JP2000026468A - Human immunodeficiency virus proliferation inhibitors and their production - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new compound that has the HIV proliferation inhibitory activity and is useful as a proliferation inhibitor against human immuno- deficiency virus. SOLUTION: This is represented by formula I (R1 is H or hydroxy; R2 is methyl, or ethyl; R3 and R4 represent each H or they are incorporated to represent an oxygen atom). The compound of formula I is prepared by culturing a microorganism in Streptomyces capable of producing the compound of formula I, for example, Streptomyces aburaviensis Mer-5504 strain (FERM P-16648) under aerobic conditions.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a human immunodeficiency virus (Human Immun) produced by a microorganism belonging to the genus Streptomyces.
Ovidity Virus (HIV).

[0002]

2. Description of the Related Art Acquired immunodeficiency syndrome (Acquire)
d Immuno Defiency Syndr
ome: AIDS) is a human immunodeficiency virus (Human Immunodef, a kind of retrovirus).
immunity Virus (HIV). With the progress of chemotherapeutic research on HIV, reverse transcriptase inhibitors such as azidothymidine (AZT) and protease inhibitors such as indinavir have been put to practical use. However, they have problems such as toxicity in long-term administration and emergence of a virus that has acquired resistance, and the development of new anti-HIV drugs has been desired (A. Molla et al.
Nature Medicine 2,760 (19
96)).

[0003]

DISCLOSURE OF THE INVENTION The present invention relates to a novel compound having an activity of inhibiting the growth of HIV, a method for producing the same,
An object of the present invention is to provide an HIV growth inhibitor containing them as an active ingredient and a novel microorganism having an ability to produce these compounds.

[0004]

Means for Solving the Problems In order to solve the above-mentioned problems, the present inventors have isolated microorganisms from soils in various places and studied metabolites produced by the microorganisms. As a result, newly isolated Streptomyces (Streptomyce
s) It was found that microorganisms belonging to the genus produce substances exhibiting HIV growth inhibitory activity in the culture medium. The active substance was separated and purified from the culture solution, and its physicochemical properties were examined. As a result, it was found that the obtained active substance was different from any known substances and had an excellent HIV growth inhibitory action. Completed the invention.

The novel substance provided by the present invention and having the activity of inhibiting the growth of HIV is a compound represented by the following general formula (I).

Embedded image (Wherein, R ¹ represents a hydrogen atom or a hydroxyl group;
² represents a methyl group or an ethyl group, and R ³ and R
⁴ each represents a hydrogen atom, or R ³ and R ⁴
Together represent an oxygen atom. )

The present inventors have found that among the compounds represented by the general formula (I), the following formulas (Ia), (Ib) and (Ib)
-C) and the compound represented by the formula (Id) were converted to Mer-5504A1, Mer-5504A2, and Me, respectively.
They were named r-5504A3 and Mer-5504A4. Hereinafter, in the present specification, these compounds will be described using the above names. In addition, when these compounds are collectively referred to, they will be described using the names of the Mer-5504A substance group.

[0007]

Embedded image

[0008]

Embedded image

[0009]

Embedded image

[0010]

Embedded image

The Mer-5504A substance group is Oligom reported as an antibacterial or antitumor substance.
ycin substance group or 44-Homooligomyc
in structurally similar to the in substance group (S. Mas
amune et al. J. A. C. S. , 80,6
092 (1958); Yamazaki eta
l. The Journal of Antibio
tics, 45, 171 (1992)). However, due to their molecular formula, physicochemical properties and structural features, the Mer-5504A substance group is a known compound Oli.
gomycin group substance or 44-Homooligo
It is a novel substance that is distinguished from mycin group substances.

Further, the present invention provides a method for preparing Streptomyces (S)
A method for producing a Mer-5504A substance group, which comprises culturing a microorganism belonging to the genus Treptomyces and having the ability to produce a Mer-5504A substance group exhibiting HIV growth inhibitory activity and collecting the substance group from the culture, HIV growth inhibitor as active ingredient, and Mer-5504A
Streptomyces (St) with the ability to produce substance groups
The present invention provides a microorganism belonging to the genus (reptomyces).

[0013] The microorganism used in the present invention may be any strain belonging to the genus Streptomyces and having the ability to produce the group of substances for inhibiting the proliferation of HIV-5 of the present invention, Mer-5504A. Can be used. The search for such a microorganism can be performed, for example, as follows. In cultured cells infected with HIV, cell growth inhibition and appearance of giant cells are observed. The culture solution of various microorganisms was added to the culture solution of HIV-infected cells, and the proliferation of HIV-infected cells and the appearance of giant cells were observed.
A comparison is made with HIV-infected cells to which no microbial culture has been added. By isolating and confirming an active substance from a culture of a microorganism that has inhibited the growth of HIV-infected cells and suppressed the appearance of giant cells, a microorganism capable of producing the target HIV growth-inhibiting substance can be obtained.

[0014] As the microorganisms thus found,
For example, Mer- belonging to the genus Streptomyces which the present inventors isolated from soil.
5504 strains, but not limited to this strain, Streptomyces
If the strain belongs to the genus and has the ability to produce the HIV-5 growth inhibiting substance Mer-5504A substance group, those mutants, for example, artificial mutants and natural mutants that can be obtained by mutagenizing ultraviolet rays, X-rays, radiation, drugs, etc. All of them, including mutants, can be used in the present invention.

