by adding 7~ aqueous
ammonia and the reaction mixture was extracted twice with
benzene (200 ml). The benzene layer was dehydrated with
anhydrous magnesium sulfate and dried up in vacuo to obtain
2'-acetyl-SF-837 substance (RfA = 0.66, RfB = 0.33)(4.15 g,
yield: 98.6~).
To this substance dissolved in acetone (40 ml) was
added dry pyridine (1.34 ml) an* there was added dropwise
dichloroacetylchloride (1.07 ml) under cooling, then stirred
for 1 hour and 20 minutes under cooling. The reaction mixture
was added to ice-water (400 ml) and its pH adjusted to 9.5
by 7% aqueous ammonia. The precipitate was filtered, washed
and dried completely in vacuo to obtain a powder of 2'-acetyl-
9-dichloroacetyl-SF-837 substance (RfA = 0.83, RfB = 0.71,
RfC = 0 45) (4.13 g).
To 2'-acetyl-9-dichloroacetyl-SF837 substance (1 g)
dissolved in dry ethyl acetate (10 ml) was added y-collidine
(1.5 ml) and there was added dropwise acetylchloride (0.73 ml)
under ice-cooling. Stirring was continued for 2 hours at room
temperature, then at 70C for 48 hours. The reaction mixture
was poured in ice - water (50 ml), adjusted to pH 5.7 by
adding 7% aqueous ammonia and extracted twice with chloroform
(50 ml).
- 49 -
1~33~7S
The extract was dehydrated by anhydrous magnesium
sulfate and concentrated in vacuo. The residue was dissolved
in acetone (10 ml), ice-water (100 ml) was added and the pH
was adjusted to 9.5 by adding aqueous ammonia. The precipi-
tate was filtered, washed with water and dried to obtain theproduct (850 mg). This was chromatographed on a silica gel
column by developing with benzene - acetone (20 : 1). The
eluate showing RfC = 0.71 was concentrated in vacuo to obtain
2', 3"-diacetyl-9-dichloroacetyl-SF-837 substance (RfB = 0.87,
RfC = 0.71) (550 mg).
The compound was dissolved in ammonia saturated methanol
solution (10 ml), allowed to stand for 2 hours at room tempera-
ture, dried up in vacuo, dissolved in methanol (20 ml) and
heated at 70C for overnight. The reaction mixture was
dried up in vacuo and the residue was chromatographed on a
silica gel column by eluting with benzene - acetone (5 : 1).
The eluate showing RfA = 0.58 was concentrated in vacuo to
obtain the product (420 mg).
RfA = 0-58, RfB = 0-22
Mass (m/e): 855 (M ), 796 (M -59), 782 (M -73)
NMR (CDC13, 100 MHz): 1.43 (3"-CH3), 2.01 (3"-Ac),
2.57~3'-N(CH3)2], 3.58 (4-CH3),
9.72 (CHO) ppm.
Example 10.
3-acetyl-3"-propionyl leucomycin A5:
In Example 2, 2'-acetyl leucomycin A3 was replaced by
2'-acetyl leucomycin A5 to prepare 2'-acetyl-9-dichloroacetyl
leucomycin A5.
- 50 -
1133475
To 2'-acetyl-9-dichloroacetyl leucomycin A5 (2 g)
dissolved in dry acetone (10 ml) there was added dry pyridine
(1.6 ml)~further added acetylchloride (1.3 ml) under ice-
cooling, and reacted for 2.5 hours at 45C. The reaction
mixture was added to ice-water (100 ml), its pH was adjusted
to 9.5 by adding aqueous ammonia, and the reaction mixture
was extracted twice with chloroform (50 ml). The chloroform
layer was dehydrated by anhydrous sodium sulfate and dried
up to obtain 3,2"-diacetyl-9-dichloroacetyl leucomycin A5
(2.02 g).
