A novel compound of the formula ##STR1## and the salts thereof, derived from dianemycin, are useful as antiprotozoal and antibacterial agents.

BACKGROUND 
Dianemycin, from which the novel compound of the present invention is 
prepared, is a polyether antibiotic described in U.S. Pat. No. 3,577,531, 
Journal of Antibiotics, 22, 161 (1969), and ibid., 33, 137 (1980). This 
antibiotic has activity against protozoa such as coccidiosis and against 
Gram positive bacteria, and is represented by the formula 
##STR2## 
DESCRIPTION AND PREFERRED EMBODIMENTS 
The present invention relates to a novel derivative of dianemycin. More 
particularly, the present invention is concerned with the novel compound 
(hereinafter referred to as compound I) of the formula 
##STR3## 
and the salts thereof. 
The salts of compound I include the corresponding alkali metal salts such 
as sodium and potassium salts, alkaline earth metal salts, and ammonium 
salt. Most preferred are the sodium salt and potassium salt in view of 
ease in handling. 
As a result of various studies in attempts to find compounds having 
improved properties as compared with dianemycin of which the use in 
therapy is extremely restricted by the high toxicity, compound I has been 
found to show the diminished toxicity together with valuable antiprotozoal 
activity and antibacterial activity against Gram positive bacteria. The 
present invention is based on the above finding. 
Accordingly, an object of the present invention is to provide compound I 
and the salts thereof which have desired inhibitory activity and show much 
less toxicity than dianemycin. 
Compound I or a salt thereof may be prepared as follows: In an organic 
solvent such as acetone, acetonitrile or chloroform is dissolved 
dianemycin or a salt thereof such as an alkali metal, an alkaline earth 
metal or ammonium salt. The resulting solution, after adjustment of the pH 
to about 3 with p-toluenesulfonic acid, is allowed to stand at room 
temperature to give compound I. Compound I is converted to its sodium salt 
in a conventional manner and extracted with an organic solvent such as 
benzene, ethyl acetate or chloroform. The extract is concentrated to 
dryness and then again dissolved in a suitable organic solvent (i.e., 
chloroform, methanol and a mixture thereof). Purification by appropriately 
combined procedures of silica gel column chromatography and gel 
filtration, and recrystallization from a mixed solvent of methanol and 
water give compound I sodium salt as prismatic crystals. 
Compound I may be obtained by treating the sodium salt thus obtained in a 
conventional manner. The desired salt of compound I may be obtained by 
treating compound I in a conventional manner. 
Dianemycin, the starting material for preparation of compound I, may be 
produced by culturing Streptomyces hygroscopicus, for example, 
Streptomyces hygroscopicus TM-531 described in Journal of Antibiotics, 33, 
137 (1980) and U.S. Pat. No. 4,269,971, which has been deposited under the 
name of Streptomyces TM-531 at Fermentation Research Institute, Agency of 
Industrial Science and Technology, Japan, as FERM-P 4737 on Nov. 29, 1978 
and at American Type Culture Collection, as ATCC 31590. 
Compound I and the salts thereof thus obtained have the excellent activity 
against protozoa, especially toxoplasma, and against Gram positive 
bacteria. Decreased toxicity is an important advantage provided by 
compound I and the salts thereof. Thus, compound I and the salts thereof 
can be used as antiprotozoal agents and antibacterial agents in animals 
including humans. 
For the control of coccidiosis in poultry, a nontoxic anticoccidial amount 
of the compound of the present invention is administered to the birds, 
preferably orally on a daily basis. In case of the oral administration, 
compound I or the salts thereof may be supplied with a substance capable 
of being consumed by the birds, preferably the feed of the birds. 
The rate of administration, which is effective against infection of 
coccidiosis, is generally in the range of from 100 to 700 ppm, preferably 
250 to 500 ppm, by weight of unmedicated feed. The compounds of the 
present invention may be used optionally together with other anticoccidial 
agents.

The following "Test Examples" are illustrative of the physiological effects 
of the compound of the present invention. 
TEST EXAMPLE 1 
Normal ICR mice were intraperitoneally injected with 1 ml of a sterilized 
0.2% solution of glycogen in saline solution and, after 5 days, 
intraperitoneally injected with 5 ml of a Hank's solution containing 
heparin to collect macrophage. 