Hereinafter, the mycological properties of the strain Mer-5504 will be described. 1. Morphological properties Straight or curved (Rect
eflexibiles). A spore chain consisting of 10 to 50 cylindrical spores is formed before the mature aerial hyphae. Spore sac is not observed. The size of the spores is about 0.8 × 0.8-1.0 μm, the surface of the spores is smooth, and no flagella are observed.

2. Growth conditions in various media All cultures were performed at 28 ° C. The description of the color tone is given by Container Corporation of America (Container
er Corporation of American
a) Color Harmony Manual (Color)
Harmony Manual) is displayed in parentheses.

(1) Yeast / malt agar medium Growth is good, aerial hyphae abundantly grow on the surface, and red (5dc) spores are observed. The back of the culture is ocher to brown. Produces slightly brown soluble pigment. (2) Oatmeal agar medium Growth is good, aerial hyphae are formed on the surface, and gray (7fe) spores are observed. The back of the culture is brown.
Produces slightly brown soluble pigment. (3) Starch / inorganic salt agar medium Growth is good, aerial hyphae abundantly grow on the surface, and red (5dc) spores are observed. The back of the culture is cream-colored. No soluble dye is produced. (4) Glycerin / asparagine agar medium Growth is good, aerial hyphae abundantly grow on the surface, and red (5 cb) spores are observed. The back of the culture is cream to ocher. No soluble dye is produced. (5) Tyrosine agar medium Growth is good, aerial hyphae are formed on the surface, and red (5ec or 6ec) spores are observed. The back of the culture is cream-colored. No melanin-like pigment is produced in the medium.

3. Utilization of Various Carbon Sources Various carbon sources were added to Pridham-Gottrie agar medium (ISP9), and growth at 28 ° C. was observed. The results are as follows. L-arabinose-D-xylose ± D-glucose + D-fructose ± sucrose-inositol-L-rhamnose-D-mannitol-raffinose- + used, ± used somewhat,-not used.

4. Properties of cell wall components The cells were hydrolyzed and analyzed by thin-layer chromatography on cellulose. As a result, diaminopimelic acid (diamino pimeric acid), a cell wall component of the present strain, was analyzed.
The isomer form of cid) was the LL-form.

From the above mycological properties, it is clear that this strain belongs to the genus Streptomyces, and the International Journal of Systematic Bacteriology (Inter)
national Journal of System
The International Streptomyces Project (Internationa), Vol. 18, No. 2 (1968).
Streptomyces (Streptomyces Project) approved by Streptomyces (Streptomyces Project)
s) Compared to the described properties of the genus strain, Streptomyces abraviensis (Streptomyces)
aburaviensis).

Therefore, the present inventors set the strain to Streptomyces abraviensis (Streptomyces).
aburaviensis) Mer-5504, which was deposited on February 19, 1998 with the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology under the number FERMP-16648.

The Mer-5504A substance group of the present invention is produced by inoculating the above strain into a nutrient medium and culturing aerobically. The method for culturing the above-mentioned strains is basically the same as the method for culturing general microorganisms, but it is generally preferable to carry out the culture under aerobic conditions such as shaking culture by liquid culture and aeration-agitation culture.

The medium that can be used for the culture includes
Any medium containing a nutrient source that can be used by microorganisms belonging to the genus Streptomyces may be used, and any of various synthetic media, semi-synthetic media, natural media, and the like can be used. As the medium composition, for example, glucose, sucrose, starch syrup, molasses, dextrin, starch, glycerol, animal oil, vegetable oil, etc. can be used alone or in combination as a carbon source. , Wheat germ, corn steep liquor, cottonseed meal, meat extract, peptone, yeast extract, urea, ammonium sulfate and the like can be used alone or in combination. Also, if necessary,
Sodium chloride, magnesium sulfate, calcium carbonate,
Inorganic salts such as phosphates and metal salts can be added.
In addition, organic substances that promote the growth of the strain or the production of the Mer-5504A substance group, such as vitamins and amino acids, can be added. If necessary, an antifoaming agent, for example, adecanol (trade name: manufactured by Asahi Denka Kogyo KK), silicon, or the like can be added.

The culturing conditions are such that the above strain grows well and
The er-5504A substance group may be appropriately selected within a range where it can be produced. For example, the pH of the medium is preferably about 6 to 8, usually around neutral. The culturing temperature is a temperature at which the microorganisms grow well, usually 15 to 30 ° C, preferably 2
It is good to keep at 5-28 ° C. The culture time may be about 2 to 10 days, preferably 3 to 5 days. Of course, the various culture conditions described above can be appropriately changed according to the type and characteristics of the microorganism used, external conditions, and the like, and the optimal conditions can be selected and adjusted from the above range accordingly. At that time, the culture solution obtained by stopping the culture when the amount of accumulation of the Mer-5504A substance group in the culture solution reaches the maximum may be used in the following purification step.

The Mer-5504A substance group accumulated in the culture solution obtained as described above is separated from the bacterial cells by general solid-liquid separation means such as filtration and centrifugation after culturing, and the filtrate or supernatant is obtained. Can be recovered from It can also be recovered from the isolated cells.

