AU440552B2 - Method of producing derivatives of antibiotic b-5050 - Google Patents

Description

30,734/71 This invention relates to pharmaceutically useful

esters of antibiotics B-5050 and tetrahydro-B-5050, and a

method for producing them.

It has been found by some of the present inventors

that a group of new antibiotics is produced by a micro-

organism belonging to the genus Streptomyces and is

accumulated in the culture broth (cf. Belgian Patent

754,819). The new antibiotics named as B-5050A, B, C,

D,.E and F are of macrolide group inhibitory to gram-

positive bacteria and represented by the general formula

CH 3 CH 3 H2 HO HO JkCH 3

HO 9 7 5 5 OH 1 CHO\CQ-0 _0 H /CH 0R2 0 46' H3

14 1

R1 R2 B-5050-A. Propionyl i-Valeryl

B-5050-B Acetyl i-Valeryl

B-5050-C Propionyl Propionyl

B-5050-D Acetyl Propionyl

B-5050-E Propionyl Acetyl

B-5050-F Acetyl Acetyl

and have been collectively named as "B-5050" (hereafter in

the specification as well as in claims each of these

2 440552

309 73 4/71 antibiotics and a mixture of tw~o or mnor'e of them are collectively referred to as simply 3 50 5 0", 1 unless

otherwise noted.) The structural formula of tetrahydro-B-5050 is represented as follows:

'3

CH3

w~herein P,1and R2are as defined hereinbefore.

30,734/71 B-5050 has two secondary hydroxyl groups at positions

9 and Let these two secondary hydroxyl groups be

simply designated as a'and -hydroxyl group, respect-

ively in specifications and claims. Then, if one or both

of those «-and -hydroxyl group be esterified select-

ively, in one or more steps under certain conditions, the

-monoesters, B-monoesters or a,$-diesters of B-5050 can

be produced.

B-5050 also has one-unsaturated double bond at

position 10, 11 and one epoxy group at positions 12, 13.

It has been found by the present inventors, too, that

when the B-5050 is catalytically reduced, the unsatur-

ated double bond is saturated and simultaneously the epoxy

group is reductively cleaved to yield the corresponding

tetrahydro-B-5050 having a newly formed hydroxyl group

at position 13.. In the specification as well as in the

claims, each of tetrahydro-B-5050 A, B, C, D, E and E,

and a mixture of two or more of them are collectively

referred to as simply "tetrahydro-B-5050", unless other-

wise noted. "Tetrahydro-B-5050" is more stable than

"B-5050" against acids. Tetrahydro-B-5050, thus

obtained, has three secondary hydroxyl groups at positions

9, and 13, respectively.

Let these three secondary hydroxyl groups be simply

designated as and r -hydroxyl groups, respectively

in specifications and in claims. Then if one or more of

30,734/71 e either those and X-hydroxyl groups are esterified, or more steps, selectively or non-selectively and in one a,8-diesters, under certain conditions, the P-monoesters,

a-,r -diesters, -triesters of tetrahydro-B-5050

can be produced. can be These esters of B-5050 and tetrahydro-B-5050 -ka-ee recovered directly or by chromatography or other show improved conventional means in desired purity and pages. therapeutic effects as disclosed on the following This invention is the culmination of the above

findings. invention Thus, the principalob.ject of the present or tetra- is to provide new and useful esters of B-5050 of one or more' hydro-B-5050, collectively, or a mixture of the members. Another object is to provide a method for producing

said useful esters of B-5050 or tetrahydro-B-5050. tetra- Said objects are realized by reacting B-5050, with an hydro-B-5050 ora-e ters of tetrahydro-B-5050 esterifying agent/ a af~ r group of 2 to 7

carbon atoms derived from an organic acid or an organic

carboxylic acid.

Among the starting materials of the present invention,

B-5050 is obtained, as disclosed in afore-cited Belgian micro- Patent, by cultivating an antibiotic B-5050 producing in a culture organism belonging to the genus Streptomyces medium containing assimilable carbon sources and digestible

nitrogen sources under aerobic conditions until antibiotic

B-5050 is substantially accumulated in the culture broth,

and recovering the accumulated B-5050 therefrom. Among the

3 0,73 4/71 B-5050 producing microorganisms to be employed in the process aforesaid, there is Streptomyces,, 4 aod (IFO 12995) as a typical example.

Another starting material tetrahydro-B-5050 is

produced by subjecting the abovementioned B-5050 to a

catalytic reduction which is conventional -re The antibiotic B-5050 is fat-soluble and, accordingly,

soluble in such organic solvents as methanol, ethanol and

ethyl acetate. Therefore, when B-5050 is first dissolved in such a solvent and, then, catalytically reduced with hydrogen gas in the presence of a catalyst such as palladiu/charcoal( platinum oxide, e4reTtetrahydro-B- 5050 is obtained in a good yield. The other starting material, -esters of tetrahydro-B-5050, is produced by esterifying B-5050 through a method of the present

invention and then subjecting thus-obtainedcL-esters of B-5050 to a catalytic reduction as mentioned above.

It is to be noted that, while B.5050 and esters thereof

are catalytically reduced at positions 10 and 12 of

formula as described above, they are not affected in any other positions.

In the esterification of this invention, various

conventional esterifying agents may be employed. The ester group are the residue of organic carboxylic acids which may for example be the groups represented by general

formula R'CO-(R means as organic residue) which may be derived from organic acids or the groups represented by the general formula R'O*CO- (R is as defined above) which are formed on removal of hydroxyl radicals from organic

carbonic acids and esterifying agents containing such

30,734/71 radicals can be employed. While the simplest of such

esterifying agents are organic acids as such, the

corresponding acid halides and acid anhydrides may also

be employed. Among such organic acids are saturated

monocarboxylic acids such as acetic acid, propionic acid,

n-butyric acid, i-butyric acid, n-valeric acid, i-valeric acids acid and palmitic acid, unsaturated monocarboxylic

such as crotonic acid; polybasic acids such as succinic

acid; aromatic carboxylic acids such as benzoic acid,

p-aminobenzoic acid/-etc-; aryl carboxylic acids such as

lk. phenylpropionic acid, and the-4 Among these organic

acids, those acids which contain an ester group of 1 to

especially 2 to 7 carbon atoms are favorably employed

acetic acid, propionic acid, i, and tert.-

butyric acid, n- and i-valeric acid, crotonic acid, succinic acid/ benzoic acidr-e4e). Those organic acids

may have one or more substituents such as halogen, alkyl, alkoxy, amino, hydroxyl and other groups. When using such an esterifying agent, it is preferable

to employ a solvent which will not adversely affect the

esterification reaction, such as pyridine, tetrahydro-

furan, acetone which are routinely used as reaction solvents

or a mixture of such solvents.

Especially when it is desired to selectively synthesize

the t-esters, it is advisable to react the starting

material with an organic acid halide at about 500 or a

lower temperature in the presence of a reaction solvent

such as pyridine, quinoline or picoline. It has been

found tnat by doing so, the d-hydroxyl group alone its

.selectively esterified in a good yield. Therefore, the

30,734/71 desired product can be isolated very easily and in a

good yield from the reaction mixture without resort to

procedures for separating the 0-ester, -ester or/

and unreacted starting material.

To synthesize thej-monoesters, it is desirable to

react the starting material B-5050, tetrahydro-B-5050

with an organic acid anhydride or halide at about 50 G

or a lower temperature in the presence of such a solvent

as acetone, ethyl acetate, methanol or tetrahydrofuran.

Incidentally, when it is desired to introduce an

aromatic carboxylic acid residue, for example, that of

benzoic anhydride, into the_position of P-hydroxyl, the

reaction is desirably carried out at about 1500 or a

higher temperature in the presence of, for instance,

pyridine.

To obtain ct, P -diesters, the reaction is preferably

conducted at about 150C or a higher temperature for a

longer time 10 30 hours) than for the production

of c-esters. For instance, the desired result may be

achieved by conducting the reaction overnight in the

neighborhood of 200C.

