CA1133475A - 3"-acylated macrolide antibiotics - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
The present invention as disclosed provides compounds of the formula:

[1]

wherein R1 is a hydrogen atom or a (C2-C3) alkanoyl group, R2 is a hydrogen atom or a (C2-C4) alkanoyl group, at least one of R1 and R2 being a hydrogen atom, at least one of R' and R" is an R3 group and the other is an R4 group, where R3 is a (C2-C6) alkanoyl group; and pharmaceutically acceptable salts. These compounds are macrolide antibiotics having enhanced antibacterial activity as compared with known antibiotics.

Description

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~

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 Espinomycin A2 " COCH(CH3)2 Espinomycin A
^'~;'.^.'~

1133~7'~

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 -.

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]

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;

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 ~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 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 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 ~ ' 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 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 , 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 ~ . .

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~

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~

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 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 ~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.

, 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 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 ~.

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, , 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 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>~

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 11339~75 of the compound [17] of the formula 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 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 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 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 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.

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~ -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.

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.

'~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~

~ ,~

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.

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 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 .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 ~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 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 ~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, 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 ~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 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

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).

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.

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.

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 ~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:

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~, 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 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 ~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 , ~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 ~ ' ~,,, ~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

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 ~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 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).

Claims (7)

Claims

1. A process for the production of a compound of the formula [1]
wherein R1 is a hydrogen atom or a C2-3 alkanoyl group and one of R' and R" being R3 and the other R4, R3 being a C2-6 alkanoyl group and R4 being a C2-5 alkanoyl group, and R2 is a hydrogen atom or a (C2-C4) alkanoyl group, and at least one of R1 and R2 is a hydrogen atom; or a pharmaceutically acceptable salt thereof; characterized in that a compound of the formula wherein R1 is a hydrogen atom or an R11 group, R11 being a C2-3 alkanoyl group, R4 is a C2-5 alkanoyl group, R5 is a halogenated acetyl, p-nitrobenzoyl or silyl group, and R6 is hydrogen or R61, in which R61 is a C2-4 alkanoyl group, is acylated with a carboxylic acid halide of formula R3X, wherein R3 is a C2-6 alkanoyl group and X is halogen, or with a (C2-C6) aliphatic carboxylic acid anhydride, under heating to obtain a mixture of a compound of the formula wherein R8 is R61 or R3, and R', R", R11, R3, R4, R5 and R61 have the same meanings as hereinbefore, and a compound of the formula wherein R', R", R11, R3, R5 and R8 have the same meanings as hereinbefore, then the said mixture is treated, when R5 is a halogenated acetyl or p-nitrobenzoyl group, with ammonia in an alkanol to remove the protective group at position 9, then treated by heating in methanol which may optionally contain water to remove the acyl group at position 2', and when R5 is a silyl group, the said mixture is treated, to remove the protective group at position 9 and acyl groups at position 18 and 2', by one of the processes of:
(a) removing the silyl and acyl groups at positions 18 and 2' by heating in aqueous lower alkanol which can con-tain a base;
(b) removing the silyl and acyl groups at position 18 by treating at ambient temperature with aqueous lower alkanol in the presence of a base, thereafter removing the acyl group at position 2' by heating in methanol which can contain water;
(c) removing the acyl group at position 18 by treating with methanol containing ammonia and further removing the silyl and acyl groups 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 group at position 2' by heating with methanol and re-moving the silyl group by treating with aqueous acid; and when desired, forming a pharmaceutically acceptable salt of the compound of formula [1] so produced.