The separation and purification of the Mer-5504A substance group can be performed by selecting and combining various known methods. For example, solvent extraction using ethyl acetate, methanol, n-butanol or the like, Amberlite XAD (manufactured by Rohm and Haas), Diaion HP-20
A column chromatography method using a carrier such as polystyrene-based adsorption resin such as (manufactured by Mitsubishi Chemical Corporation), silica gel, alumina, or activated carbon can be used.
The method of eluting the target substance from these carriers differs depending on the type and properties of the carrier. For example, in the case of a polystyrene-based adsorption resin, a hydrated alcohol, hydrated acetone, or the like can be used as an elution solvent. Further, gel filtration using Sephadex LH-20 (manufactured by Pharmacia), Bio-Gel P-2 (manufactured by Bio-Rad), thin-layer chromatography using silica gel, alumina, or the like, a normal phase or reverse phase column. Preparative high performance liquid chromatography (preparative HPL
C) and the like can be used, and these methods can be separated or purified by using these methods alone or in an appropriate combination, and in some cases, repeatedly using them. The isolated cells can be extracted from the cells by a solvent extraction method using an appropriate organic solvent or an elution method by crushing the cells, and isolated and purified in the same manner as described above.

Mer-550 obtained as described above
The 4A substance group has the following physicochemical properties. 1. Physicochemical properties of Mer-5504A1 (1) molecular weight: 793.04 (2) molecular formula: C₄₄H₇₂O₁₂  (3) Melting point: 136 to 137 ° C. (4) Specific rotation: [α]²⁵ _D−70.76 ° (c
0.516, CH₃OH) (5) Ultraviolet absorption spectrum: measured with methanol solution
The results are shown in FIG. The characteristic absorption is as follows
It is as follows. UV λmax nm, (ε) in CH₃OH: 22
5 (37516); 233 (34687); 243 (s
h. )

(6) Infrared absorption spectrum: The result of measurement by the KBr method is as shown in FIG. The characteristic absorptions are as follows. IRν _max (KBr) cm ⁻¹ : 3449,296
8, 1703, 1643, 1458, 1386, 128
2,1095,993 (7) Solubility: soluble in methanol and dioxane, slightly soluble in chloroform, and insoluble in water. (8) Color reaction: Color is formed with a phosphomolybdic acid reagent and iodine vapor. The ninhydrin reagent has no color. (9) Rf value of thin layer chromatography: 0.58 (chloroform: methanol = 3: 1) on silica gel plate 15715 manufactured by Merck Ltd. (10) Color and shape: white powder.

(11) ¹ H-NMR spectrum (400
MHz, CD ₃ OD): As shown in FIG. The chemical shift, multiplicity, and spin coupling constant of each signal are as follows. δ (ppm, based on TMS) 6.82 (1H, d
d, J = 15.4, 9.9 Hz), 6.07 (1H, d
d, J = 14.9, 10.3 Hz), 6.00 (1H,
dd, J = 14.5, 10.3 Hz), 5.82 (1
H, d, J = 15.4 Hz), 5.42 (1H, dd)
d, J = 14.9, 10.6, 4.0 Hz), 5.16
(1H, dd, J = 14.5, 10.0 Hz), 4.8
8 (1H, dd, J = 11.3, 4.8 Hz), 4.5
8 (1H, brd, J = 10.3 Hz), 4.11 (1
H, brd, J = 10.3 Hz), 4.09 (1H,
m), 3.99 (1H, m), 3.92 (1H, br)
s), 3.76 (1H, dd, J = 9.2, 1.8H)
z), 3.67 (1H, brd, J = 9.5 Hz),
3.04 (1H, dd, J = 14.3, 5.5 Hz),
2.85 to 2.82 (2H, m), 2.50 (1H,
m), 2.45 (1H, m), 2.21 (1H, m),
2.16 (1H, dd, J = 14.3, 2.0 Hz),
2.13 to 1.98 (3H, m), 1.90 to 1.80
(3H, m), 1.74 to 1.60 (3H, m), 1.
51 to 1.38 (4H, m), 1.28 (1H, m),
1.24 (3H, d, J = 6.6 Hz), 1.14 (3
H, d, J = 6.6 Hz), 1.07 (1H, m),
1.06 (3H, s), 1.03 (3H, d, J = 7.
0 Hz), 1.01 (3H, d, J = 6.6 Hz),
0.95 (3H, d, J = 7.0 Hz), 0.89 (3
H, d, J = 6.8 Hz), 0.87 (3H, t, J =
7.4 Hz), 0.84 (3H, d, J = 6.8H)
z), 0.84 (3H, d, J = 6.5 Hz)

(12) ¹³ C-NMR spectrum (10
0 MHz, CD ₃ OD): As shown in FIG. The chemical shift and multiplicity of each signal are as follows. δ (ppm, based on TMS) 220.0 (s),
203.9 (s), 166.8 (s), 152.3
(D), 137.5 (d), 133.6 (d), 13
2.8 (d), 132.4 (d), 122.6 (d),
101.3 (s), 84.3 (s), 81.3 (d),
80.4 (d), 77.4 (d), 73.8 (d), 7
1.9 (d), 68.7 (d), 68.0 (d), 6
5.1 (d), 47.7 (d), 45.0 (t), 4
4.6 (t), 42.5 (d), 42.5 (t), 4
2.1 (t), 39.8 (t), 38.4 (d), 3
7.3 (d), 36.7 (d), 34.6 (d), 3
2.7 (d), 32.6 (t), 32.2 (t), 3
0.0 (t), 25.0 (q), 22.3 (q), 1
8.4 (q), 15.6 (q), 13.1 (q), 1
2.5 (q), 12.2 (q), 9.7 (q), 6.7
(Q), 4.7 (q) (13) Distinguishing between acidic, basic and neutral: neutral

