JPH06100588A - Hiv protease inhibitor mer-n5075a and its production - Google Patents

Abstract

PURPOSE:To obtain the subject new enzymic inhibitor useful as an anti-human immunodeficiency virus(anti-HIV) agent for a therapeutic agent, etc., for acquired immunodeficiency syndrome by culturing a microorganism, belonging to the genus Streptomyces and capable of producing an HIV protease inhibitor Mer-N5075A. CONSTITUTION:The objective human immunodeficiency virus(HIV) protease inhibitor Mer-N5075A, expressed by the formula and usable as an anti-HIV agent is obtained by inoculating a microorganism, belonging to the genus Streptomyces and capable of producing an HIV protease inhibitor Mer-N5075A [e.g. Streptomyces.chromofuscus Mer-N5075 (FERM P-13134)] into a culture medium, culturing the microorganism at 28 deg.C for 3 days on a rotary shaking incubator, providing a seed culture solution, then inoculating the resultant seed culture solution into a culture medium in a jar fermenter, culturing the culture solution at 28 deg.C for 88hr according to the spinner culture with aeration, filtering the culture solution, collecting a filtrate, extracting the filtrate with n-butanol, concentrating the extract solution under reduced pressure, dissolving the prepared residue in methanol and purifying the resultant solution according to the silica get chromatography.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel HIV protease inhibitor Mer-N5075A and a method for producing the same.

[0002]

2. Description of the Related Art Acquired Immunodeficiency Syndrome
Deficiency Syndrome (AIDS) is a human immunodeficiency virus (Human Immunodeficiency) that is a type of retrovirus.
It is an immunodeficiency disease caused by Virus: HIV.

[0003] With the progress of research on HIV, enzyme groups specific to this virus have been clarified, and inhibitors aimed at anti-HIV drugs have been developed. In particular, the reverse transcriptase inhibitor azidothymidine (AZT) is one of the few anti-HIV drugs currently in clinical use, but it has serious side effects and the emergence of resistance-acquiring virus has become a problem.

[0004] HIV protease is an enzyme essential for mature HIV infection, its structure has been largely elucidated, and it has been clarified that it belongs to acidic proteases. By finding an inhibitor, a new anti-HIV drug. Is under development [eg Sham et al., Biochemical and Biophysica].
l Research Communications, vol.175, 914-919 (1991), etc.].

[0005]

DISCLOSURE OF THE INVENTION The present inventors aim to develop a new anti-HIV drug, and
While continuing the search for an inhibitor of IV protease, we found that a substance having HIV protease inhibitory activity was produced in the culture of a certain strain of actinomycetes, and the active substance was isolated. The present invention was completed by separating and determining its physicochemical properties and structure.

[0006]

SUMMARY OF THE INVENTION Provided by the present invention.
The HIV protease inhibitor Mer-N5075A has the following formula (I)
Indicated by.

[0007]

[Chemical 2]

The physicochemical properties of this compound are as follows. (1) Color and shape: white powder. (2) Molecular formula: C ₃₀ H ₄₃ N ₇ O ₆ (3) Mass spectrum (FAB-MS, matrix: glycerin) Positive: 598 [(M + H) ⁺ ] Negative: 596 [(MH) ^- ] (4) Melting point: 198-199 ℃ (5) Specific rotation: [α] ²⁸ _D -27.6 ° (c0.11, acetic acid)

(6) Ultraviolet absorption spectrum: shown in FIG. λMeOH max (nm): 268.3 (ε231), 264.6 (ε281), 258.7
(ε328), 253.4 (ε289), 248.1 (ε245) (7) Red external absorption spectrum (KBr method): Shown in FIG. The major absorptions are shown below. (cm-1): 3382 (br), 2963, 2934, 2874, 1642, 1611, 15
53, 1499, 1470, 1454, 1395, 1250, 1107, 1044, 700 (8) Solubility: Soluble in dimethyl sulfoxide, acetic acid, slightly soluble in methanol, insoluble in water. (9) Color reaction: Phosphomolybdic acid reagent, Rydon-Smith reagent, and Sakaguchi reagent give color. It does not develop color with ninhydrin reagent. (10) Rf value of thin layer chromatography: 0.27 (n-butanol: acetic acid: water = 10: 1: 1) on silica gel plate F-254 manufactured by Merck Ltd. 0.49 (n-butanol: methanol: water = 4: 1) : 1)