RfA = 0-84, RfB = 0-67, RfC = 0-36
To the 3,2"-diacetyl-9-dichloroacetyl leucomycin A5
(1.5 g) dissolved in dry dioxane (15 ml) was added ~-collidine
(2.25 ml), there was added propionylchloride (1.39 ml) under
cooling and reacted at 100C for 44 hours. The reaction
mixture was added to ice-water (150 ml) and extracted with
benzene (150 ml). The benzene layer was washed with diluted
aqueous ammonia and concentrated in vacuo. The residue was
dissolved in a small amount of benzene and chromatographed
on a silica gel column by eluting with benzene - acetone
(25 : 1). The eluate showing RfC = 0 74 was dried up to
obtain 3,2"-diacetyl-9-dichloroacetyl-3"-propionyl leucomycin
A5 (562 mg).
RfB = 0-83, RfC = 0 74
NMR (CDC13j 100 MHz): 1.43 (3"-CH3), 2.03 (2'-Ac),
2.29 (3-Ac), 2.46 [3'-N(CH3)2~,
3.52 (4-OCH3), 6.04 (9-COCHC12),
9.76 (CHO) ppm.
- 51 -
1133~75
The product hereinabove was dissolved in ammonia
saturated methanol solution (10 ml) and allowed to stand for
1 hour. The reaction mixture was concentrated in vacuo and
the residue was dissolved in methanol (20 ml), refluxed for
16 hours, then dried in vacuo. The residue was dissolved in
small amount of benzene and chromatographed on a silica gel
column by eluting with benzene - acetone (10 : 1). Fractions
showing RfA = 0 57 were dried up in vacuo to obtain the
_ .
product (420 mg).
RfA = 0 57~ RfB = 0.19
Mass (m/e): 869 (M ), 796 (M -73), 782 (M -87)
NMR (CDC13, 100 MHz): 1.44 (3"-CH3), 2.30 (3-Ac), 2.60
l3'-~(CH3)2]~ 3-59 (4 OC 3
9.75 (CHO) ppm.
Example 11.
3-acetyl-3"-propionyl leucomycin A5:
In Example 10, 2'-acetyl-9-dichloroacetyl leucomycin A5
was replaced by 2'-acetyl-9-chloroacetyl leucomycin A5 to
obtain the product (400 mg).
The above 2'-acetyl-9-chloroacetyl leucomycin A5 was
prepared by acetylation of 9-chloroacetyl leucomycin obtained
by the process described in Japanese Patent Open. No.
50-96584, at 2' position (2'-acetylation) described in Japanese
Patent Publ. NQ. ~3-7434.
Example 12.
9-acetyl-3"-propionyl leucomycin A5:
9,18,2'-triacetyl-3, 18-0-cyclo-leucomycin A5 (10 g)
obtained by the process in Example 3 was dissolved in ethyl
- 52 -
~133~S
acetate (100 ml), ~f-collidine (16 ml) was added, then there
was further added propionylchloride (9.69 ml) under cooling,
and the reaction mixture was stirred at 70C for 4 days.
Chloroform (200 ml) was added to the reaction mixture and
washed twice with water (200 ml) and once with diluted
aqueous ammonia (200 ml). The chloroform layer was dehydrated
by adding anhydrous sodium sulfate and dried up in vacuo.
The residue was dissolved in a small amount of benzene and
chromatographed on a silica gel column by eluting with
benzene - acetone (17 : 1).
Fractions showing RfB = 0 75 were dried up in vacuo to
obtain 9, 18, 2'-triacetyl-3, 18-0-cyclo-3"-propionyl
leucomycin A5 (RfB = 0 75~ RfC 0.56) (4.02 g).
The said substance was dissolved in ammonia saturated
methanol solution (40 ml), allowed to stand for 17 hours and
dried up in vacuo. The residue was dissolved in methanol
(50 ml), refluxed for 17 hours, then dried up in vacuo.
The residue was chromatographed on a silica gel column
by eluting with benzene - acetone (7 : 1). Fractions showing
RfA = 0.67 were collected and dried up in vacuo to obtain
the product (3.1 g).
RfA = 0.67, RfB = 0 33' RfC = 0-13
Mass (m/e): 869 (M ), 796 (M -73), 783(M -87)
NMR (CDC13, 100 MHz): 1.42 (3"-CH3), 2.00 (9-Ac), 2.54
[3'-N(CH3)2~, 3.53 (4 OCH3),
(CHO) ppm.
Example 13.
