CA1051801A - Bestatin, an enzyme inhibitor from streptomyces - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

The invention relates to bestatin inhibiting aminopeptidase B, leucine aminopeptidase and bleomycin hydrolase, enhancing the antitumor effect of bleomycin and having the following structure and a process for the production thereof:

Description

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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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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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 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;

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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.

- 4~a) -~, lOS1801 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 _ - 5 O
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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~:

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 105180~L

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 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 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 ~_ *Trade Mark ~j ,~,. -.~ .. . . ..

-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.

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 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, 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).

-- *Trade Mark -. ~.

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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 ~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 lOS1801 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.

_ *Trade Mark -Bi 1051~301 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.

- *Trade Marks 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 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 ~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.

Claims (15)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A process for the production of bestatin which inhibits aminopeptidase 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; [.alpha.]?2=-15.1° (c=0.861, methanol);
positive Rydon-Smith, ninhydrin; negative Sakuguchi;

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, and recovering bestatin from the nutrient medium.

2. The process of Claim 1 wherein bestatin is recovered from the nutrient medium containing bestatin by adsorption on an ion exchange resin and subsequent elution.

3. The process of Claim 1 wherein bestatin is recovered from the nutrient medium containing bestatin by adsorp-tion on carbon and subsequent elution.

4. The process of Claim 1, wherein bestatin is recovered from the nutrient medium containing bestatin by extraction with a water immiscible solvent in which bestatin is more soluble than in water.

5. The process of Claim 4 wherein said water immiscible solvent is selected from the group consisting of propanol, butanol and amylalcohol.

6. The process of Claim 1 wherein bestatin is produced in the nutrient medium in a crude solid form, and the bestatin is extracted with an organic solvent in which bestatin is soluble.

7. The process of Claim 6 wherein said organic solvent is a lower alkyl alcohol.

8. The process of Claim 6 wherein said organic solvent is selected from the group consisting of methanol, ethanol and butanol.

9. The process of Claim 1, for preparing an ester of bestatin, further comprising reacting bestatin with the corresponding alcohol to form an ester bond between the carboxyl group of bestatin and the alcohol.

10. The process of Claim 9 wherein said alcohol is methanol.

11. The process of Claim 1, for preparing an acid addition salt of bestatin, further comprising reacting bestatin with the corresponding acid.

12. Bestatin which inhibits aminopeptidase 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; [.alpha.]?2=-15.1° (c=0.861, methanol); positive Rydon-Smith, ninhydrin; negative Sakaguchi;
whenever prepared by the process of Claim 1, 2 or 3 or by an obvious biological or chemical equivalent thereof.

13. An ester of bestatin whenever prepared by the process of Claim 9 or an obvious biological or chemical equivalent thereof.

14. The methyl ester of bestatin whenever prepared by the process of Claim 10 or by an obvious biological or chemical equivalent thereof.

15. An acid addition salt of bestatin whenever prepared by the process of Claim 11 or by an obvious biological or chemical equivalent thereof.