(11) ¹ H-NMR spectrum (400 MHz, DMSO-d ₆ ):
It is shown in FIG. δTMS (ppm): 0.77 (3H, d, J = 7.0Hz), 0.78 (3H, d, J = 6.6Hz),
1.43 (3H, m), 1.69 (1H, br s), 1.90 (1H, sextet, J = 6.6Hz),
2.63 (1H, dd, J = 13.9,8.1Hz), 2.82 (1H, m), 2.84 (1H, dd, J = 1
3.9,5.9Hz), 2.96 (2H, br dd, J = 13.2,5.1Hz), 3.07 (1H, m),
3.92 (1H, m), 4.00 (1H, m), 4.07 (1H, dd, J = 8.8,7.0Hz), 4.14
(1H, m), 6.08 (1H, br d, J = 7.0Hz), 6.66 (1H, brs), 7.18 (10
H, m), 7.46 (1H, br s), 7.64 (1H, br d, J = 8.4Hz), 7.84 (1H, b
rd, J = 6.6Hz), 8.84 (1H, br s)

(12) ¹³ C-NMR spectrum (100 MHz, DMSO-
d ₆ ): As shown in FIG. δTMS (ppm): 17.88q, 19.05q, 24.70t, 29.11t, 30.51d, 36.
22t, 38.33t, 40.15t, 52.17d, 52.57d, 56.51d, 57.70d, 62.2
3t, 125.38d, 125.74d, 127.55d (2C), 127.94d (2C), 128.93d
(2C), 129.36d (2C), 138.92s, 139.31s, 157.24s, 157.89s, 1
Data on the multiplicity of 70.25s, 172.02s, 175.61s signals were obtained by DEPT test. (13) Distinction between neutral, acidic and basic: It shows basic when developed by silica gel thin layer chromatography and then colored with a pH indicator.

HIV protease inhibitor of the present invention Mer-N50
75A is a compound having a peptide skeleton as shown in the above formula (I). This compound is a known peptide compound MAPI obtained from a culture of a microorganism belonging to the genus Streptomyces as an alkaline protease inhibitor.
(S. Murao; Agricultualan
d Biological Chemistry, 42, 2209-2215 (1980)). Also M
API is also known to have HIV protease inhibitory activity (Journal of Antibiotics, 44, 1019-1022 (1991))
.

However, the Mer-N5075A substance is a novel substance which is distinguished from the known compound MAPI by its molecular formula, physicochemical properties and structural characteristics. Of the present invention
The HIV protease inhibitor Mer-N5075A can be produced, for example, by culturing a microorganism belonging to the genus Streptomyces and having the ability to produce Mer-N5075A in a nutrient medium, and collecting Mer-N5075A from the culture.

The search for the microorganism capable of producing the Mer-N5075A substance can be carried out, for example, as follows. 8
Synthetic peptide consisting of 4 amino acids (Val-Ser-Gln-Asn-T
yl-Pro-Ile-Val) Substrates are hydrolyzed by HIV protease expressed in Escherichia coli into the reaction system, and the culture solution of various microorganisms is added to detect the progress of the hydrolysis reaction by high performance liquid chromatography or the like. Compared to the hydrolysis reaction when no microbial culture is added, by isolating and confirming the active substance from the microbial culture in which the reaction is inhibited,
It is possible to obtain a microorganism capable of producing the target enzyme inhibitor.

One of the microorganisms capable of producing the Mer-N5075A substance thus discovered is the actinomycete Mer-N5075 strain isolated from the soil of Taketomi-cho, Yaeyama-gun, Okinawa Prefecture. The mycological properties of this Mer-N5075 strain are as follows.

(1) Morphological properties In ISP-4 medium, helicoids are formed from well-branched base hyphae.
(spiral) aerial hyphae extend. L0 on top of mature aerial mycelium
Form a spore chain consisting of ~ 50 elliptic to cylindrical spores. No sporangia is observed. Spore size is 0.7-l.
At 0 × 1.0 to 1.5 μm, the surface of the spores is spiny or hairy (spiny to hairy) with no flagella.

(2) Growth state in various media All cultures were performed at 28 ° C. The color tone is described by Container Corpo
(ration of America) Color Harmony Manual
It was displayed based on (Color Harmony Manual). 1) Yeast and malt agar medium Growth is good and aerial hyphae are well developed. The color of the aerial mycelium is cream, but produces gray (g) spores on it.
The reverse side of the culture is brownish orange (3ea). No soluble dye is visible.

