JPS5925366A - Novel physiologically active substance diprotin and its preparation - Google Patents

Abstract

NEW MATERIAL:A compound shown by the formula (R<1>, R<2>, and R<3> are H, methyl, or ethyl) or its salt, or ester. EXAMPLE:Diprotin A (namely, L-isoleucyl-L-prolyl-L-isolycine). USE:Having enhancing action on cell-medicated immunity, useful as an immune activator. Useful also as a drug auxiliary for chemotherapy agent of various kinds of cancers. Showing an enzyme inhibitory action on dipeptidylamino- peptidase IV. PROCESS:A culture filtrate of a bacterium (FERM-P 6623) capable of producing diprotin separated from the soil collected at Kugayama, Suginami-ku, Tokyo is fractionated by chromatograpy of Dowex 50 to give a compound shown by the formula. For example, diprotin A having the following properties. Melting point: 178-180 deg.C. Molecular weight: 341. Values of elemental analysis: C=59.42%, H=8.90%, and N=12.05%. Molecular formula: C17H31N3 O4.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention discloses a novel physiologically active substance, diglotin, which exhibits enzyme inhibitory activity against dipeptidylaminozotidase.
Dlprotln) Regarding A, B and C,
It also relates to a method for producing Noprotin A and B.

The present inventors cultivated a bacterium isolated from the soil of Kugayama, Suginami-ku, Tokyo, named strain BMF 673-RFI, and found that the resulting culture had dipeptidyl aminopeptidase IY inhibitory activity. It was discovered that two tripeptide substances exist.

We investigated methods for collecting the fermentation-blocking substances, and succeeded in separating these substances into 4i molecules.We confirmed that these substances were new substances and named them diprotene A and noprotin B. In addition, we synthesized diglotin C as a related compound,
The invention has been completed.

The present inventors further investigated the usefulness of diglotin as a medicine and found that this substance has a role in cell-mediated immunity.

It has been found that it is useful as an immunostimulant.

A study on the efficacy of diprotin A, B, and C in inhibiting the degradation of glycylzololine-nonaphthylamide by sobeptizol aminopeptetase IV.
As shown in the test examples described below, it was confirmed that it had anti-diR7'' tedylaminobeptidase activity.

Therefore, the gist of the first invention is the following general formula: % formula % () (However, in diprotin A, R is an ethyl group, R2 is water X, R3 is a methyl group, and zoprotin B Now, R'
is a methyl group, R,2 is a methyl group, R3 is a water volume,
In diprotin C, R is a methyl group - R2 is a water volume, R3
dizolotine A, B, and C, or salts or esters thereof, have anti-nopezotedylaminopeptimase activity, represented by the following formula: (1) is an ethyl group.

Diprotins A, B and C of the present invention each have the following structures.

(L) (L) (L) Noprotin people (
That is, L-isoleucyl-L-prolyl-L-isoleucine).

H3 匡(L) (L) (L) Diprotin B (i.e. L-) 9ly-L-prolyl-L-
leucine).

1 (T,) (L) (L), P, II Lotin C ([fJchi L-paryl L-70 loli-L-isoleucine).

Diprotin A and B can be obtained as colorless powders by fractionating the cultured P solution of schiflotin-producing bacteria using DOWEX 50W chromatography or the like, as shown in Examples below.

The physical properties of the compound of the present invention are as follows.

That is, Diprotin A has a melting point of 178~! The molecular weight obtained by 80°C spectroscopy is 341. The elemental analysis value is
C59, 42 section,) I8.90%, N 12.0
5. Obtain the molecular formula of C+ 7T-I31N304. The infrared absorption spectrum of diprotin (potassium bromide tablet) is as shown in Figure 1 of the attached drawing.
34CI0°2960, 2875. +650.
1590. 1460. 1390°+240. 1
200. 1145. 1100.9qo, qos
, gso.

680 (t“m) shows a specific absorption band. Proton nuclear magnetic resonance spectrum of noproten A (heavy water solution,
90MHz) is l・I'3~2.06 (CH3X
4. CH2X 2 ).

2.06-3.00 (CH2X2.CHX2), 3.8
6-4-43 (CH2), 4.43-4.83 (
CII X 2 ) and 4-83 to 5.33 (CH
) Shows absorption at ppm.

