Note: Descriptions are shown in the official language in which they were submitted.
1051801
BACXGROUND OF THE INVENTION
~1) Field of the Invention
This invention relates to an inhibitor called
bestatin of aminopeptidase B, leucine arninopeptidase
and bleomycin hydrolase as produced by aerob:ic culti~
vation of streptomyces and includes processes for
production by fermentation and for extraction.
(2) Description of the Prior Art
Bestatin is a sùbstance found and isolated from
culture broth of a streptomyces and has interesting
biological and physiological activities. Namely
bestatin has substantially no antibacterial activity,
but exhibits a weak inhibition of growth of mammalian
cells including cancer cells and a strong inhibition
EC 3, 4. /~, /
of aminopeptidase B, leucine aminopeptidase~and bleo-
mycin hydrolase. Based on inhibition of bleomycin
hydrolase, it exhibits a strong synergistic activity
with bleomycin in inhibiting tumors. Therefore, bestatin
is very important not only in treatment of squamous
cell carcinoma by bleomycin, but also in analysis of
biological functions and disease processes.
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105180~
SUMMARY OF THE INVENTION
This invention relates to a new and useful
microbial produc~ named bestatin inhibiting amino-
peptidase B, leucine aminopeptidase and bleomycin
hydrolase. It also relates to processes for its pro-
duction by fermentation and methods of its recovery
and purfication. This invention embrases this enzyme
inhibitor and its salts as crude concentrates, crude
solids, as purified solids and pure forms. This sub-
stance and its salts are effective in inhibiting
aminopeptidase B, leucine aminopeptidase and bleomycin
hydrolase, in inhibiting mammalian cancer cells weakly
and in increasing bleomycin activity agianst squamous
cell carcinoma. They have low toxicity and are useful
for treatment of squamous cell carcinoma in its use
combined to bleomycin.
There is now proved, according to the present
invention, an anti-aminopeptidase B compound (and its
salts) effective in inhibiting hydrolysis action of
aminopeptidase B, leucine aminopetidase and bleomycin
hydrolase and in increasing antitumor activity of
bleomycin, said compound being soluble in water, acetic
acid, pyridine, dimethylsulfoxide, methanol and ethanol,
slightly soluble in propanol, and butanol, practically
insoluble in ethyl acetate, butyl acetate, ethyl ether,
hexane, petroleum ether, benzene and chloroform, melting
at 203 - 206C, exhibiting levoration of [~]D2= -15.10
(c=0.861, methanol), exhibiting strong endabsorption
with maxima at 241.5 nm (ElCrn-3.8), 248 nm (E1%m=4.0);
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105180~
253 nm (ElCm-5.0); 258 nm ~ElCm-6.0), 254.5 nm (ElCm-
4.6) and 268 nm (El~m-2.7), gi~ing positive Rydon-Smith
and ninhydrin reactions and negative Sakaguchi reaction r
exhibiting following bands in the infrared region of
the spectrum when pelleted with potassium bromide:
3400, 3300, 3200, 2920, 2850, 1685, 1635, 1530, 1400,
1315, 1265, 1245, 1175, 1125, 1100, 850, 735, 700
cm~l, having the formula of C16H24N2O4
by the mass spectrum and elemental analysis (found:
C 60.86%, M 7.79~, N 8.61~), yielding L-leucine and
a new amino acid ~(2S,3R)-3-amino-~-hydroxy-4-phenyl-
butyric acid) which structure is shown by X ray crystal
analysis of its hydrobromide, yielding acid salts which
are more soluble in water than bestatin, e~hibiting
inhibition of aminopeptidase B, leucine aminopeptidase
and bleomycin hydrolase, having the following structure
shown by nmr, structures of hydrolysis products, chemical
synthesis:
~ , 27 H
CH?-,C- C- CO-NH-C-COOH
~==7 H OH CH2
CH
-
There are included within the scope of the present
invention bestatin and its salts. Acid salts of bestatin
can be easily obtained by addition of equimolar acid
and the salts such as hydrochloride, acetate etc are
more soluble in water than bestatinn
1051801
In one aspect of this invention there is provided
a process for production of bestatin which inhibits amino-
peptidase B, leucine aminopeptidase and bleomycin hydrolase and
increases the effect of bleomycin against squamous cell carcinoma
and which has the following properties and structure: soluble
in water, methanol, ethanol and insoluble in ethyl acetate,
ether, petroleum ether; [~]D2= -15.1 (c=0.861, methanol);
positive Rydon-Smith, ninhydrin; negative Sakaguchi;
2 ~ ~ .