To synthesize the cf-i-diesters of tetrahydro-B-

5050, it is advantageous to react tetrahydro-B-5050 or

a-esters of tetrahydro-B-5050 with an organic acid halide

at about 5 0 C or a lower temperature in the presence of a

reaction solvent such as, for example, pyridine. To

synthesizec-, ~-triesters of tetrahydro-B-5050 from

tetrahydro-B-5050 or mono- or di-esters, it is advantage-

ous to conduct the reaction at about 15°C or a higher

temperature than for the production of the mono- and di- f3 4/71 esters. For instance, -esters may be obtained by carrying out the reaction overnight at about 15 C or a-higher temperature. In case of the production for di- or tri-esters, the esterification procedure can be carried out in two or three steps. By way of illustration, if a- or f-mono esterification is carried out and, then, a further ester- ification is conducted with the use of a group different from the organic acid residue thus introduced, there will be obtained a diester in which the hydrogen atoms of the 4- and p- hydroxyl groups have been replaced by different organic residues. The various esters of B-5050 or tetrahydro-B-5050 which are obtainable according to this invention can be identified by their shift of NMR signals (100 MC, in CDC13) as will be seen from the following table.

30,73 4/71 Table 1

NMR ppm)

B-5050 5.66, 6.03 (olefinic proton)

i esters of B-5050 ester 4.00--) approx. S-ester 3.52-- approx. 3.52-- approx. 4.00

tetrahydro-B-5050 disappearance of olefinic

protons

esters of tetrahydro-B- 5050

a-ester. approx. a-ester approx. 2.4 ct, X -diester approx. 3.9--approx. c, P, -triester 3.5----approx. 3.9----4approx. approx. 3.95-- approx. 2.4

Another test is afforded by the fact that whereas

the PKa' of B-5050 or tetrahydro-B-5050, which is a basic

substance, is approximately 6.6 to 6.9, the PKa' of the

-esters is approximately 4.7 to 4.8, as shown in Table 2.

30,734/71 Table 2 PKa'

B-5050 approx. 6.9

esters of B-5050

c,-ester approx. 6.6

P-ester approx. 4.7 ct,P-diester approx. 4.7

tetrahydro-B-5050 approx. 6.8

esters of tetrahydro-B-5050

t-ester approx. 6.6.

P-ester approx. 4.8

a,Y-diester approx. 6.6

a, y-triester approx. 4.8

The various esters which can be obtained according

to this invention give different Rf values on thin-layer

chromatography and, therefore, the reaction product can be

easily fractionated. For instance, when chloroform-

methanol is used as a developer solvent system, B-5050, its

6-propionate, A-propionate, and d, g-dipropionate emerge

at Rf values of 0.1 0.2, 0.37 0.52, 0.25 0.30, and 0.67 0.80, respectively.

If a mixture of B-5050, its and/or diester

is obtained, they may be separated by column chromatography

on silica gel or alumina using a solvent system of chloro-

form-methanol, benzene-acetone or benzene-ethyl acetate,

for instance.

The 3ters of B-5050 and tetrahydro-B-5050 obtainable

according to this invention is highly inhibitory to Gram-

30,734/71 positive bacteria, in vitro and also, have shown excellent

therapeutic effects in experimental infected animal tests.

It has also been found by the assay of their blood

concentrations in animals that those esters are efficiently

absorbed into the body fluid. Some of those esters have

also become more stable against acids than the starting B-

5050 and more fat-soluble, thereby lending themselves to the

preparation of an ointment.

The following table indicates the improved physico-

chemical and biological properties of some esters of B-5050

or tetrahydro-B-5050 which are obtained in the method of

the present invention.

Table 2

Thera- Blood con- Acute stabili- Compound peutic centration toxic- ty for effect in rats ity acid against in Staphylo- mice coccus (IP) aureus

B-5050(control) 1 1 1 1

B-5050 cc-acetate 2 times 2-3 approxi. times same

B-5050 t-propionate 2-3 times 2-3 a little times more stable

B-5050 a-n-butyrate 2-3 times 2 times 2 times

B-5050 l-i-butyrate 2 times 2 times

B-5050 a-crotonate 2 times 2 times

B-5050 a,p -diacet- ate? 2 times 2 times A or approxi. less same

B-5050 a-propionyl- a-acetate 2 times 2 times

B-5050 fc,p-dipropio- or 1.5-2.0 nate 2 times 3 times less times

30,73 4/71 retrahydro-B-5050 1 1 2 times

Tetrahydro-B-5050 3 times 3 times l or 3 times 't,P,8-triacetate less

Tetrahydro-B-5050 a little same 3 times F p, -tripropionate stronger

Besides the above-merits, the objective esters in

the present invention are more favorable for the-

administration due to the less bitterness in comparison

with the starting compounds.

The following references and examples are given to

further illustrate this invention, but it is to be under-

stood, however, that the 6cope of this invention is by no

means limited thereto.

The relationship between part(s) by weight and part(s)

by volume is the same as that between gram(s) and milli-

liter(s).

Reference 1

500 ml. each of a pre-seed culture medium (pH

consisting of glucose soluble starch soybean

flour .cornsteep liquor "Polypepton" (trade

name of a casein hydrolyzate, produced by Daigo Nutritive

Chemicals, Ltd. Osaka, Japan) sodium chloride

calcium carbonate and water is charged in 2000 ml.-

Sakaguchi flasks, and sterilized. The sterilized medium

is inoculated with a loopful of Streptomyces hygroscopicus

(IFO-12995) from an agar slant culture which has been

cultivated for at least 4 days, and incubated at 2800°C on

a recil-'cal shaker of 115 r.p.m. with 10 cm. stroke for

48 hours. The resulting broth in an amount of 1000 ml. is

used as the pre-seed culture.

.440552

30,734/71 The pre-seed culture is inoculated into 100 L. of

a culture medium (pH 7.0) charged and sterilized in a

200 L-stainless steel tank, the medium consisting of

glucose cornsteep liquor defatted

soybean meal sodium chloride magnesium

sulfate calcium carbonate and water.

Incubation is conducted at 280C with aeration of 100 L.

per minute and agitation of 200 r.p.m. for 24 hours.

100 L. of the resulting seed culture is transferred

into 1000 L. of a sterilized main culture medium of the

same composition as that of the culture medium for seed

culture, charged in a 2000 L-stainless steel tank.

Incubation is continued at 280C with aeration of 1000 L.

per minute and agitation by impeller of 120 r.p.m. for 66

hours. During the incubation, "Actocol" (trade name of

a polyoxypropylene trial mixture of OH number 56, sold

by Takeda Chemical Industries, Ltd., Osaka, Japan) is

added as/ an agent at a concentration of about

0.05% relative to the whole medium. The culture broth

thus obtained exhibits an antimicrobial potency of 350

units in terms of dilution units against Bacillus subtilis

as the assay microorganism.

The broth is filtered. The filtered broth (950 L.)

is adjusted to pH 8.5 with a 2N-aqueous NaOH solution, and

extracted with ethyl acetate (300 The ethyl

acetate layer is separated, washed with water, and

extracted twice with 70 L. each of O.005N-HC1. The

aqueous extracts are combined, adjusted to pH 8.5 with

diluted aqueous ammonia and again extracted twice with 45 L.

each of ethyl acetate. The extracts are washed with

30,734/71 .water, dehydrated and concentrated. in vacuo to 50 ml.

To the concentrate is added 750 ml. of n-hexane to obtain

of crude powdery Antibiotic B-5050 mixture.