Diglotin B has a melting point of 158-160 DEG C. and a molecular weight of 327 as determined by quality fii analysis. Elemental analysis value is C5
8.20%, HI3.88 q6. N 12.52
, and obtain the molecular formula of C+6HIN304.

The infrared absorption spectrum of Diprotin B (potassium bromide tablet) is as shown in Figure 2.
21375. 1650. 1585. 1520.
1475°+455. 1400. +370. 1
240. 1200. 1165°+110. 105
0. 1,000. 940. 880. 760.
7101 Ca1r) has a unique absorption band. Proton nuclear magnetic resonance spectrum of noprotin B (heavy water solution, 9QMIIz
) is 1.16~I・73 (CH3x 4), '
1.86-2-20 (ClI2.C)t),
2.20~3.03 ((J12 X 2°CH),
3.86~4.36 (CH2), 4.46~4
劃0(CHX2), and 4.80~5.33 (
CII) Shows absorption in ppm.

Diprotin C can be produced from L-isoleucine by a conventional peptide synthesis method as shown in the Examples below.

The physical properties of dizolotine C are as follows. The molecular weight obtained by mass spectrometry is 327. Elemental analysis value is C58,
Section 37, H8, 82%, N 12.73, giving a molecular formula of CI 6H29N3040. Infrared absorption Nuvector (potassium bromide tablets) is as shown in Figure 3, and has a rating of 3430. 2975. 2890. +65
Q, 1530°1410, 1245. 1210
．． 1150. 1060. +000°8651
It has a specific absorption band at 690 CCm). Proton nuclear magnetic resonance spectrum of nogrotin C (heavy water solution,
90MHz) kl:1.16~2.06 (CH3
X4.

CH2), 2.06-3.06 (CH2X 2.
CI(X2).

3.86-4.43 (CH2), 4.43-4.
86 (CH x 2) and 4.86 to 5.33 (CH
) Shows absorption at ppm.

The second gist of the present invention is to culture a noprotin-producing bacterium that inhibits Bacillus, produce noprotin A or/and non-lotin B in the culture, accumulate S', and then use these alone. A method for producing a novel physiologically active substance diprotin or/and B, characterized in that it is collected as a mixture or as a mixture.

In the method of the present invention, Nobrotene Nama'BzWi is:
It means a bacterium that produces diprotinoid or B, or both. Raw W used for production of noprotin of the present invention
An example of 1ffi is a bacterium named strain BMF 673-RFI, which was isolated by the present inventors from soil collected in Kugayama, Suginami Ward, Tokyo. Is this strain the same as the Institute of Industrial Science and Technology? r9
On July 15, 1983, the entrusted storage was entrusted to R, and entrusted No. 6623 (FEltM-P 6623) was transferred to f-1 and stored at °C.

The Dutch characteristics of this 100-type body are described below.

IIMF673-RF Mycological Ig of strain I-autumn/form Yu (11,7111 cells are culm'I & J x size tyu 0.6
~1.OXI.5 to 2.5 microns.

(2) No particular cell pleomorphism was observed.

(3) Shows active motility and has periflagella.

(4) Contains spores. Its shape is oval and its size is 0.6
~0.7 x 1.4~1.6 micron position is neutral (c
internal), no swelling of bacterial cells was observed.

It is also heat resistant.

, (5) Durham staining is positive.

(6) It is non-acid-resistant.

Growth status in various media All tests were conducted at 30°C except for broth gelatin puncture culture.

(1) Colonies cultured on broth agar plates are dull, opaque, circular, with irregular edges, and pale yellow to pale yellow brown in color.
shows.

After 40 to 48 hours of culturing, the colony develops wrinkled edges. No diffusible pigments are observed.

(21 Gravy agar-based one-sided medium L3 C grows uniformly along the seed line, is opaque and lacks luster,
Pale yellow (pale yellow) Garausu yellow tea C
pale yellow brown). Diffusible dyes cannot be stored.

(：++Nikki9+'1. t・To culture required・24 hours after incubation, a fungal film is formed on the surface of the medium (p), and on the 3rd day, a fungal film is formed on the surface of the medium, and Y appears at the bottom of the test tube. ,''' bodies settle and the medium comes to γ;,j.

(4) Meat juice gelatin;) Chicken sting culture When cultured at 20°C, 1t4 growth was visible on the surface and along the sting line, and liquefaction was observed on the 5th day after 6 u of culture. It is one layer in the type of 0E.