3"-propionyl leucomycin A5:
- 53 -
1'~33~75
18, 2'-diacetyl-9-dichloroacetyl-3, 18-0-cyclo-leucomycin
A5 (5 g) obtained in Example 4 was dissolved in dry dioxane
(50 ml), there was added dry y-collidine (7.S ml), then
propionylchloride (4.5 ml) was added dropwise under ice-
cooling and the mixture was stirred at 90C for 20 hours.
Benzene (500 ml) was added thereto, washed twice with water
(50 ml) and once with diluted aqueous ammonia (500 ml). The
benzene layer was dehydrated by adding anhydrous sodium
sulfate and dried up in vacuo to obtain the powder (5.1 g).
This powder was chromatographed on a silica gel column by
eluting with benzene - acetone (18 : 1). Fractions showing
RfB = 0.78 were dried up to obtain 18, 2'-diacetyl-9-
dichloroacetyl-3, 18-0-cyclo-3"-propionyl leucomycin A3
(2.8 g).
Recrystallization was made by benzene - n-hexane to
yield colorless crystals. m.p.: 177 - 179C.
The crystals hereinabove were dissolved in ammonia
saturated methanol solution (50 ml), allowed to stand for 4
hours at room temperature and dried up in vacuo.
The residue was dissolved in methanol (50 ml), refluxed
for 16 hours then dried up in vacuo. The residue was dis-
solved in a small amount of benzene and chromatographed on a
silica gel column by eluting with benzene - acetone (6 : 1).
Fractions showing RfA = 0 47 were dried up to obtain the
product (2.1 g).
RfA = 0 47~ RfB = 0-14
Mass (m/e): 827 (M ), 754 (M -73), 740 (M -87)
NMR (CDC13, 100 MHzO: 1.44 (3"-CH3), 2.57 [3'-N(CH3)2~,
- 54 -
34~75
3.56 (4-OCH3), 9.86 (CHO) ppm-
Example 14.
3"-butyryl leucomycin A5:
To 18, 2'-diacetyl-3,18-0-cyclo-9-dichloroacetyl
leucomycin A5 (5 g) obtained in Example 4 dissolved in dry
dioxane (50 ml) was added dry ~-collidine (7.51 ml), then
butyrylchloride (5.37 ml) was added under ice-cooling and
the mixture stirred at 90C for 16 hours. Benzene (500 ml)
was added to the reaction mixture, washed twice with water
and once with diluted aqueous ammonia (500 ml). The benzene
layer was dehydrated by adding anhydrous sodium sulfate and
dried up. The residue was chromatographed on a silica gel
column by eluting with benzene - acetone (20 : 1). Fractions
showing RfC = 0.65 were dried up in vacuo to obtain 18, 2'-
diacetyl-3, 18-0-cyclo-9-dichloroacetyl-3"-butyl leucomycin
A5 (2.72 g).
RfB = 0.80, RfC = 0.65
NMR (CDC13, 100 MHz): 1.43 (3"-CH3), 2.06 (2'-Ac), 2.11
(18-Ac~, 2.48 [3'-N~CH3)2], 3-46
(4-OCH3), 6.36 (9-COCHC12)
m.p.: 196 - 198C. (recrystallized colorless crystals
from benzene - n-hexane)
The above product was dissolved in ammonia saturated
methanol solution (20 ml), allowed to stand for 16 hours and
dried up in vacuo. The residue was dissolved in methanol
(50 ml) refluxed for 16 hours then dried up. The residue
was chromatographed on a silica gel column by eluting with
benzene - acetone (6 : 1). Fractions showing Rf = 0.48 were
- 55 -
113347S
dried up to obtain the product (1.7 g).
RfA = 0-48, RfB = 0-15
Mass (m/e): 841 (M ), 754 (M -87)
NMR (CDC13, 100 MHz): 1.42 (3"-CH3), 2.57[3'-N(CH3)2],
3.55 (4-OCH3), 9.84 ~CHO) ppm.
Example 15.
4"-acetyl-3,3"-dipropionyl leucomycin V:
2'-acetyl-SF-837 substance (1 g) obtained in Example 9
was dissolved in dry dichloromethane (10 ml), there was added
dry pyridine (0.23 ml) and p-nitrobenzoylchloride (480 ml),
then the mixture was stirred at room temperature for 17 hours.