2. A process for the production of a compound of the formula [1c]

wherein R3 is a C2-6 alkanoyl group and R4 is a C2-5 alkanoyl group, characterized in that a compound of the formula R4 has the same meaning as hereinabove, wherein R54 is a chlorinated acetyl or silyl group, and R61 is C2-4 alkanoyl, is acylated with a carboxylic acid halide of formula R3X, wherein R3 has the same meaning as hereinabove and X
is halogen, under heating in the presence of an inert organic solvent to prepare a mixture of the compound of the formula wherein R3, R4, R54 and R61 have the same meanings as herein-before, and a compound of the formula wherein R3, R4, R54 and R61 have the same meanings as here-inbefore, and the mixture is treated, when R54 is a chlorin-ated acetyl group, with ammonia in methanol, then removing 2'-acyl by treating with methanol under heating, and when R54 is a silyl group the mixture is treated as follows:
(a) removing the silyl and acyl groups at positions 18 and 2' by heating in aqueous lower alkanol which can con-tain a base;
(b) removing the silyl and acyl groups at position 18 by treating at ambient temperature in 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 and 2'-acyl groups by heating with aqueous lower alcohol;
or (d) removing the acyl group at position 18 by treating with methanol containing ammonia, thereafter removing the 2'-acyl group by heating with methanol and removing the silyl group by treating with aqueous acid.

3. A process for the production of a compound of the formula [1c]
wherein R3 is a C2-6 alkanoyl group and R4 is a C2-5 alkanoyl group, or a pharmaceutically acceptable salt thereof, characterized by that a compound of the formula wherein R51 is a chlorinated acetyl group, R6 is hydrogen or R61 in which R61 is a C2-4 alkanoyl group, and R4 has the same meanings as above, is reacted with a (C2-C4) aliphatic carboxylic acid anhydride in the presence of an inorganic base to prepare a compound of the formula wherein R7 is R61 or R21, R51, R61 and R4 have the same meanings as hereinbefore, and R21 is a (C2-C4) alkanoyl group;
and the said compound is acylated with a carboxylic acid halide of formula R3X, wherein R3 is a C2-6 alkanoyl group and X is halogen, under heating in the presence of an inert organic solvent to prepare a compound of the formula wheein 21' R3, R4, R51 and R7 have the same meanings as hereinbefore, and the said compound is treated with ammonia in methanol to remove protective groups at positions 3 and 18, and the said compound is heated in methanol which can contain water to remove the acyl group at position 2'; and where desired, forming a pharmaceutically acceptable salt of the compound so produced.

4. A process for the production of a compound of the formula wherein R21 is a C2-4 alkanoyl group and one of R' and R"
being R3 and the other R4, R3 being a C2-6 alkanoyl group and R4 being a C2-5 alkanoyl group, characterized in that a compound of the formula wherein R2 is hydrogen or R21, R6 is hydrogen or R61, in which R61 is a C2-4 alkanoyl group and R21 and R4 have the same meanings as hereinbefore, is reacted with a (C2-C4) aliphatic carboxylic acid anhydride, in the presence of an inorganic base, to prepare a compound of the formula wherein R7 is R61 or R21, R4 and R61 have the same meanings as hereinbefore, and the said compound is acylated with a carboxylic acid halide of formula R3X, wherein R3 is a C2-6 alkanoyl group and X is halogen, or with a (C2-C6) aliphatic carboxylic acid anhydride under heating to prepare a compound of the formula wherein R21, R', R" and R7 have the same meanings as herein-before, and the protective group at position 18 is removed by treating with a methanol solution of ammonia or an ethanol solution of an aqueous alkaline carbonate, and then the 2' acyl group is removed by heating in methanol.

5. A compound of the formula wherein R1 is a hydrogen atom or a (C2-C3) alkanoyl group, R2 is a hydrogen atom or a (C2-C4) alkanoyl group at least one of R1 and R2 being a hydrogen atom, and one of R' and R"
being R3 and the other R4, R3 being a (C2-C6) alkanoyl group and R4 being a (C2-C5) alkanoyl group; or a pharmaceutically acceptable salt thereof, when prepared by the process of claim 1 or by an obvious chemical equivalent thereof.

6. A compound of the formula [1c]

wherein R3 is a (C2-C6) alkanoyl group and R4 is a (C2-C5) alkanoyl group, when prepared by the process of claim 2 or by an obvious chemical equivalent thereof.

7. A compound of the formula [1e]

wherein R21 is a (C2-C4) alkanoyl group and one of R' and R"
being R3 and the other R4, R3 being a (C2-C6) alkanoyl group and R4 being a (C2-C5) alkanoyl group, when prepared by the process of claim 4 or by an obvious chemical equivalent thereof.