Cultured at 30°C for 24 hours, bacteria H3 are formed on the surface, and wrinkles appear on the crotch from around the 3rd day.

Liquefaction of gelatin (: [, about 28 months after culture, 1
On the 44th day, tl was completed.〇(5) Mill When cultured in BCP milk medium, complete clots 1 were observed on the second day after culture. Almost completed. The reaction is alkaline.

3. Physiological properties (unless otherwise noted, all culture temperatures were 3.
0°C) (1) Reduction of nitrate: Positive (2) Denitrification reaction (Komagata et al.'s method: Takeharu Hoso Hase; Classification and identification of microorganisms, 223 pages, University of Tokyo Press, 197
5 years): Negative + 31 MR test: Negative + 4) V-P test: Positive (5) Indole production: Negative (61 Hydrogen sulfide production: Negative (71 Starch hydrolysis: Positive (8) Utilization of citric acid: Koser's medium, Ch
Grows with the medium of rist-ense.

(9) Use of inorganic nitrogen sources: Both sodium nitrate and ammonium sulfate are used.

Production of aci color x (King A and King B media.

Eiken): In King A medium, a slight yellowish tinge was observed, and in King B medium, a yellow soluble pigment was observed.

0υ Urease (urea medium, Sakae?ijF): Yin
Sex a side Okitume 1-ase: Negative OJ Catalase: Positive A6 growth range: pH 4,4 to pH 8・B Nooi
The optimum pH is 7.0 to 8.0. Growth was observed in the range of 10°C to 45°C, and the optimum temperature was 24°C to 30°C.

fls Attitude towards oxygen: Facultative anaerobic [10-F test (Hugh-Lelfaon method) two shotsjj
? It is a type.

aη Production of acids and gases from sugars (Hugh -L
(using Elfaon's medium) The production of acid production gas from sugars was all negative.

θυ Resonarm Sensitive u tl Upper Delta Ground (Peptone 10g, Beef Extract 10g
, NaCJ 5g, pH 7,4) and separately sterilized lysozyme at 0.00! In addition to being in charge, 13M
The F673-RFI strain was inoculated, and shake culture was performed at 30°C for 124 hours on a rotary shaker (180 rpm, +n) to inhibit bacterial growth.

01 Recitopitelin (LV) reaction (Ueva (reaction)
Nirecito-vitellin agar medium (BBlllln and
Luclchurst: J, appl, Bic
On the second day, the area around the ingested bacteria became transparent, and a clear layer was observed. LV reaction is positive.

Considering the above characteristics, the BMF 673-RFI strain is a facultatively anaerobic, Gram-159, 159-sex spore-spore bacillus, has periflagellae, and exhibits a continuous 1Jll sex. Spores are oval and neutral (
central ) + heat resistance. No swelling of bacterial cells was observed, and the bacteria were non-acid-fast. Growth on a large medium is opaque, with irregular edges, and grows uniformly along tangent lines, and later spreads slightly over the surface of the medium and proliferates. It liquefies gelatin, coagulates milk, and converts it into bezoton. Reduces nitrate, denitrification reaction negative, MR test negative, v-p test 1%
It is gender. Indole is not detected and does not produce sulfide water bulk. It decomposes zero starch and utilizes citric acid.

The urease reaction is negative, the oxidase reaction is IS, and the catalase reaction is positive. It grows in the range of 1l1114.4 to 8.8, and the optimum pH is 7.0 to 8.O. Also,
Grows in the range of 106°C to 45°C, and the optimum water temperature is 240°C.
C to 30"C. Glucose is fermentatively broken down to produce enemies. Production of acids from sugars is L-arabinos, D
-xylose, D-mannose, D-galactose, maltose, 'A2k.) L//゛o-su, D-sorbitol, Inojit 1 D-mannite, glycerin, and starch.

No gas formation is observed from either sugar.

It is resistant to lysozyme and the lecitopitelin (egg yolk) reaction is positive.