N OH CH2
~C~
H3C CH3
which comprises cultivating a bestatin-producing streptomyces
which is Streptomyces olivoreticuli having the identifying
characteristics of A.T.C.C. 31159 in a nutrient medium containing
carbon and nitrogen sources under aerobic conditions until a
substantial amount of bestatin is produced by said organism in
said medium.
The bestatin is then recovered from the nutrient
medium.
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Fig. 1 is the ultraviolet spectrum of bestatin in
methanol.
Fig. 2 is the infrared absorption spectrum of bestatin
pelleted in potasslum bromide.
The present inventors have thought that microorga-
nisms produce protease inhibitors, and after discoveries
of leupeptin inhibiting trypsin, chymostatin inhibiting
chymotrypsin, pepstatin inhibiting acid proteases and
phosphoramidon inhibiting metalloendopeptidases, by the
continuation of the study, bestatin was discovered in
culture filtrates of a streptomyces. Moreover, bestatin
was found to inhibit enzymatic inactivation of bleomycin
in mammalian cells and to increase the bleomycin thera-
peutic effect on squamous cell carcinoma. As described
by H. Umezawa, one of the inventors,in his booX "Enzyme
Inhibitors of Microbial Origin" published by the University
of Tokyo Press, Bunkyo-ku, Tokyo in 1972, proteases
i~hibitors are obtained from various species of strepto-
myces. This is the same in bestatin. In this invention,
characters of a typical bPstatin-producing strain are
described. The description in brackets t 1 follows
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the color standard shown in Color Harmony Manual of
Container Corporation of America.
The characters of the strain MD976-C7:
The strain was isolated from a soil sample collected
at Raitamura-Nishino, Kisogun, Nagano~ It was deposited
in Kogyo Gijutsuin Hakko Kenkyusho on May 18 of 1974
and the deposit number is 2590. This strain is deposited
in American Type Culture Collection and the number
ATCC ~1159 was given, and therefore it is now available
for scholars. Microscopically, substrate mycelia are
well branched and extend ae~ial hypae. On aerial hypae,
spirals are not observed but whorls are observed. Surface
of spores is smooth.
The characters on various media are as follows:
(1) On sucrose nitrate agar medium (cultured at 27C):
growth is poor and colorless, and white [a, ~hite]
aerial mycelium develops slightly; no soluble pigment.
(2) On glucose asparagine agar medium (cultivated at
27C): growth is pale yellowish brown ~1 1/2 ic, Lt
Antique Gold], and white [a, White] to grayish white
(b, Oyster White] aerial mycelium develops; no soluble
pigment.
(3) On glycerol-asparagine agar medium (ISP-5, cul~.ured
at 27C): growth is pale yellowish brown 11 1/2 ic, Lt
Antique Gold], and around the growth white [a, White]
to grayish white [b, Oyster White] aerial mycelium
develops; no soluble pigment.
(4) Inorganic salts-starch agar medium (ISP-4, cultured
105180~L
at 27C): growth is pale yellowish hrown [1 1/2 ic,
Lt Antique Gold] to yellowish brown [2 lc, Gold], and
on the growth light brownish gray ~4 ec, Bisque Lt
Rose Beige] aerial mycelium develops; no soluble pigment.
(5~ On tyrosine agar medium (ISP-7, cultured at 27C~:
growth is pale yellow [1 1/2 ia, Sunlight Yellow,
Daffodil, Forsythia, Jonquil] to pale yellowish brown
tl 1/2 ic, Lt Antique Gold], and on the growth grayish
white lb, Oyster White] aerial mycelium develops; no
soluble pigment.
(6) On peptone-meat extract agar meaium (culture~
at 27C~: growth is pale yellowish brown [1 lJ2, Lt
Antique White], and around the colony grayish white
[b, Oyster White] aerial mycelium develops; pale
yellowish brown soluble pigment is produced slightly.