The powdery product is dissolved in 450 ml. of

benzene under warming. After the addition of 2 g. of activated carbon to the solution being warmed, the

mixture is filtered, and the filtrate is allowed to cool

on standing to separate 19.0g. of Antibiotic B-5050

mixture as colorless needles.

g. of the crystalline Antibiotic B-5050 mixture

is dissolved in 50 ml. of ethyl acetate, and is subjected

to a column chromatography on silica gel (450g., produced

by Merck Germany, the granular size ranging 0.05 to 0.2 The charged materials are developed and eluted with a mixture of ethyl acetate and benzene (2 1 by volume). The first 600 ml. of the eluate showing anti- microbial activity is concentrated to dryness and the

residue is dissolved in hot benzene. On standing, the solution separates 1g. of colorless crystals which consist

mainly of Antibiotic B-5050-A.

After the column is eluted with 4 L. of the ethyl

acetate-benzene mixture, it is further eluted with 3 L.

of ethyl acetate to collect the eluates, which are then

concentrated in vacuo to leave a residue. The residue is dissolved in hot benzene. On standing, the solution separates 550 mg. of colorless crystals which consist

mainly of Antibiotic B-5050-F.

'00 mg. of the crystals consisting mainly of Anti-

biotic 3 5050-A are dissolved in 1.5 ml. of ethyl acetate, and again chromatographed on a column of silica gel (50 g.;

30,734/71 see above). The charged material is eluted with a

mixture of ethyl acetate and benzene (1 1 by volume)

to obtain fractions. Each fraction is tested for the

active ingredients contained, by means of thin layer

chromatography on silica gel (sold by Merck A.G.,

Germany under the name of "HF 2 54 with the use of a

mixture of benzene and acetone (3 2) by volume as the

developer. The fractions which contain solely Anti-

biotic B-5050-A.as the active ingredient are collected,

and concentrated in vacuo to leave a residue. On

crystallization of the residue from benzene, 88.5 mg. of

Antibiotic B-5050-A.is obtained as colorless prisms.

On the other hand, 350 mg. of the crystals consist-

ing mainly of Antibiotic B-5050-F are subjected to a

column chromatography on the silica gel (50 in the

same manner as above. After the column is eluted with

700 ml. of ethyl acetate-benzene (2 1 by volume), it is

further eluted with a mixture of, ethyl acetate and

benzene (3 1 by volume), followed by the tests by

thin layer chromatography for the active ingredients on

each fraction. The fractions which contain solely

Antibiotic B-5050-F as the.active ingredient are collected,

and concentrated in vacuo to leave a residue. The

residue is crystallized to obtain 75.6 mg. of B-5050-F

as colorless prisms.

Reference 2

In 2 L.-Sakaguchi flasks, 500 ml. each of a steril-

ized pre-seed culture medium of the same composition as

used in'Example 1 is inoculated with Streptomyces

hygroscopicus (IFO-12995), and the inoculated flask is

16 440552

30,734/71 incubated in the same manner as in Example 1 to prepare

a pre-seed culture. In 500 L.-stainless steel tank, there is prepared 200 L. of a culture medium of the same composition as

used in Example 1 for the seed culture. After being sterilized at 121 C for 20 minutes, the culture is

inoculated with 2 L. of the above obtained pre-seed

culture. Incubation is effected at 28 C with aeration of 200 L. per minute and agitation of 200 r.p.m. for 24 hours to prepare the seed culture for main fermentation.

200 L. of thus prepared seed culture is transferred into 4000 L. of a main culture medium of the same composi-

tion as above, charged in a 6000 L.-stainless steel tank, and the cultivation is conducted at 28 0 C with aeration of

2400 L. per minute and agitation by impeller of 180 r.p.m.

for 48 hours. During the incubation, "Actocol" (see

above) is used as an antifoaming agent. The culture broth is filtered. The filtered broth (4000 L.Y-is adjusted to pH 9 with 2N-NaOH, and extracted with 1330 L. of ethyl acetate, followea by washing with

water to obtain 1052 L. of ethyl acetate extract. The ethyl acetate solution is concentrated in vacuo to 100 -L.

The concentrated solution is extracted twice with 50 L.

each of a M/l.5-aqueous KH 2 PO 4 solution previously

adjusted at pH 4.0 with phosphoric acid. The extracts are combined, adjusted to pH 8.5 with a diluted aqueous

ammonia solution, and extracted with 50 L. of ethyl

aceta,. The ethyl acetate solution is washed with water, dehydrated and concentrated in vacuo to leave a

syrup. Addition of 3 L. of n-hexane to the syrup gives

30,734/71 330 g. of crude powder. 300 g. of the powder is

dissolved in 1.1 L. of benzene under warming, and the

solution is allowed to cool on standing to give 117 g.

of crystals, which consist mainly of Antibiotics

B-5050-C, -D and (Crystal I).

On the other hand, the ethyl acetate layer which

was extracted with the phosphate solution is further

extracted twice with 50 L. each of N/200 H2S0 4 and the

aqueous layers are combined, adjust to pH 8 with a

diluted aqueous ammonia solution and extracted three

times with 30 L. each of ethyl acetate. The ethyl

acetate extracts are combined, washed with water,

dehydrated and concentrated in vacuo to leave a syrup.

Addition of 1.5 L. of n-hexane to the syrup gives 160 g:

of a crude powder. 150 g. of the powder is crystallized

from hot benzene to give 72.5 g. of crystals, which

consist mainly of Antibiotic B-5050-A.and -B together

with a small amount of Antibiotic B-5050-C (Crystal II).

of Crystal II is dissolved in 20 ml. of ethyl

acetate, followed by the addition of 14 ml. of benzene.

The solution is subjected to column chromatography on

silica gel (Merck Germany, granular size ranging

from 0.05 to 0.2 mm.) which is previously filled with an

equivolume mixture of benzene and ethyl acetate. The

chromatogram is developed firstly with ethyl acetate-

benzene (1 1 by volume) to collect 200 ml. of the eluate,

then with ethyl acetate-benzene (3 2 by volume) to obtain

fractions exhibiting antimicrobial activity against

Bacillus subtilis. The fractions of the same ingredients

are respectively combined, concentrated and crystallized

30,734/71 from benzene to give 435 mg. of crystals of Antibiotic

B-5050-A, on one hand, and 185 mg. of crystals of Anti-

biotic B-5050-B, on the other hand.

g. of Crystal I is dissolved in 50 ml. of ethyl

acetate, followed by the addition of 25 ml. of benzene.

The solution is chromatographed on silica gel (400g.) in

the same manner as explained above. The column is eluted firstly with 3 L. of ethyl acetate-benzene (1 1 by volume), then with ethyl acetate-benzene (2 1 by volume). The eluate is fractionated by 500 ml. The fractions obtained by the 1 1 mixture contain Antibiotics B-5050-A and The first fraction obtained by the 2 1 mixture contain Antibiotics B-5050-B and the second fraction Antibiotics B-5050-C and the third fraction Anti- biotics B-5050-D and and the last fraction Antibiotics B-5050-E and F, respectively.

2 g. of the crystals obtained from the fraction

containing Antibiotics B-5050-B and -C is again dissolved

in 15 ml.-of ethyl acetate, followed by the addition of

ml. of benzene. The solution is subjected to column chromatography with 250 g. of silica gel, and the column

is eluted with ethyl acetate-benzene (3 2 by volume). The eluate is fractionated by 20 ml. each. The fractions of the same component are combined, concentrated and

crystallized from benzene or a mixture of ethyl acetate

and n-hexane to give 83 mg. of crystals of Antibiotic B-

5050-B, 940 mg. of crystals of Antibiotic B-5050-C and

mg. of crystals of Antibiotic B-5050-D. In the same way, 1.2 g. of the fraction containing

Antibiotics B-5050-D and -E is subjected to column chromato-

30,734/71 graphy on silica gel, to give 600 mg. of crystals of

Antibiotic B-5050-E.