To the reaction mixture was added an equal amount of water,
stirred well and the separated dichloromethane layer was
washed with water (10 ml) and saturated aqueous sodium
bicarbonate (10 ml). After dehydrating with anhydrous sodium
sulfate, the solution was dried up to obtain 2'-acetyl-9-p-
nitrobenzoyl-SF-837 substance (RfB = 0.72, RfC = 0 44) This
substance was dissolved in dry pyridine, acetic anhydride
(1.2 ml) was added, and the mixture was reacted at 90C for
3 days. The reaction mixture was concentrated in vacuo and
dissolved in chloroform (10 ml) which was washed with diluted
hydrochloric acid (10 ml), water (10 ml) and saturated aqueous
sodium bicarbonate (10 ml), in this order. The residue was
dissolved in a small amount of benzene and chromatographed
on a silica gel column by eluting with benzene - acetone
(20 : 1). The main eluate was concentrated in vacuo and
dissolved in ammonia saturated methanol (15 ml), allowed to
stand at room temperature for 2 days, dried up in vacuo,
, - 56 -
. -
~133475
methanol (20 ml) was added and the mixture was refluxed for18 hours. The reaction mixture was dried up in vacuo and
chromatographed on a silica gel column by eluting with
benzene - acetone (7 : 1). The eluate showing RfA = 0.56
was dried up to obtain the product (280 mg).
RfA = 0.56, RfB = 0.18
Mass (m/e): 855 (M ), 796 (M -59), 782 (M -73).
Example 16.
3"-butyryl-4"-propionyl leucomycin V:
18, 2-diacetyl-9-dichloroacetyl-3, 18-0-cyclo-leucomycin
A5 (5 g) obtained in Example 4 was dissolved in dry pyridine
(30 ml), propionic anhydride (15 ml) was added, and the
reaction mixture reacted at 100C for 40 hours. The reaction
mixture was poured into ice-water (500 ml), its pH was
adjusted to 9.5 by adding aqueous ammonia, and it was ex-
tracted twice with chloroform (300 ml!. The chloroform layer
was washed with water, dehydrated by adding anhydrous sodium
sulfate, then dried up in vacuo to obtain the powder (5.13 g).
; This powder was chromatographed on a silica gel column by
eluting with benzene - acetone (18 : 1). The eluate showing
RfC = 0.63 was dried up to obtain 18, 2'-diacetyl-3"-butyryl-
3, 18-0-cyclo-9-dichloroacetyl-4"-propionyl leucomycin V
(1.5 g)-
This substance was dissolved in ammonia saturated
methanol solution (30 ml), allowed to stand for 16 hours,and dried up in vacuo. The residue was dissolved in methanol
(50 ml), refluxed for 20 hours, then dried up in vacuo. The
thus formed residue was chromatographed on a silica gel column
~133475
by eluting with benzene - acetone (6 : 1). The eluate showing
RfA = 0.46 was dried up in vacuo to obtain the product (1.2
g)-
RfA = 0-46, RfB = 0.11
Mass (m/e): 827 (M ), 754 (M -73), 740 (M -87).
NMR (CDC13, 100 MHz): 1.43 (3"-CH3), 2.57 ~3'-N(CH3)2],
3.54 (4-OCH3), 9.87 (CHO) ppm.
Example 17.
3"-isovaleryl leucomycin A5:
To 2'-acetyl leucomycin A5 (5 g3 dissolved in dry di-
chloromethane (25 ml) was added dry pyridine (1.64 ml)
dichloroacetylchloride (1.77 ml) was added dropwise under
ice-cooling and the mixture stirred for 1 hour. Cold water
was added to the reaction mixture and extracted with dichloro-
methane (25 ml). After drying the dichloromethane layer by
anhydrous magnesium sulfate, dried up in vacuo to obtain
quantitatively 2'-acetyl-3, 9-dichloroacetyl leucomycin A5.
