Enzyme inhibitor and method of producing the same

Novel peptide analogues which exhibit inhibitory activity against aspartic proteinases, a novel species of actinomycetous microorganism which produces said novel peptide analogues, a process for producing said novel analogues by culturing said species and a pharmaceutical composition containing said analogues.

FIELD OF THE INVENTION 
The present invention relates to novel biologically active compounds which 
exhibit an inhibitory activity against enzymes such as aspartic 
proteinases, e.g., pepsin, renin and the like, as well as to a 
microorganism of the genus Kitasatosporia having the ability to produce 
said compounds, and a method of producing the compound using the 
microorganism. 
Representatives of aspartic proteinase which is known to exist in living 
organisms include pepsin, which is found in the stomach, renin, which is 
found in the liver, and so forth. Pepstatin (Umezawa et al., J. 
Antibiotics, 23, 259 (1970) or Japanese Patent Laid-Open 29582/1972) and 
pepstanone (Miyano et al., J. Antibiotics, 25, 489 (1972) or Japanese 
Patent Laid-Open No. 88281/1973) are well known as low-molecular weight 
peptide inhibitors which act on such aspartic proteinase. Examples of 
pepstatin analogues include hydroxypepstatin (Umezawa et al., J. 
Antibiotics, 27, 615 (1973)) in which the alanine residue is replaced by a 
serine residue and analogues in which the acyl group at the N-terminal is 
a straight chain group having 2 (acetic acid) to 20 (alginic acid) carbon 
atoms or a branched aliphatic group (isolated by Aoyagi et al., J. 
Antibiotics, 26, 539 (1973)). SP-1 (Murao et al, Agric. Biol. Chem., 34, 
1265 (1970)) and pepsinostreptin (Kakinuma et al., J. Takeda Res. Lab., 
35, 123 (1976)) are identical to the substances included in this 
classification of pepstatin compounds. All of such compounds are 
characterized by being pentapeptides in which the third residue from the 
N-terminal and the C-terminal contains an abnormal amino acid statin ((3S, 
4S)-4-amino-3-hydroxy-6-methylheptanoic acid) or, in the case of 
pepstanone, the C-terminal contains stanone 
((3S)-3-amino-5-methyl-hexane-2-one), and in which a straight or branched 
aclyl group is bonded to the N-terminal. Although many known low-molecular 
weight peptide inhibitors which act on aspartic proteinases are produced 
by microorganisms, these inhibitors are not readily dissolved in solvents 
because of the nature of their structures. Therefore, they cannot be 
easily purified and, in addition, the permeability of these inhibitors 
through biological systems is not particularly good. From this viewpoint, 
there is a demand for novel biologically active substances which not only 
have inhibitory activity against aspartic proteinases but also have good 
compatibility with solvents. 
SUMMARY OF THE INVENTION 
The object of the present invention is to provide novel biologically active 
compounds which exhibit inhibitory activity against aspartic proteinases 
such as pepsin, renin and the like. 
A further object of the present invention is to provide novel statin 
analogues which have inhibitory activity against aspartic proteinases as 
well as good compatibility with various solvents such as methanol, 
ethanol, dimethyl sulfoxide and butanol etc., and hence can be expected to 
have good permeability through biological systems. 
A still further object of the present invention is to provide 
Kitasatosporia kyotoensis, a novel actinomycetous species of the genus 
Kitasatosporia, which is capable of producing novel statin analogues 
having inhibitory activity against aspartic proteinases as well as having 
good compatibility with various solvents. 
A yet further object of the present invention is to provide a method for 
producing novel statin analogues of the invention by cultivation of a 
strain of Kitasatosporia Kyotoensis followed by isolation and purification 
of said analogues from the culture medium. 
Still another object of the invention is to provide a pharmaceutical 
composition which comprises at least one compound of the general formula 
I, together with a pharmaceutically acceptable carrier. 
These and other objects of the invention will be apparent from the 
following description.

DETAILED DESCRIPTION OF THE INVENTION 
As a result of extensive investigations conducted by the inventors with a 
view to discovering a novel microorganism which is capable of producing 
biologically active compounds having a novel structure and exhibiting 
inhibitory activity against the enzyme function of aspartic proteinases, 
the inventors found that a group of biologically active compounds 
exhibiting such inhibition against various aspartic proteinases can be 
produced from a culture solution of Kitasatosporia kyotoensis strain 
SAM0107, which was isolated from a soil sample collected in Kyoto, Japan, 
and which belongs to actinomycetous microorganisms. The inventors also 
succeeded in isolating and purifying these biologically active compounds 
in a pure state. Such novel compounds of the present invention are 
significantly different from the known pepstatin analogues in terms of the 
number and arrangement of the amino acid residues and the acyl groups at 
the N-terminals. 