Based on the following properties, BMF 673-RFI strain was classified as B.
erg-cya Manual of Doterml
natlve Bacteriology 8thed
(R, E, Buchanan & N,
E. Gibbons, TheWlliamm & Wi.
lklns Company+ Baltlmore,
When searching for Bacillus (1974), 531 pages of Bacillus (B.
It is thought that it belongs to the genus Nclllus). Furthermore, when searching for species, L-Ara produces acetoin from glucose, and no swelling of the bacterial body during spore formation was observed, indicating that L-Ara is North-Ara.
A raccoon that does not produce acid from D-xylose, D-mannite, namely Baetllua Oereua, n,
thuringi-ens1g+ B・mIg&t
I think llrlum town is near green. Among these, H. thuringlenilg is a pathogenic bacterium of insects (see 5 of the above B@rgeyh Manual).
36-e)) Therefore, BMF 673-RF
It is clearly distinguished from the I strain. Characteristics of BMF 673-RFI strain, 13. Hr-eu8 and B megate
A comparison of the descriptions of rlum in literature is as shown in the following table.

As is clear from the table, l3Ml1'673-RFI
The strain is viable in anaerobic culture, has a positive lecitovitellin reaction, and is resistant to lysozyme.
Unlike at-erium, - should B-cereu
It shows a very similar character to s.

Therefore, the BPViF' 673-RF I strain was transformed into Bacillus cereus.
) It was identified as BMF673-RF I.

Next, the method of the present invention will be specifically explained.

When carrying out the method of the present invention, any known nutrient source for bacteria can be appropriately used as the nutrient source used to culture the dizolotine-producing bacteria.

For example, commercially available glycerin, glucose,
Rough 1゛-su, sucrose, starch.

Carbohydrates such as maltose, molasses, fats, etc. are used as carbon sources to produce commercially available Peptide, meat extract, corn staple liquor, cottonseed flour,
Soybean flour, corn gluten + fishmeal +
Yeast extract, NZ amine.

Sodium casein, ammonium nitrate, ammonium sulfate, etc. can be used as nitrogen sources, and salt, phosphate, calcium carbonate, magnesium sulfate, etc. can be used as inorganic nutritional sources. Particularly preferred medium components include V'1, carbon sources such as glycerin, potato starch, and glucose, and nitrogen sources such as N'seripezoton.

There are also meat extracts. Polypeptone 0.5%, Gelcoast 0%, Potato Starte 0, Glycerin 1
．． 0%, meat extract 0.5%, salt 0.5%, calcium carbonate 0.32 width, antifoaming agent ■170 (manufactured by Shin-Etsu Chemical Co., Ltd.)
It is preferable to use a medium containing 0.01.

Liquid culture is preferred for the production of diprotin.

As for the culture temperature, a temperature within the range where dizolotine-producing seeds grow and diprotin can be applied, but a particularly preferable temperature is 25 to 35°C. Cultivation is normally continued in a trench where the substance is produced and accumulates sufficiently in the culture.

For example, 7]? Lipeptone 0.5%, Dalcoast 0
%, potato starch 0%, glycerin 1.0%,
Meat extract 0.5%, salt 0.5%, calcium carbonate 0.32φ, antifoaming agent KM 70 (registered trademark) 0.0
After sterilizing the medium containing 1%, it was inoculated with one white ear of a slant culture of diprotin live blood bacteria and cultured aerobically with shaking at 270C.
After some time, accumulation of diprotin was observed. Dibrotin is produced as well by the talc culture method as by the shaking culture method. For example, put 100t of culture medium into a 200t fermentation tank, sterilize it, and add 3tw:'1J of pre-cultured seed culture.
Sterile air was passed through the tube at 1 hour per minute, and the mixture was stirred at 250 revolutions per minute. Under these conditions, the production of this substance reached its maximum in 65 hours.

Tracking during the cultivation and purification processes of diprotin
The anti-dibegutidylaminobezotidase activity was determined based on the following method. Measurement of anti-dibezotinolaminopeptidase IV activity was performed using OYA (H,
OYA, 1. NAGATSU, i', NAGA
TSU, Bloa-himlca et Blopb
yaica 258.591.1972) This was done using an improved method of the method listed in the id. That is, 0.002 M glycylproline-β-naphthyl amyl' (O,
IΔT Trinu ~ Marein I ¥9! M solution (pH'7.2
) 0.591, a mixed solution containing 0.25 ml of the sample was heated for 37°03 minutes, and then the ammonium sulfate fractionation method (55~ Add 50 μL of di-pear petinolaminopeptidase ■v solution purified at 80°C (ly saturated) at 37°.
After reacting for 30 minutes, 1. containing first garnet GBC at a concentration of Irrq/d and containing the surfactant Twaen 20 at a concentration of 10. OM vinegar mild vinegar solution (pH 4-2) l rrt 'a
: Add L, room temperature kc I S min af'# % & 52
What is the absorbance (a) of 5nnl? It has been decided. At the same time, only a buffer solution containing nobrotin was used.
The absorbance (b) of the sample was measured, and the inhibition rate of Noveptidyl aminopeptidase IV was calculated using the formula C(b-a)/b ]
00yo? [1'! f: I did.