To the above product (5 g) dissolved in dry dioxane
(50 ml) was added y-collidine (7.87 ml), isovalerylchloride
(6.62 ml) was added dropwise under cooling, then the mixture
was stirred at 90C for 80 hours. The reaction mixture was
poured in cold water (500 ml) and extracted twice with
benzene (400 ml). The benzene layer was dehydrated by adding
anhydrous magnesium sulfate and dried up in vacuo. The
residue was dissolved in methanol (20 ml), ammonia saturated
methanal solution (20 ml) was added thereto and the mixture
was stirred at room temperature for 30 minutes. The reaction
mixture was poured into cold water (500 ml), extracted twice
- 58 -
~1339,~7~
with benzene (400 ml), dehydrated by anhydrous magnesium sul-
fate and dried up in vacuo. The residue was chromatographed
on a silica gel column by gradient elution with benzene -
acetone (15 : 1, 7 : 1 and 5 : 1). The main fractions were
collected and dried up in vacuo to obtain crude 2'-acetyl-3"-
isovaleryl leucomycin A5 (1.25 g). This was dissolved in
methanol (20 ml), refluxed by heating for 20 hours and dried
up in vacuo. The residue was again chromatographed on a
silica gel column by eluting with benzene - acetone (9 : 1).
Fractions showing RfA = 0.48 were dried up to obtain the
product (830 mg).
RfA = 0.48
Mass (m/e): 855 (M ), 768 (M -87), 754 (M -101).
NMR (CDC13, 100 MHz): 1.42 (3"-CH3), 9.89 (CHO) ppm.
Example 18.
3"-acetyl leucomycin Al:
To 2'-acetyl leucomycin Al (5 g) dissolved in dry
dichloromethane (25 ml) was added dry pyridine (1.64 ml),
then dichloroacetylchloride (1.77 ml) was added dropwise
under ice-cooling and the mixture stirred for 1 hour. To
the reaction mixture was added cold water (25 ml) and the
mixture was extracted with dichloromethane (25 ml). The
dichloromethane layer was dehydrated by anhydrous magnesium
sulfate and dried up in vacuo to obtain the quantitative
amount of 2'-acetyl-3, 9-dichloroacetyl leucomycin Al.
To the above product (5 g) dissolved in dry ethyl
acetate (50 ml) was added ~-collidine (9.4 ml) under ice-
cooling, acetylchloride (4.6 ml) was added and the mixture
,
- 59 -
~133475
was stirred for 72 hours at 70C.
The reaction mixture was poured into cold water (250
ml) and extracted twice with chloroform (150 ml). The
chloroform layer was washed with diluted hydrochloric acid
(pH 2), water and saturated aqueous sodium bicarbonate, in
this order, dehydrated by anhydrous sodium sulfate and dried
up in vacuo. The residue was dissolved in methanol (10 ml),
ammonia saturated methanol solution (10 ml) was added, then
the mixture was stirred at room temperature for 30 minutes.
The reaction mixture was poured into cold water (250 ml) and
extracted twice with chloroform. The chloroform layer was
dehydrated with anhydrous magnesium sulfate and dried up in
vacuo. The residue was chromatographed on a silica gel
column by eluting with benzene - acetone (8 : 1) and the
eluate was concentrated to obtain the crude powder (1.4 g)
of 2', 3"-diacetyl leucomycin Al. This crude powder was
dissolved in methanol (20 ml), refluxed for 20 hours and
dried up in vacuo. The residue was again chromatographed
on a silica gel column by eluting with benzene - acetone
(6 : 1). The eluate showing RfA = 0.46 was dried up in
vacuo to obtain the product (716 mg).
RfA = 0.46.
Mass (m/e): 827 (M ), 768 (M -59), 726 (M -101).
NMR (CDC13, 100 MHz): 1.43 (3"-CH3), 200 (3"-OAc), 9.89
(CHO) ppm.
Example 19.
3"-acetyl leucomycin A5:
To 2'-acetyl leucomycin A5 (65 g) dissolved in dry
~ '
~,,,
- 60 -
~133~75
dichloromethane (300 ml) was added dry pyridine (44 ml) and
trimethylchlorosilane t39 ml), then the mixture was stirred
for l hour at room temperature. The reaction mixture was
poured into water (2 lit.) and extracted with chloroform
(500 ml!. The extract was washed with 0.1 N HCl, water and
3% aqueous ammonia, in this order, dehydrated by anhydrous
magnesium sulfate and dried up in vacuo to obtain the crude
2'-acetyl-3, 9-di(trimethylsilyl) leucomycin A5 (69 g).