The compounds of the present invention which have inhibitory activity 
against the enzyme function of aspartic proteinases have a structure 
represented by the general formula I: 
##STR1## 
wherein A denotes --OH or --O--CH.sub.3 and B denotes 
##STR2## 
The Kitasatosporia kyotoensis SAM0107 which is a novel microorganism 
capable of producing the biologically active substances of the general 
formula I has the following morphology and cultural characteristics: 
1) Morphological character 
The SAM0107 strain forms a straight chain with a spiral end at the end of 
an aerial hypha which is not so long. The straight chain of mature spore 
comprises 20 to 50 or more spores. Each of the spores has a size of (0.4 
to 0.8) .mu.m.times.(0.8 to 1.2) .mu.m and a smooth surface. No 
fragmentation is to be observed in substrate mycelium. 
______________________________________ 
2) Cultural character (culture at 28.degree. C. for 14 days) 
Sucrose-nitrate agar medium: 
Aerial mycelium; None 
Reverse side color; White to light yellow 
Soluble pigment; None; 
Glucose-asparagine agar medium: 
Aerial mycelium; Thin, white to grey 
Reverse side color; Yellowish brown 
Soluble pigment; None 
Glycerin-asparagine agar medium: 
Aerial mycelium; Thin, white to grey 
Reverse side color; Yellowish brown 
Soluble pigment; Brown 
Inorganic salts-starch agar medium: 
Aerial mycelium; Dense, white 
Reverse side color; Dark yellowish brown 
Soluble pigment; None 
Tyrosine agar medium: 
Aerial mycelium; Thin, white to grey 
Reverse side color; Brown 
Soluble pigment; Dark brown 
Nutrient agar medium: 
Aerial mycelium; None 
Reverse side color; Light yellow 
Soluble pigment; None 
Yeast extract-malt extract agar medium: 
Aerial mycelium; Dense, white to grey 
Reverse side color; Yellowish brown 
Soluble pigment; None 
Oatmeal agar medium: 
Aerial mycelium; Thin, white to grey 
Reverse side color; Yellowish brown 
Soluble pigment; None 
Peptone-yeast extract-iron agar medium: 
Aerial mycelium; None 
Reverse side color; Light yellow 
Soluble pigment; None 
3) physiological property 
(1) Growth temperature range (culture in a CYC liquid 
medium for 3 days) 
Viable temperature: 18 to 31.degree. C. 
Optimum growth temperature: 21 to 24.5.degree. C. 
(2) Liquefaction of gelatin 
Negative 
(3) Hydrolysis of starch Positive 
(4) Coagulation of milk Negative 
(5) Peptonization of milk Negative 
(6) Production of melanoid pigment 
Peptone-yeast extract-iron agar medium 
Negative 
Tyrosine agar medium (0.2% glucose, 
Negative 
1.0% yeast extract (Difco), 0.05% 
L-tyrosine, 0.5% NaCl, 2.0% agar, 
pH 7.0 
Trypton-yeast extract agar medium 
Negative 
(7) Reduction of nitrate Positive 
(8) Utilization of carbon source (culture in Pridham 
and Gottlieb medium at 28.degree. C. for 14 days) 
D-glucose + 
D-xylose + 
L-arabinose 
+ 
L-rhamnose - 
D-fructose .+-. 
D-galactose 
+ 
Raffinose - 
D-mannitol - 
Inositol - 
Salicin .+-. 
Sucrose + 
______________________________________ 
(+, utilized; .+-., doubtful whether the carbon source is utilized or not 
-, not utilized). 
4) Chemical Properties 
(1) Cell Wall 
a. Amino Acid 
As a result of examination of the hydrolysate of whole cells and the cell 
wall in accordance with the method of Stanek, J. L. and Roberts, G. D. 
(Applied Microbiology, 28, 226 (1974), the existence of two isomers, 
meso-2,6-diaminopimelic acid and L,L-2,6-diaminopimelic acid, as well as 
glycine was observed. 
b. Sugar 
Ribose, mannose, glucose and galactose exist in the hydrolysate of whole 
cells. 