2) Starch / inorganic salt agar medium Growth is good, many aerial mycelia and spores are settled, and the color of the surface of the colony, the color of the back surface and the soluble pigment are the same as those on the yeast / malt agar medium. Is. 3) Tyrosine agar medium Growth is good. Set aerial hyphae and spores. It produces melanoid pigments in the medium.

(3) Assimilation of various carbon sources Various carbon sources were added to the Pridham-Gotrieve agar medium, and growth was observed. 1) L-arabinose + 2) D-xylose + 3) D-glucose + 4) D-fructose + 5) Sucrose + 6) Inositol + 7) L-rhamnose + 8) Raffinose-9 ) D-Mannier ++ assimilates, -does not assimilate

(4) Properties of cell wall components Hydrolyzed cells were analyzed by cellulose thin layer chromatography to find that the isomeric form of diamino pimelic acid, which is the cell wall component of this bacterium,
It was LL-type and had no characteristic sugar component.

From the above mycological properties, it is clear that the Mer-N5075 strain is a bacterium of the genus Streptomyces, and International Journal of Systematic Bacteriology, Vol. 18, No. 2, 1968. (Inte
rnational Journal of Systematic Bacteriology, vol.1
8, No. 2, 1968) in the International Streptomyces Proj
(ect) was compared with the mycological properties of Streptomyces strains approved by (ect), except that the color tone of the back surface of the culture is slightly different, and that this strain assimilates sucrose and cannot utilize raffinose. , And the description of Streptomyces chromofuscus was almost the same. In addition, the Mer-N5075 strain is the Mer-N5075A.
It has the characteristic of producing MAPI at the same time as the substance.

Therefore, the present inventors have confirmed that this strain is Streptomyces chromovuscus Mer-N5075 (Streptomyces).
chromofuscus Mer-N5075), and on August 31, 1992, at the Institute of Microbiology, National Institute of Advanced Industrial Science and Technology, Microorganisms Research Institute No. 13134
It has been deposited with the number of (FERM P-13134).

The Mer-N5075 strain is liable to change its shape as in the case of other actinomycetes, and can be mutated by an artificial mutagenesis means using, for example, ultraviolet rays, X-rays, radiation, chemicals and the like. , Whichever mutant,
Any material capable of producing the Mer-N5075A substance can be used in the present invention.

Culturing of the Mer-N5075 strain can be carried out, for example, as follows. As a culture source of the medium, for example,
As the carbon source, glucose, galactose, maltose, dextrin, starch, starch syrup, soybean oil and the like can be used alone or in combination, and as the nitrogen source, ammonium chloride, urea, ammonium sulfate, ammonium nitrate can be used. , Sodium nitrate, peptone, meat extract,
Yeast extract, dry yeast, corn steep liquor, soybean meal, oatmeal, casamino acid, bacto soyton and the like can be used alone or in combination. Inorganic salts such as sodium chloride, magnesium sulfate, calcium carbonate, phosphates and metal salts can be added as required. In addition, organic substances such as vitamins and amino acids that promote the growth of this strain and the production of the Mer-N5075A substance can be added. Further, if necessary, a defoaming agent, for example, Adecanol (trade name: manufactured by Asahi Denka Kogyo Co., Ltd.), silicon or the like can be added.

As the culture method, liquid culture under aerobic conditions, especially deep culture method, is suitable as well as the known culture method used for the production of antibiotics by actinomycetes. The temperature suitable for culturing is within the range of 20 to 30 ° C., and the pH of the culture solution at that time is preferably around neutral. In liquid culture,
Usually, when the culture is performed for 3 to 5 days, the Mer-N5075A substance is accumulated in the medium. When the amount of the Mer-N5075A substance accumulated in the culture solution reaches the maximum, the culture is stopped, and the Mer-N5075A substance, which is the target substance, may be separated and purified from the culture solution obtained by filtering the bacterial cells.

In collecting the Mer-N5075A substance from the culture, a conventional separation means utilizing its properties, for example,
Synthetic adsorption resin such as Diaion HP-20 (manufactured by Mitsubishi Kasei), activated carbon, Diaion SK106 (Mitsubishi Kasei)
(Manufactured by K.K.), ion-exchange resins such as Sephadex, gel filtration agents such as Sephadex and biogel, or column chromatography with silica gel can be appropriately combined and used.