2. Physicochemical properties of Mer-5504A2
Quality (1) Molecular weight: 807.06 (2) Molecular formula: C₄₅H₇₄O₁₂  (3) Melting point: 130 to 131 ° C. (4) Specific rotation: [α]²⁵ _D−76.58 ° (c
3.03, CH₃OH) (5) Ultraviolet absorption spectrum: measured with methanol solution
The result is shown in FIG. The characteristic absorption is as follows
It is as follows. UV λmax nm, (ε) in CH₃OH: 22
5 (38167); 233 (35400); 243 (s
h. )

(6) Infrared absorption spectrum: The result of measurement by the KBr method is as shown in FIG. The characteristic absorptions are as follows. IRν _max (KBr) cm ⁻¹ : 3458,296
8, 1703, 1641, 1460, 1386, 128
2,1097,989 (7) Solubility: soluble in methanol and dioxane, slightly soluble in chloroform, and insoluble in water. (8) Color reaction: Color is formed with a phosphomolybdic acid reagent and iodine vapor. The ninhydrin reagent has no color. (9) Rf value of thin layer chromatography: 0.58 (chloroform: methanol = 3: 1) on silica gel plate 15715 manufactured by Merck Ltd. (10) Color and shape: white powder.

(11) ¹ H-NMR spectrum (400
MHz, CD ₃ OD): as shown in FIG. The chemical shift, multiplicity, and spin coupling constant of each signal are as follows. δ (ppm, based on TMS) 6.81 (1H, d
d, J = 15.6, 10.1 Hz), 6.07 (1H,
dd, J = 14.7, 10.6 Hz), 5.99 (1
H, dd, J = 14.5, 10.6 Hz), 5.83
(1H, d, J = 15.6 Hz), 5.42 (1H, d
dd, J = 14.7, 10.6, 4.0 Hz), 5.1
6 (1H, dd, J = 14.5, 9.6 Hz), 5.0
6 (1H, dd, J = 11.9, 5.0 Hz), 4.5
6 (1H, brd, J = 9.9 Hz), 4.09 (1
H, brd, J = 10.3 Hz), 4.03 (1H,
m), 3.99 (1H, m), 3.92 (1H, s),
3.76 (1H, dd, J = 9.2, 1.8 Hz),
3.64 (1H, brd, J = 9.6 Hz), 3.03
(1H, dd, J = 14.1, 5.5 Hz), 2.85
-2.83 (2H, m), 2.51 (1H, m), 2.
30 (1H, ddd, J = 11.9, 5.9, 3.7H
z), 2.23 (1H, m) 2.17, (1H, dd,
J = 14.1, 2.2 Hz), 2.12 to 1.98 (3
H, m), 1.90 to 1.81 (3H, m), 1.74
~ 1.62 (3H, m), 1.52-1.37 (5H,
m), 1.32 to 1.25 (2H, m), 1.23 (3
H, d, J = 6.6 Hz), 1.13 (3H, d, J =
6.6 Hz), 1.07 (3H, s), 1.05 (1
H, m), 1.03 (3H, d, J = 6.6 Hz),
1.01 (3H, d, J = 6.6 Hz), 0.97 (3
H, d, J = 6.8 Hz), 0.93 (3H, t, J =
7.2 Hz), 0.89 (3H, d, J = 6.8H)
z), 0.86 (3H, t, J = 7.2 Hz), 0.8
4 (3H, d, J = 6.6 Hz)

(12) ¹³ C-NMR spectrum (10
0 MHz, CD3OD): As shown in FIG. The chemical shift and multiplicity of each signal are as follows. δ (ppm, based on TMS) 220.0 (s),
204.2 (s), 166.6 (s), 152.6
(D), 137.4 (d), 133.6 (d), 13
2.8 (d), 132.4 (d), 122.7 (d),
102.4 (s), 84.4 (s), 81.3 (d),
80.4 (d), 77.7 (d), 73.9 (d), 7
1.8 (d), 68.6 (d), 68.0 (d), 6
5.1 (d), 47.7 (d), 44.9 (t), 4
4.6 (t), 42.6 (d), 42.6 (d), 4
2.1 (t), 39.8 (t), 38.5 (d), 3
7.5 (d), 36.8 (d), 34.6 (d), 3
2.6 (d), 32.6 (t), 32.1 (t), 3
0.0 (t), 25.0 (q), 23.2 (t), 2
2.3 (q), 18.5 (q), 15.6 (q), 1
3.7 (q), 13.3 (q), 12.5 (q), 9.
7 (q), 6.8 (q), 4.7 (q) (13) Distinguishing between acidic, basic and neutral: neutral

3. Physicochemical properties of Mer-5504A3
Quality (1) Molecular weight: 793.08 (2) Molecular formula: C₄₅H₇₆O₁₁  (3) Melting point: 129 to 130 ° C (4) Specific rotation: [α]²⁵ _D−65.56 ° (c
2.29, CH₃OH) (5) Ultraviolet absorption spectrum: measured with a tanol solution
The results are as shown in FIG. The characteristic absorption is as follows
It is as follows. UV λmax nm, (ε) in CH₃OH: 22
5 (38209); 233 (35646); 243 (s
h. )