(2) Quinone System 
It contains MK-9(H.sub.6) and MK-9(H.sub.8) as major components. 
The morphology and cultural characteristics of this strain are summarized 
below. 
Aerial hyphae of the SAM0107 strain are relatively short and have spore 
chains which are straight and have a spiral end. The spore chain comprises 
20 to 50 or more spores. The surface of the spore is smooth. Aerial hyphae 
which are white to yellowish grey adhere to substrate hyphae which are 
light yellow to brown, in various mediums. Soluble pigments of dark brown 
are produced in tyrosine agar medium but no melanoid pigment is produced. 
Meso-2,6-diaminopimelic acid, L,L-2,6-diaminopimelic acid, glycine, ribose, 
mannose, glucose and galactose were observed in the hydrolysate of whole 
cells. The quinone system has MK-9(H.sub.6) and MK-9(H.sub.8) as major 
components. 
From the viewpoint described above, particularly the fact that two isomers 
of 2,6-diamminopimelic acid exist in the hydrolysate of whole cells, it 
can be concluded that the strain SAM0107 belongs to the genus 
Kitasatosporia (J. Antibiotics, 35, 1013 (1982); The Actinomycetologist 
(The society for Actinomycetes, Japan), 45, 12, 1984). 
Examples of actinomycetes belonging to the genus Kitasatosporia include 
Kitasatosporia setalba KM-6054 reported by OMURA et al. in J. Antibiotics, 
35, 1013 (1982); Kitasatosporia phosalacinea KA-338 and Kitasatosporia 
griseola AM-9660 reported by TAKAHASHI et al. in J. Gen. Appl. Microbiol., 
30, 377 (1984); Kitasatosporia melanogena K-55-G-32 reported by SHIMAZU et 
al. on page 9 in the summary of the annual meeting of the Society for 
Actinomycetes Japan in Osaka, 1984; Kitasatosporia sp. SANK60684 reported 
by INAOKA et al in Japanese Patent Laid-Open No. 088884/1986; 
Kitasatosporia sp. RK-419 reported by ISONO et al. in Japanese Patent 
Laid-Open No. 146188/1986; Kitasatosporia setae MF730-N6 reported by 
UMEZAWA et al. in Japanese Patent Laid-Open No. 285992/1986; 
Kitasatosporia kifnense 9482 reported by IWAMI et al. in J. Antibiotics, 
40, 612 (1987) and Kitasatosporia clausa 33.35-1 reported by Liu, Z. et 
al. in Acta Microbiologica Sinica, 26, 87 (1986). 
As compared with these strains, the SAM0107 strain is clearly different 
from these strains in the fact that the SAM0107 strain forms spore chains 
having ends with a spiral form, while Kitasatosporia setalba KM-6054, 
Kitasatosporia phosalacinea KA-338, Kitasatosporia griseola KM-9660, 
Kitasatosporia melanogena K55-G-32 and Kitasatosporia setae MF-730-N6 
displays spore chains having an end with a rectusflexibilis form and the 
spore chains of Kitasatosporia sp. SANK60684 have a straight or curved 
form. 
Although the Kitasatosporia kifnense 9482 forms spore chains with ends 
having a hooked or spiral form, this strain is clearly distinguishable 
from the strain SAM0107 of the present invention with respect to the 
production of soluble pigments in a glycerin-asparagine agar medium, the 
reduction of a nitrate and the utilization of D-xylose and D-mannitol. 
The Kitasatosporia sp. RK-419 forms spore chains having ends with an open 
spiral form but is clearly distinguished from the strain SAM0107 of the 
present invention with respect to the formation of aerial mycelium in a 
sucrose-nitrate agar medium, the production of soluble pigments in a 
glycerin-asparagine agar medium and in a tyrosine agar medium, the 
formation of aerial mycelium in a nutrient agar medium, growth in a 
peptone-yeast extract-iron agar medium, utilization of D-xylose, 
L-arabinose and raffinose, coagulation of milk, peptonization of milk and 
the sugar composition of the hydrolytic product of whole cells. 
The Kitasatosporia clausa 33.35-1 is clearly distinguished from the strain 
SAM0107 with respect to the fact that the substrate hyphae are fragmented. 
In view of the above-described consideration, the inventors concluded that 
the strain SAM0107 belongs to a new species of the genus Kitasatosporia 
and named the species Kitasatosporia kyotoensis. 
This strain was deposited at the Fermentation Research Institute, Agency of 
Industrial Science and Technology on Sept. 10, 1987, and given accession 
number FERM P-9580. The deposit was transferred to a deposit under the 
Budapest Treaty as of Sept. 9, 1988, and given accession number FERM 
BP-2045. 
It can be inferred that the biologically active compounds of the present 
invention can be produced by a person skilled in the art using a known 
method of synthesizing peptides. Although the compounds synthesized by 
such synthetic methods are intended to be included in the present 
invention, the biologically active compound can be more conveniently 
produced on a commercial scale by culturing actinomycetous microorganisms 
belonging to the genus Kitasatosporia and having the ability to produce 
biologically active compounds of the formula I in an appropriate medium, 
followed by separation of the compounds from the culture medium and 
purification thereof. 
The medium used for producing the biologically active compounds in the 
present invention may be either liquid or solid, but shaking culture or 
aerobic agitating culture in a liquid medium is generally convenient. Any 
medium which allows the growth of the microorganism producing the 
compounds of the present invention and accumulation of the product therein 
may also be used. In other word, for example, glucose, lactose, glycerin, 
starch, sucrose, dextrin, molasses and organic acids are used as carbon 
sources, and protein hydrolysate such as peptone and casamino acid, meat 
extract, yeast extract, soybean meal, corn steep liquor, amino acids, 
ammonium salts, nitrates and other organic and inorganic nitrogen 
compounds are used as nitrogen sources. Various phosphates, magnesium 
sulfate or sodium chloride may be added as an inorganic salt to the 
medium, and vitamins and compounds relevant to nucleic acids may be added 
thereto for the purpose of accelerating the growth of the microorganism. 
The addition of silicone, polypropylene glycol derivatives or soybean oil 
which all serve as an antifoamer to the medium is in some cases effective 
for increasing the amount of the compounds accumulated in the medium in 
accordance with the present invention. 
Preferably, pre-culture on a small scale is first performed and the 
pre-cultured organisms are then inoculated in the medium rather than 
starting the production culture directly. Although the conditions such as 
the culture temperature, culture period and the properties of the culture 
solution are appropriately selected and adjusted so that the accumulation 
of the compounds of the invention will be the maximum possible, the 
culture in many cases is preferably performed at 25.degree. C. to 
35.degree. C. for 1 to 3 days under aerobic conditions, with the pH value 
of the culture solution being kept at 4.0 to 9.5. 
Such culture enables the compounds of the invention to be accumulated in 
the culture mixture. In the case of culture using a liquid medium, the 
desired compounds are mainly accumulated in the liquid portion, and it is 
hence preferable that the culture mixture is first filtered or centrifuged 
so that the microorganism is removed, and the desired compounds are then 
separated from the filtrate or supernatant. Alternatively, the compounds 
can also be isolated directly from the culture mixture without the 
microorganisms being removed. The compounds can be separated from the 
culture mixture and purified by using various methods based on the 
chemical characteristics of the compounds of the present invention. 
Examples of methods that may be effectively used include precipitation by 
addition of ammonium sulfate or the like; extraction with an organic 
solvent such as n-butanol which does not freely mix with water and which 
is capable of dissolving the compounds of the invention therein; 
dissolution in polar solvents such as methanol and ethanol; removal of 
impurities by treatment with hexane; gel filtration using a matrix of 
Sephadex types; ion exchange chromatography using various types of iron 
exchangers such as ion-exchange resin, ion-exchange cellulose and 
ion-exchange Sephadex; and adsorption chromatography using adsorbents such 
as activated charcoal, alumina, silica gel, Amberlite XAD-1 or 2. The 
compounds of the invention can be isolated in a white amorphous form by 
appropriately combining these methods. Any other methods which 
appropriately utilize the characteristics of the compounds of the 
invention may also be suitably used. Examples of particularly preferred 
adsorbents include Diaion HP-20, Sephadex LH-20, TSKG-3000S, Cosmosil 
10C18 and DEAE-cellulose. 
The biologically active compounds produced by the method of the present 
invention exhibit the ability to inhibit aspartic proteinases such as 
pepsin. For example, as shown in the examples described below, it was 
confirmed that the degradation of hemoglobin by pepsin is inhibited by the 
compounds of the present invention. 
The present invention is described in detail below with reference to 
examples, but the invention is not limited to these examples. 