(7) On yeast extract-malt extract agar medium ~ISP-2,
cultured at 27C~: growth is dull yellow [2 lc, Gold]
to yellowish brown [2 pg, Mustard Gold~, and on the
growth grayish white [b, Oyster White] to light bxownish
gray [4 ec, Bisque, Lt Rose Beige] aerial mycelium
develops; pale yellow brown soluble pigment slightly.
(8~ On oatmeal agar medium (ISP-3, cultured at 27C):
growth is pale yellow (1 1/2 ia, Sunlight Yellow,
Daffodil, Forsythia, Jonquil], and on the growth grayish
white [b, Oyster White~ aerial mycelium develops
slightly; no soluble pigment.
(9) On calcium malate agar medium (cultured at 27C~:
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1051801
growth is colorless, and on the growth white [a, ~ite]
aerial mycelium develops; no soluble pigment.
(l0) On glucose peptone gexatin agar stab: growth
is yellowish brown 12 lc, Gold], and on the gr~wth
light brownish gray [4 ec, Bisque, Lt Rose Beige] aerial
mycelium develops; brownish black soluble pigment is
produced.
(ll) On milk medium (cultured at 2ioC): growth is pale
yellow [l 1/2 ia, Sunlight Yellow, Daffodil, Forsythia,
Jonquil] to pale yellowish brown [l l/2 ic, Lt Antique
~old]; no aerial mycelium; p~le yellowish brown pigment
is produced.
(12) On peptone-yeast extractiron ag~r medium (ISP-6,
cultured at 27C): growth is dark brownish gray [3 li,
Beaver~, and on the growth grayish white lb, Oyster
White] aerial mycelium develops slightly; brownish
black pigment is produced.
Physiological and biochemical properties:
(l) Growth temperature: it grew on maltose yeast
extract agar medium in the range of from 15C to 37C
and the optimum temperature was 22 - 32C. -
(2) Liquefaction of gelatin (tested on glucose peptone
gelatin medium at 20C); no liqùefaction of gelatin.
(3) Hydrolysis of starch (tested on inorganic salts-
starch agar medium-ISP-4 at 27C): hydrolysis was
observed after 4 days and the hydrolytic strength was
medium.
(4) Coagulation and peptonization of milk (tested
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at 37C): complete coagulation occurred after 3 days
and peptonization was observed thereafter. The strength
was medium.
(5) Production of melanoid pigment (tested on tryptone
yeast extract broth (ISP-l~, peptQ~e-yeast extract
iron agar ~edium (ISP-6), tyrosine agar medium (ISP-7)
at 27C): melanoid pigment was produced in these media
except tyrosine agar medium.
(6) Utilization of carbon sources (tested on Pridham
Gottlieb medium): glucose and fructose were utilized,
yielding good growth; L-arabinose, ~-xylose, sucrose,
L-rhamnose, raffinose, and D-mannitol were not utilized.
Inositol utilization was doubtful.
(7) Hydrolysis of calcium malate (tested on calcium
malate agar medium cultured at 27~C): no hydrolysis.
(8) Nitrate reduction (tested onl.O% nitrate ~eptone
water at 27C): negative reduction.
Characters described above can be summarized as
follows: the strain MD976-C belongs to streptomyces
which forms whorls when grown on sucrose nitrate agar
medium and glucose-added Pridham Gottlieb medium, but
not spirals; its spore surface is smooth; growth on
various media is pale yellow, pale yellowish brown or
dull brown; and aerial mycelium on the growth is white,
light brownish gray or grayish white; pale yellowish
brown pigment is produced occasionally; melanoid pigment
is produced in organic nitrogen media but not in tyrosine
agar; a wea~ proteolytic activity; medium grade of starch
1051801
hydrolysis. If these characters are compared with
those of known species, then 7 the strain MD9 76~C is
most closely related to Streptom~ces olivor ~iculi
Arai et al described in Antib.iotics and Chemotherapy 7,
435-442, 1957. Description of this spe~ies, the ISP
Type Culture-5105 and the strain MD976-- were directly
compared, and'it was confirmed tbat there is none of
significant difference between the strain MD976-C4
and Streptomyces olivoreticuli. ThUS r this strain was
:
classified to this species.