(6) Infrared absorption spectrum: The result of measurement by the KBr method is as shown in FIG. The characteristic absorptions are as follows. IRν _max (KBr) cm ⁻¹ : 3445,296
6,1701,1643,1458,1393,128
2,1089,989 (7) Solubility: soluble in methanol and dioxane, slightly soluble in chloroform, and insoluble in water. (8) Color reaction: Color is formed with a phosphomolybdic acid reagent and iodine vapor. The ninhydrin reagent has no color. (9) Rf value of thin layer chromatography: 0.58 (chloroform: methanol = 3: 1) on silica gel plate 15715 manufactured by Merck Ltd. (10) Color and shape: white powder.

(11) ¹ H-NMR spectrum (400
MHz, CD3OD): As shown in FIG. The chemical shift, multiplicity, and spin coupling constant of each signal are as follows. δ (ppm, based on TMS) 6.77 (1H, d
d, J = 15.8, 9.9 Hz), 6.09 (1H, d
d, J = 14.7, 10.3 Hz), 5.99 (1H,
dd, J = 14.5, 10.3 Hz), 5.82 (1
H, d, J = 15.4 Hz), 5.42 (1H, dd)
d, J = 14.7, 10.3, 3.7 Hz), 5.18
(1H, dd, J = 14.5, 9.6 Hz), 5.07
(1H, dd, J = 11.4, 9.9 Hz), 4.05
-4.02 (2H, m), 3.95 (1H, m), 3.
93 (1H, s), 3.85 (1H, brd, J = 8.
8 Hz), 3.75 (1H, dd, J = 9.5, 1.5)
Hz), 3.61 (1H, brd, J = 9.6 Hz),
2.86 to 2.83 (2H, m) 2.51 (1H,
m), 2.20 to 1.97 (4H, m), 1.96 to
1.69 (4H, m), 1.69-1.38 (10H,
m), 1.34 to 1.20 (4H, m), 1.19 (3
H, d, J = 6.3 Hz), 1.12 (3H, d, J =
6.6 Hz), 1.07 (3H, s), 1.04 (3
H, d, J = 7.0 Hz), 1.02 (3H, d, J =
6.6 Hz), 0.97 (1H, m), 0.94 (3
H, d, J = 7.1 Hz), 0.90 (3H, d, J =
6.6 Hz), 0.85 (3H, t, J = 7.4H
z), 0.84 (3H, d, J = 7.3 Hz)

(12) ¹³ C-NMR spectrum (10
0 MHz, CD3OD): As shown in FIG.
The chemical shift and multiplicity of each signal are as follows. δ (ppm, based on TMS) 219.9 (s),
166.6 (s), 152.1 (d), 137.6
(D), 133.6 (d), 132.5 (d), 13
2.2 (d), 122.8 (d), 101.1 (s),
84.2 (s), 81.3 (d), 80.4 (d), 7
7.7 (d), 73.7 (d), 69.7 (d), 6
8.5 (d), 67.9 (d), 64.9 (d), 4
7.6 (d), 45.5 (d), 44.4 (t), 4
3.6 (t), 42.4 (d), 41.9 (d), 3
9.8 (t), 37.5 (d), 36.6 (d), 3
4.6 (d), 32.7 (t), 32.4 (t), 3
1.6 (d), 30.0 (t), 27.6 (t), 2
7.3 (t), 25.1 (q), 22.6 (t), 2
2.3 (q), 18.5 (q), 15.7 (q), 1
4.8 (q), 12.5 (q), 11.8 (q), 9.
8 (q), 6.8 (q), 4.8 (q) (13) Distinguishing between acidic, basic and neutral: neutral

4. Physicochemical properties of Mer-5504A4
Quality (1) Molecular weight: 777.08 (2) Molecular formula: C₄₅H₇₆O₁₀  (3) Melting point: 128-130 ° C (4) Specific rotation: [α]²⁵ _D−61.49 ° (c
1.25, CH₃OH) (5) Ultraviolet absorption spectrum: measured with methanol solution
The result is shown in FIG. The characteristic absorption is below
It is as described. UV λmax nm, (ε) in CH₃OH: 22
5 (38236); 233 (35804); 243 (s
h. )

(6) Infrared absorption spectrum: The result of measurement by the KBr method is as shown in FIG. The characteristic absorptions are as follows. IRν _max (KBr) cm ⁻¹ : 3439,296
6,1701,1641,1458,1383,128
2,1091,989 (7) Solubility: soluble in methanol and dioxane, slightly soluble in chloroform, and insoluble in water. (8) Color reaction: Color is formed with a phosphomolybdic acid reagent and iodine vapor. The ninhydrin reagent has no color. (9) Rf value of thin layer chromatography: 0.70 (chloroform: methanol = 3: 1) on silica gel plate 15715 manufactured by Merck (10) Color and shape: white powder.