The fraction, obtained subsequent to the fraction

containing Antibiotics B-5050-B and contains Anti-

biotic B-5050-C as the main ingredient. 770 mg. of the

resultant crude crystals are again dissolved in 5 ml. of

acetone, and the solution is subjected to column chrom-

atography with 100 g. of silica gel in a similar way to

the above explained, whereupon 220 mg. of Antibiotic

B-5050-C is obtained as crystals. Reference 3

4000 L. of the filtered broth obtained by the main

fermentation as described in Reference 2 is adjusted to

pH 8.0, and is extracted with its one third volume of

ethyl acetate. The ethyl acetate extract is concentrated

to about 100 L. The concentrate is washed with 50 L.

of water, and extracted three times with 50 L. each of

an M/3-aqueous KH 2 PO4 solution previously adjusted to

pH 3.0 with phosphoric acid. The aqueous extracts are

combined, adjusted to a pH of 9 to 10 with an 8N-aqueous

ammonia solution, and extracted with 75 L. of ethyl

acetate. The ethyl acetate extract is washed three

times with 25 L. each of water, and then concentrated to

about 1.5 L. Addition of about 30 L. of n-hexane to the

concentrate gives 522 g. of crude powder. Crystalliz-

ation of the powder from benzene gives 272 g. of crystals

of Antibiotic B-5050 mixture.

In the above procedures, the use of a 0.2N-aqueous

acetic acid solution in place of the KH 2 PO 4 solution

gives substantially the same result.

30,734/71 19 g. of thus obtained Antibiotic B-5050 mixture is

dissolved in 75 ml. of acetone. The solution is subjected

to column chromatography with 800 g. of silica gel (Merck

A. see above). The column is eluted with 3000 ml. of

ethyl acetate-benzene (1 1 by volume) to give 7.25 g.

of crystals consisting mainly of Antibiotics B-5050-A and

Then, the column is eluted with ethyl acetate-benzene

(2 1 by volume). The first fraction eluted with 1100 ml.

of the developer is similarly treated to give 2.22 g. of

crystals consisting mainly of Antibiotics B-5050-B and -C.

The fraction obtained by the subsequent 200 ml. gives

0.90 g. of crystals whichconsist mainly of Antibiotic

B-5050-C. From the further subsequent 300 ml. is obtained

1.4 g. of a mixture consisting mainly of Antibiotics

B-5050-C and From the last 600 ml. fraction is

obtained 1.99 g. of crystals consisting mainly of Anti-

biotics B-5050-D and The column is then eluted with

600 ml. of ethyl acetate-benzene (3 1 by volume) to give

0.645 g. ef crystals consisting mainly of Antibiotics

B-5050-D and From the last fraction eluted with

1600 ml. of ethyl acetate is obtained 2.09 g. of crystals

consisting mainly of Antibiotic B-5050-F.

The above obtained Antibiotics B-5050-A.and -B

crystals (7.25 are dissolved in 40 ml. of acetone, and

subjected to column chromatography with 400 g. of silica

gel, followed by elution with ethyl acetate-benzene 1 1

by volume), whereby 1.5 g. of crystals of Antibiotics

B-5050- and 0.65 g. of crystals of Antibiotic B-5050-B

are obtained.

The afore-obtained Antibiotics B-5050-B and -C

3 0,73 4/71 crystals (2.22 are dissolved in 20 ml. of acetone, and

column-chromatographed on 300 g. of silica gel, followed

by elution with.b.enzene-acetone (3 1 by volume) to give

0.398 g. of crystals of B-5050-B. Further elution with

benzene-acetone (2 1 by volume) gives 0.269 g. of

crystals of Antibiotic B-5050-C.

The crystals consisting mainly of Antibiotic B-

5050-C (0.90 are dissolved in 5 ml. of acetone, and

column chromatographed on 100 g. of silica gel, followed

by elution with ethyl acetate-benzene (2 1 by volume),

to give 0.258 g. of crystals of Antibiotic B-5050-C.

The Antibiotics B-5050-C and -D crystals (1.40 g.)

are dissolved in 10 ml. of acetone, and chromatographed

on a column packed with '150 g. of silica gel, followed

by elution with benzene-acetone (2 1 by volume), to

give 0.59b g. of Antibiotic B-5050-C and 0.187 g. of

crystals of Antibiotic B-5050-D.

The Antibiotics B-5050-D and -E crystals (1.99 g.)

are dissolved in 30 ml. of acetone, and column-chromato-

graphed on 300 g. of silica gel,.followed by elution with

benzene-acetone (2 1 by volume), to give 0.304 g. of

crystals of Antibiotic B-5050-D and 0.187 g. of crystals

of Antibiotic B-5050-E.

The crystals consisting mainly of Antibiotics B-

5050-F (2.09 are dissolved in 15 ml. of acetone, and column-chromatographed on 250 g. of silica gel, followed by elution with ethyl acetate-benzene (3 1 by volume),

to give 0.585 g. of crystals of Antibiotic B-5050-F.

Reference 4

(Preparation of tetrahydro-B-5050)

22 440552

30,734/71 In 400 ml. of ethanol are dissolved 40.g. crystals

of B-5050, and 12 g. of a 10% palladium/charcoal catalyst

(manufactured by Engelhard) is added. Upon this catalytic reduction, about 2 moles of hydrogen is absorbed in

7 hours.

The reaction mixture is filtered to remove the

catalyst and, then, concentrated under reduced.pressure.

To the residue is added n-hexane, whereupon a powdery

precipitate is obtained. Yield 36.6 g. Elementary analysis

Calcd. for C4 1 H 7 1 N0 1 6 C, 59.05; H, 8.58; N, 1.68 Found C, 58.88; H, 8.72; N, 1.60

3 -57.00 (c=1.0 in CHCl D2 3

Reference

(Preparation of t-esters of tetrahydro-B-5050)

In 40 ml. of ethanol is dissolved 900 mg. ofe propionyl-B-5050, and 300 mg. of a 10% palladium/charcoal

catalyst (manufactured by Merck) is added. Upon this catalytic reduction, 2 moles of hydrogen is absorbed in about 8 hours. The reaction mixture is filtered to remove the catalyst and the filtrate is concentrated under reduced pressure. The residue is dissolved in benzene, followed by the.addition of n-hexane. The procedure yieldso 465 mg. ofi -prdpionyl tetrahydro-B-5050 in a powdery form.

Elementary analysis

Caocd. for C 4 4 H 7 5 NO 1 C, 59.37; 7 H, 8.49; N, 1.58 Found C, 59.05; II, 8.5.7; N, 1..55 a 0 D -64 EtOH)

30,734/71 In the similar manner, the other c-esters of tetrahydro- B-5050 can be also produced.

Reference 6

(Preparation of 4,P-diesters of tetrahydro-B-5050)

In 60 ml. of ethanol are dissolved 1.9 g. of a&,p- dipropionyl-B-5050, and 600 mg. of a 10% palladium/ charcoal catalyst is added. Upon catalytic reduction, about 2 moles of hydrogen is absorbed in about 7 Hours. The reaction mixture is filtered and the filtrate is concentrated to dryness under reduced pressure. The residue is dissolved in a small amount of acetone and, with the addition of 10 times'its volume of n-hexane, the solution is concentrated under reduced pressure. The procedure yields 1.53 g. of a, -dipropionyl tetrahydro-

B-5050. Elementary analysis Calcd. for C47 H9 N018 C, 59.68; H, 8.36; N, 1.48 Pound C, 59.98; H, 8.88; N, 1.86

-77.2° (c=0.99, EtOH)

In the similar manner, the other at. -diesters of tetra-

hydro-B-5050 can be also produced.

Reference 7 (Preparation of a-esters of tetrahydro-B-5050) In 40 ml. of ethanol is dissolved 900 mg. of t- propionyl-B-5050, and a catalytic reduction is carried out with hydrogen gas in the presence of 90 mg. platinum oxide. By this procedure, 2 moles of hydrogen is absorbed in about hours and a half. The reaction mixture is filtered to remove the catalyst

30,734 71 and the filtrate is concentrated under reduced pressure.