Since inhibitors of all proteases are produced by
various species of s~,reptomyces and the production is
not limited to a sinc~le species, ~n tllis invention,
instead o. names of the above species, bestatin-producing
streptomyces is used -for description~
Method of testing the activi~y of bestatin inhibit.i-ny
aminopeptidase B:
The method desc.r:ibed by V. K. Hopusu, K. K. Makinen,
G. G. Glenner in Archives of Biochemistry and Biophysics
114, 557, 1966 was modified. To the mixture of 0.3 ml
of 1 mM arginine ~-naphthylamide and 1.0 ml of 0.1 M
Tris hydrochloride buffer (pH 7.0j, 0.7 ml of distilled
water with or without a test material is added and
warmed at 37C for 3 minutes. The reaction is started
by addition of 0.2 ml of aminopeptidase B solution which
is prepared by Sephadex 100 chromatography as described
by Hopusu et al, After 30 minutes at 37C, 0.6 ml of
1.0 M acetate buffer (pH ~.2) containing diazonium salt
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1051801
of o-aminoazotoluene at 1.0 mg/ml and Tween 20 at 1.0% is
added. Fifteen minutes at room temperature thereafter,
absorbance (a) at 530 nm is measured by spectrophotometer.
As the control, by similar means, the absorbance (b)
after the reaction in the absence of bestatin is measured.
The inhibition percent is calculated as follows: ~b-a)/b x
100. The 50% inhibition dose (ID50) of bestatin crystal
was 0.1 yg.
A bestatin-producing strain when grown under
suitable conditions produces bestatin. For production
of bestatin cultivation on a solid medium is possible,
but for production of large quantities cultivation in
a liquid medium is preferred. Any fermentation tempera-
ture can be employed to produce bestatin w~thin the
range in which bestatin-producing organisms can grow,
although 25 - 35C is preferred. Media containing known
nutritional sources for actinomycetes are useful for
production of bestatin. For example, commercial products
such as peptone, meast extract, yeast extract, corn
steep liquor, cotton seed flour, soybean flour, N-Z amine,
casein, sodium nitrate, ammonium nitrate, ammonium
sulfate and other nitrogenous materials such as wheat
bran, xice bran; fish meal etc are useful for nitrogen
source. The commercially available products such as
lactose, glycerol, sucrose, starch, glscose, maltose,
molasses and other carbohydrates or fats in pure or
crude state are useful as the carbon source. Sodium
chloride, sodium or potassium phosphate, calcium carbonate
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105~801
or magnesium ion can also be added. Any material which
have been known for cultivation of actinomycetes are
useful.
The fermentation is continued until bestatin is
substantially accumulated. For example, the MD976-C
strain was inoculated to media containing NaCl 0.3%,
MgSO4-7H2O 0.1%, K2HPO4 0.1%, metal solution 0.1 ml/100 ml,
organic nitrogen source and carbon source. The nitrogen
sources added to media were as follows: meat extract
0.75% and peptone 0.75%; N-Z amine 1.0~ and yeast extract
0.2%; soybean meal 1.5%. The carbon sources added to
the media were as follows: glycerol 2.0%; lactose 2. a%;
glucose 1.0% and lactose 1.0%. The metal solution
consisted of CuSO4-5H2O 700 mgl FeSO4-7H2O 100 mg,
MnC12 4H2O 800 mg, ZnSO4 7H2O 200 mg in 100 ml of distilled
water. Shake flasks of 500 ml volume were used and
100 ml of each medium was placed. It was shake-cultured
at 27 - 29C on a reciprocating shaking machine (amplitude
8 cm, 200 strokes/min~te). Bestatin production was
recognized after 2 days of the shaking culture and the
maximum yield was obtained on 2 - 6 day of the culture.
Among nitrogen sources used, the better yield was obtained
in media containing meat extract 0.75% and peptone 0.75%,
N-Z amine 1.0% and yeast extract 0.2% or soybean meal
1.5% than in the medium containing corn steep li~uor 1.5%.
Bestatin was produced in any media containing glycerol,
lactose, or glucose and the yield was not significantly
different in media containing one of these carbon sources.
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1051801
During fermentation, there was no decrease of bestatin
in the media, suggesting its stable property.
For production o~ bestatin by fermentation, the
ordinary methods used for antibiotics can be employed.
