"Compounds, named the ""Leustroducsins"", their preparation and their therapeutic uses"

New compounds, which we have named the "Leustroducsins" have the formula (I): ##STR1## in which R represents a 5-methylhexanoyloxy group, a 6-methyloctanoyloxy group or a 7-methyloctanoyloxy group, and salts thereof, and may be prepared by fermentation using a microorganism of the genus Streptomyces, especially a strain of the species Streptomyces platensis, such as strain SANK 60191 (FERM BP-3288). These compounds may be used for the treatment or prophylaxis of: adverse reactions resulting from cancer chemotherapy or radiotherapy; infections; cancer; cerebral dysfunction; and fungal infections.

BACKGROUND TO THE INVENTION 
The present invention provides a series of new compounds which we have 
named "Leustroducsin A", "Leustroducsin B" and "Leustroducsin C". These 
compounds have the formula (I), shown hereafter. The invention also 
provides methods of preparing these compounds by fermentation using a 
microorganism of the genus Streptomyces, and especially a strain of the 
species Streptomyces platensis, which is also new and also forms part of 
the present invention. The compounds of the invention have a variety of 
therapeutic effects, and, thus, the invention also provides compositions 
and methods of therapy or prophylaxis using these compounds. 
The leustroducsins of the present invention are novel compounds which 
stimulate the production of hematopoietic factors, such as granulocyte 
colony-stimulating factor (hereinafter abbreviated to "G-CSF") and 
granulocyte-macrophage colony-stimulating factor (hereinafter abbreviated 
to "GM-CSF"); they also stimulate the production of nerve growth factor 
(hereinafter abbreviated to "NGF"); moreover, they exhibit an antifungal 
effect, e.g. against Tricophyton mentagrophytes. 
Various kinds of cytokines having a hematopoietic activity, such as 
colony-stimulating factors (hereinafter abbreviated to "CSF"), and several 
kinds of glycoproteins (generally named the "interleukins") have to date 
been prepared by the techniques of gene manipulation. These substances 
have been used clinically in various ways to reduce the adverse side 
effects commonly caused by cancer chemotherapy and radiotherapy and to 
block infection. The effectiveness of these substances has recently become 
clear [Br. J. Cancer 59, 2-5 (1989) ]. 
It has been found that the administration of these factors themselves to 
humans by various routes results in clear pharmacological effects, which 
leads to the possible use of these factors in therapy. However, it is 
thought that these factors are essentially produced in vivo by certain 
kinds of cells (e.g. lymphocytes, monocytes, fibroblasts, vascular 
endothelial cells and stromal cells) through a complicated regulatory 
system, and that they play homeostatic roles in the production of various 
kinds of blood cells. Accordingly, if these factors are administered 
without any consideration for the delicate balance of this regulatory 
mechanism, several side effects may be observed, which may be caused by 
the imbalance of this regulatory mechanism; examples of such side effects 
include inflammation at the site of injection, bone pain, fever and rigor. 
On the other hand, instead of administering the hematopoietic factors 
themselves, it might be possible to administer certain substances which 
are known to stimulate the production of these hematopoietic factors in 
the body. For example, it is known that interleukin 1 (hereinafter 
abbreviated to IL-1) and tumor necrosis factor (hereinafter abbreviated to 
TNF) can induce the production of CSF etc. by various kinds of cells. 
These factors, however, cannot always act as specific inducers of 
hematopoietic factors because they also have various other biological 
activities. 
It is also known that various kinds of low-molecular weight 
immunoactivators, such as lipopolysaccharides (hereinafter abbreviated to 
LPS) and muramyldipeptides (hereinafter abbreviated to MDP) can produce 
various kinds of CSF's by activation of monocytes and macrophages. 
However, it is known that other physiological effects caused by the 
activation of macrophages, for example the production of monokines such as 
IL-1 and TNF occurs at the same time, which can result in various side 
effects, such as fever [Nippon Acta Radiologica 48, (4), 514 (1988)]. 
Phorbol esters and calcium ionophores are known to induce CSF production 
synergistically [Kohama et al.: Experimental Hematology 16, 603-608 
(1988)], however, they are also known not only to stimulate the production 
of hematopoietic factors but also to stimulate the production and 
secretion of the whole of the secreting proteins, including hormones such 
as insulin [Y. Nishizuka: Science 225, 1365 (1984)]. 
Although the precise mechanism has not yet been clarified, it is thought 
that, in the formation of blood cells, various kinds of mature blood cells 
can be formed from common cell precursors, called hematopoietic stem 
cells, through the action of various hematopoietic factors and through 
cell to cell interaction. It has been established that the site where 
blood cells are formed in normal adults is limited only to the inside of 
the bone marrow, that cells called stromal cells existing in the bone 
marrow play an important role in formation of blood cells [Dexter et al.: 
J. Cell. Physiol. 91, 335 (1977)], and that stromal cells in the bone 
marrow produce various hematopoietic factors [Harigaya et al.: proc. Natl. 
Acad. Sci. USA 82, 3477 (1985); Kohama et al.: Experimental Hematology 16, 
603-608 (1988)]. 
Therefore, if a substance which stimulates the production of hematopoietic 
factors by stromal cells can be found, this substance may not only play a 
very important role in analyzing the action of the hematopoietic mechanism 
in physiological conditions and in the pathology of hematologic diseases 
but it may also find considerable clinical use. 
European Patent Publication No. 329 361 discloses certain new 2-pyranone 
derivatives which resemble the compounds of the present invention except 
that they differ in the nature of the group "R", defined hereafter. Those 
prior art compounds are also only said to be agricultural biocides and are 
not shown in the published art to have the valuable and unexpected 
therapeutic and prophylactic activities of the compounds of the present 
invention. Although the prior art compounds are produced, like the 
compounds of the present invention, by a microorganism of the species 
Streptomyces platensis, the strain described in the prior document is 
believed to be clearly different from that described herein. 
Very similar compounds and microorganisms, having essentially the same 
disclosed utility, are described in Japanese Patent Application Kokai Hei 
2-186, and these are likewise thought to be clearly distinct from the 
compounds and microorganism disclosed herein. 
The Journal of Antibiotics, Vol. XLII, No. 9, page 1331 discloses a novel 
antitumor compound, which the authors call "Phospholine", and which is 
produced by a microorganism of the genus Streptomyces, which was then 
newly isolated. However, the microorganism is clearly said to be 
Streptomyces hygroscopicus and is distinguished from the Streptomyces 
platensis, which is used in the present invention. Moreover, although the 
prior art phospholine, like the compounds of the present invention, has 
both an amino group and a phosphoric group, it has a different molecular 
formula is thus clearly distinguished. 
BRIEF SUMMARY OF INVENTION 
It is, therefore, an object of the present invention to provide a series of 
new phosphoric acid compounds having a variety of pharmacological 
activities. 
In particular, it is believed that the compounds of the present invention 
have the following activities: they reduce adverse reactions resulting 
from cancer chemotherapy or radiotherapy; they protect against infections; 
they activate macrophages and thus have an anticancer effect; they improve 
cerebral function; and, in addition, they act as antifungal agents. 