(11) ¹ H-NMR spectrum (400
MHz, CD3OD): As shown in FIG. The chemical shift, multiplicity, and spin coupling constant of each signal are as follows. δ (ppm, based on TMS) 6.77 (1H, d
d, J = 15.4, 9.6 Hz), 6.12 (1H, d
d, J = 14.7, 10.6 Hz), 6.00 (1H,
dd, J = 14.7, 10.6 Hz), 5.82 (1
H, d, J = 15.4 Hz), 5.40 (1H, dd)
d, J = 14.7, 10.3, 4.4 Hz), 5.19
(1H, dd, J = 14.7, 10.3 Hz), 5.0
6 (1H, dd, J = 11.8, 5.2 Hz), 4.1
0 (1H, m), 4.04 (1H, brd, J = 8H
z), 3.95 (1H, m), 3.89 (1H, br)
d, J = 11.0 Hz), 3.85 (1H, m), 3.
75 (1H, dd, J = 9.5, 1.5 Hz), 3.6
2 (1H, brd, J = 10.3Hz), 2.73 ~
2.69 (3H, m), 2.50 (1H, m), 2.1
9 to 2.03 (4H, m), 1.98 to 1.75 (3
H, m), 1.70-1.64 (4H, m), 1.59
-1.39 (7H, m), 1.35-1.23 (4H,
m), 1.19 (3H, d, J = 6.6 Hz), 1.1
2 (3H, d, 6.8 Hz), 1.00 (1H, m),
0.99 (3H, d, J = 6.6 Hz), 0.97 (3
H, t, J = 7.2 Hz), 0.95 (3H, d, J =
6.6 Hz), 0.90 (3H, d, J = 6.8H)
z), 0.88 (3H, d, J = 6.6 Hz), 0.8
7 (3H, d, J = 6.6 Hz), 0.86 (3H,
t, J = 7.2 Hz), 0.84 (3H, d, J = 6.
6Hz)

(12) ¹³ C-NMR spectrum (10
0 MHz, CD3OD): As shown in FIG.
The chemical shift and multiplicity of each signal are as follows. δ (ppm, based on TMS) 216.2 (s),
166.7 (s), 152.0 (d), 137.6
(D), 133.7 (d), 132.6 (d), 13
1.7 (d), 122.8 (d), 101.1 (s),
81.4 (d), 80.4 (d), 77.9 (d), 7
2.4 (d), 69.8 (d), 68.5 (d), 6
7.8 (d), 65.0 (d), 51.0 (d), 4
9.3 (t), 47.7 (d), 45.6 (d), 4
3.6 (t), 42.6 (d), 41.6 (d), 3
7.9 (t), 37.6 (d), 36.7 (d), 3
4.9 (d), 32.9 (t), 32.5 (t), 3
1.7 (d), 30.1 (t), 27.7 (t), 2
7.4 (t), 25.1 (q), 22.6 (t), 1
8.6 (q), 14.8 (q), 14.0 (q), 1
3.2 (q), 12.5 (q), 11.8 (q), 9.
7 (q), 6.9 (q), 4.7 (q) (13) Distinguishing between acidic, basic and neutral: neutral

The Mer-5504A substance group of the present invention has excellent HIV growth inhibitory activity, and is expected to be used as an anti-HIV agent. The HIV growth inhibitory activity of the compound of the present invention can be measured, for example, by the method described below.

HIV used for assaying HIV growth inhibitory activity
An infected MT-4 cell solution is prepared by the following method. First, the cultured MT-4 cells (Miyoshi)
I. et al, GANN Monogr. Can
cer Res. 28, 219 to 228) by the trypan blue method, 3 × 10 ⁶ cells were measured and centrifuged at 1000 rpm for 5 minutes, and then 10% FCS was added to a culture solution (RPMI1640 (manufactured by Gibco)).
100 units / ml penicillin G and 100 μg
/ Ml of Streptomycin).

Here, the TCID value was set to 1000 so that the TCID50 value (50% tissue culture infectious dose (Takashi Kitamura, tissue culture technology for virus test, Modern Publishing, Tokyo, 1976)) was 1000.
IV is added and the cells are cultured at 37 ° C. for 1 hour in a CO 2 incubator to allow HIV to adsorb to MT-4 cells. Here 9
Add ml of the culture solution and use as HIV-infected MT-4 cell solution. 100 μl of the HIV-infected MT-4 cell solution prepared by the above method and 100 μl of a test sample diluted to an appropriate concentration with a culture solution are added to one well of a 96-well plate. At the same time, 100 μl of the HIV-uninfected MT-4 cell solution and 100 μl of the diluted test sample are added to one well on the same plate as a control. The cells on the 96-well plate are cultured in a CO ₂ incubator at 37 ° C. for 3 days, and the growth and shape change of the cells are observed under a microscope every day. In addition, the cells on the third day are suspended, 50 μl of the cells are transferred to a new plate, 150 μl of a culture solution containing a test sample is added, and culturing and observation are further performed for 3 days.

In HIV-infected cells, cell proliferation is suppressed and the appearance of giant cells is observed. HI in the test sample
When a component effective for V growth suppression is contained, the appearance of giant cells is suppressed, and normal cell growth is observed. Also, the change in the cell findings with respect to the concentration of the test sample determines the effective concentration range of the test sample. Mer-5 of the present invention
504A1 substance, Mer-5504A2 substance, Mer-
The 5504A3 and Mer-5504A4 substances are:
In each case, the appearance of giant cells was suppressed at 10 ng / ml.