With this fixed sealing method, 1・1μ&/Me's Noburochi/A,
Nobrotin B &i novezotinolaminopenonadase IV was inhibited by 50% (IC5
0) Shita.

Dinolotin A and B exist in the culture solution of nopronan-producing madder and in the i'J form.When collecting noprotin from the culture, there is a method of adsorbing and desorbing it from the culture medium UF solution to an adsorbent. It can be collected with good yield.

As the adsorbent, activated carbon, ion exchange resin, etc. can be used. For example, dizolotine is adsorbed on activated carbon, 9
It is eluted with 0% methanol water (hydrochloric acid acidity-12). Adsorb siderotine by putting activated carbon in culture P in solution 2 and stirring for 4 rt. After filtering, wash the activated carbon with water and add 174 volumes of 90 methanol to the used culture 4>F solution. water(
It is eluted with hydrochloric acid (pal 2 ).

Approximately 50% of diprotin in the medium F solution is eluted.

A crude substance can be obtained by concentrating this 90% methanol water under reduced pressure. Deep ion exchanger chromatography is also used for purification. Especially DOWEX 50
W chromatography is effective, and birinone-vinegar [
Dizolotine A, J: and B can be mutually separated by elution with Doyokutsukuda J solution.

On the other hand, noprotin C is produced starting from L-inleucine by a conventional peptide synthesis method.

Namely, Cal? L with benyl group protected by C
-inleucif (11e 0Bzl), and the amine group is replaced by t-butoxycar+1? Proline (Boe-Pro) and a racemization inhibitor such as 1-hydroxybenzotriazole (n
ffQ using an active esterifying agent, e.g. cyclohexylcalcinide (DCC), in the presence of
Let me @.

The reaction product was Idanobedeted (l3oc-Pro).
-11e 0Bzl ) amino terminal retention i1
After converting the group (B o c ) with trifluoroacetic acid, an amino group was added to this with pennoloxycarbonyl W.
The harvested L-valine (Z-Val) is synthesized using the same peptide synthesis method. (f) IJ-e butide reaction production! 1o(Z-Val-Pro-rla-
0Bzl ), it's amazing! LL is now a protected old name! ; When (2 and T3zl) are deheated, nobrotin C is produced (Acts 3 and 11).

The pharmacological effects of the proboscis activity of the nobrotin of the present invention were investigated. As a result, nopronan A.

It was found that B and C have an enhancing effect on cell-mediated immunity.

The cell-mediated immunostimulatory activity of Nobrotin of the present invention will be explained using test examples.

Test Example 1 This example shows the effect on cell-mediated immunity in normal mice. The effect of nobrotin on cell-mediated immunity was determined by using conjugated hypersensitivity CD, T, Il, which was obtained by glazing sheep red corpuscle (abbreviated as 5RBC) to a mouse leg kick as an antigen. Reference J, Exp, λhdyyi, 1529-1539.1974).

That is, FN!, in which 10 5RBCs were suspended in O-05rlle physiological saline! JCL the turbid liquid: ICR()4N
At the same time, diprotin A was inoculated subcutaneously into the leg of a mouse (6 weeks old), and at the same time, 0.2 kg of diprotin A was administered.
nno 27 kg, 0.05 my/kg.

0.0125++f/9. 0,003131'+g
1 in the form of diprotin A saline solution for administration of /kg
After 0 administration A El administered intraperitoneally, the other leg i
(2) A suspension of 5RBC10' cells suspended in 0.05 ml of physiological saline was subcutaneously administered as a suspension of (HH) for secondary sensitization. 24 hours after (B), the bulge observed in the foot of the foot was subcutaneously administered. (Increase in foot kick thickness) was measured in a caliper. Control animals received subcutaneous injections of SR11C and physiological saline without administration of the test compound. (σ) Foot force point thickening degree was corrected to 100. The cell-mediated immunity enhancing effect of the test compound was determined by comparing the degree of footpad thickening of the treated test animals.

Sideronane B and C were similarly tested.