For instance, 100 - 1~0 liters of a medium were placed
in a stainless steel fermenter of 200 liters volume,
and sterilized, bestatin-producing organisms were
inoculated, and fermentation was carried out under
aeration of 200 liters of sterile air per minute with
200 rpm stirring. ~hen, production of bestatin reached
the maximum after 48 - 72 hours.
Bestatin is a stable compound. No decrease of
the activity was observed when a culture filtrate
containing bestatin was made pH 2.0 or p~ 9.~ and heated
at 60C for 30 minutes.
In culture broth, both the liquid part and the
solid part contain bestatin. Bestatin in the solid
part, that is, in the mycelium cake, can be extracted
with methanol. When the fermentation yield of bestatin
is increased, then, the rate of bestatin in the mycelium
cake to that in the culture filtrate increases.
Bestatin is a stable compound, therefore, culture
filtrate itself can be concentrated by distillation,
preferably under reduced pressure, and from the concentrated
solution or the dried residue it is extracted with
organic solvents such as methanol, ethanol, butanol etc
in which bestatin is enough soluble. The cultured beer
containing mycelium can be subjected to extraction with
1051801
a water-immiscible solvent such as propanGl, butanol
or amylalcohol to extract bestatin in the liquid part
and in the mycelium part at one time. When large amount
of culture filtrate is extracted with organic solvent,
butanol is preferably used. ~ counter-current method
can be used to purify bestatin. Crude material of
bestatin can be obtained by concentration under reduced
pressure of organic solvent extracts. Usually this
crude material contains more than 1% of bestatin. This
purity varies according to the amount of bestatin
produced in culture filtrate.
Adsorption method is also useful for extraction
and purification of bestatin. For this purpose, active
earbon, ion exchange resins, alumina, silica gel ete
are useful. For instance, bestatin in culture - filtrates
ean be adsorbed by active carbon and after washed with
water, bestatin is eluted with methanol or aqueous
methanol. The raise of the temperature increases the
elution yield. For instance, active carbon was added
to culture filtrate at 2.0%, the carbon was treated
twice with 20 times volume of methanol at 40C under
stirring, and bestatin was eluted. The yield from the
eulture filtrate was about 80%. Crude powder of bestatin
can be obtained by concentration under reduced pressure
of the methanol extract. Chromatography of alumina
or silica gel can be utilized for purification of
bestatin. Especially, silica gel column chromatography
is useful in the final purification step. In this case,
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10518~L
n-butanol-acetic acid-water-butyl acetate (4~ 6
in volume) is an example used for final purification
of bestatin by a silica gel chromatography. Bestatin
obtained as described above can be crystallized with a
suitable organic solvent such as methanol benzene.
Ion exchange resins are useful in purification of
bestatin. Strong and weak cation~exchange resins can
be used for this purpose.
Properties of bestatin are here described. Bestatin
crystallizes as white needle crystals and melts at
203 - 206C. It is optically active, and -15.1 was
obtained for [d]2 in 0.861% methanol solution. The
elemental analysis gave the following results: calcd.
for C16H24N2O4: C, 62.32; H, 7.82; N, 9.08; O, 20-75;
found C, 60.86; H, 7.79; N, 8.61; O, 21.06. This
molecular formula was supported by the mass spectrum.
Ultraviolet absorption spectrum of bestatin at a con-
centration of 500 ,ug/ml in methanol is shown in Fi~. 1.
The infrared absorption spectrum is shown in Fig. 2,
in which the following bands are observed: 3400, 3300,
3200, 2920, 2850, 1685, 1635, 1530, 1400, 1315, 1265,
1245, 1175, 1125, 1100, 850, 735, 700 cm . The
nuclear magnetic resonance spectrum of bestatin was
taken by Varian HA-100 equipment in tetradeuteromethanol
using trimethylsilane as the internal standard and
the following signals were ob~erved: 0.9 - 1.05 (6H),
1.6 - 1.8 (3~), 2.9 - 3.15 (2H), 3.6 - 3.9 (lH), 4.13 -
4.2 (lH), 4.3 - 4.55 (lH), 7.35 (5H).