The residue is dissolved in a small amount of ether, followed

by the addition of n-hexane. The procedure yields 547 mg.

of 4-propionyl tetrahydro-B-5050 in a powdery form.

The properties of this product, such as Rf value of

thin-layer chromatography on silica gel, infrared absorption

spectrum and nuclear magnetic resonance spectrum, are in

well accord with those of the 4-propionyl tetrahydro-B-

5050 obtained in Example 2.

Reference 8

(Recovering process of individual ingredients

from a reaction product)

450 g. of t-propionate of B-5050 obtained in claim 1 is dissolved in and recrystallized from a mixed solvent of

500 ml. acetone and 1 litre n-hexane to give 73.7 g. of

crystals The mother liquid is concentrated.

The concentrate is dissolved in 1.8 litre of methanol and

kept standing after addition of 1.1 litre water. This procedure yields 159 g. of crystals Further, the second mother liquid is concentrated, and the concentrate

is recrystallized from a mixed solvent of n-hexane ana

petroleum ether to give 183 g. of powder.

70 g. each of crystals and (II) is dissolved in 300 ml. of benzene and chromatographed on a column packed

with 6 kg of silica gel (Merck, 0.05 0.20 mm) in benzene, and eluted by a mixed solvent system of benzene and

acetone.

30,734/71 Solvent system Volume Ingredient Yield benzene: of elut- (Ct-propio- (g) acetone ing, sol- nate of) .v vent (L)

rystals 5 1 49.3 B-5050-B 4 1 26 -D 23.5 3 1 40 -F

rystals (II) 5 1 46.1 B-5050-A. 15.8 4 1 22 -C 16.2 3 :1 32 -E 3.3

The physicochemical properties of thus obtained. ingredients are as follows:

C -propio- nate of B-505 -F -E -D -C -A -B

m.p. (C) 186-90 179-810 S1 8 0 (decomp.) 3o 191-3° 140-2° 198-201 Solvent for di- diethyl- ace- ethyl di- recrystalli- diethyl ethyl ether- tone ace- ethyl ether zation ether n- tate- ether- hexane n- n- hexane hexane

[4D -67.0 -63.9 -64.3 -61.3 -66.3 -62.5 in CHC1 3 c=0.97 c=1.00 c=0.99 c=1.00 c=1.01 c=1.00 Elementary analysis (calcd.) C 58.79 59.23 59.23 59.66 60.05 60.44 H 7.87 7.98 7.98 8.02 8.18 8.27 N 1.63 1.61 1.61 1.58 1.56 1.53 (found) C 58.89 59.14 58.26 59.75 59.69 60.28 H 8.05 7.81 7.81 8.20 8.23 8.36 N 1.49 1.59 1.51 1.57 1.31 1.40 Chemical C42 67 C formul a C4 3 H6 9 C4 3 H6 9 44H71 C45 73 C4 6 H7 N 17 I N 17. NO1 7 -wc174 SNO1 7 SN0O f 17 o

30,734/71 Example 1 (Preparation of g-propionate of B-5050)

In 480 parts by volume of pyridine are dissolved 120 parts by weight of crystals of antibiotic B-5050 and

parts by volume of propionyl chloride is gradually added under cooling with ice and stirring. The mixture is further stirred at 0° 500 for 20 minutes and allowed to stand in a refrigerator for 2 hours. It is then poured into 5000 parts by volume of ice-water and

extracted twice with 2500 parts by volume each.of ethyl

acetate. The ethyl acetate layer is washed with water,

1% aqueous solution of sodium bicarbonate and water in

the order mentioned, followed by dehydration and concentration

under reduced pressure.

The residue is dissolved in 100 parts by volume of

acetone, followed by the addition of 5000 parts by volume

of petroleum ether, whereupon 108 parts by weight of a

white crystalline powder is obtained. This powder is recrystall-ized from a mixture of acetone and n-hexane.

Elementary analysis

Calcd. for C 4 4 H 7 1N0 1 7 C, 59.65; H, 8.08; N, 1,58

Found C, 59.39; H, 8.07; N, 1.73

-61.40 _D2 CHCl) -68.60 EtOH) In the same manner as above except for the ester-

ifying agents employed, the following compounds are

obtained.

1. t -a etate of B-5050

Esterifying agent: acetyl chloride Elementary analysis:

30,734/71 Calcd. for C4 3 H 6 9 N0 1 7 C, 59.23; H, 7.98; N, 1.61

Found C, 58.94; H, 8.04; N, 1.86

[c23 -62.60 CHC13 2. O-n-butyrate of B-5050

Esterifying agent: n-butyryl chloride

Elementary analysis:

Calcd. for C4 5 H 7 3 N0 C, 60.05; 17 H, 8.18; N, 1.56 Found C, 60.05; H, 8.15; N, 1.75

[,X23 0 -64.3 CHCl 3

3. d--isobutyrate of B-5050

Esterifying agent:,-i-butyryl chloride

Elementary analysis:

Calcd. for 04 5 H 7 3 N0 60.05; 17 H, 8.18; N, 1.56 Found 59.69; H, 8.23; N, 1.57

[3 -62.8 CHC1 3 4. c -n-valerate of B-5050

Esterifying agent: n-valeroyl chloride

Elementary analysis:

Calcd. for C4 6 H 7 N0 C, 60.44; 5 17 H, 8.27; N, 1.53 Found C, 60.46; H, 8.37; N, 1.76 2123 -59.90 CHC13)

c-i-valerate of B-5050

Esterifying agent: i-valeroyl chloride

Elementary analysis:

Calcd. for 4 6 H 7 5 N01 7 C, 60.44; H, 8.27; N, 1.53 Found C, 60.38; H, 8.07; N, 1.63 3

[2 -58.00 CHC1

30,734/71 6. a-acetate of B-5050-A

Esterifying agent: acetyl chloride

Elementary analysis:

Calcd. for C 4 5 H 7 3 N01 7 C, 60.05; H, 8.18; N, 1.56

Found C, 59.61; H, 8.48; N, 1.40 5

[t2 -62.50 CHC13).

7. a-propionate of B-5050-Ak

Esterifying agent: propionyl chloride

Elementary analysis:

Galcd. for C46H 7 5 N01 7 C, 60.44; H, 8.27; N, 1.53

Found C, 59.80; H, 8.46; N, 1.59 3

[4g _62.70 (c=l.00, CHC1 3

8. et-propionate of B-5050-B Esterifying agent: propyonyl chloride

Elementary analysis:

Calcd. for C4 5 H 7 3 N0 1 7 C, 60.05; H, 8.18; N, 1.56

Found C, 59.29; H, 8.17; N, 1.55 ri23 -65.80 CHC1 3 9. CC-acetate of B-5050-A and -B Esterifying agent: acetyl chloride Elementary analysis: Found C, 58.97; H, 8.34; N, 1.67 59.04 8.52 1.65

-63.10 (c=1.02, CHCeI)

ct-propionate of B-5050-A.and -B Es-erifying agent: propionyl chloride

Elementary analysis:

30,734/71 Found C, 59.54; H, 8.16; N, 1.60

59.58 8.38 1.62

ja]23 -61.2° (c=0.98, CHC1)

11. e-propionate of B-5050-C

Esterifying agent: propionyl chloride

Elementary analysis:

Calcd. for C4 4 H 7 1 N01 7 C, 59.66; H, 8.02; N, 1.58

Found C, 59.34; H, 8.23; N, 1.59

aj22 -60.80 (c=0.97, CHCl3

12. 4-acetate of B-5050-C Esterifying agent :,Caetyl chloride

Elementary analysis:

Calcd. for C 4 3 H 6 9 N0 1 7 C, 59.23; H, 7.98; N, 1.61

Found C, 59.47; H, 8.09; N, 1.77

Example 2 (Preparation of 4-crotonate of B-5050)

In the same manner as in Example 1, crotonate of B-5050 is obtained. Elementary analysis

Calcd. for C 4 5 H 7 1 N0 1 7 C, 60.19; H, 7.97; N, 1.56

Found C 59.81; H, 7.98; N, 1.51 3 -73.10 Sj CHCl 3 Example 3

(Preparation of P-acetate of B-5050)

In 30 parts by volume of acetone is dissolved

parts by weight of crystals of antibiotic B-5050 followed

by the gradual addition of 2 parts by volume of acetic

30,734/71 anhydride under cooling with ice and stirring. The mixture

is allowed to stand in a refrigerator for 17 hours. The

reaction mixture is added to a mixture of 500 parts by

volume of 3% sodium hydrogen carbonate and 300 parts by

volume of ice-water and adjusted to pH 7.5 with an aqueous

solution of sodium hydrogen carbonate. It is then

extracted twice with 450 parts by volume each of ethyl

acetate. The ethyl acetate solution is washed well with

water, dehydrated and concentrated under reduced pressure.