The test results are shown in Table 1.

From the above results, it was recognized that diprotin A, B, and C are substances that enhance the cellular immune capacity of normal animals. Therefore, the compounds obtained according to the present invention are useful as immunopotentiators. It is also useful as an anti-tumor immune stimulant and as an adjunct to various glaze layer chemotherapeutic agents.

In acute toxicity tests on mice, 5o of diprotin A
No deaths were observed when mice were intravenously administered o trby/kp or when 500 μ/kg of diprotin B and C were intravenously administered to mice. Therefore, diprotin is recognized as a substance with low toxicity.

The above drug containing diprotin as an active ingredient and L-C+J,
Diprotin or any of its pharmaceutically acceptable radicals or d-esters can be mixed with conventional carriers, or even mixed with various chemotherapeutic agents. Examples of salts of diprotin include C, a linguistically acceptable cation in the carboxyl group of nozolotine, such as Vj, an alkali metal cation such as sodium or potassium, or an alkaline earth metal cation such as calcium or magnesium. Acid addition salts of nozolotine with amino acids, such as pharmaceutically acceptable salts such as λ-ge hydrochloride, or with organic acids, such as vinegar, are also included. Examples of esters include alkyl esters, acetoxymethyl esters, and piparoyloxymethyl esters, which are easily metabolized and decomposed in vivo.

The compound or drug of the present invention is administered orally.

Either injection or rectal suppository may be used. When preparing an injection, add a pH adjuster and a mild ionic acid ('ij) to the above-mentioned ringing component compound.
A lyophilized injection can be prepared by adding a stabilizer, a stabilizer, 9 excipients, and performing lyophilization in a conventional manner to prepare a lyophilized injection. j adjustment agent, niappentsu agent.

Subcutaneous, intramuscular, or intravenous injections can also be prepared in a conventional manner by adding a stabilizer, an isotonic agent, a local narcotic, etc. When preparing oral solid preparations, use Yagura Eisei S, ? After adding excipients and, if necessary, binders/disintegrants, lubricants, coloring agents, flavoring agents, and odor agents to the compound, it is processed into tablets, rolled tablets, and granules using conventional methods.・Can make powders, capsules, etc. When preparing oral liquid preparations, add auxiliary compounds and flavoring agents.

By adding laxatives, stabilizers, flavoring agents, etc., syrups and dry syrups can be made by the C1 method.

When preparing rectal herbal medicine preparations, add active ingredient compounds such as
After adding excipients and, if necessary, a surfactant, it can be made into a suppository by a conventional method.

Diprotin administration to rats varies depending on the symptoms, but in adults, 1
0.02-2Q Orr4 as diprotin once a day
It is best to administer twice. Furthermore, when used in combination with a cancer chemotherapeutic agent or an immune enhancer, diprotin may be used in combination with the usual use of the cancer chemotherapeutic agent or immune enhancer and in an amount within the above range.

Next, an example will be shown regarding the production of dizolotine of the present invention, but since the physical and chemical properties of this substance, the production method, and its production method have been clarified by the present inventors, they are shown in this book J 1YII1 An. It is easy to modify the method described, and the invention is not limited to the examples described below. For example, in this study, Nozolotine A and B were produced in a bM 19j manner, but chemical synthesis of these is also possible.
It can be synthesized very easily by emitting light from L-inleucine and appropriately using a known method for synthesizing the compound.

Example: Bacillus cereus BMF'6 as a nobrotin-producing bacterium
73-1 Seven F1 stock (fine] Niken Yayori No. 6623) 1 ton of platinum from the diagonal ifi'ij Miyayo, +20 in advance
'c.

The culture medium was incubated for 20 minutes (dispense 1los+f! into a 500r fart rotary flask at a volume of 0.5 tons).

Gluco-71%+ +le Tetostana 1%, Glycerin 1%, Meat Extract 0.5% 1 Salt 0.5? bT calcium carbonate 0.32%, antifoaming agent (KM-70) 0.01
%75°) was inoculated. 27°C1 180 per minute
Noprotin production was examined over time under rotating culture conditions.

As a result, after 48 hours of culture, diprotene Wti lt1
The concentration of diprotin reached a plateau and did not decrease until the next 6 days of culture, when diprotin reached a constant level due to the inhibitory activity of dibeptinol ε-nopeptidase IV.