EXAMPLE 1 
A. Production of Biologically Active Compounds by Culture of Kitasatosporia 
kyotoensis 
Pure seed culture of Kitasatosporia kyotoensis strain SAM0107 was 
inoculated in 3 l of a synthetic medium (pH 7.0) comprising glucose, 
peptone, corn starch, yeast extract, dry yeast and dipotassium phosphate, 
followed by aerobic culture under agitation in a small fermentor for 24 
hours at 28.degree. C., an aeration rate of 3 l/min and a speed of 300 
revolutions/min. The pre-culture (two batches in small fermentors) was 
inoculated in 300 l of the same synthetic medium as that described above, 
followed by aerobic culture under agitation in a tank for 17 hours at 
28.degree. C., an aeration rate of 210 l/min and a speed of 100 
revolutions/min. 
The culture mixture was centrifuged, and the supernatant (250 l) was 
adsorbed on a column (25 l) of Diaion HP-20 (Mitsubishi Chemical 
Industries Co., Ltd.). The column was washed with 100 l of water and then 
eluted with 150 l of methanol whereby the fractions having antipepsin 
activity were collected. The active fractions were pooled, concentrated 
under reduced pressure and then again adsorbed on a column of the same 
type (4 l) as that described above. This column was washed with 20 l of 
water and then subjected to elution with 8 l of each of 40%, 60% and 80% 
methanol. 
The fraction eluted with 60% methanol was concentrated under reduced 
pressure, and the dry residue was dissolved in 50 ml of 50% methanol. The 
resulting solution was then introduced to a column (55 mm.times.2100 mm) 
of Sephadex LH-20 (Pharmacia Co., Ltd.). Then the eluate was fractionated 
into fractions of 15 ml each, the enzyme inhibitory activity was eluted in 
Fraction Nos. 119 to 176. Fraction Nos. 119 to 139 contained compounds I, 
III and IV and Fraction Nos. 140 to 176 contained compounds V and VI. The 
fractions (Nos. 119 to 139) eluted from the HP-20 column in the first half 
were concentrated under reduced pressure, the dry residue was dissolved in 
100 ml of 20% methanol and then introduced to a HP-20 column which had 
previously been equilibrated with 20% methanol. The column was subjected 
to elution with 600 ml of 20% methanol and then with a linear gradient of 
20 to 80% methanol (a total volume of 1200 ml). When the eluate was 
fractionated into fractions of 10 ml each, enzyme inhibitory activity was 
recovered in the fractions of 20% methanol (containing compounds III and 
IV) and in Fraction Nos. 81 to 100 (containing compound I). The fractions 
containing the compounds III and IV were pooled and again separated on a 
Sephadex LH-20 column (30 mm.times.1710 mm, 50% methanol). Fraction Nos. 
80 to 92 from the fractionation of 15 ml each contained the inhibitory 
activity against the enzyme, which were pooled and then concentrated under 
reduced pressure. The dry residue was dissolved in 50% methanol and then 
loaded on column of high performance liquid chromatography (Cosmosil 10C18 
packed column, 20 mm.times.250 mm). The elution was performed at a flow 
rate of 2 ml/min. The fractions were collected for each of 0.25 min while 
being monitored by absorption at 235 nm. 0.1% trifluoroacetic acid (TFA) 
was used as the solvent for the first 4 minutes and then changed to 0.1% 
TFA-60% acetonitrile in a linear manner over a time of 20 minutes which 
was then caused to flow for 10 minutes. As a result, 7 mg of pure compound 
III and 3 mg of pure compound IV were recovered from Fraction Nos. 34 to 
36 and Fraction Nos. 56 to 59, respectively (FIG. 1). 
Fraction Nos. 80-100 from the foregoing sephadex LH-20 column containing 
the compound I were pooled, concentrated under reduced pressure, the dry 
residue was dissolved in 90 ml of 30% ethanol and then introduced to a 
TSKG 3000S (Toyo Soda Co., Ltd.) column (15 mm.times.285 mm) which had 
previously been equilibrated with 30% ethanol. The column was washed with 
900 ml of 30% ethanol and then subjected to elution with 50% ethanol. When 
the eluate was fractionated into fractions of 10 ml each, Fraction Nos. 
106 to 123 contained the compound I. These fractions were collected and 
introduced to a Cosmosil 10C18 packed column as used for the purification 
of the above-described compounds III and IV. 4 mg of pure compound I was 
recovered in Fraction Nos. 21 to 25 (FIG. 2). 