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The hydrolysis of bestatin in 6N HCl at 100C
for 18 hours yields L-leucine and an unusual amino
acid. The ratio of these amino acids is 1:1.
Bestatin gives positive Rydon-Smith and ninhydrin reac-
tions. The amino acid sequence of bestatin shown in the
structure was elucidated by fragmentation pattern in
mass spectrum of bestatin methyl ester hydrochloride.
Thus, the structure of bestatin is (2S,3R)-3-amino-2-
hydroxy-4-phenylbutyryl-L-leucine.
Based on the carboxyl group, the esters of bestatin
can be easily synthesized by treating bestatin with
alcohols under usual conditions. Bestatin amide is
also synthesized by a usual method. N-acyl derivatives
can be synthesized by treating bestatin with acid
anhydride or acid chloride. Bestatin is soluble in
acetic acid, pyridine, dimethylsulfoxide, methanol,
ethanol and water, less soluble in propanol and butanol,
hardly soluble in ethyl acetate, butyl acetate, ethyl
ether, hexane, petroleum ether, benzene and chloroform.
Equimolecular addition of acid gives acid salt of
bestatin which is more soluble in water than bestatin.
In thin layer chromatographies using silica gel G
the following Rf values are observed for bestatin in
the following solvents: 0.24, butyl acetate-butanol-
acetic acid-water (4:4:1:1 in volume); 0.13, butyl
acetate-butanol-acetic acid-water (6:4:1:1 in volume).
In high voltage electrophoresis using acidic solvent
such as formic acid-acetic acid-water (25:75:900 in
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~051801
volume) under 3500 V for 15 minutes bestatin moves
to cathode showing Rm value O.6~ taking L-alanine as
1Ø
Bestatin shows 50~ inhibition on aminopeptidase B
of rat liver and leucine aminopeptidase of swine kianey
at concentrations of 0.055 ~g/ml and 0.01 yg/ml res-
pectively. However, bestatin shows only weak inhibition
against aminopeptidase A of human serum: 26.4~ inhibi-
tion at 100 ~ug/ml. Bestatin has low toxicity and
intraperitoneal injection of 295 mg/kg of bestatin
causes no toxicity in mouse.
Type o~ inhibition of aminopeptidase B and leucine
aminopeptidase by bestatin is competitive with the
substrate such as L-arginyl ~-naphthylamide or L-leuc~l
~-naphthylamide and Ki was 6 x 10 8M and 2 x 10 8~
respectively. Addition of bestatin at 6.25 ~g/ml to
tissue culture medium increased the activity of bleomycin
in inhibiting Yoshida rat sarcoma cells 4 - 8 times.
Simultaneous subcutaneous daily injection of 5 mg/kg
of bestatin with 5 mg/kg of bleomycin increased the
bleomycin effect on methylcholanthrene-induced s~uamous
cell carcinoma in rats 4 times.
The following examples are described to illustrate
this invention; however, our invention should not be
limited to the example. Since the characteristics
and the structure of bestatin and the activities of
bestatin, its salts and its esters are now clear and
bestatin which inhibits aminopeptidase B is widely
10518~1
distributed among actinomycetes, it is easily possible
to make various modifications of this invention. In
the light of the foregoing disclosure, this invention
covers a novel product, bestatin and its acid salt,
and processes for production, extraction purification
thereof.
Example 1
A hundred ml of medium containing 2.0% glucose,
2.0~ starch, 2 0% soybean meal, 0.5% yeast extract,
0.25% NaCl, 0.32% CaCO3, 0.0005% CuSO4 5H2O, 0.0005~
MnC12-4H2O and 0.05% ZnSO4 7H2O was placed in a shaking
flask of 500 ml volume and sterilized at 120~C for
20 minutes. PH was adjusted to become 7.0 after the
sterilization. One loopful amount of spores and
mycelium of the strain MD976-C7 on the ayar medium was
inoculated and shake-cultured at 27C on a shaking
machine (180 rpm/minutes). PH was 6.5 on the first
day of the shaking culture and the same pH was main-
tained thereafter. The determination of reducing sugar
by anthrone method indicated the optisal density of
0.855/0.01 ml on the second day, 0.79/0.01 ml on the
fifth day. Maximum production of bestatin was attained
in 5 days and maintained for 10 - 12 days thereafter.