Example 2 BMF673-
46 tons of the culture medium a obtained by culturing the RFI strain was subjected to a continuous centrifuge to obtain 454 supernatants. Above this 1ft liquid o
) Nopegutynolaminopeptidase TV (!Ii harmful activity (IC50) was 39 μt/dile.

900 g of culture charcoal was added to the obtained culture supernatant liquid 451, stirred, and adsorbed. The activated carbon was filtered through P paper, and after washing with water, p[l
2 liters of 9056 methanol water under acidic conditions
I put out 64 in OT. I put this eluate under reduced pressure. +A I did. Dilute this concentrate to 2t with water and use DOWEX 5.
0W (H”7) adsorbed on a 1.4 t column,
After washing with water, it was eluted with IN aqueous ammonia. The eluate was concentrated to dryness under reduced pressure to obtain 56 g of powder. The 2-peptinolaminopezotidase inhibitory activity (IC5o') of this crude powder was 143 μ9/rrl.

The obtained crude powder was dissolved in 2 tons of 0.2 M pyrinone-acetic acid solution VC at pH 3.2, and acetic acid was added to the solution to pH 3.
,2. It was adsorbed onto a DOWEX 50W (pyridinium type, x4, 100-200 mesh) I-4t column equilibrated with the same buffer solution in advance, and eluted with the same mild+mj solution. The active fraction was condensed to dryness under reduced pressure to obtain 9.8 g of crude powder. The crude powder had an inhibitory activity (IC5o) of Tsubabuchituru aminopeptidase mV of 67 μg/m
It was l. Next, this crude powder was dissolved in 50 trl of water, applied to a 1.6 t Sephadex G25 column, and developed with water. The active fraction was concentrated to dryness under reduced pressure to obtain a crude powder of 2.67y()-treated bunanolaminopezotidase IV.
jil harmful activity IC5o = 20yg/mt).

This coarse powder 2.671 was dissolved in 28 lod of ammonia water, 50% of ammonia water (6%) was added, and adsorbed on 300ml of silica. A mixture of I) was developed and the active fraction was concentrated to dryness under reduced pressure to obtain a crude powder of 620 my (dibeptenolaminobeptedase IV inhibitory activity IC50 = 5.75 μ9/mt
) was obtained. Next, this crude powder was dissolved in 30 tnl of 0.2 M birinone-acetate buffer (pH 3.2), acetic acid was added to adjust the pH to 3.2, and DOWEX 50W (pyridinium type, x4 ,100～
Two active fractions were obtained by applying the mixture to a column of 200 mesh) and developing it with the same mild glaze J solution.

By concentrating and drying the active fraction eluted first, 2
A crude powder of diprotin B of 20vv (dipeptidyl aminopeptidase IV inhibitory activity ICAO = 7.5μ9/me) was converted to IIOmy (dipeptidyl aminopeptidase IV) by condensing and drying the active fraction eluted later. A crude powder of diprotin A with ZeIV inhibitory activity IC50 = 6 μg/mt was obtained.

10 f of each of the resulting coarse powders of diprotene A and B
f+7! of water, added 5 g of dissolved silica dal, dried under reduced pressure, applied to a 50 vrl silica silica column, and prepared chloroform: methanol: acetic acid: water (
By developing with a mixture of 60:8:2:l) and concentrating each active fraction to dryness under reduced pressure, a crude powder of Diproten A of 43.6 vry (IC5o=1.4μ9/
art ) and coarse powder of diprotene B +45.5η
(IC50 = 5.5 μ9/ml was obtained.

(4 ml each of diprotin A and 130 No. 11 powder) pH 3, 21) 0.2 M pyrinone-vinegar r;
2, each with 50 ml of Downic 7 = 50 W (pyridinium type l) equilibrated in advance with the same buffer.
The active fractions were divided into G% 2i' under reduced pressure.
22.5 g of white powder (di4putinolaminobezotidase IV l) to pure protin by drying.
l harm f(5j4 ICAO=I, lμg/at
) and 64.8p of pure Soprotin B white powder! 7
(Nobeptinolaminopeptidase IV inhibitory activity IC5
o = 5.5 μ97 mrt) was obtained.