The fractions (Fraction Nos. 146 to 176) containing the compounds V and VI 
were concentrated under reduced pressure, the dry residue was dissolved in 
100 ml of 20% methanol and then absorbed on a HP-20 column (30 
mm.times.300 mm) which had previously been equilibrated with 20% methanol. 
This column was washed with 600 ml of 20% methanol and then subjected to 
elution using a linear gradient changing from 20 to 80% methanol (a total 
volume of 1200 l). As a result of fractionation into fractions of 10 ml 
each, the enzyme inhibitors were recovered in Fraction Nos. 71 to 111 
(FIG. 3). 
These active fractions were collected and introduced to a Cosmosil 10C18 
packed column in the same way as that described above. 10 mg of pure 
compound VI and 40 mg of pure compound V were recovered in Fraction Nos. 
28 to 31 and 36 to 44, respectively (FIG. 4). 
The fractions eluted with 80% methanol from the first Diaion column HP-20 
were concentrated under reduced pressure, and the dry residue was 
dissolved in 50 ml of 50% methanol and then passed through a Sephadex 
LH-20 column (55 mm.times.2100 mm). As a result of fractionation into 
fractions of 15 ml each, the enzyme inhibitory activity was eluted in 
Fraction Nos. 110 to 140. These fractions were concentrated under reduced 
pressure, and the dry residue was dissolved in 100 ml of 50% methanol. 
After the pH value of the thus-obtained solution was adjusted to 8, the 
solution was adsorbed on a DEAE cellulose (Whatman DE23) column (26 
mm.times.110 mm) which had previously been equilibrated with 50% methanol 
solution in 50 mM ammonium acetate buffer (pH 5.6). As a result of elution 
by the same solution and fractionation into fractions of 10 ml each, the 
inhibitory activity was recovered in Fraction Nos. 16 to 18 (FIG. 5). 
These active fractions were collected and concentrated under reduced 
pressure, and the dry residue was dissolved in 50% methanol and then 
loaded on a packed column for high performance liquid chromatography as 
described above. As a result, 22 mg of pure compound II was recovered in 
Fraction Nos. 6 to 10 and 12 to 18 (FIG. 6). 
The pure compounds I to VI which were isolated from the culture of 
Kitasatosporia kyotoensis and purified in the above-described steps were 
respectively named SUAM-20007, SUAM-20008, SUAM-20009, SUAM-20010, 
SUAM-20011 and SUAM-20012. 
B. Physicochemical Properties of the Compounds I-VI 
(1) Compound I (SUAM-20007) 
Form: white powder 
Solubility: easily soluble in methanol, ethanol and dimethyl sulfoxide; 
soluble in butanol; sparingly soluble in water, benzene, ether, petroleum 
ether, chloroform, carbon tetrachloride, hexane and ethyl acetate 
Molecular formula: C.sub.44 H.sub.79 N.sub.7 O.sub.14 
Molecular weight: 930.1 
Mass spectrum: 930 [M+H].sup.+ 
Proton NMR spectrum: TMS standard 
0.80-1.10 (30H, m), 1.20-1.40 (9H, m), 1.50-1.80 (9H, m), 2.00-2.10 (2H, 
m), 2.30-2.50 (6H, m), 4.00 (6H, m), 3.65 (1H, q, J=5.0), 4.05-4.20 (2H, 
m), 4.30-4.50 (2H, m). 
Color reaction: negative in ninhydrin reaction, position in the Rydon-Smith 
reaction and hydrochloric acid-ninhydrin reaction 
(2) Compound II (SUAM-20008) 
Form: white powder 
Solubility: easily soluble in methanol, ethanol and dimethyl sulfoxide; 
soluble in butanol; sparingly soluble in water, benzene, ether, petroleum 
ether, chloroform, carbon tetrachloride, hexane and ethyl acetate 
Molecular formula: C.sub.43 H.sub.77 N.sub.7 O.sub.12 
Molecular weight: 884.1 
Mass spectrum: 884 [M+H].sup.+ 
proton NMR spectrum: TMS standard 
0.80-1.10 (30H, m), 1.20-1.24 (9H, m), 1.50-1.58 (9H, m), 2.10 (3H, s), 
2.00-2.10 (2H, m), 2.30-2.50 (4H, m), 4.00-4.40 (9H, m). 