After centrifugation and washing, the macrophage was diluted with a medium 
of TC-199 containing 20% of calf serum so that the resulting solution 
contained about 10.sup.6 cells/ml of macrophage. 1 ml of this suspension 
was pipetted into a cultivation Petri dish containing circular cover glass 
of 1.5 cm in diameter, followed by cultivation for 24 hours in an 
incubator containing 5% of carbon dioxide. 
5.times.10.sup.5 toxiplasma RH strain collected on the 2nd day after 
intraperitoneal infection of mice were brought into contact with the thus 
cultivated macrophage at 37.degree. C. for about 1 hour, and non-infecting 
toxiplasma RH strain were washed away using the same medium followed by 
adding thereto the same medium. 
A drug-containing group was prepared by adding compound I sodium salt to 
the medium so that the final concentration of the compound became 0.1 ppm. 
Medical effects were evaluated as follows; After cultivation at 37.degree. 
C. for 48 hours, May-Grunwald/Giemsa double straining was conducted to 
determine the percentage of macrophage cells containing therein 1 to 5 
toxoplasma or 6 or more toxoplasma, thus the infected control group was 
compared with the drug-added group. 
The results thus obtained are shown in Table 1. 
TABLE 1 
______________________________________ 
Anti-toxoplasma Effect 
Percentage of Toxoplasma- 
infected Macrophage 
1 to 5 Toxoplasma/ 
6 or more Toxo- 
cell plasma/cell 
______________________________________ 
Infected Control 
30.8 31.8 
Group 
Compound I Sodium 
0 0 
Salt-Added Group 
______________________________________ 
(Note) 
Observation under an optical microscope revealed no cytotoxic effect by 
the addition of compound I sodium salt. 
TEST EXAMPLE 2 
MIC (Minimum Inhibitory Concentration) was measured according to the method 
specified by the Chemotherapeutic Society using a heat infusion agar 
medium containing compound I sodium salt, to examine antibacterial effects 
of the compound. The results thus obtained are shown in Table 2. 
TABLE 2 
______________________________________ 
Antibacterial Effects 
Tested Bacteria MIC (mcg/ml) 
______________________________________ 
Staphylococcus aureus FDA 209 P 
25 
Staphylococcus aureus Smith 
50 
Staphylococcus aureus TPR-23 
50 
Staphylococcus epidermidis TPR-25 
50 
Staphylococcus epidermidis IID 866 
50 
Streptococcus faecalis ATCC 8043 
50 
Bacillus subtilis ATCC 6633 
25 
Bacillus licheniformis 
12.5 
Escherichia coli NIHJ C-2 
&gt;200 
______________________________________ 
TEST EXAMPLE 3 
Compound I sodium salt and the control drug of dianemycin sodium salt were 
respectively suspended in a 5% solution of gum arabic in saline solution. 
Two groups of 7 ddY strain male mice (4-week old) were intraperitoneally 
injected with each of the above-described drug suspension. LD.sub.50 
values (mg/kg) of the compounds were determined from the total mortality 
per group for 7 consecutive days to examine the acute toxicity. 
The results thus obtained are shown in Table 3. 
TABLE 3 
______________________________________ 
Tested Drug LD.sub.50 (mg/kg) 
______________________________________ 
Dianemycin Sodium Salt 
7.2 
Compound I Sodium Salt 
191 
______________________________________ 
The following "Reference Example" and "Examples" are illustrative of the 
source material dianemycin and the compounds of the present invention, 
respectively. 
REFERENCE EXAMPLE 
A sterilized liquid medium comprising 2% glucose, 2% oatmeal, 0.3% beef 
extract, 0.3% sodium chloride, 0.25% calcium carbonate, 0.04% ferric 
sulfate and 0.04% manganese chloride was inoculated with TM-531 strain, 
and the inoculated strain was aerobically cultivated at 30.degree. C. for 
72 hours with stirring to produce a seed culture. 
200 l of a sterile medium having the same composition as above charged in a 
250 l-fermentation tank was inoculated with 5 l of the seed culture 
prepared as above and the culture was aerobically cultivated at 30.degree. 