The Mer-N5075A substance of the present invention has excellent HIV protease inhibitory activity and is expected to be used as an anti-HIV agent.

The HIV protease inhibitory activity of the compound of the present invention can be measured, for example, by the method described below. Add 4 μl of test sample to the substrate solution (final concentration of each substrate 1 m
g / ml, 50mM MES (pH6.0), 2M NaCl, 1mM EDTA, 1mM DTT, 0.1
% Triton X-1). More about
Add 0.2 U of HIV protease crude enzyme solution to make a total of 40 μl, and incubate at 37 ℃ for 20 minutes. Then, heat in boiling water for 3 minutes to inactivate the enzyme, and then add 0.1 M Tris-HCl (pH 7.
5) After adding 80 μl of the buffer and centrifuging, the substrate concentration in the high performance liquid chromatography reaction solution is analyzed, and the enzyme activity may be calculated from the reduced amount. At this time, the substrate is the synthetic peptide Val-Ser-Gln-Asn-Tyr-Pro-Ile-Val or Ser-Gln-Asn.
-Tyr-Pro-Ile-Val can be used, especially Val-Ser-Gln-
Asn-Tyr-Pro-Ile-Val is preferred. Also the HIV to use
The protease is preferably one obtained by genetic engineering, for example, synthetic HIV is provided downstream of the promoter of pKP1500.
It is possible to use HIV protease produced by a transformant obtained by introducing the vector pMAK105 obtained by inserting the protease gene into the host E. coli KP3998 (T. Miki et al. Protein Engineering 4,3
27 (1987)).

For high performance liquid chromatography, a reverse phase column (TSK-gel ODS120-T (manufactured by Tosoh Corporation)) was used.
The peptide substrate was detected by absorption at 210 nm using 23% aqueous acetonitrile (containing 0.1% trifluoroacetic acid) as a mobile phase. Under these conditions, the substrate was eluted at around 5-6 minutes and the enzymatic degradation products were eluted earlier. The peak area of the substrate on the chromatogram was measured, and the enzyme inhibition degree (%) was calculated according to the following formula. [1- (Substrate peak area reduction due to enzyme when sample is added / Substrate peak area reduction due to enzyme when sample is not added)]
× 100 (%) The HIV protease inhibitory activity of the Mer-N5075A substance of the present invention had an IC ₅₀ value (concentration for 50% inhibition) of 40 μg / ml.

The compounds of the present invention can be administered orally, topically or parenterally as HIV protease inhibitors, wherein the compounds are used alone or in combination with one or more pharmaceutically acceptable carriers or excipients. It can be administered in combination with a dosage form. Depending on the intended dosage form, the compounds of the invention may be formulated in solid, semi-solid or liquid dosage forms such as tablets, pills, capsules, powders, liquids, suspensions and the like. it can. The preparation containing the compound of the present invention contains a conventional pharmaceutical carrier or excipient, and may further contain other pharmaceutical compounds and the like.

Examples of the present invention will be shown below, but these are merely examples and do not limit the present invention.

[0032]

【Example】

Example 1 From a slant medium (potato-dextrose agar medium) of the Mer-N5075 strain, one platinum loop was added to 50 ml of a seed medium (potato starch).
2%, glucose 1%, soybean powder 2%, potassium phosphate
0.1%, magnesium sulfate 0.05%, unadjusted pH)
A 500 ml Erlenmeyer flask was inoculated and cultured at 28 ° C. for 3 days on a rotary shaker to obtain a seed culture solution. 200 ml of this seed culture was added to the production medium (glycerol 2.5%, meat extract
0.5%, polypeptone 0.5%, yeast extract 1%, sodium chloride 0.2%, magnesium sulfate 0.05%, potassium phosphate 0.05%, calcium carbonate 0.32%, antifoam agent 0.05
%, PH 7.4) Inoculate two 10-l capacity jar fermenters containing 5 liters, and culture with aeration and stirring at 28 ° C for 88 hours (aeration rate of 5 l / mi).
n., stirring 300 rpm).