The cultured broth on the fifth day of 50 shaking flasks
were com~ined and filtered, and adjusted to pH 2.0
with 2NHCl. The precipitate was removed and the
filtrate was extracted with 2400 ml and 2000 ml of
n-butanol successively. The butanol extracts were
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combined and evaporated under reduced pressure, yielding
brown crude powder of 2.~ g. Fifty ~ inhibition of
aminopeptidase B was shown by adding 57 ~g of this
powder to the test solution.
Example 2
The strain MD976-C7 was shake-culturea in the
same medium as described in Example 1 for 3 days. One
liter of culture beer thus obtained was inoculated
into 15 liters of the medium placed in a 30-liter jar
fermentor. In this case the medium used for production
contained 1.5~ maltose, 0.3% yeast extract, 1.0% N-Z
amine, 0.3% NaCl. After 2 days at 30C under aeration
(15 liters/min.) and stirring (250 rpm), 0.04 ml of
culture filtrate produced 50% inhibition of aminopeptidase
B. The culture broth on the second day of 4 jar fer-
mentors were combined and filtered. The filtrate was
passed through a column (4 liters, 10 cm in diameter)
of Amberlite XAD -4. After the column was washe~ with
distilled water, bestatin was eluted with 4 liters of
methanol. The active eluate was evaporated under reduced
pressure. The dried active material was dissolved in
2 liters of distilled water, and after adjusted to
pH 2.0 this solution was extracted with 2 liters of
n-butanol. After washed with distilled water, the
butanol solution was evaporated under reauced pressure,
yieiding crude powder of 30 g. Addition of 21 ~g of
this powder to the reaction mixture produced 50% inhibi-
tion of aminopeptidase B.
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Example 3
Bestatin was further purified from a crude powder
which was obtained as described in Example 2. The
powder of 30 g was dissolved in 3 liters of 0.2M
pyridine-acetic acid buffer p~ 3.0 and charged on
500 ml of Dowex 50 X8 (100 - 200 mesh) equilibrated
with 0.2M pyridine-acetic acid pH 3.0 and washed with
2 liters of the same buffer. Gradient elution was
made between 1.0 liter of 0.2M pyridine-acetic acid
(pH 3.0) and 1.0 liter of l.OM pyridine-acetic acid
(pH 4.75). Active fractions were collected and
concentrated under reduced pressure, yielding crude
powder of 2.6 g. .Addition of 2 pg to the test solution
produced 50% inhibition of aminopeptiaase B.
Example 4
The crude powder prepared by the procedure described
in Example 3 was dissolved with S ml of methanol and
charged on 1.5 liters of Sephadex LH-20 column and
eluted with methanol. Active fractions were collected
and concentrated under reduced pressure, yielding light
yellowish powder Pf 0.5 g. Addition of 0.4 ~g produced
50% inhibition of aminopeptidase B.
Example 5
A light yellowish powder prepared by the procedure
described in Example 4 was subjected to silica gel
column chroamtography, using butyl acetate-butanol-
acetic acid-water (6:4:1:1). Active fractions were
collected and concentrated under reduced pressure.
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105~801
A white powder thus obtained was dissolved in a small
amount of methanol and crystal:Lized by adding ethyl
acetate dropwise. White needle crystals of bestatin
was obtained. Addition of 0.10 ,ug to the test solution
produced 50~ inhibition of aminopeptidase B.
Example 6
Bestatin (20 mg) was dissolved in 20 ml of methanol
and 0.5 ml of conc hydrochloric acid was introduced
under stirring for 6 hours at 40C. This solution was
concentrated under reduced pressure and dissolved with
samll amount of methanol and subjected to Sephadex L~-20
chromatography using methanol as solvent. Active fraction
were collected and concentrated under reduced pressure,
yielding white crystals. White crystals thus obtained
was dissolved in a small amount of methanol and crystal-
lized by adding ethyl acetate dropwise. White needle
crystals (m.p. 213 - 216C) (15 mg) was thus o~tained and
showed 50~ inhibition of aminopeptidase B at 8.4 ,ug.
Methyl ester of bestatin was confirmed by the infrared
spectrum and NMR spectrum.