Color reaction: negative in ninhydrin reaction negative, positive in the 
Rydon-Smith reaction and hydrochloric acidninhydrin reaction 
(3) Compound III (SUAM-20009) 
Form: white powder 
Solubility: easily soluble in methanol, ethanol and dimethyl sulfoxide; 
soluble in butanol; sparingly soluble in water, benzene, ether, petroleum 
ether, chloroform, carbon tetrachloride, hexane and ethyl acetate 
Molecular formula: C.sub.34 H.sub.61 N.sub.5 O.sub.11 
Molecular weight: 715.9 
Mass spectrum: 716 [M+H].sup.+ 
Proton NMR spectrum: TMS standard 
0.80-0.95 (12H, m), 0.98 (6H, d, J=5.2), 1.02 (6H, d, J=5.2), 1.37 (2H, m), 
1.38 (3H, d, J=5.0), 1.40 (3H, d, J=5.0), 1.50-1.70 (4H, m), 2.20 (2H, m), 
2.30-2.50 (4H, m), 3.62 (3H, s), 3.63 (1H, q, J=5.0), 4.00 (4H, m), 4.10 
(1H, d, J=5.2), 4.18 (1H, d, J=5.2), 4.30 (1H, d, J=5.0) 
Color reaction: negative in ninhydrin reaction, positive in Rydon-Smith 
reaction and hydrochloric acid-ninhydrin reaction 
(4) Compound IV (SUAM-20010) 
Form; white powder 
Solubility: easily soluble in methanol, ethanol and dimethyl sulfoxide; 
soluble in butanol; sparingly soluble in water, benzene, ether, petroleum 
ether, chloroform, carbon tetrachloride, hexane and ethyl acetate 
Molecular formula: C.sub.33 H.sub.59 N.sub.5 O.sub.11 
Molecular weight: 701.9 
Mass spectrum: 702 [M+H].sup.+ 
Proton NMR spectrum: TMS standard 
0.80-0.95 (12H, m), 0.98 (6H, d, J=5.2), 1.02 (6H, d, J=5.2), 1.35 (2H, m), 
1.38 (6H, m), 1.50-1.70 (4H, m), 2.10-2.25 (2H, m), 2.30-2.50 (4H, m), 
3.63 (1H, q, J=5.0), 4.00 (4H, m), 4.10 (1H, d, J=5.0), 4.18 (1H, d, 
J=5.0), 4.30 (1H, d, J=5.0) 
Color reaction: negative in ninhydrin reaction, positive in the Rydon-Smith 
reaction and hydrochloric acid-ninhydrin reaction 
(5) Compound V (SUAM-20011) 
Form: white powder 
Solubility: easily soluble in methanol, ethanol and dimethyl sulfoxide; 
soluble in butanol; sparingly soluble in water, benzene, ether, petroleum 
ether, chloroform, carbon tetrachloride, hexane and ethyl acetate 
Molecular formula: C.sub.33 H.sub.59 N.sub.6 O.sub.9 
Molecular weight: 669.9 
Mass spectrum: 670 [M+H].sup.+ 
Proton NMR spectrum: TMS standard 
0.80-1.10 (24H, m), 1.20-1.24 (6H, m), 1.50-1.70 (6H, m), 2.15 (3H, s), 
2.00-2.10 (2H, m), 2.30-2.45 (2H, m), 3.63 (3H, s), 3.70 (1H, m), 4.00 
(3H, m), 4.10-4.25 (2H, m), 4.25-4.30 (2H, m) 
Color reaction: negative in ninhydrin reaction positive in the Rydon-Smith 
reaction and hydrochloric acid-ninhydrin reaction 
(6) Compound VI (SUAM-20012) 
Form: white powder 
Solubility: easily soluble in methanol, ethanol and dimethyl sulfoxide; 
soluble in butanol; sparingly soluble in water, benzene, ether, petroleum 
ether, chloroform, carbon tetrachloride, hexane and ethyl acetate 
Molecular formula: C.sub.32 H.sub.57 N.sub.5 O.sub.9 
Molecular weight: 655.8 
Mass spectrum: 655 [M+H].sup.+ 
Proton NMR spectrum: TMS standard 
0.80-1.10 (24H, m), 1.20-1.24 (6H, m), 1.50-1.70 (6H, m), 2.00-2.10 (2H, 
m), 2.30-2.45 (2H, m), 3.63 (3H, s), 3.70 (1H, m), 4.00 (3H, m), 4.10-4.25 
(2H, m), 4.25-4.30 (2H, m) 
Color reaction; negative in ninhydrin reaction, positive in the Rydon-Smith 
reaction and hydrochloric acid-ninhydrin reaction 
The solubility of all of these compounds I-VI in methanol was 10 times as 
high as that of previously known pepstatin analogues (for example, 
pepstatin). 