Thus, the present invention provides compounds of formula (I): 
##STR2## 
in which R represents a 5-methylhexanoyloxy group, a 6-methyloctanoyloxy 
group or a 7-methyloctanoyloxy group, and salts thereof. These compounds 
have been named by us "Leustroducsin A", "Leustroducsin B" and 
"Leustroducsin C", respectively. 
The invention also provides a process for preparing the leustroducsins, 
which comprises cultivating a leustroducsin-producing microorganism of the 
genus Streptomyces and collecting at least one leustroducsin from the 
culture. 
The invention also provides a pharmaceutical composition comprising at 
least one leustroducsin or a salt thereof in admixture with a 
pharmaceutically acceptable carrier or diluent. 
The invention still further provides a method for the treatment or 
prophylaxis of: adverse reactions resulting from cancer chemotherapy or 
radiotherapy; infections; cancer; cerebral dysfunction; and fungal 
infections, which method comprises administering an effective amount of at 
least one leustroducsin or a salt thereof to a meal, which may be human, 
suffering from or susceptible to such reactions, infections, cancer or 
dysfunction. 
DETAILED DESCRIPTION OF INVENTION 
The three leustroducsins of the present invention are as follows: 
##STR3## 
It is clear from the above formulae that the leustroducsins of the present 
invention contain a number of asymmetric carbon atoms and several double 
bonds. They can therefore form various optical and geometric isomers. 
Although these are all represented herein by a single molecular formula, 
the present invention includes both the individual, isolated isomers and 
mixtures, including racemates thereof. Where stereospecific synthesis 
techniques are employed or optically active compounds are employed as 
starting materials, individual isomers may be prepared directly; on the 
other hand, if a mixture of isomers is prepared, the individual isomers 
may be obtained by conventional resolution techniques. 
The leustroducsins of the present invention may be prepared by culturing a 
leustroducsin-producing microorganism of the genus Streptomyces, and then 
collecting one or more of the leustroducsins from the culture medium. 
In particular, we especially prefer to employ as the microorganism a newly 
isolated strain of the genus Streptomyces, which we have established 
belongs to the species Streptomyces platensis and to which we have 
assigned the designation SANK 60191 (FERM BP-3288). 
This microorganism was deposited under the terms of the Budapest Treaty at 
the Fermentation Research Institute, Agency of Industrial Science and 
Technology, on 20th February 1991 with the accession no. FERM BP-3288. 
Details of the microbiological properties of Streptomyces platensis SANK 
60191 (FERM BP-3288) are shown below. 
1. Morphological characteristics 
Streptomyces platensis SANK 60191 was cultured for 14 days at 28.degree. C. 
on each of the agar media defined by the ISP (International Streptomyces 
Project). On microscopic observation after culture for 14 days, it was 
found that the substrate mycelium of this strain elongated and branched 
well and was colored yellowish gray or pale yellowish orange. However, 
neither the fragmentation nor the zig-zag elongation observable in 
Nocardioform actinomycetes were observed. Branching of the aerial mycelium 
was simple. The spore chains were a loose spiral in shape, and 10 to 50 or 
more spores formed a chain. Observation with a scanning electromicroscope 
showed the surface structure of the spores to be smooth. Spores were ovoid 
or oval, and 0.5-0.6.times.0.6-1.3 .mu.m in size. Whirl of aerial 
mycelium, sclerotium, fragmentation of hyphae and specific organs, such as 
sporangia, could not be found. 
2. Properties on various kinds of culture media 
Table 1 shows the properties of the microorganism after culture for 14 days 
at 28.degree. C. on various kinds of culture media. The colors are 
indicated by the color tip number given in the "Guide to Color Standard" 
edited by Nippon Shikisai Kenkyusho. This strain moistens and its color 
changes to black with the passage of time. 
In the Table, the following abbreviations are used: 
G: Growth; AM: Aerial mycelium; R: Reverse; 
SP: Soluble pigment; SC: Specific character 
TABLE 1 
______________________________________ 
Medium Item Property of SANK 60191 
______________________________________ 
Sucrose- G: Good, smooth, pale yellowish orange 
nitrate agar (2-9-9) 
AM: Good, velvety, light brownish gray 
(2-7-8) 
R: Pale brown (2-8-9) 
SP: No formation 
Glucose- G: Good, smooth, pale yellowish orange 
asparagine agar (2-9-9) 
AM: Moderate, velvety, white 
R: Pale brown (2-8-9) 
SP: No formation 
Glycerol- G: Very good, smooth, pale yellowish orange 
asparagine agar (2-9-9) 
(ISP 5) AM: Moderate, velvety, brownish gray 
(2-6-8) 
R: Pale yellowish light brown (4-8-9) 
SP: No formation 
Inorganic G: Good, smooth, pale yellowish orange 
salts- (2-9-9) 
starch agar 
AM: Good, velvety, brownish white (1-6-6) 
(ISP 4) R: Brownish gray (2-5-9) 
SP: No formation 
SC: Aereal hyphae moisten and their color 
changes to black 
Tyrosine G: Very good, smooth, pale yellowish orange 
agar (ISP 7) (2-9-9) 
AM: Good, velvety, white, light brownish gray 
(2-7-8) spots 
R: Pale yellowish brown (4-8-9) 
SP: No formation 
Nutrient G: Good, smooth, pale yellowish orange 
agar (DIFCO) (2-9-9) 
AM: Moderate, velvety, white 
R: Pale yellowish brown (4-8-9) 
SP: No formation 
Yeast extract- 
G: Very good, smooth, pale yellowish orange 
malt extract (2-9-9) 
agar (ISP 2) 
AM: Abundant formation, velvety, light 
brownish white (1-7-6) 
R: Pale yellowish brown (6-7-9) 
SP: No formation 
SC: Aerial hyphae moisten and their color 
changes to black 
Oatmeal G: Good, smooth, pale yellowish orange 
agar (ISP 3) (2-9-9) 
AM: Good, velvety, dark brownish gray 
(1-4-6) 
R: Yellowish brown (4-6-9) 
SP: No formation 
Water agar 
G: Moderate, smooth, yellowish gray 
(1-9-10) 
AM: Moderate, velvety, brownish gray 
(2-5-8) 
R: Light brownish gray (2-7-8) 
SP: No formation 
Potato extract- 
G: Moderate, smooth, yellowish gray 
carrot extract (1-9-10) 
agar AM: Moderate, velvety, brownish gray (2-5-8) 
R: Light brownish gray (2-7-8) 
SP: No formation 
______________________________________ 
3. Physiological properties 
The physiological properties of strain SANK 60191 observed over the period 
of from day 2 to day 21 after the beginning of cultivation at 28.degree. 
C. are shown in Table 2. 
TABLE 2 
______________________________________ 
Hydrolysis of starch Positive 
Liquefaction of gelatin Negative 
Reduction of nitrates Negative 
Coagulation of milk Negative 
Peptonization of milk Negative 
Production of melanoid pigment 
(Medium 1)* Negative 
(Medium 2) Negative 
(Medium 3) Negative 
Decomposition of substrate 
Casein Negative 
Tyrosine Positive 
Xanthine Positive 
Temperature range for growth (Medium 4) 
9-35.degree. C. 
Optimum temperature for growth (Medium 4) 
20-26.degree. C. 
Sodium chloride tolerance 10% 
______________________________________ 
*Medium 1; Tryptoneyeast extract broth (ISP 1) 
2; Peptoneyeast extract iron agar (ISP 6) 
3; Tyrosine agar (ISP 7) 
4; Yeast extractmalt extract agar (ISP 2) 
Strain SANK 60191 was also cultivated at 28.degree. C. using 
Pridham-Gottlieb agar (ISP 9) as the cultivation medium. The utilization 
pattern of carbon sources observed after cultivation for 14 days is shown 
in Table 3. 
TABLE 3 
______________________________________ 
.sub.--D-Glucose 
+ .sub.--D-Fructose 
+ 
.sub.--L-Arabinose 
- .sub.--L-Rhamnose 
- 
.sub.--D-Xylose 
- Sucrose + 
Inositol + Raffinose + 
.sub.--D-Mannitol 
+ Control - 
______________________________________ 
+ Utilization positive; 
- Utilization negative. 
4. Cellular components 
The cell walls of strain SANK 60191 were analyzed by the method of B. 
Becker et al. [Applied Microbiology, 12, 421-423 (1984)]. Since 
LL-diaminopimelic acid was detected, the cell walls was confirmed to be of 
Type I. Furthermore, the whole cell sugar components of strain SANK 60191 
were analyzed by the method of M. P. Lechevalier [Journal of Laboratory & 
Clinical Medicine, 71, 934 (1968)]. No particular pattern was observed. 
From these microbiological properties, this strain was confirmed to belong 
to the genus Streptomyces of the Actinomycetes. In comparison with the 
strains described in the ISP by E. B. Shirling and D. Gottlieb 
[International Journal of Systematic Bacteriology 18, 68-189 (1968); 18, 
279-392 (1968); 19, 391-512 (1969); 22, 265-394 (1972)], and the strains 
described in other literature, such as "The Actinomycetes, Vol. 2" by S. 
A. Waksman, "Bergey's Manual of Determinative Bacteriology, 8th Edition 
(1974)" edited by R. E. Buchanan and N. E. Gibbons, "Bergey's Manual of 
Systematic Bacteriology, Vol. 4 (1989)", and in other recent literature 
about the Actinomycetes, this strain was considered to be very close to 
Streptomyces plantensis. 
Furthermore, after liquid culture using a yeast-dextrose medium, strain 
SANK 60191 produced a soluble pigment having a fresh reddish brown color. 
On the addition of 0.05N aqueous hydrochloric acid, its color turned 
yellow, and, on the addition of 0.05N aqueous sodium hydroxide, no change 
was observed. 
Streptomyces platensis produces a reddish or yellowish pigment after 
culture on yeast extract--malt extract agar, oatmeal agar, inorganic 
salts--starch agar, and glycerol--asparagine agar media. On the other 
hand, strain SANK 60191 hardly produced any of these pigments, which 
indicates a difference between this strain and the known strains of 
Streptomyces platensis. Since, however, the new strain and the known 
strains could only be distinguished by differences in the production of 
soluble pigments, SANK 60191 was identified as a new strain of 
Streptomyces platensis. 
It has been established that strain SANK 60191 produces the leustroducsins. 
However, as is well known, the properties of fungi in general, and 
actinomycetous microorganisms in particular, can vary considerably and 
such fungi can readily undergo mutation, both through natural causes and 
as the result of induction by artificial means (for example, ultraviolet 
irradiation, radioactive irradiation, chemical treatment, etc.). 
Accordingly, the present invention embraces the use any microorganism 
which can be classified within the genus Streptomyces and which shares 
with strain SANK 60191 the characteristic ability to produce the 
leustroducsins. The new microorganism, strain SANK 60191, is not expected 
to be exceptional, and the term "SANK 60191" thus is used to include all 
mutants of this strain which share with strain SANK 60191 the 
characteristic ability to produce the leustroducsins. Moreover, these 
mutants include those obtained by means of genetic engineering techniques, 
for example, recombination, transduction, transformation or the like. It 
is a matter of simple experimentation to determine, on the basis of the 
information given herein regarding the properties of the leustroducsins, 
whether any given strain produces these compounds or produces them in 
sufficient quantity to render that strain of potential commercial 
interest. 
In addition to the new strain of Streptomyces platensis described above, we 
have also found that a known strain, namely Streptomyces platensis 
SAM-0654 (deposited at the Fermentation Research Institute, Japan under 
the accession number FERM BP-1668 on 22nd Jan. 1988) also produces the 
leustroducsins of the present invention. This known strain is fully 
described in European Patent No. 329 361, the disclosure of which is 
incorporated herein by reference. 
The leustroducsins of the present invention may be prepared by the culture 
of these strains of fungus in culture media of the type conventionally 
used for the production of other fermentation products from similar 
microorganisms. Such media necessarily contain microbiologically 
assimilable sources of carbon and of nitrogen as well as inorganic salts, 
as is well known to those skilled in the art. 
Preferred examples of carbon sources include: glucose, fructose, maltose, 
sucrose, mannitol, glycerol, dextrin, oats, rye, corn starch, potato 
starch, corn flour, soybean meal, cottonseed cake, cottonseed oil, 
molasses, citric acid, tartaric acid and the like. Such compounds can be 
used alone or in any suitable combination. In general the amount used may 
vary in the range of from 1 to 10% by weight of the culture medium. 
Preferred nitrogen sources are normally protein-containing materials such 
as are commonly used in a fermentation process. Examples of such nitrogen 
sources include: soybean meal, wheat bran, peanut meal, cottonseed cake, 
cottonseed oil, cottonseed meal, casein hydrolyzates, fermamine, fish 
meal, corn steep liquor, peptone, meat extract, yeast, yeast extract, malt 
extract, sodium nitrate, ammonium nitrate, ammonium sulfate and the like. 
These nitrogen sources may be used alone or in any suitable combination. 
In general, we prefer to employ them at a concentration between 0.2 and 6% 
by weight of the culture medium. 
The nutritive inorganic salts that my be incorporated into the culture 
medium are conventional salts that are capable of providing various ions 
necessary to the growth of microorganisms, such as sodium, ammonium, 
calcium, phosphate, sulfate, chloride and carbonate ions. In addition, the 
medium should contain minor amounts of essential trace elements, such as 
potassium, calcium, cobalt, manganese, iron and magnesium. 
When the process of the present invention is carried out by a liquid 
culture technique, an antifoaming agent, such as a silicone oil, vegetable 
oil or surface-active agent, is preferably used in the culture medium. The 
pH of the culture medium for producing the leustroducsins by the 
cultivation of microorganisms of the genus Streptomyces, especially strain 
SANK 60191, preferably varies within the range of from 5.0 to 8.0, more 
preferably from 5.0 to 7.0. 
The cultivation may be carried out at any temperature within the range of 
from 9.degree. C. to 37.degree. C. However, growth proceeds well at a 
temperature within the range of from 20.degree. to 35.degree. C. and such 
a temperature is preferred. A temperature of from 22.degree. to 30.degree. 
C. is preferred in order to optimise the production of the leustroducsins. 
These compounds are produced under aerobic culture conditions and 
conventional aerobic culture methods, such as solid culture, shaking 
culture and aeration-stirring (submerged) culture methods, my be used. In 
the case of small scale cultivation, shaking culture for a few days at 
28.degree. C. is typically employed. In such a small scale culture method, 
the culture may be initiated with 1 or 2 proliferation steps, producing 
seed cultures in, for example, Erlenmeyer flasks, fitted with baffle 
plates, which serve as a liquid flow regulator. The medium for the seed 
culture steps preferably contains both carbon and nitrogen sources. In the 
preferred sequence of operations for such small scale cultivation, the 
seed culture flasks are shaken in a constant temperature incubator at 
28.degree. C. for 3 days or until sufficient growth is achieved. The grown 
seed culture is then transferred to a second seed medium or to the 
production medium. When an intermediate growth phase is used, essentially 
the same method is used for growth and an aliquot of the resulting 
intermediate product is inoculated into the production medium. The 
inoculated flask may be incubated for several days whilst shaking, and, 
after completion of the incubation, the contents of the flask may be 
centrifuged or filtered. 
In the case of large scale production, the use of an appropriate fermentor 
equipped with a stirrer and an aeration apparatus is preferred. In this 
case, the nutritive medium can be prepared inside the fermentor. The 
medium is preferably sterilized by elevating the temperature to a suitable 
temperature, for example from 120.degree. C. to 125.degree. C.; after 
cooling, the sterilized medium may be inoculated with the previously 
prepared seed culture. The culture then proceeds under stirring and 
aeration at a suitable temperature, for example 28.degree. C. This method 
is suitable for obtaining the compounds of the present invention in a 
large amount. 
The change in the amount of leustroducsins produced with the passage of the 
culture time can be monitored by any suitable means, such as are well 
known in the fermentation art, for example high performance liquid 
chromatography. Most commonly, the amount of leustroducsins produced 
reaches a maximum after culturing for a period of from 48 hours to 96 
hours. 
After completion of the culture, the leustroducsins remaining in the liquid 
part of the medium liquid and in the bacterial cells can be extracted and 
purified by conventional means, making use of the physicohemical 
properties of the leustroducsins. For example, the leustroducsins present 
in the filtrate or in the supernatant can be extracted with a 
water-immiscible organic solvent, such as ethyl acetate, chloroform, 
ethylene chloride, methylene chloride or butanol (or with a mixture of any 
two or more of these solvents) under acidic conditions; after this it may 
be purified by conventional means. It is also possible to remove any 
impurities by extraction with one or more of the aforementioned solvents 
under basic conditions, after which it may be purified by conventional 
means. Alternatively, impurities can be removed by adsorption by passing a 
liquid containing the leustroducsins through a layer of a suitable 
adsorbent, or the leustroducsins can be adsorbed on a suitable adsorbent 
and then eluted using an appropriate eluent, such as aqueous methanol, 
aqueous acetone or aqueous butanol. Examples of suitable adsorbent s which 
may be used in these procedures include active carbon and adsorbent 
resins, such as Amberlite XAD-4 (a trade name for a product of Rohm & 
Haas) or Diaion HP-10, HP-20, CHP-20, HP-50 (trade names for products of 
Mitsubishi Chemical Industries Co.). Leustroducsins which are present in 
the cells can be obtained by extraction with a suitable solvent, such as 
50-90% by volume aqueous acetone or aqueous methanol, followed by removal 
of the organic solvent; after this, the product can be subjected to 
similar extraction and purification procedures to those described above 
for the filtrate. 
The leustroducsins thus obtained can be further purified by well known 
techniques, for example: adsorption column chromatography using a carrier, 
such as silica gel or magnesium-silica gel, for example that sold under 
the trade name "Florisil"; partition column chromatography using an 
adsorbent such as Sephadex LH-20 (a trade name for a product of 
Pharmacia); or high performance liquid chromatography using a normal phase 
or reverse phase column. As is well known in the art, these isolation and 
purification procedures may be carried out alone or in any suitable 
combination, and, if desired, repeatedly, to isolate and purify the 
desired leustroducsins. 
The leustroducsins of the present invention are novel compounds not 
previously reported in the literature. They stimulate the production of 
hematopoietic factors, such as G-CSF and GM-CSF, in animals (such as 
humans, dogs, cats and rabbits), and are, therefore, useful as therapeutic 
agents for reducing the side effects caused by cancer chemotherapy and 
radiotherapy; they also prevent infections and exhibit an anticancer 
effect through the activation of macrophages. In addition, the 
leustroducsins are expected to be useful for improving cerebral metabolism 
by stimulating the production of NGF. Furthermore, they are useful as 
antifungal agents and have demonstrated an antifungal effect against 
Tricophyton metagrophytes. 
The ability of the leustroducsins to stimulate the production of 
hematopoietic factors in accordance with the present invention can, in 
principle, be assayed by the method by Kohama et al. [Experimental 
Hematology 16, 603-608 (1988)]. In this method, a production system for 
various hematopoietic factors and an assay system for various 
hematopoietic factors are combined. KM-102 cells, for example, which 
originated from human bone marrow stromal cells, my be employed to produce 
various hematopoietic factors. However, any cells capable of the 
production of various hematopoietic factors may be used instead, for 
example primary cultured bone marrow stromal cells, vascular endothelial 
cells, lymphocytes, or macrophages which exist in the bone marrow. A test 
sample of the compound whose activity is to be assessed is diluted to a 
suitable concentration and added the culture system containing the cells 
which produce various hematopoietic factors ("HF-producing cells"). After 
a suitable period, usually 24 hours, a part of the supernatant of the 
culture medium is taken, and added to a culture system of cells which are 
dependent on various hematopoietic factors, for example, TF-1 cells and 
NFS-60 cells, ("HF-dependent cells") at a suitable concentration. After a 
certain time, the amount of hematopoietic factors can be measured by 
measuring the growth of the HF-dependent cells, and thus the ability of 
the compound to stimulate the production of various hematopoietic factors 
can be assayed. The growth of the HF-dependent cells may be determined by 
any conventional method, such as the incorporation of tritium-thymidine by 
the cells or using an MTT 
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay 
method (Chemicon International Inc., USA). The assay of various 
hematopoietic factors may be carried out by any conventional method, such 
as the colony-forming method or the ELISA method. 
The leustroducsins of the present invention contain both an acidic group 
(the phosphoric acid group) and a basic group (the amino group) and can 
thus form salts. There is no particular restriction on the nature of these 
salts, provided that, where they are intended for therapeutic use, they 
are pharmaceutically acceptable. Where they are intended for 
non-therapeutic uses, e.g. as intermediates in the preparation of other, 
and possibly more active, compounds, even this restriction does not apply. 
The compounds of the present invention can form salts with bases. Examples 
of such salts include: salts with an alkali metal, such as sodium, 
potassium or lithium; salts with an alkaline earth metal, such as barium 
or calcium; salts with another metal, such as magnesium or aluminum; 
organic base salts, such as a salt with dicyclohexylamine; and salts with 
a basic amino acid, such as lysine or arginine. The compounds of the 
present invention can also form acid addition salts. Examples of such acid 
addition salts include: salts with mineral acids, especially hydrohalic 
acids (such as hydrofluoric acid, hydrobromic acid, hydroiodic acid or 
hydrochloric acid), nitric acid, perchloric acid, carbonic acid, sulfuric 
acid or phosphoric acid; salts with lower alkylsulfonic acids, such as 
methanesulfonic acid, trifluoromethanesulfonic acid or ethanesulfonic 
acid; salts with arylsulfonic acids, such as benzenesulfonic acid or 
p-toluenesulfonic acid; salts with organic carboxylic acids, such as 
acetic acid, fumaric acid, tartaric acid, oxalic acid, maleic acid, malic 
acid, succinic acid or citric acid; and salts with amino acids, such as 
glutamic acid or aspartic acid. 
When these compounds are intended for therapeutic use, they may be 
administered alone or in a suitable pharmaceutical formulation containing, 
in addition to the active compound, one or more conventional diluents, 
carriers, excipients or adjuvants. The nature of the formulation will, of 
course, depend on the intended route of administration. However, for the 
oral route, the compound is preferably formulated as powders, granules, 
tablets, capsules or syrups. For parenteral administration, it is 
preferably formulated as an injection (which may be intravenous, 
intramuscular or subcutaneous) or as drops or suppositories. These 
preparations can be prepared by known means by adding such additives as 
vehicles, binders, disintegrators, lubricants, stabilizers, corrigents, 
solubilizing agents, suspending agents or coating agents. Although the 
dosage may vary depending upon the symptoms and age of the patient, the 
nature and severity of the disease or disorder and the route and manner of 
administration, in the case of oral administration to an adult human 
patient, the compounds of the present invention may normally be 
administered at a daily dose of from 1 mg to 1000 mg. The compounds may be 
administered in a single dose, or in divided doses, for example two or 
three times a day. 
The preparation of the compounds of the present invention is further 
illustrated by the following non-limiting Examples.

EXAMPLE 1 
Culture and isolation of leustroducsins 
1 (A) Culture 
One platinum loopful of spores of Streptomyces platensis SANK 60191 was 
inoculated into a 500 ml Erlenmeyer flask fitted with baffle plates and 
containing 100 ml of a previously sterilized culture medium (having the 
composition described below), and the microorganism was cultured for 3 
days at 28.degree. C. and at 200 rpm (a rotation radius of 7 cm), using a 
rotary shaker. 
______________________________________ 
Culture Medium: 
______________________________________ 
Soluble starch 30 g 
Raw yeast 10 g 
Soy bean powder 7 g 
Fish meal 5 g 
Corn steep liquor 2 g 
Meat extract 1 g 
Calcium carbonate 1 g 
Water to 1000 ml 
pH 7 (before sterilization). 
______________________________________ 
15 liters of the same culture medium as was used for the seed culture was 
charged into each of four 30 liter stainless jal fermenters, and was 
sterilized by heating it at 120.degree. C. for 30 minutes. 150 ml of the 
seed culture liquid prepared as described above were then added. The 
mixture was cultivated for B days at 28.degree. C. with aeration at an air 
flow rate of 1S liters/minute, whilst stirring. In order for the oxygen 
concentration in the liquid to be maintained at 5 ppm, the stirring rate 
was automatically controlled within the range from 100 to 300 rpm. 
1 (B) Isolation 
2.4 kg of Celite 545 (a trade name for a product of Johns & Manville 
Project Corporation, USA) were added as a filter aid to 60 liters of the 
culture liquid obtained as described in 1 (A) above, and the mixture was 
filtered. After filtration of the culture liquid, 7.2 kg of bacterial 
cells were obtained. The cells were extracted once with 30 liters of 50% 
v/v aqueous acetone, and twice with 20 liters of 80% v/v aqueous acetone 
each time. These extracts were combined and the organic solvent was 
distilled off using a rotary evaporator. Sufficient aqueous hydrochloric 
acid was added to the residue to adjust its pH to a value of 2.0, and then 
the mixture was extracted twice, each time with 10 liters of ethyl 
acetate. The extracts were combined, and 10 liters of a 1% w/v aqueous 
solution of sodium hydrogencarbonate were added to the combined extracts. 
The active fractions were transferred into the aqueous layer and the ethyl 
acetate layer was removed. This ethyl acetate layer was again extracted 
with 5 liters of a 1% w/v aqueous solution of sodium hydrogencarbonate. 
The sodium hydrogencarbonate solutions were combined and the pH of the 
combined solution was adjusted to a value of 2.0 by the addition of 
aqueous hydrochloric acid. The solution was extracted twice, each time 
with 10 liters of ethyl acetate. The organic extracts were combined, 
washed with water and then with a saturated aqueous solution of sodium 
chloride, and then dried over anhydrous sodium sulfate. During continuous 
addition of methanol, the solution was then condensed by evaporation under 
reduced pressure, using a rotary evaporator, to obtain 10 ml of an oily 
substance. This oily substance was dissolved in 100 ml of 60% v/v aqueous 
methanol, and the resulting solution was adsorbed on Sep-Pak Vac 20 cc 
C.sub.18 Cartridges (a trade name for a product of Waters Co., USA). 
Impurities were eluted with 30 ml of 60% v/v aqueous methanol. The 
leustroducsins were then eluted with 15 ml of 100% methanol, and the 
eluate was condensed to obtain 800 mg of an oily substance. This oily 
substance was dissolved in 10 ml of methanol, and subjected to high 
performance liquid chromatography. The fractions showing peaks near 13 
minutes and 24 minutes were collected and are referred to as "Raw Fraction 
A" and "Raw Fraction B", respectively. The conditions used for the 
chromatography are shown below. 
Preparative liquid chromatography 
Column: Radial-Pak 25.times.10 (Waters, USA) 
Eluting solvent: 50% by volume aqueous acetonitrile, containing 0.5% 
triethylamine - phosphate buffer, pH 3.0 
Flow rate: 9 ml/min. 
Wave length: 230 nm 
After condensation of all of these peaks, the resulting fractions were 
subjected to preparative high performance liquid chromatography. Raw 
Fraction A was subjected to preparative chromatography to collect the peak 
near 56 minutes under the following conditions; it was then desalted and 
condensed using Sep-Pak to obtain 11.66 mg of leustroducsin A. 
Preparative conditions for Raw Fraction A 
Column: Cosmosil 5C 18-AR 20.times.250 mm (Nakaraitesque Inc.) 
Eluting solvent: 42% v/v aqueous acetonitrile, containing 0.5% 
triethylamine - phosphate buffer, pH 3.0 
Flow rate: 9 ml/min. 
Wave length: 230 nm. 
Raw Fraction B was subjected to preparative chromatography to collect the 
peaks near 47 and 51 minutes under the following conditions; it was then 
desalted and condensed using Sep-Pak to obtain 9.83 mg of leustroducsin B 
and 5.22 mg of leustroducsin C. 
Preparative conditions for Raw Fraction B 
Column: Cosmosil 5C 18-AR 20.times.250 mm (Nakaraitesque Inc.) 
Eluting solvent: 47% v/v aqueous acetonitrile, containing 0.5% 
triethylarnine - phosphate buffer, pH 3.0 
Flow rate: 9 ml/min. 
Wave length: 230 nm. 
The leustroducsins thus obtained had the following properties: 
Leustroducsin A 
1) Chemical structure: formula (Ia), shown above. 
2) Nature: Acidic and fat soluble. 
3) Color: Pale yellow oil. 
4) Molecular formula: C.sub.32 H.sub.52 O.sub.10 NP. 
5) Molecular weight: 641, determined by the FAB-MS method ("FAB-MS" is Fast 
Atom Bombardment Mass Spectrometry). 
6) Ultraviolet absorption spectrum: 234 rim (maximum absorption in 
methanol). 
7) Infrared absorption spectrum: the infrared spectrum showed the following 
absorption maxima (liquid film, .nu..sub.max cm.sup.-1): 2933, 2867, 1728, 
1464, 1383, 1248, 1176, 1056, 969. 
8) .sup.1 H-Nuclear magnetic resonance spectrum: the Nuclear magnetic 
resonance spectrum (270 MHz) in heavy methanol, using trimethylsilane as 
the internal standard, is shown below: 
7.08 (1 H, doublet of doublets, J=9.8 & 4.9 Hz); 
6.21-6.35 (2 H, multiplet); 
6.06 (1 H, doublet of doublets, J=15.6 & 6.1 Hz); 
6.02 (1 H, doublet of doublets, J=9.8 & 1.4 Hz); 
5.94 (1 H, doublet, J=15.6 Hz); 
5.46 (1 H, multiplet); 
5.31 (1 H, multiplet); 
5.10 (1 H, doublet of doublets, J=6.1 & 4.4 Hz); 
4.94 (1 H, multiplet); 
4.72 (1 H, multiplet); 
4.29 (triplet of doublets, J=10.1, 10.1 & 2.4 Hz); 
2.93-3.15 (2 H, multiplet); 
2.50-2.71 (2 H, multiplet); 
2.25 (2 H, triplet, J=7.6 Hz); 
2.16 (1 H, multiplet); 
0.99-2.01 (18 H, multiplet); 
0.95 (3 H, triplet, J=7.6 Hz); 
0.89 (6 H, doublet, J=6.8 Hz). 
9) .sup.13 C-Nuclear magnetic resonance spectrum: (.delta.: ppm): the 
Nuclear magnetic resonance spectrum (270 MHz) in heavy methanol, using 
trimethylsilane as the internal standard, is shown below: 
11.4 (quartet), 22.7 (triplet), 22.9 (quartet), 
22.9 (quartet), 24.0 (triplet), 24.7 (triplet), 
28.9 (doublet), 32.4 (triplet), 33.1 (triplet), 
34.3 (triplet), 35.7 (triplet), 36.1 (doublet), 
37.1 (triplet), 39.4 (triplet), 39.5 (triplet), 
40.6 (doublet), 40.6 (triplet), 64.7 (doublet), 
73.9 (doublet), 77.8 (singlet), 78.5 (doublet), 
82.3 (doublet), 121.1 (doublet), 123.7 (doublet), 
124.3 (doublet), 127.7 (doublet), 135.3 (doublet), 137.3 (doublet), 138.2 
(doublet), 
152.7 (doublet), 166.3 (singlet), 175.0 (singlet). 
10) Solubility: 
Soluble in alcohols, such as methanol, ethanol or butanol; and soluble in 
acetone, chloroform, ethyl acetate and dimethyl sulfoxide; limited 
solubility in water; insoluble in hexane. 
ii) Color reactions 
Positive to sulfuric acid, iodine, ninhydrin and ammonium 
molybdate-perchloric acid reactions. 
12) High performance liquid chromatography: 
Separating column: Cosmosil 5C18-AR 
(Column size, 4.6.times.250 mm, Product of Nakaraitesque Inc.) 
Solvent: 45% v/v aqueous acetonitrile, containing 0.5% triethylarnine - 
phosphate buffer (pH 3) 
Flow rate: 1 ml/min. 
Wave length: 230 nm 
Retention time: 9.06 min. and 9.16 min. 
Leustroducsin B 
1) Chemical structure: formula (Ib), shown above. 
2) Nature: Acidic and fat soluble. 
3) Color: Pale yellow oil. 
4) Molecular formula: C.sub.34 H.sub.56 O.sub.10 NP. 
5) Molecular weight: 669, determined by the FAB-MS method. 
6) Ultraviolet absorption spectrum: 234 nm (maximum absorption in 
methanol). 
7) Infrared absorption spectrum: the infrared spectrum showed the following 
absorption maxima (liquid film, .nu..sub.max cm.sup.-1): 2927, 2855, 1729, 
1463, 1380, 1250, 1172, 1056, 968. 
8) .sup.1 H-Nuclear magnetic resonance spectrum: the Nuclear magnetic 
resonance spectrum (270 MHz) in heavy methanol, using trimethylsilane as 
the internal standard, is shown below: 
7.09 (1 H, doublet of doublets, J=9.8 & 4.9 Hz); 
6.21-6.35 (2 H, multiplet); 
6.07 (1 H, doublet of doublets, J=15.6 6.1 Hz); 
6.02 (1 H, doublet of doublets, J=9.8 1.5 Hz); 
5.94 (1 H, doublet, J=15.6 Hz); 
5.46 (1 H, multiplet); 
5.31 (1 H, multiplet); 
5.10 (1 H, doublet of doublets, J=6.1 4.4 Hz); 
4.94 (1 H, multiplet); 
4.72 (1 H, multiplet); 
4.29 (1 H, triplet of doublets, J=9.9, 9.9 & 2.6 Hz); 
2.93-3.15 (2 H, multiplet); 
2.50-2.71 (2 H, multiplet); 
2.27 (2 H, triplet, J=7.3 Hz); 
2.17 (1 H, multiplet); 
1.00-2.01 (22 H, multiplet); 
0.95 (3 H, triplet, J=7.5 Hz); 
0.87 (3 H, triplet, J=6.8 Hz); 
0.86 (3 H, doublet, J=6.6 Hz). 