The above substance was dissolved in dry ethyl acetate
(200 ml), tribenzylamine (227 g) was added thereto, there
was further added acetylchloride (51 ml) under ice-cooling,
then the mixture was stirred at 70C for 40 hours. The
reaction mixture was poured into water (2 lit.), adjusted to
pH 9.5 by adding aqueous ammonia, and extracted with chloro-
form (1 lit.). The extract was washed with water, dehydrated
by adding anhydrous magnesium sulfate and concentrated in
vacuo to obtain 2',3"-diacetyl-3,9-di(trimethyl silyl)
leucomycin A5, in which a small amount of 18, 2', 3"-triacetyl-
3, 9-di(trimethylsilyl)-17-dehydro leucomycin A5 and a large
amount of tribenzylamine were contained. Thereto was added
ammonia saturated methanol solution (250 ml) under ice-cooling
and the mixture was stirred for 1.5 hours at room temperature.
The reaction mixture was poured into water (2 lit.) and
extracted with chloroform (1 lit.). The extract was washed
with water, dehydrated by anhydrous magnesium sulfate and
dried up in vacuo. The residue was dissolved in cold methanol
(600 ml) and the insoluble tribenzylamine was filtered off.
The filtrate was refluxed by heating for 20 hours, then dried
B
- 61 -
11334~75
up in vacuo to obtain the crude 3"-acetyl-3,9-di(trimethyl-
silyl) leucomycin A5 (69 g).
The substance was dissolved in acetic acid - tetrahydro-
furan - water (3 : l : 1) (250 ml) and the solution was
stirred for 1 hour at room temperature. To the reaction
mixture was added chloroform (l lit.) and the mixture was
washed with 3~ aqueous ammonia. The chloroform layer was
dehydrated by anhydrous magnesium sulfate and dried up in
vacuo to obtain the crude 3"-acetyl-leucomycin A5 (64 g),
which was dissolved in a small amount of benzene and charged
on a column of silica gel. Elution was gradually carried
out by benzene - acetone (10 : 1 - 8 : 1 - 6 : 1). The
fractions were checked by silica gel thin layer chromatography
and the fractions showing RfA = 0 45 were collected and dried
up in vacuo to obtain the purified product (45.5 g. yield
70.0%)-
RfA = 0 45' ~fB = 0.10
Mass (m/e): 813 (M ), 754 (M -59), 726 (M -87)
Potency: 2850 y/mg (as compared with standard
leucomycin).
Example 20
3"-propionyl leucomycin A5:
To 2'-acetyl leucomycin A5 (65 g) dissolved in dry
dichloromethane (300 ml) was added dry pyridine (44 ml) and
trimethylchlorosilane (39 ml) and the mixture was stirred
for 1 hour at room temperature. The reaction mixture was
poured into water (2 lit.) and extracted with chloroform
(500 ml). The extract was washed with 0.1 N HCl, water and
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~133475
3% aqueous ammonia, in this order, dehydrated by anhydrous
magnesium sulfate and dried up in vacuo to obtain the crude
2'-acetyl-3,9-di(trimethylsilyl) leucomycin A5 (70 g).
To the above crude substance dissolved in dry dioxane
(200 ml) was added tribenzylamine (230 g), propionylchloride
(65 ml) was further added under ice-cooling and the mixture
was stirred at 90C for 24 hours. The reaction mixture was
poured into water (2 lit.), adjusted to pH 9.5 by adding
aqueous ammonia, and extracted with chloroform (1 lit.).
The extract was dehydrated by anhydrous magnesium sulfate
and dried up to obtain 2'-acetyl-3,9-di(trimethylsilyl)-3"-
propionyl leucomycin A5, in which there was contained a small
amount of 2'-acetyl-3,9-di(trimethyl silyl)-18, 3'-
dipropionyl-17-dehydroleucomycin A5 and a large amount of
tribenzylamine. Thereto was added ammonia saturated methanol
solution (250 ml), and the mixture was stirred at room
temperature for 1.5 hours. The reaction mixture was poured
into water (2 lit.) and extracted with chloroform (1 lit.).