C. Analysis of Amino Acids of Compounds I-VI 
2 to 3 .mu.g of each of the SUAM-20007, 20008, 20009, 20010, 20011 and 
20012 was dissolved in 0.1 ml of 6N-hydrochloric acid, vacuum-sealed in a 
tube and then hydrolyzed at 105.degree. C. for 48 hours. After hydrolysis, 
each of the reaction mixtures was dried under vacuum and dissolved in 0.3 
ml of 0.02N-hydrochloric acid which was added thereto. A 0.225 ml aliquot 
of the solution was used in the analysis of amino acids and alanine and 
valine were thereby detected from all of the compounds. In consideration 
of the molecular weight and the molar ratio of the constituent amino acids 
of each of the compounds, the values described in the next table were 
obtained. 
______________________________________ 
Number of valine 
Number of alanine 
SUAM No. molecules molecules 
______________________________________ 
20007 2 2 
20008 2 2 
20009 2 1 
20010 2 1 
20011 2 1 
20012 2 1 
______________________________________ 
From the results of the aforementioned analysis, the structures of the 
compounds (SUAM-20007 to 20012) were determined as follows: 
##STR3## 
EXAMPLE 2 
Enzyme Inhibitory Activity of the Compounds 
The enzyme inhibitory activity of each of the compounds SUAM-20007, 20008, 
20009, 20010, 20011 and 20012 of the present invention was measured by the 
following method: 
(a) Inhibition against Pepsin from Bovine Pancreas 
A given .mu.l aliquot (a .mu.l), which ranged from 0-50 .mu.l, was taken 
from an aqueous solution of each of the compounds prepared in Example 1 at 
various set concentrations and was mixed with an aqueous solution of 
pepsin (50 .mu.g/50 .mu.l). To this mixture was added (150-a) .mu.l of 
0.06N-hydrochloric acid to adjust the pH value of the mixture to 3 to 5. 
Each of the solutions was then mixed with a substrate solution to start 
the reaction. The substrate for the reaction comprised 2.0% hemoglobin in 
800 .mu.l of 0.06N-hydrochloric acid and the reaction was conducted at 
35.degree. C. for 10 minutes, the total volume of the reaction mixture 
being 1 ml. 3 ml of 5% trichloroacetic acid was then added to the reaction 
mixture to terminate the reaction. After the precipitates formed had been 
filtered off, the amount of protein in the trichloroacetic acid fraction 
which contained the components of hemoglobin hydrolyzed with pepsin was 
measured from the value of ultraviolet absorption at 280 nm. The 
inhibitory activity which was defined as the concentration of the compound 
required to achieve 50% inhibition (IC.sub.50) of each of the compounds 
was determined by comparison with a control solution. The resulting values 
obtained were as follows: 
______________________________________ 
SUAM No. IC.sub.50 (M) 
______________________________________ 
20007 2.91 .times. 10.sup.-8 
20008 1.42 .times. 10.sup.-8 
20009 2.24 .times. 10.sup.-8 
20010 1.43 .times. 10.sup.-8 
20011 2.54 .times. 10.sup.-8 
20012 2.60 .times. 10.sup.-8 
Pepstatin 1.83 .times. 10.sup.-8 
______________________________________ 
The biologically active compounds of the present invention have a 
characteristic structure on the N-terminal side thereof and are highly 
soluble in solvents (particularly methanol) and thus can be easily 
purified. Since the compounds also have enzyme inhibitory activity against 
aspartic proteinases such as pepsin and the like, they will be useful for 
inhibiting the occurrence of gastric ulcer which is thought to be caused 
by aspartic proteinase, shortening the time of recovery therefrom, 
preventing proliferation of granuloma, preventing hepatic hypertension and 
preventing formation of virus disease lesion. Since the compounds of the 
present invention can also be used as ligands to form affinity columns, 
they are useful in application as reagents for purifying aspartic 
proteinases and for research on the mechanism of enzyme reactions.