After completion of the culture, the culture broth (about 10 liters) was separated into cells and filtrate by filtration to obtain about 8 liters of culture filtrate. The culture filtrate was extracted twice with 4 l of n-butanol without adjusting the pH. The extract was concentrated under reduced pressure to obtain 7.5 g of an oily substance. This oil-like substance was washed with n-hexane to remove the oily substance, and 4.5 g of reddish brown powder was obtained. The whole amount of this reddish brown substance was dissolved in 50 ml of methanol, and the insoluble matter was removed by centrifugation.

Next, this supernatant solution was divided into two portions and purified. Sephadex LH-20 column (manufactured by Pharmacia, 4φ x 45 cm), half of which was previously filled with methanol
And developed with methanol. Fractions of 18 ml were collected, and fractions 7 to 14 were collected and concentrated to dryness. The same operation was performed for the remaining half amount to obtain a concentrate.
Next, the concentrates were combined, dissolved in 20 ml of methanol, 10 g of silica gel (Merck, Art.7734) was added, concentrated to dryness, and the solvent was removed by a vacuum pump for 18 hours at room temperature. . Silica gel column (Merck, Art.7734, 4) pre-filled with n-butanol: acetic acid mixture (5: 1).
The adsorbed silica gel prepared above was attached to the upper part of (φ × 45 cm). The column packing solvent and the same solvent were used for development, and 14 g each was collected. The active portion was roughly divided into two, and MAPI was eluted in fractions 37 to 80 and Mer-N5075A substance was eluted in fractions 90 to 140. Concentrate fractions 90-140 to dryness,
120 mg of crude Mer-N5075A material was obtained.

Then 120 mg of crude Mer-N5075A substance was added to methanol.
After dissolving with 2.0 ml of acetic acid and a few drops, preparative thin layer silica gel chromatography (Merck & Co., Art. 5744, solvent: n-butanol: acetic acid = 5: 1) was performed. For the detection of the Mer-N5075A substance, a part of the thin-layered chroma plate after development was colored by the Lydon-Smith reaction, and the silica gel of the corresponding part was scraped off and eluted with methanol. The eluate was concentrated, dissolved in 2 ml of methanol, and then applied again to a Sephadex LH-20 column (1.1φ × 90 cm) packed with methanol and developed with methanol. Fractions 17 to 20 were collected and concentrated to dryness to obtain 24.2 mg of white powder.

The Mer-N5075A substance and the MAPI substance were measured by the above-mentioned HIV protease inhibitory activity or silica gel thin-layer chromatography using butanol: acetic acid = 5: 1 as a developing solvent (Merck, Art.5715, Lydon).・ Smith reaction color development, Rf value: Detected using Mer-N5075A0.11, MAPI 0.26).

[0037]

EFFECTS OF THE INVENTION The Mer-N5075A substance has an inhibitory effect on HIV protease and can be expected to be used as an anti-HIV drug or a conversion material for them.

[0038]

[Brief description of drawings]

FIG. 1 shows an ultraviolet absorption spectrum of a Mer-N5075A substance in methanol.

FIG. 2 shows an infrared absorption spectrum of the Mer-N5075A substance by the KBr method.

Fig. 3 400MHz ¹ HN of Mer-N5075A substance in heavy DMSO solution
The MR spectrum is shown.

FIG. 4 100 MHz ¹³ C- of Mer-N5075A substance in heavy DMSO solution
The NMR spectrum is shown.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI technical display location (C12P 21/02 C12R 1: 465) (72) Inventor Masaru Terazawa 3-13 Hatori, Fujisawa City, Kanagawa Prefecture -1 Mercian Apartment 201 (72) Inventor Hiroyuki Chiba 4-7-26-102 Fukami Nishi, Yamato City, Kanagawa Prefecture (72) Inventor Satoshi Mizuno 2-10-35 Kamiosaki, Shinagawa-ku, Tokyo National Institute of Preventive Health (72) Inventor Hisayoshi Akagawa 2-10-35 Kamiosaki, Shinagawa-ku, Tokyo National Institute of Preventive Health and Safety Research

Claims (2)

[Claims] 1. An HIV protease inhibitor, Mer-N5075A, represented by the following formula: [Chemical 1] 2. An HIV protease inhibitor, Mer-N5075A, which comprises culturing an HIV protease inhibitor, Mer-N5075A, which belongs to the genus Streptomyces, and collecting the HIV protease inhibitor, Mer-N5075A, from the culture. Production method.