As described above, the Mer-5504A substance group of the present invention represented by the general formula (I) suppresses the appearance of giant cells in cells infected with HIV and promotes normal cell proliferation. Pharmaceutical preparations containing the 5504A substance group are expected to be applied to the treatment of diseases caused by HIV, ie, acquired immunodeficiency syndrome.

The Mer-5504A substance group is useful as an HIV growth inhibitor, and its pharmaceutical preparations include known fillers, bulking agents, binders, humectants, disintegrants, disintegration inhibitors, surfactants, lubricants, and the like. It can be prepared using a diluent such as a powder or an excipient. Various forms can be selected as the pharmaceutical preparation depending on the purpose of treatment, and typical examples are tablets, pills, powders, solutions, suspension syrups, emulsions, granules, capsules, suppositories, injections Agents (solutions, suspensions, etc.).

For molding into tablets, known carriers can be widely used, for example, lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silica, etc. Agents, water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, binders such as carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinylpyrrolidone, dried starch, sodium alginate, agar powder, laminaran powder, Disintegrating agents such as sodium bicarbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, stearic acid monoglyceride, starch, lactose, sucrose, cocoa butter, disintegrating inhibitors such as hydrogenated oil,
Absorption promoters such as quaternary ammonium bases and sodium lauryl sulfate; humectants such as glycerin and starch; adsorbents such as starch, lactose, kaolin, bentonite and colloidal silicic acid; purified talc, stearates, and boric acid powder And a lubricant such as polyethylene glycol.

Further, the tablets can be made into tablets coated with a usual coating, if necessary, such as sugar-coated tablets, gelatin-coated tablets, film-coated tablets, double tablets and multilayer tablets. In molding into a pill form, those conventionally known in the art can be widely used as carriers, such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, kaolin, excipients such as talc, gum arabic powder, Examples thereof include binders such as tragacanth powder, gelatin, and ethanol, and disintegrants such as laminaran agar powder.

For molding into a suppository form, as the carrier, those conventionally known in the art can be widely used, and examples thereof include polyethylene glycol, cocoa butter higher alcohol, esters of higher alcohol, gelatin, semisynthetic glyceride and the like. Can be.

When prepared as injections, the liquid preparations and suspensions are preferably sterilized and isotonic with blood. When molded into the form of these liquid preparations and suspensions, they may be used as diluents. Any of those commonly used in this field can be used, and examples thereof include water, ethanol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, and polyoxyethylene sorbitan fatty acid esters. In this case, a sufficient amount of salt, glucose or glycerin to prepare an isotonic solution may be contained in the preparation, and a normal solubilizing agent, a buffer, a soothing agent and the like may be used. Is also good. For oral administration, a coloring agent, a preservative, a flavoring agent, a flavoring agent, a sweetening agent or the like and other pharmaceuticals may be contained in the preparation as required.

The amount of the Mer-5504A substance group to be contained in the HIV growth inhibitor of the present invention is not particularly limited, and is selected widely. The administration method is not particularly limited,
The drug is administered according to various formulations, the age and sex of the patient, other conditions, and the degree of the disease. For example, tablets, pills, solutions, suspension syrups, emulsions, granules and capsules are orally administered. In the case of an injection, it may be administered intravenously, alone or as a mixture with a normal replenisher such as glucose or amino acid, and if necessary, intramuscularly, intradermally, subcutaneously or intraperitoneally. In the case of suppositories, they are administered rectally. In addition, the dose can be appropriately increased or decreased depending on the usage, age and sex of the patient, other conditions, degree of the disease, and the like.

Hereinafter, the present invention will be described in more detail by way of examples.

EXAMPLES Example 1 Potato starch 2.0%, glucose 2.0.
0%, soybean flour 2.0%, yeast extract 0.5%, sodium chloride 0.25%, copper sulfate (pentahydrate) 5ppm, manganese chloride (tetrahydrate) 5ppm, zinc sulfate (heptahydrate) 5pp
m, and a medium having a composition of calcium carbonate of 0.32% was sterilized by dispensing 50 ml each into a 500 ml Erlenmeyer flask. This was mixed with a slant medium (I-5) of Mer-5504 strain.
One loopful of platinum loop was inoculated from SP-2 agar medium) and incubated at 28 ° C.
The seed culture was obtained by culturing on a rotary shaker for days. As a production medium, glucose 2%, dextrin 1.1
%, Dry yeast 0.9%, meat extract 0.5g, sodium chloride 0.5g, polypeptone 0.5%, calcium carbonate 0.4%, using a medium having a composition of 10L jar fermenters. 5 L was dispensed and sterilized. Inoculate 50 ml of the seed culture into a jar fermenter,
Aeration and agitation culture (aeration rate 5 L / min, agitation 400 rpm) was performed at 28 ° C for 96 hours to obtain a culture solution.

7.5 L of the culture filtrate was separated by filtration into a supernatant and cells. The supernatant was extracted with 6 L of ethyl acetate. The cells were extracted with 4 L of methanol, and the extract was concentrated under reduced pressure. 1 L of water and ethyl acetate were added to this concentrate, respectively.
After thorough stirring, the ethyl acetate layer was removed, and the ethyl acetate extract from the supernatant was combined and dried under reduced pressure to obtain 9.3 g of a brown oily substance. This oily substance was dissolved in a small amount of chloroform, and flash chromatograph apparatus (manufactured by Biotage, FLASH 40M, 4.0c) was used.
mφ × 30 cm) and chloroform: methanol =
Eluted at 20: 1, 10: 1, 5: 1. The active fraction was concentrated to dryness, and 6.3 g of the obtained yellow substance was further purified by a high-performance liquid chromatograph.