9) .sup.13 C-Nuclear magnetic resonance spectrum: (.delta.: ppm): the 
Nuclear magnetic resonance spectrum (270 MHz) in heavy methanol, using 
trimethylsilane as the internal standard, is shown below: 
11.4 (quartet), 11.7 (quartet), 19.6 (quartet), 
22.7 (triplet), 24.7 (triplet), 26.4 (triplet), 
27.6 (triplet), 30.6 (triplet), 32.4 (triplet), 
33.1 (triplet), 34.1 (triplet), 35.5 (triplet), 
35.5 (doublet), 36.1 (doublet), 37.1 (triplet), 
37.3 (triplet), 39.4 (triplet), 40.6 (doublet), 
40.6 (triplet), 64.7 (doublet), 73.9 (doublet), 
77.8 (singlet), 78.5 (doublet), 82.3 (doublet), 
121.0 (doublet), 123.7 (doublet), 124.3 (doublet), 127.7 (doublet), 135.2 
(doublet), 
137.4 (doublet), 138.2 (doublet), 152.7 (doublet), 166.4 (singlet), 175.1 
(singlet). 
10) Solubility: 
Soluble in alcohols, such as methanol, ethanol or butanol; and soluble in 
acetone, chloroform, ethyl acetate and dimethyl sulfoxide; limited 
solubility in water; insoluble in hexane. 
Color reactions: 
Positive to sulfuric acid, iodine, ninhydrin and ammonium 
molybdate-perchloric acid reactions. 
12) High performance liquid chromatography: 
Separating column: Cosmosil 5C18-AR (Column size, 4.6.times.250 mm, Product 
of Nakaraitesque Inc.) 
Solvent: 45% v/v aqueous acetonitrile, containing 0.5% triethylamine - 
phosphate buffer (pH 3) 
Flow rate: 1 ml/min. 
Wave length: 230 nm 
Retention time: 20.62 min. and 20.87 min. 
Leustroducsin C 
1) Chemical structure: formula (Ic), shown above. 
2) Nature: Acidic and fat soluble. 
3) Color: Pale yellow oil. 
4) Molecular formula: C34H.sub.56 O.sub.10 NP. 
5) Molecular weight: 669, determined by the FAB-MS method. 
6) Ultraviolet absorption spectrum: 234 nm (maximum absorption in 
methanol). 
7) Infrared absorption spectrum: the infrared spectrum showed the following 
absorption maxima (liquid film, .nu..sub.max cm.sup.-1): 2930, 2856, 1728, 
1464, 1382, 1252, 1192, 1056, 968. 
8) .sup.1 H-Nuclear magnetic resonance spectrum: the Nuclear magnetic 
resonance spectrum (270 MHz) in heavy methanol, using trimethylsilane as 
the internal standard, is shown below: 
7.08 (1 H, doublet of doublets, J=9.8 & 4.9 Hz); 
6.21-6.35 (2 H, multiplet); 
6.07 (1 H, doublet of doublets, J=15.6 & 6.1 Hz); 
6.02 (1 H, doublet of doublets, J=9.8 & 1.5 Hz); 
5.94 (1 H, doublet, J=15.6 Hz); 
5.46 (1 H, multiplet); 
5.31 (1 H, multiplet); 
5.10 (1 H, doublet of doublets, J=6.1 & 4.9 Hz); 
4.94 (1 H, multiplet); 
4.72 (1 H, multiplet); 
4.28 (1 H, triplet of doublets, J=10.1, 10.1 & 2.4 Hz); 
2.93-3.15 (2 H, multiplet); 
2.50-2.71 (2 H, multiplet); 
2.27 (2 H, triplet, J=7.3 Hz); 
2.16 (1 H, multiplet); 
1.00-2.01 (22 H, multiplet); 
0.95 (3 H, triplet, J=7.3 Hz); 
0.88 (6 H, doublet, J=6.3 Hz). 
9) .sup.13 C-Nuclear magnetic resonance spectrum: (.delta.: ppm): the 
Nuclear magnetic resonance spectrum (270 MHz) in heavy methanol, using 
trimethylsilane as the internal standard, is shown below: 
11.4 (quartet), 22.7 (triplet), 23.1 (quartet), 23.1 
(quartet), 24.7 (triplet), 26.2 (triplet), 28.2 
(triplet), 29.1 (doublet), 30.4 (triplet), 32.4 
(triplet), 33.1 (triplet), 34.2 (triplet), 35.4 
(triplet), 36.1 (doublet), 37.1 (triplet), 39.4 
(triplet), 40.0 (triplet), 40.6 (doublet), 40.6 
(triplet), 64.6 (doublet), 73.9 (doublet), 77.8 
(singlet), 78.4 (doublet), 82.3 (doublet), 121.1 
(doublet), 123.7 (doublet), 124.3 (doublet), 127.7 
(doublet), 135.2 (doublet), 137.3 (doublet), 138.2 
(doublet), 152.7 (doublet), 166.4 (singlet), 175.0 (singlet). 
10) Solubility: Soluble in alcohols, such as methanol, ethanol or butanol; 
and soluble in acetone, chloroform, ethyl acetate and dimethyl sulfoxide; 
limited solubility in water: insoluble in hexane. 
11) Color reactions: Positive to sulfuric acid, iodine, ninhydrin and 
ammonium molybdate-perchloric acid reactions. 
12) High performance liquid chromatography: 
Separating column: Cosmosil 5C18-AR 
(Column size, 4.6.times.250 mm, Product of Nakaraitesque Inc.) 
Solvent: 45% v/v aqueous acetonitrile, containing 0.5% triethylamine - 
phosphate buffer (pH 3) 
Flow rate: 1 ml/min. 
Wave length: 230 nm 
Retention time: 21.90 min. and 22.19 min. 
BIOLOGICAL ACTIVITY 
The following Test Examples will explain the effect of the compounds of the 
present invention in more detail. 
TEST EXAMPLE 1 
Stimulation of GM-CSF production: 
Determination of the stimulation of GM-CSF production by the compounds of 
the present invention was carried out, in principle, using a combination 
of the method of Kohama et al. [Experimental Hematology 16, 603-608 
(1988)] and that of Kitamura et al. [Journal of Cellular Physiology 140, 
323-334 (1989)]. In more detail, KM-102 cells originating from human bone 
marrow stromal cells, which served as to produce GM-CSF, were mixed with a 
test sample solution diluted to a suitable concentration (the "GM-CSF 
producing system"). After incubation for 24 hours, a part of the culture 
supernatant was taken and was added to a culture system of 
GM-CSF-dependent human TF-1 cells. After between 48 and 72 hours, the 
amount of GM-CSF was measured by the growth of TF-1 cells (the "GM-CSF 
assay system") to obtain a measure of the stimulation of GM-CSF 
production. The growth of TF-1 cells was determined by tritium thymidine 
pulse-labelling for 4 hours. Similar results were obtained also by means 
of an MTT kit (Chemicon International Inc. USA) to assay the growth of 
TF-1 cells and by means of an ELISA kit (Genzyme Inc. USA) to assay the 
GM-CSF. Whether the GM-CSF production inducing system functioned normally 
or not was assayed by means of recombinant interleukin 1.beta. 