C. for 72 hours with stirring. After completion of the cultivation, the 
resulting fermentation broth was centrifuged to separate the broth into a 
supernatant and microbial cells. The supernatant thus obtained was 
extracted with three portions of ethyl acetate and the combined extracts 
were set aside. The microbial cells were extracted with two 15 l-portions 
of acetone and the combined acetone extract was concentrated until acetone 
was distilled out and further extracted with ethyl acetate. The resulting 
extract was combined with the above ethyl acetate extract and the combined 
extracts were concentrated under reduced pressure to obtain about 112 g of 
a brown syrup. The whole amount of the syrup was dissolved in 500 ml of 
benzene and the solution was adsorbed on a silica gel column [Wako Gel 
C-200 (trade name; made by Wako Junyaku K.K.)] which had been treated with 
benzene. 
The column was eluted first with benzene and then with benzene-acetone 
(90:10) and the eluate was discarded. The column was subsequently eluted 
with benzene-acetone (50:50) and the fractions thus obtained were 
collected and concentrated to a dryness under reduced pressure. The 
resulting crude product was dissolved in acetone. The acetone solution was 
gel-filtered on Sephadex LH-20 (trade name; made by Pharmacia Co.) with 
acetone and the resulting active fraction was concentrated to dryness 
under reduced pressure to obtain 34 g of a light yellow powder. 
Recrystallization of the product from acetone-water (2:1) yielded 26 g of 
dianemycin as needle crystals. 
EXAMPLE 1 
1 g of dianemycin was dissolved in 70 ml of acetonitrile, and 250 mg of 
p-toluenesulfonic acid was added thereto. After stirring the mixture to 
the point of dissolution, the resulting solution was allowed to stand at 
room temperature to react overnight. 
An aqueous solution of sodium bicarbonate was added to the reaction 
solution to adjust the pH to 8 and, after allowing to stand for 30 
minutes, the solution was extracted twice with 50 ml of benzene. The 
extracts were combined, washed with water, dried over anhydrous sodium 
sulfate, and concentrated to dryness to obtain 0.86 g of a crude powder of 
compound I sodium salt. 
EXAMPLE 2 
1 g of dianemycin sodium salt was dissolved in 70 ml of acetonitrile, and 
300 mg of p-toluenesulfonic acid was added thereto. After stirring the 
mixture to the point of dissolution, the solution was allowed to stand at 
room temperature to react overnight. An aqueous solution of sodium 
bicarbonate was added to this reaction solution to adjust the pH 8 and, 
after allowing to stand for 30 minutes, the solution was extracted twice 
with 50 ml of benzene. The extracts were combined, washed with water, 
dried over anhydrous sodium sulfate, and concentrated to dryness to obtain 
0.83 g of a crude powder of compound(I) sodium salt. 
EXAMPLE 3 
The crude products of compound I sodium salt obtained in Examples 1 and 2 
were combined, dissolved in 5 ml of a mixed solvent of chloroform-methanol 
(49:1), adsorbed on 100 ml of silica gel column [Wako Gel C-200 (trade 
name; made by Wako Junyaku K.K.)] packed with the mixed solvent described 
above, and eluted with the solvent. 
Eluate fractions from 250 to 400 ml [confirmed by thin layer chromatography 
(chloroform:methanol=9:1)] were collected and concentrated to dryness. The 
resulting residue was dissolved in a small amount of methanol and was 
subjected to gel filtration with methanol using Sephadex LH-20 (trade 
name; made by Pharmacia Co.). 
Active fractions were collected and concentrated to dryness, and 
crystallization of the residue from a mixed solvent of methanol-water 
(2:1) gave 0.55 g of compound I sodium salt as colorless prismatic 
crystals. 
m.p. 203.degree.-205.degree. C. 
Specific rotatory power: [.alpha.].sub.D.sup.26 =+42.degree. (c=0.5, 
methanol) 
UV absorption spectrum (methanol): E.sub.1 cm.sup.1% (232 nm)=163.8 
Elemental analysis: Calcd. for C.sub.40 H.sub.65 O.sub.12 Na: C, 63.16; H, 
8.55; Na, 3.03. Found: C, 63.10; H, 8.66; Na, 2.99.