The extract was washed with water, dehydrated by anhydrous
magnesium sulfate and dried up in vacuo. The residue was
dissolved in cold methanol and the insoluble tribenzylamine
was filtered off. The filtrate was refluxed for 20 hours
and dried up in vacuo to obtain the crude 3,9-di (trimethyl-
silyl)-3"-propionyl leucomycin A5. This substance was
dissolved in acetic acid - tetrahydrofuran - water (3 : 1 : 1)
(250 ml) and stirred for 1 hour at room temperature. To
the reaction mixture was added chloroform (1 lit.), and the
mixture was washed with 3% aqueous ammonia. The chloroform
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layer was dehydrated with anhydrous magnesium sulfate and
dried up in vacuo to obtain the crude 3"-propionyl leucomycin
A5 (60 g), which was dissolved in a small amount of benzene
and charged on a silica gel column. Elution was carried
by mixtures of benzene - acetone (15 : 1 - 10 : 1 - 8 : 1).
Each fraction was checked by silica gel thin layer chromato-
graphy and the fractions showing RfA = 0 57 were collected
and dried up in vacuo to obtain the purified product (52.5 g,
yield: 79.4%).
RfA = 0-57~ RfB = 0-14
Mass (m/e): 827 (M ), 754 (M -73), 740 (M -87)
Potency: 2850 y/mg (as compared with standard leuco-
mycin)
Example 21.
3"-acetyl leucomycin A4 (3,3"-diacetyl leucomycin A5):
To 2'-acetyl leucomycin A5 (30 g) dissolved in dry
dichloromethane (150 ml) was added dry pyridine (6 ml) under
ice-cooling and trimethylchlorosilane (5.69 ml), and the
mixture was stirred for 1 hour at room temperature. The
reaction mixture was washed with 0.1 N HCl, water and 3%
aqueous ammonia, in this order, dehydrated by anhydrous
magnesium sulfate and dried up in vacuo to obtain the crude
2'-acetyl-9-trimethylsilyl leucomycin A5 (30.2 g).
The above crude material was dissolved in dry dioxane
(50 ml), tribenzylamine (113 g) and acetylchloride (25 ml)
were added thereto, and the mixture was stirred for 40 hours
at 80C. The reaction mixture was poured into water (1 lit.),
its pH was adjusted to 9.5 by aqueous ammonia, and the
" t
: '
1133~7S
mixture extracted with chloroform (500 ml). The extract
was dehydrated by anhydrous magnesium sulfate and concentrated
in vacuo. To the residue was added ammonia saturated methanol
solution (50 ml) and the mixture was stirred for 2 hours at
room temperature. The reaction mixture was poured into water
(1.5 lit.) and extracted with chloroform (500 ml). The
chloroform layer was dehydrated by anhydrous magnesium sulfate
and dried up in vacuo. The residue was dissolved in cold
methanol (200 ml), and the insoluble tribenzylamine was
filtered off. The filtrate was refluxed for 20 hours and
dried up to obtain the crude 3,3"-diacetyl-9-trimethylsilyl
leucomycin A5.
This crude material was dissolved in acetic acid -
tetrahydrofuran - water (3 : 1 : 1) (125 ml) and the solution
was stirred for 2 hours at room temperature. To the reaction
mixture was added chloroform (500 ml), and the mixture was
washed with 3~ aqueous ammonia. The chloroform layer was
dehydrated by anhydrous magnésium sulfate and dried up in
vacuo to obtain the crude 3,3"-diacetyl leucomycin A5
(29.8 g). This substance was dissolved in a small amount of
benzene, charged on a column of silica gel and gradiently
eluted by benzene - acetone (10 : 1 - 8 : 1 - 6 : 1).
Fractions were checked by silica gel thin layer chromatography,
and the fractions showing RfA = 0.58 was collected, and
dried up in vacuo to obtain the purified product (21.2 g.
yield: 67.2%).
RfA = 0.58, RfB = 0-15
Mass (m/e): 855 (M ), 796 (M -59), 768 (M -87).
B - 65 -