Example 7
Acetic anhydride (2 ml) was added to a solution
of bestatin (20 mg) in methanol (4 ml~, and stirred
for two days at room temperature. After the reaction
was stopped by adding of distilled water (10 ml), the
reaction mixture was dried in reduced pressure yielding
N-acetyl-bestatin methyl ester. This crude N-a^etyl-
b~statin methyl ester was dissolved in methanol and
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1051801
passed through a column of Sephadex LH-20, obtained
pure N-acetyl-bestatin methyl ester. This structure
was ascertained by IR, NMR and mass spectrometry.
The pure material inhibited aminopeptidase B (ID50-
8 pg)~ The modification of C and/or N-terminal of bestatin
reduced the activity of inhibition of aminopeptidase B.
Example 8
A medium (300 liters) containing glycerin 2%,
polypeptone 1~, yeast extract 0.2~, NaC1 0.3~, K2HPO4
0-1%~ MgSO4 7H2O 0- 1%! CuSO4~5H2O 0.0007%, FeSO4~7H2O
0.0001%, MnC12 4H2O 0.0008%, ZnSO4;7H2O 0.0002%, L-
leucine 0.1%, L-phenylalanine 0.1%, anti-foaming agent
(KM-72) 0.01~ was placed stainless steel tunk (570 liters)
and sterilized at 115C for 30 minutes~ Culture beer
(6 liters) of a strain MD976-C7 shake cultured for
3 days was inoculated and incubated at 29C for 96
hours with aeration (300 liters~min) and agitation
(230 rpm). The culture broth was filtered, yeilding
285 liters of broth filtrate (pH 6.3, ID50=0.05 ml),
and adjusted to pH 2.8 with HCl. The broth filtrate
was extracted with 150 liters of butanol. The butanol
extract was concentrated to 98 liters at reduced pressure
and washed with water (p~ 8.45). The butanol layer
was dried in reduced pressure, yielding 101.9 g of
crude powder (ID50=26 ,ug).
Example 9
-
The crude powder which was obtained as
described in Example 8 was purified by the similar
procedure as shown in Example 3 and 4. The crude
10518V~
powder of 90.5 g was dissolved in 150 ml of methanol
and this solution was chromatoc3raphed on Dowex 50 X8
(1500 ml, 100 - 200 mesh) which was equilibrated with
0.2M pyridine-acetic acid at pH 3Ø A linear gradient
between 3 liters o~ l.OM pyridine-acetic acid (pH 3.0)
and 3 liters of l.OM pyridine-acetic acid (pH 4.75)
was carried out. Active fractions were collected and
concentrated under reduced pressure, yielding the
crude powder of 3~89 g. It showed 50% inhibition o~
aminopeptidase B at 1.35 ,ug. Further purification,
this solution was chromatographed on Sephadex LH-20
(2.6 liters) which was equilibrated with methanol,
and eluted with methanol. Active fractions were collected
and concentrated under reduced pressure, yielding light
I yellowish powder of 1.66 g. It showed 50~ inhibition
of aminopeptidase B at 0.52 ,ug.
Example 10
The crude powder which was obtained as described in
Example 9 was purified by the similar procedure as shown
in Example 5. The light yellowish powder of 1.6~ y
was dissol~ed in 150 ml of methanol and mixed with silica
gel. After removal of methanol by evaporation
under reduced pressure, the powder was placed on silica
gel (5 x 40 cm) which was equilibrated with butyl
acetate-butanol-acetic acid-water (6:4:1~1), and eluted
with the same solvent. Active fractions were collected
and concentration under reduced pressure and recrystal-
lized from methanol-ethyl acetate yielded white
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~051801
needle crystals of 185 mg. The purified bestatin
showed 50% inhibition of aminopeptidase B at 0.1 ,ug.
Included within the scope of this invention are
bestatin, acid addition salts of bestatin with inorganic
acids and organic acids such as hydrochloric acid,
sulfuric acid, acetic acid, succinic acid etc and
esters of bestatin such as methyl, ethyl, butyl,
isobutyl etc. For therapeutic purpose, salts which
are more soluble in water than bestatin are useful
and salts and esters which are more insoluble in water
than bestatin but more soluble in oryanic solvents
are useful for extraction and purification.
When desired, there may be admixed with bleomycin
to increase the effect of this antibiotic.
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