The residue is dissolved in a small amount of ethyl acetate,

followed by the addition of 90 parts by volume of n-hexane.

The procedure yields 4.5 parts by weight of a white

crystalline powder.

Elementary analysis

Calcd. for C 4 3 H 6 9 N0 1 7 C, 59.23; H, 7.98; N, 1.61

Found C, 59.01; H, 8.07; N, 1.67

-92.2o CHCl)

In the same manner as above, except for the esterifying

agent, following compounds are obtainable.

1. B -propionate of B-5050

Esterifying agent: propionic anhydride

Elementary analysis:

Calcd. for C 4 4 7 1N 1 7 C, 59.65; H, 8.08; N, 1.58

Found C, 59.11; H, 8.22; N, 1.42

.4i 3 _87.00 CHCl)

2. p-n-butyrate of B-5050

Ecterifying agent: n-butyric anhydride

Elementary analysis:

3 0,734/71 Calcd. for C4 5 H7 3 N0 C, 60.05; 17 H, 8.18; N, 1.56 Found C, 59.89; H, 8.74; N, 1.40

LL 25 -90.90 CHC13)

3. P-i-valerate of B-5050 Esterifying agent: i-valeric anhydride Elementary analysis Calcd. for C4 6 H 7 5N01 C, 60.44; 7 H, 8.27; N, 1.53 Found C, 59.76; H, 8.27; N, 1.49 3

[a]1 -89.20 CHC1 4. P-acetate of B-5050-A. and -B Esterifying agent: acetic anhydride Elementary analysis Found C, 58.21 H, 8.07 N .1.37 58.42; 8.11 N, 1.50 D3 -84.30 EtOH)

0-acetate of. B-5050-A. Esterifying agent: acetic anhydride Elementary analysis Calcd. for C4 5 H73N0 C, 60.05; 17 H, 8.18; N, 1.56 Found C, 59.76; H, 8.32; N, 1.52

[dj24 -91.30 EtOH) 6. P-propionate of B-5050-C, Esterifying agent: propionic anhydride Elementary analysis: o Calcd. for C44 H71 N01 C, 59.66; H, 8.02; 7 N, 1.58 Found 58.82 H 8.81 N, 1.60 58.85 8.66' 1.95 a2 3 -93.50 (c=1.02, EtOH)

32 440552

30,734/71 7. s-acetate of B-5050-C. Esterifying agent: acetic anhydride

Elementary analysis:

Calcd. for GC43H 6 9 NO 1 7 C, 59.23; H, 7.98; N, 1.61

Found C, 58.88; H, 8.12; N, 1.66

-90.60 _cE323 CHCl 3

Example 4

(Preparation of 0-succinate of B-5050)

In 4 parts by volume of acetone are dissolved

part by weight of crystals of antibiotic B-5050, followed

by the gradual addition of-0 -175 part by weight of succinic

anhydride at room temperature with stirring. The mixture is stirred at room temperature for 5 hours and concentrated

under reduced pressure. The residue is extracted with 150 parts by volume of ethyl acetate and the extract is washed

well with water, dehydrated, concentrated and allowed to stand. The resulting crystals are recovered by filtration

and washed with ethyl acetate and ether. Yield 0.375 part by weight.

Elementary analysis

Calcd. for C45H IN019 CH3COOC2HH2Q

C, 56.81; H, 7.83; N, 1.35 Found C, 57.15; H, 7.92; N, 1.55

[E26 -81.7° CHCl 3

Example

5 parts by volume of pyridine is dissolved 0.9 part

by weight of crystals of antibiotic B-5050 and while the

solution is cooled with ice-water, 0.4 part by weight of

30,734/71 succinic anhydride is gradually added.

The mixture is allowed to stand at room temperature

for 16 hours, at the end of which time 200 parts by volume

of ice-water is added. The mixture is extracted three times with 100 parts by volume each of ethyl acetate, washed with water, dehydrated and concentrated under

reduced pressure. The residue is dissolved in 10 parts by volume of ethyl acetate-chloroform(l:l), followed by

the addition of 100 parts by volume of n-hexane. The procedure yields 0.704 part by weight of a crystalline

powder. The characteristics of this'powder are in accord 0

with those of the sample obtained in Example 4.

In the same manner as above, except for the ester- ifying agents, the following compounds are obtainable:

-benzoate of B-5050 Esterifying agent: benzoic anhydride

Elementary analysis: Calcd. for C4 8 H 7 1 N01 7 61.72, H, 7.66; N, 1.50

Found C, 61.55; H, 7.59; N, 1.45 \MemaxOH 23m 230 (c-13,600) 3 -76.7 0 CHCI3

Example 6 (Preparation of af-diacetate of B-5050)

In 10 parts by volume of pyridine are dissolved

parts by weight of crystals of antibiotic B-5050 and, at

room temperature and under stirring, 5.0 parts by volume

of acetic anhydride is added. The mixture is allowed to stand for 15 hours, at the end of which time it is poured

into 300 parts by volume of ice-water. The resulting pre- cipitate is recovered by filtration and washed with water.

30,734/71 The mother fluid is combined with the washing and the pool

is extracted twice with 200 parts by volume each of ethyl

acetate. The extract is washed with water, a 2N-aqueous

solution of sodium bicarbonate and water inthe order

mentioned, dehydrated and concentrated under reduced

pressure. The precipitate previously recovered is

dissolved in methylene chloride and the solution is The dehydrated and concentrated under reduced pressure.

two concentrates are combined and recrystallized from

methylene chloride-n-hexane, acetone-n-hexane or methylene

chloride-n-hexane-petroleum ether. The procedure yields

4.65 parts by weight of B- 050 %,p-diacetate. m.p.127

132 0 C (decomp.) Elementary analysis:

Calcd. for C 4 5 H 7 1 N0 1 8 C, 59.13; H, 7.83; N, 1.50

Found C, 59.25; H, 7.93; N, 1.49

3 _84.90 CHCl3

In the same manner as above, except for the esterify-

ing agent, the following compounds are obtained:

1. a.,p-dipropionate of B-5050

Esterifying agent: propionic anhydride

m.p.1320 C(decomp.)