The high-performance liquid chromatograph is manufactured by Shimadzu Corporation
C-8A was used as a separation column by GL Sciences,
Inertsil ODS-3 (5.0cmφ × 25c
and eluted with acetonitrile: water = 7: 3 using m). At this time, the flow rate was 40 ml / min and UV 220 n
The absorbance was measured in m and detected. Retention time 25.4 minutes,
The peaks at 28.2 minutes, 44.6 minutes and 58.1 minutes were collected, and each fraction was separated by gel filtration chromatography (Sephadex LH-20, 100 manufactured by Pharmacia).
and then dried under reduced pressure to obtain Mer-5504A1 and Mer-5504.
A2, Mer-5504A3, and Mer-5504A4 substances were 18.6 mg, 98.6 mg, and 118.1, respectively.
mg and 65.6 mg of the purified product.

[0057]

EFFECT OF THE INVENTION The Mer-5504A substance group has an inhibitory effect on HIV proliferation and can be expected to be used as an anti-HIV drug or a material for converting to it.

[Brief description of the drawings]

FIG. 1 is an ultraviolet absorption spectrum of Mer-5504A1 substance in methanol.

FIG. 2 is an infrared absorption spectrum of Mer-5504A1 substance obtained by a KBr method.

FIG. 3 is a ¹ H-NMR spectrum (400 MHz) of a Mer-5504A1 substance in a heavy methanol solution.

FIG. 4 is a ¹³ C-NMR spectrum (100 MHz) of a Mer-5504A1 substance in a heavy methanol solution.

FIG. 5 is an ultraviolet absorption spectrum of Mer-5504A2 substance in methanol.

FIG. 6 is an infrared absorption spectrum of Mer-5504A2 substance by a KBr method.

FIG. 7 is a ¹ H-NMR spectrum (400 MHz) in a heavy methanol solution of Mer-5504A2 substance.

FIG. 8 is a ¹³ C-NMR spectrum (100 MHz) of a Mer-5504A2 substance in a heavy methanol solution.

FIG. 9 is an ultraviolet absorption spectrum of Mer-5504A3 substance in methanol.

FIG. 10 is an infrared absorption spectrum of a Mer-5504A3 substance by a KBr method.

FIG. 11 is a ¹ H-NMR spectrum (400 MHz) of a Mer-5504A3 substance in a heavy methanol solution.

FIG. 12 is a ¹³ C-NMR spectrum (100 MHz) of a Mer-5504A3 substance in a heavy methanol solution.
It is.

FIG. 13 is an ultraviolet absorption spectrum of Mer-5504A4 substance in methanol.

FIG. 14 is an infrared absorption spectrum of Mer-5504A4 substance by a KBr method.

FIG. 15 is a ¹ H-NMR spectrum (400 MHz) of a Mer-5504A4 substance in a heavy methanol solution.

FIG. 16 ¹³ C-NMR spectrum (100 MHz) of Mer-5504A4 substance in deuterated methanol solution
It is.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) // (C12N 1/20 C12R 1: 465) (C12P 17/18 C12R 1: 465) (72) Inventor Emiko Tsuji 7053 Tsujido, Fujisawa-shi, Kanagawa Prefecture (72) Inventor Reiko Izui 322-10, Natsumizu, Atsugi-shi, Kanagawa Prefecture (72) Inventor Kazuyuki Dobashi 3-4-1, Nagagaoka, Hadano-shi, Kanagawa Prefecture 5-204 (72) Inventor Takeo Yoshioka 1782-10 Yoshioka Yoshioka, Ayase City, Kanagawa Prefecture BH10 CA04 DA02 4B065 AA50X CA18 CA44 4C071 AA04 BB02 CC13 EE07 FF17 GG01 GG03 HH05 HH08 HH09 KK17 LL01 4C086 AA01 AA02 AA03 AA04 GA02 MA17 MA22 MA23 MA31 MA35 MA37 MA41 MA52 MA60 MA66 NA66 09 ZB33 ZC55

Claims (8)

[Claims] 1. A compound represented by the following general formula (I). Embedded image (Wherein, R ¹ represents a hydrogen atom or a hydroxyl group;
² represents a methyl group or an ethyl group, and R ³ and R
⁴ each represents a hydrogen atom, or R ³ and R ⁴
Together represent an oxygen atom. ) 2. A compound represented by the following formula (Ia). Embedded image 3. A compound represented by the following formula (Ib): Embedded image 4. A compound represented by the following formula (Ic). Embedded image 5. A compound represented by the following formula (Id): Embedded image 6. Streptomyces (Streptomyces)
yces) culturing a microorganism belonging to the genus and capable of producing the compound according to any one of claims 1 to 5, and collecting the compound according to any one of claims 1 to 5 from the culture. A method for producing the compound according to claim 1. 7. A human immunodeficiency virus growth inhibitor comprising one or more substances selected from the group consisting of the compounds according to claim 1 as an active ingredient. 8. Streptomyces aburavisis having the ability to produce the compound according to any one of claims 1 to 5.
ensis) Mer-5504 strain (FERM P-16)
648).