(IL-1.beta.: Genzyme Inc. USA). IL-1.beta. induced GM-CSF production in a 
dose-dependent manner within the range from 1 to 100 units/ml, and, at a 
maximum, the production of GM-CSF induced was from 10 to 20 times the 
production without IL-1.beta., showing a normal functioning of the 
experimental system. 
After each addition of leustroducsin A, B or C to the KM-102 cells at a 
certain concentration, GM-CSF production was found to be induced in a 
dose-dependent manner. At the maximum, the amount of GM-CSF produced was 
from 10 to 20 times the amount produced without the addition. The 
ED.sub.50 values were found to be 180+50, 50+15 and 50+15 ng/ml for 
leustroducsins A, B and C respectively. 
TEST EXAMPLE 2 
Stimulation G-CSF production: 
Stimulation of G-CSF production was assayed by a system in which the GM-CSF 
assay system employed inTest Example 1 was replaced by a G-CSF assay 
system [as described by Shirafuji et al.: Experimental Hematology 17, 
116-119 (1989)]. In more detail, KM-102 cells originating from human bone 
marrow stromal cells, which served to produce G-CSF, were added to a 
leustroducsin solution diluted to a suitable concentration (the "G-CSF 
producing system"). After incubation for 24 hours, a part of the culture 
supernatant was taken, and added to a culture system of G-CSF-dependent 
NFS-60 cells. After between 24 and 48 hours, the amount of G-CSF was 
assayed from the growth of NFS-60 cells (the "G-CSF assay system") to 
obtain a measure of the stimulation of G-CSF production. The growth of 
NFS-60 cells was determined by tritium-thymidine pulse-labelling for 4 
hours. Similar results were also obtained by means of an MTT kit to assay 
the growth of NFS-60 cells and by means of an ELISA kit to assay the G-CSF 
[Motojima et al.: Journal of Immunological Methods 118, 187-192 (1989)]. 
Whether the G-CSF production inducing system functioned normally or not 
was assayed by means of recombinant interleukin 1 .beta. (IL-1.beta.: 
Genzyme Inc. USA). IL-1.beta. induced GM-CSF production in a 
dose-dependent fashion within a range from 1 to 100 units/ml, and at the 
maximum the production of G-CSF induced was from 10 to 20 times the 
production without IL-1.beta., showing normal functioning of the 
experimental system. 
After each addition of leustroducsins A, B and C to KM-102 cells at a 
certain concentration, G-CSF production was found to be induced in a 
dose-dependent fashion. At the maximum, the amount of G-CSF produced was 
from 10 to 20 times the amount produced without the addition. The 
ED.sub.50 values were found to be 200+50, 50+15 and 50+15 ng/ml for 
leustroducsins A, B and C respectively. 
TEST EXAMPLE 3 
Stimulation of NGF production 
Furukawa et al. reported that fibroblast-forming L-M cells derived from 
mouse connective tissue produce and secrete a relatively large amount of 
NGF, and that catecolamines stimulate this production and secretion [J. 
Biol. Chem. 261, 6039-6047 (1986)]. We examined whether the leustroducsins 
stimulate NGF production and secretion or not. 
L-M cells were cultivated using 199 medium containing 0.5% peptone. Into 
each well of a 24-well culture plate, 5.times.10.sup.4 of L-M cells were 
inoculated, and cultured in a CO.sub.2 incubator (37.degree. C., 5% 
CO.sub.2) until confluent. The culture liquid was removed, and the cells 
were washed once with 199 medium containing 0.5% bovine serum albumin 
(Sigma). One of the leustroducsins was added at a certain concentration to 
199 medium containing 0.5% bovine serum albumin, and then the L-M cells 
were treated with this mixture. The L-M cells were then cultured in a 
CO.sub.2 incubator for 24 hours. After collecting the culture liquid, the 
NGF in the liquid was assayed. 
The NGF was assayed by enzyme immunoassay [Proc. Natl. Acad. Sci. USA 80, 
3513-3516 (1983)]. Into each well of a polystyrene 96-well plate, 75 .mu.l 
of anti-mouse-.mu.NGF antibody (Boehringer) solution (0.3 .mu.g/ml, pH 
9.6) was poured, and allowed to stand for 1 hour at room temperature. 
After removal of the antibody, all of the wells were washed with a wash 
solution three times. 50 .mu.l of the standard .beta.NGF (Wako Pure Chem. 
Ind.) or of the standard solution was poured into each well, and the 
mixture was allowed to stand for from 6 to 8 hours at room temperature. At 
the end of this time, the standard .beta.NGF or standard solution was 
removed, and all of the wells were washed three times. 50.mu.l of labelled 
anti-.beta.NGF monoclonal antibody (Boehringer) solution (100 mU/ml, pH 
7.0) was poured into each well, and the solution was allowed to stand for 
from 15 to 18 hours at 4.degree. C. At the end of this time, the 
enzyme-labelled antibody was removed, and all of the wells were washed 
three times. 100 .mu.l of chlorophenol-.beta.-D-galactoside (Boehringer) 
solution (1 mg/ml, pH 7.3) was then poured into each well. After a proper 
color had developed (after 2 to 3 hours at room temperature), the 
absorbance at 570 nm was determined. The amount of NGF was calculated from 
the standard curve, and this was related as a percentage to the amount of 
NGF produced and secreted from the cells without leustroducsin treatment. 
The leustroducsins were all found to stimulate the production of NGF in a 
dose-dependent fashion. At 5 .mu./ml of each leustroducsin, 2 times or 
more NGF production was induced compared with that in the cells without 
treatment. 
TEST EXAMPLE 4 
Antifungal activity 
In order to examine the antifungal activity of leustroducsins against fungi 
pathogenic to animals, the activity was tested against Tricophyton 
mentagrophytes. For each of leustroducsins, an inhibitory circle was 
observed at a concentration of 1 .mu.g/disc. 
TEST EXAMPLE 5 
Acute toxicity 
According to the conventional procedure, the acute toxicity was tested in 
five ddY mice (male). After intraperitoneal administration of a dose of 
0.2 mg/kg of leustroducsin A, no toxicity was observed over a period of 5 
days. Similarly, no toxicity was observed after administration of 
leustroducsin B, leustroducsin C and their related derivatives. 
From the results reported above, it is apparent that leustroducsins, A, B 
and C stimulate the production of hematopoietic factors such as 
granulocyte colony-stimulating factor and granulocyte macrophage 
colony-stimulating factor. Accordingly, the leustroducsins are useful as 
therapeutic agents to reduce side effects caused by cancer chemotherapy 
and radiotherapy. They also protect against infection and have an 
anticancer effect through activation of macrophages. In addition, the 
leustroducsins are useful to improve cerebral metabolism by stimulating 
production of NGF. Furthermore, the leustroducsins are useful as 
antifungal agents as shown by their antifungal effect against Tricophyton 
mentagrophytes.