Elementary analysis: Calcd. for C 4 7 H 7 5 N0 1 8 59.92; H, 8.02; N, 1.49

Found C, 60.01; H, 8.31; N, 1.46

3 -81.70 8 CHCl 3

2. d.,-dicrotonate of B-5050 Esterifying agent: crotonic anhydride

30,734/71 Elementary analysis:

Calcd. for C4 9 H 7 5 N0 1 8 C, 60.92; H, 7.82; N, 1.45

Found C, 60.55; H, 7.76; N, 1.41

3 .00 CHC13 j 92

3. rt,p-di-n-butyrate of B-5050

Esterifying agent: n-butyric anhydride

Elementary analysis:

Calcd. for C4 9 H 7 9 N0 1 8 C, 60.66; 1, 8.21; N, 1.44

Found C, 60.37; 1, 8.30; N, 1.53 3 D2 -74.90 CHC13)

4. ex,p-diacetate of B75650-A.and -B Esterifying agent: acetic anhydride

m.p.131-135C(decomp.) Elementary analysis: Found C59.01 H 8.05. 1.58 S59.26; '7.91' 1.68

2 _83.70 CHUC1 3) X,,-dipropionate of B-5050-A, -B Esterifying agent: propionic anhydride

m.p.104-1070 C(decomp.) Elementary analysis:

Found C, 60.09. H 8.24. N 1.45 60.26' 8.32' 1.63

3 2[c -70.70 CH013)

6. 4a,P-diacetate of B-5050-A

Esterifying agent: acetic anhydride

Elementary analysis:

Calcd. for C 4 7 H 7 5 N0 1 8 C, 59.92; H, 8.02; N, 1.49 Found C, 59.49; H, 8.21; N, 1.62

30,734/71. -77.4o EtOH)

7. a,h-dipropionate of B-5050-A

Esterifying agent: propionic anhydride

Elementary analysis:

Calcd. for C 49H79N01 8 C, 60.66; i, 8.21; N, 1.44

Found C, 60.48; I 8.34; N, 1.51

[122 -83.2o (c=1.02, EtOH)

8. a,o-diacetate of D-5050-B

Esterifying agent: acetic anhydride

Elementary analysis:

Calcd. for C 4 67T 3 N018 C, 59.53; H 7.93; N, 1.51 Found C, 59.47; H 8.10; N, 1.51 9. g,O-diacetate of B-5050-C

Esterifying agent: acetic anhydride

Elementary analysis:

Calcd. for C 4 5 H 7 1 N0 1 8 C, 59.13; H 7.48; N, 1.53 Found C, 58.89. 8.10. 1.65 Y 59.02' 8.07, N, 1.57 3

[aJ -81.50 HC1 3

c,-dipropionate of B-5050-C Esterifying agent: propionic anhydride Elementary analysis: Calcd. for C47 H7 5 N01 8 C, 59.92; H, 8.02; N, 1.49 Found 59.54; H, 8.20; N, 1.50 3 j&a1 -86.90 (c=1.01, CHCI 3

Example 7 601 4t, VIap41 (Preparation of -ee~-t ee of B-5050) In 12 parts by volume of pyridine is dissolved

37 440552

30j734/71 at parts by weight of the antibiotic B-5050 -propionate volume room temperature and under cooling, 1.5 parts by of acetic anhydride is added. The mixture is allowed to is poured stand for 14 hours, at the end of which time it into 500 parts by volume of ice-water. The resulting with precipit~te is recovered by filtration and washed water. The mother fluid is combined with the washing and

the pool is extracted twice with 200 parts by volume each of ethyl acetate. The precipitate is combined with the reduced above extract and the pool is concentrated under by volume pressure, followed by the addition of 300 parts of n-hexane. The procedure__yields 5.45 parts by weight of a crystalline powder. Elementary analysis: Calcd. for C46 H7 3 NO1 8 C, 59.53; H, 7.93; N, 1.51

Found C, 58.84; H, 7.85; N, 1.57

Example 8

(Preparation of CELkfepvrsyl~-p-acetate o -00

In 10 parts by volume of pyridine is dissolved parts by weight of the antibiotic ct-propionate of B-5050 by and, at room temperature and under stirring, 5 parts volume of acetic anhydride is added. The mixture is time it allowed to stand for 15 hours, at the end of which The is poured into 300 parts by volume of ice-water. washed resulting precipitate is recovered- by filtration and with water. It is dissolved in 100 parts by volume of

ethyl acetate and the solution is dehydrated and concentrated petroleum under reduced pressure, followed by the addition of

30,734171 ether. The procedure yields 1.8 parts'by weight of a.

crystalline powder.

Elementary analysis:

Calcd. for 0 4 6 H 7 3 N01 8 C, 59.53; H, 7.93; N, 1.51

Found C, 59.58; H, 7.92; N, 1.51

3 -82.80 CHCl 3

Example 9 (Production of P-acetate of tetrahydro-B-5050)

In 1.2 part by volume of acetone is dissolved 0.2

part by weight of tetrahydro-B-5050 and with the addition

of 0.1 part by volume of-acetic anhydride, the solution

is stirred at 00C. The mixture is held at OOC for 18 hours, at the end of which time it is poured into

parts by volume of ice-water. After being adjusted to pH 8.0 with 5% sodium hydrogen carbonate, the mixture

is extracted twice with 20 parts by volume each of ethyl

acetate. The extracts are pooled, washed with water,

dehydrated and concentrated under reduced pressure. To the residue is added n-hexane, whereupon 0.161 part by weight of e~~4c tetrahydro-B-5050 is obtained as a

powder.

Elementary analysis:

Calcd. for C3H 73N17 C, 58.97; H, 8.34; N, 1.60

Found C 58.62 8.34 N 1.59 58.57' 8.46' 1.53

[423 -68.20 (c=l in EtOH)

In the same manner as above, except for the esteri- fying agent, -propionate of tetrahydro-B-5050 is obtained: Esterifying agent: propionic anhydride

734/71 Elementary analysis:

Calcd. for C4 4 H 7 5 N01 7 C, 59.37; H, 8.49; N, 1.57

Found C, 59.48; H, 8.59; N, 1.39

[]23 76 .2o in EtOH)

Example

(a,Y -dipropionate of tetrahydro-B-5050)

In 20 parts by volume of pyridine is dissolved

parts by weight of tetrahydro-B-5050 and with the addition

of 2.0 parts by volume of propionyl chloride, the

solution is stirred at 0 C. The mixture is allowed to stand for 2 hours, at the end of which time it is poured

into 500 parts by volume of ice-water. The mixture is extracted twice with 250 parts by volume of ethyl acetate,

and the extracts are pooled and washed with water, a

aqueous solution of sodium bicarbonate and water in the

order mentioned, followed by dehydration and concentration

under reduced pressure.

To the concentrate is added n-hexane-petroleum ether,

whereupon a,6 -p Foony of tetrahydro-B-5050 is obtained as powder. Yield 3.0 parts by weight. Elementary analysis:

Calcd. for C4 7 H 7 9 N01 8 C, 59.66; H, 8.42; N, 1.45

Found C, 58.96; H, 8.40; N, 1.82

ag 23 -53.50 (c=l.0 in CHC1)

In the same manner as above, except for the esteri-

fying agent,a,2 di-n-butyrate of tetrahydro-B-5050 is obtained:

Esterifying agent: n-butyryl chloride

440552.

30,734/71' Elementary analysis:

Calcd. for C 4 9 H 8 3 NO1 8 C, 60.41; H, 8.59; N, 1.44

Found C, 60.62; H, 8.49; N, 1.50

-52.90 (c=l.0 in CHC 3

Example 11

(4,-Y-triacetate of tetrahydro-B-5050) In 30 parts by volume of pyridine is dissolved

parts by weight of tetrahydro-B-5050, and while the

solution is stirred at room temperature, 15 parts by

volume of acetic anhydride is added. The mixture is

allowed to stand for 15 hours. The reaction mixture is

poured into 800 parts by volume of ice-water and extracted

twice with 270 parts by volume each of ethyl acetate.

The ethyl acetate layer are pooled, washed with water,

a 5% aqueous solution of sodium hydrogen carbonate and

water in the order mentioned, dehydrated and concentrated

under reduced pressure to about 200 parts by volume.

Then, 8.0 parts by weight of chromatographic activated

carbon is added, and after the mixture is heated to a

moderate temperature, it is filtered and the filtrate

concentrated.

To the concentrate is added a mixture of methylene

chloride and n-hexane, whereupon 4.2 parts by weight of

-triacetate of tetrahydro-B-5050 is obtained as colorless prisms. Upon addition of petroleum ether to

the mother fluid, 2.5 parts by weight of powder is

further -htained. m.p.110 0 -113 0 (decomp.)

Elementary analysis:

Calcd. for C4 7 H 7 7 N0 1 9 C, 58.80; H, 8.08; N, 1.46

30,734/71 Found C, 58.33; H, 8.08; N, 1.49

23 (c=1.0 in CHC1 1 -76.30 3

In the same manner as above, except for the esteri-

fying agent, a,pY -tripropionate of tetrahydro-B-5050 is

obtained. m.p.1180 -122 0 C. (decomp.)

Esterifying agent: propionic anhydride

Elementary analysis:

Calcd. for C 5 0 H 8 3 N01 9 C, 59.92; H, 8.35; N, 1.40

Found C, 60.24; H, 8.54; N, 1.35

[ar23 -70.10 (c=1.0 in CH01 3

Example--12

(Production of a,p,y -tripropionate of tetrahydro-B-5050)

In 4 parts by volume of pyridine is dissolved 1 patt

by weight of p-propionate of tetrahydro-B-5050 and, under cooling with ice-water, 1 part by volume of propionic

anhydride is added dropwise. The mixture is allowed to

stand at room temperature for 20'hours and then poured

into 150 parts by volume of ice-water and the resulting

precipitate is recovered by filtration, washed with water

and dried. Yield 1.04 part by weight.

Elementary analysis:

Calcd. for C 5 0 H 8 3 N 19 C, 59.92; H, 8.35; N, 1.40

Found C, 59.66; H, 8.42; N, 1.51

5 23 7 3. 10 c CH C

Example 13

Production of -triacetate of tetrahydro-B-5050)

in 3 parts by volume of pyridine is dissolved 0.3

part by weight of a,p-diacetate of tetrahydro-B-5050 and

42 440552 wi30,734/71 under cooling with ice-water, 0.3 part by volume of acetic anhydride is added dropwise. The mixture is allowed to stand at room temperature for 20 hours and then poured into 80 parts by volume of ice-water and the resulting precipitate is recovered by filtration, washed with water and dried. Yield 0.25 part by weight.

Elementary analysis:

Calcd. for C 77NO19 C, 58.80; H, 8.08; N, 1.z46

Found C, 58.45; H, 8.26; N, 1.i43

S23 -74.90 in CHC1 3

In the same manner, oC,p, Y-triacetate of tetrahydro- B-5050 is also obtained by the esterification of d, Y diacetate of tetrahydro-B-5050.

Example 14

y-tripropionate of tetrahydro-B-5050) In 2 parts by volume of pyridine is dissolved 0.3

part by weight of 4 -propionate of tetrahydro-B-5050 and,

under cooling with ice, 0.3 part by volume of propionic

anhydride is added dropwise, with stirring. The mixture

is allowed to stand at room temperature for 20 hours. The reaction mixture is poured into 100 parts by volume of ice-

water and the resulting precipitate is recovered by filtra-

tion, washed with water and dried. The procedure yields 0.25 part by weight of ax,P, K-tripropionate of tetrahydro- B-5050 as powder. The Rf value (TLC on silica gel) and other characteristics of this product are in accord with

those of The a,f, y-tripropionate of tetrahydro-B-5050 sample obtained in Example 11.

440552

30,734/71. Example

(Production of ,$-dipropionate of tetrahydro-B-5050)

In 30 parts by volume of acetone is dissolved 5 parts 0 by weight of a-propionate of tetrahydro-B-505 and, under

cooling with ice-water 2 parts by volume of propionic

anhydride is added dropwise. The mixture is allowed to

stand in a refrigerator for 18 hours and poured into 100

parts by volume of a 2% aqueous solution of sodium bicarbo-

nate and the resulting precipitates are extracted twice

with ethyl acetate, washed, dehydrated and concentrated

under reduced pressure. To the resulting syrupy substance

is added 200 parts by volume of n-hexane to give 4.6 parts

by weight of white powder.

Elementary analysis:

Calcd. for C 4 7 H 7 9 NO1 8 C, 59.66; H, 8.42; N, 1.45

Found C, 59.38; H, 8.29; N, 1.36

j 3 -78.5° CHCl 3

Example 16

(Production of ca, -dipropionate of tetrahydro-B-5050)

In 15 parts by volume of pyridine is dissolved 3

parts by weight of O-propionate of tetrahydro-B-5050 and

under cooling with ice-water, 1.0 part by volume of

propionyl chloride is added and stirred for 2 hours.

The reaction mixture is poured into 300 parts by

volume of ice water and the whole is extracted twice with

300 parts by volume each of ethylacetate. The combined

extract is washed twice with an 5% aqueous solution of

sodium bicarbonate, washed, dehydrated and concentrated

under reduced pressure and then n-hexane is added to give

30973417' a.,y -dipropionate of tetrahydro-B-5050 as powder. Yield 2.4 parts by weight. Elementary analysis: Calcd. for C47H79WC1 59.66; H, 8.42; N, 1.45

Found C, 59.41; H, 8.53; N, 1.47

23 Da 54.70 CHM 3

4405.52

Claims (19)

30,734/71 The claims defining the invention are as follows:-

1. A method of introducing an ester group into the

antibiotic B-5050 or tetrahydro-B-5050 as hereinbefore defined comprising treating the antibiotic B-5050, tetrahydro-B-5050 o an &ester of tetrahydro-B-5050 as hereinbefore defined with an esterifying agent having an acyl group of from 2 to 7 carbon atoms derived from a carboxylic acid, an acid anhydride or an acid halide.

2. A method as claimed in claim 1, wherein the end prodi is an A- or P-monoester or anL, -diester of the antibiotic B-5050 as hereinbefore defined.

3. A method as claimed in claim 1, wherein the end prodi is a P-monoester, anA,P-diester, and -diester or triester of the antibiotic tetrahydro-B-5050 as hereinbefore defined.

4. An ester of the antibiotic B-5050 as hereinbefore defined selected from the group consisting off- and g- monoesters andd, -diesters of the antibiotic B-5050 the ester groups having 2-7 carbon atoms.

Thedo-acetate of the antibiotic B-5050 as hereinbefore defined.

6. Theo(-propionate of the antibiotic B-5050 as hereinbefore defined.

7. The O-n-butyrate of the antibiotic B-5050 as hereinbefore defined.

8. The-i-butyrate of the antibiotic B-5050 as

46 440552

30,734/71 hereinbefore defined,

9. Theok-crotonate of the antibiotic B-5050 as hereinbefore defined.

The p-acetate of the antibiotic B-5050 as hereinbefore defined.

11. The P-propionate of the-antibiotic B-5050 as hereinbe fore defined.

12. Theck,-P-diacetate of the antibiotic B-5050 as hereinbe fore defined.

13. Thedk-propionate-p-acetate of the antibiotic B-5050 as hereinbefore defined.

14i. The eA,P,-dipropionate of the antibiotic B-5050 as hereinbe fore defined.

An ester of the antibiotic tetrahydro-B-5050 as hereinbefore defined selected from the group consisting of monoestersA.0- and#1 ,W,-diesters andeS,,,-triesters of tetrahyd- ro-B-5050 as hereinbefore defined.

16. Thed,,,-triacetate of the antibiotic tetrahydro-B- 5050 as hereinbefore defined.

17. ThedJ,Pj-tripropionate of the antibiotic tetrahydro- B-5050 as hereinbefore defined.

18. A method of preparing the antibiotic tetrahydro- as hereinbefore defined or esters thereof comprising reducing the antibiotic B-5050 as hereinbefore defined or

44 05 5 2

30,734 71 esters thereof catalytically.

19. A method as claimed in claim 18, wherein the reducing

agent is hydrogen gas and the catalyst is palladium carbon, nickel or platinum.

The antibiotic tetrahydro-B-5050 as hereinbefore defined.

DATED THIS 6TH DAY OF JULY, 1973

TAKEDA CHEMICAL INDUSTRIES, LTD. By their Patent Attorney:

of GRIFFITH, HASSEL FRAZER.