Example 3 (a) 1.967! 9f7) Dissolve L-isoleucine bennorester tosylate (manufactured by Protein Research Foundation) in 50 tnt of dichloromethane, add 7 ml of triethylamine while cooling with water, and then dissolve 1.076 g of t-butoxycarbonyl-cyclozoline (protein Todo 02g of 1-hydroxybenzotriazole (Protein Research Foundation) and 1.24g of dicyclohexylcarne ζ diimide were added, reacted for 2 hours under water cooling, and then further reacted for 14 hours at room temperature. I let it happen. After concentrating the reaction solution, 200 Inl of ethyl acetate was added again to dissolve in a bath, and the mixture was washed sequentially with 200 me each of 10 parts citric acid, water, 4 parts sodium carbonate and water. After washing, anhydrous sodium sulfate was added to the ethyl acetate layer, and after dehydration, IJ was removed and concentrated under reduced pressure to give 2 g of t-butoxycarbonyl-L-zorolyl-L-yne. Leucylpennorester wo ni'4 ta.

[Naka] Add trifluoroacetic acid 1 Ome to 2 g of the above-mentioned product obtained in the previous section, leave it at room temperature for 30 minutes, and then vacuum the mixture.
! ! The remaining trifluoroacetic acid was distilled off by adding chloromethane and concentrating under reduced pressure to obtain 2.8 g of a candy-like substance. Dissolve this in 150 rrrl of water and 50 tnl of ethanol and add DOWEX C.
C1-type, X4.100-200 mesh) l OO
After passing through an rnt column, it was washed with water. The passing liquid and the washing liquid were combined and freeze-dried to obtain 1.679 L-Zorolyl-L.
-Isoleucyldundyl ester hydrochloride was obtained.

e′t Reaction product obtained in the previous section 683 +119
was taken, dissolved in 20 ml of dichloromethane, and mixed with 7 ml of triethylamine O-2' and 483 ml of Caly! under water cooling. ? Add benzoxy-L-PaIJ (manufactured by Protein Research Institute) and add 392 μ of 1-hydroxybenzotriazole and 4
80η of dicyclohexylcarbodiimide was added, and the mixture was reacted for 2 hours under water cooling, and then further reacted for 14 hours at room temperature.

Concentrate the reaction solution under reduced pressure and add 200 ml of ethyl acetate again.
The solution was dissolved in 200 d of O, IN hydrochloric acid/water, 4-dose sodium bicarbonate solution, and then washed with vinegar in sequence. After washing, the vinegar h5 ethyl layer P was dehydrated by adding anhydrous sodium sulfate, and concentrated to dryness under reduced pressure to obtain 1.OI I) Cal H,4
Henzoxy L-valyl L-zorolyl L-inleucine pendyl ester was obtained.

) 700 mg of the reaction product obtained at both ends was added with 3 mL of acetic acid.
・Dissolve in a mixture of 5tnl - water 1offIg and methanol 5d, add 500μ of 10% palladium on carbon (manufactured by Yoken Fine Chemical Co., Ltd.), catalytically reduce it in a hydrogen gas stream at normal pressure for 3 hours, and then filter. The p solution was concentrated to dryness under reduced pressure. This is 100. Dowex 50W (11 type, x4.IQO-200 mesh) 40
It was adsorbed to rnl and eluted with IN aqueous ammonia. The eluate was concentrated to dryness under reduced pressure to obtain pure L-valyl-
L-prolyl-L-isoleucine (Val-Pro
1e) That is, a crystalline white powder of dizolotine C was obtained.

The dipeptidylaminobezotidase inhibitory activity (Crcso) of this diprotin C was 1.9μ9/d.

[Brief explanation of the drawing]

Figure 1 shows the infrared absorption spectrum of dizolotene, Figure 2 shows the infrared absorption spectrum of diprotin B, and Figure 3 shows the infrared absorption spectrum of diprotin C.

Claims (1)

[Scope of Claims] (1) Anti-dipeptidyl aminopeptidase represented by the general formula (wherein R, R and R each represent a hydrogen-methyl group or an ethyl group) ■A new physiologically active substance that exhibits activity Dinolotine or its salt or ester. 2. A compound according to Item 1 of the Hartt's Required Range, which is diprotene A represented by the formula %. 8. The compound according to claim 1, which is dizolotine B represented by the formula. 4. The compound according to claim 1, which is dizolotine C represented by the formula (i'H3 (L) (L) (L)). 5. Cultivating diplotene-producing bacteria belonging to the genus Bacillus and collecting both. A method for producing a new physiologically active substance diprotin or/and B, characterized in that: