WO2000000631A1 - Novel physiologically active substances inhibiting the proliferation of human immunodeficiency virus (hiv) - Google Patents

Abstract

Compounds represented by general formula (I), which have been isolated from a strain belonging to the genus Streptomyces during studies for detecting remedies for HIV infection inhibiting the activity of HIV Tat molecules and are capable of inhibiting the HIV Tat molecule activity and thus inhibiting the proliferation of HIV both in acute infection and persistent infection systems, and salts or hydrates of the same wherein R1 represents linear or branched higher (C12-18) alkyl.

Description

 Description New biologically active substance that suppresses the proliferation of human immunodeficiency virus (HIV)

 The present invention relates to a novel bioactive substance which is effective for treating HIV infection, which inhibits the activity of HIV (human immunodeficiency virus) Tat molecule and suppresses HIV proliferation. Conventional technology

 AIDS is a disease of the immune system caused by the retroviruses HIV-1 and HIV-2, which almost always results in the death of the patient. There is interest in the search for compositions that inhibit the growth of the virus for the prevention of AIDS and reduction of symptoms.

 After HIV infects cells, the gene RNA is reverse transcribed into DNA by reverse transcriptase contained in the virus particles, and integrated into host cell DNA by HIV integrase. HIV RNA is transcribed from the promoter in the LTR at the 5 'end of the integrated HIV gene into gene RNA, and at the same time, viral proteins are synthesized using the RNA as type II. Some of the synthesized viral proteins are cleaved by HIV protease to become mature proteins and form the next generation of viral particles. Based on the virus life cycle described above, reverse transcriptase inhibition, integrase inhibition, HIV LTR transcription inhibition, and protease inhibition can be cited as the action points of therapeutic drugs for HIV infection.

 To date, nucleoside and non-nucleoside reverse transcriptase inhibitors and protease inhibitors have been put to practical use as therapeutic drugs for HIV infection and have been effective in the treatment. However, the emergence of resistant strains to these anti-HIV drugs has already been reported (Gimhard H. etal., J. of Virol. 72 (3): 2422 (1998)), and a new strain has been reported. The development of anti-HIV drugs is desired. In particular, anti-HIV drugs with different action points do not cross the resistance-causing mutations, making it possible to treat HIV infections that have acquired resistance to existing drugs. Is expected to delay the appearance of.

The HIV Tat molecule binds to the TAR region at the 5 'end of HIV RNA and promotes transcription from the HIV LTR. It is known to have a promoting activity (Tat activity) (Laspia MF et al., Cell 59: 283 (1989)). Because Tat-deficient HIV cannot grow (Popik W. et a, J. of Virol. 67 (2): 1094 (1993)), drugs that inhibit Tat activity (Tat inhibitors) suppress HIV growth However, it is expected to be a new mechanism for treating HIV infection.

 So far, Ro 5-3335 and Ro 24-7429, which have reduced nephrotoxicity in rats, have been reported as compounds that inhibit Tat activity and suppress HIV proliferation. Insufficient anti-HIV activity, such as MT-4 acute infection system, which is an HIV activity evaluation system (Witvrouw M. et a, Antimicrob. Agents and Chemother. 36: 2628 (1992)). Introduced Ro 24-7429 had no effect

(Haubrich R. H. et a., J. of Infect. Disease 172: 1246 (1995)).

 ALX40-4C, an analog of the domain that binds to the TAR region of Tat, has not been reported to inhibit Tat activity in cell lines, and the reported anti-HIV activity is also based on adsorption inhibition. (O'Brien W. et al, J. of Virol. 70 (5): 2825 (1996)). Disclosure of the invention

 An object of the present invention is to isolate a novel substance that inhibits Tat activity and suppresses HIV proliferation, and to provide a therapeutic agent for HIV infection.

In view of the above situation, the present inventors conducted screening and screening for a Tat inhibitor using a microorganism culture solution as a raw material. As a result, they found that a Tat inhibitor was produced in the culture broth of a microorganism belonging to the genus Streptomyces, and the isolation and structure determination of this active substance revealed that it was a novel active substance. The active substance was shown to inhibit HIV proliferation in various acute HIV infection systems, including the ^ -4 acute infection system in which Ro 5-3335 shows no activity. In addition, as expected from the mechanism of action that suppresses the RNA transcription process, HIV proliferation was also suppressed in the persistent infection system, and the HIY gene, in which a reverse transcriptase inhibitor was ineffective, was integrated into the host cell MA. It has been shown that it can be a therapeutic agent that can be effective even afterwards. That is, the present invention provides the following general formula (I)

(In the formula, including those with a branch in § alkyl group of c _{1 8} from c ₁₂ and shows a higher alkyl group. Wherein higher alkyl.)

The present invention relates to a compound represented by the formula (I), a salt or a hydrate thereof, a production method thereof, and a medicament containing the active ingredient as a active ingredient.

More specifically, in the general formula (I), I is the following general formula (II):

(Wherein, R ₂ is a methyl group or an ethyl group)

A compound represented by the formula, a salt or a hydrate thereof,

で表される基である化合物、 その塩又はその水和物である。 In the general formula (I), is represented by the following formula (III):

Or a salt or hydrate thereof.

 Streptomyces '' sp.

Mer-2487, FERM P-16718) The cultivation of bacteria in a nutrient medium, and the collection of a compound represented by the general formula (I :, a salt thereof or a hydrate thereof) from the culture broth. Provided is a method for producing a compound represented by the formula (I), a salt thereof, or a hydrate thereof.

The present invention relates to a human immunodeficiency virus (HIY) growth inhibitor comprising a compound represented by the general formula (I), a salt thereof or a hydrate thereof as an active ingredient. A pharmaceutical composition comprising an effective amount of a compound represented by the formula (I), a salt or a hydrate thereof and a pharmacologically acceptable carrier; a compound represented by the formula (I), a salt thereof or a hydration thereof A human immunodeficiency virus (HIV) growth inhibitor; and administering an effective amount of the compound represented by formula (I), a salt thereof, or a hydrate thereof to a human to obtain a human immunodeficiency virus (HIV). The present invention provides a method for inhibiting the growth of Rus (HIV). Therefore, prevent and treat diseases caused by the proliferation of human immunodeficiency virus (HIV).

In the present specification, the compound represented by the general formula (I) is a group represented by the general formula (II), and the compound in which R ₂ in the killing formula (II) is a methyl group is referred to as EM 2487A A compound in which is an ethyl group is referred to as EM 2487B, and a compound in which the compound represented by the general formula (I) is a group represented by the formula (III) is referred to as EM 2487C. Detailed description of the invention

Bacteriological properties of producer strain

 As a typical strain producing the compound of the present invention, an actinomycete isolated from the soil of Shiga Kogen Forest, Nagano Prefecture, and a strain numbered by the present inventors as strain E.M.I. 2487 (Mer-2487) was used. No. The bacteriological properties of this Mer-2487 strain are as follows.

(1) Form

 Extends aerial hyphae of Rectiilexibiles from the base hyphae. A mature aerial mycelium forms a spore chain consisting of cylindrical spores 10 to 50 rounds ahead. Spores are not found. The size of the spores is about 0.6-0.8 mXl.0-0.2 xm, and the surface of the spores is smooth (shows SfflOOtW and no flagella are observed.

 (2) Growth status in various media

 All cultures were performed at 28. The description of the color tone is indicated by the symbols shown in parentheses in Container, Corporation, Ob, Amerika Color, Harmony Manual (Color Ηετιποην Manual of Container Corporation of America).

 1) Yeast malt agar medium

Growth is moderate, with aerial hyphae on the surface and reddish (5ca-6ec) spores. Culture back Dark green to dark brown. Produces slightly brown soluble pigment. CT / JP99 / 03428

2) Automeal agar

 It grows well, with aerial hyphae on its surface and reddish (5dc) spores. The back of the culture is dark brown. Produces slightly brown soluble pigment.

 3) Starch, inorganic salt agar medium

 The growth is good, and aerial hyphae grow abundantly on the surface, and red (5ca-6ec) spores are seen. The back of the culture is dark green to bluish purple. No soluble dye is produced.

 4) Glycerin / asparagine agar medium

 It grows moderately and does not form aerial hyphae on its surface. The reverse side of the culture is cream-colored and does not produce soluble dyes.

 5) Tyrosine agar medium

 It grows moderately and does not form aerial hyphae on its surface. Does not produce melanin in the medium. The back of the culture is dark green.

 (3) Assimilation of various carbon sources

 Various carbon sources were added to Preed Ham Götrib agar medium to observe the growth.

 1) L-arabinose I

 2) D—Xylose +

 3) D—glucose +

 4) D—Fructose soil

 5) Sucrose +

 6) Noshi

 7) L—Ramnose I

 8) D—mannitol

 9) Lahui North I

 + Assimilate, ± slightly assimilate, one does not assimilate

 (4) Properties of cell wall components

Analysis of the hydrolyzed cells by thin-layer chromatography on cellulose revealed that the isoform of diaminopimeric acid, a component of the bacterial cell wall, was L-shaped. From the above bacteriological properties, it is considered that this bacterium belongs to the genus Streptomyces.

 The present inventor has used this strain in Streptomyces 'S.P.'

(St reptomyces sp. Mer-2487) in the Institute of Biotechnology and Industrial Technology (Postal code 305-8566, Tsukuba East 1-chome 1-3, Ibaraki, Japan) with FERM P-16718 number as 1998 Deposited on March 19, 2009. FERM P-16718 was transferred to International Deposit FERM BP-6762 on June 18, 1999.

(Culture conditions)

 The physiologically active substances EM2487A, B and C of the present invention are produced by inoculating the above-mentioned strains in a nutrient-containing medium and aerobically culturing them. The microorganisms that produce the physiologically active substance EM2487 include, but are not limited to, the above-mentioned strains, as long as they have the ability to produce Streptomyces; EM2487A, B.C.

The method for culturing the microorganisms described above is basically the same as the method for culturing general microorganisms. However, it is usually preferable to carry out the culture under aerobic conditions such as shaking culture by liquid culture and aeration and stirring culture. The medium used for the culture may be any medium containing a nutrient source that can be used by microorganisms belonging to the genus Streptomyces, and any of various synthetic, semi-synthetic, and natural media can be used. As the medium composition, glucose, sucrose, fructos, glycerin, dextrin, starch, molasses and the like as a carbon source can be used alone or in combination. As a nitrogen source, an organic nitrogen source such as pharmamedia, peptone, meat extract, soybean powder, casein, amino acid, yeast extract, urea or the like can be used alone or in combination. Other salts such as sodium chloride, potassium chloride, calcium carbonate, magnesium sulfate, sodium phosphate, potassium phosphate, cobalt chloride, etc., heavy metal salts, and vitamins such as vitamin B and biotin may be added as necessary. Can be used. If foaming during culturing is remarkable, various known antifoaming agents can be appropriately added to the medium. When adding an antifoaming agent, the concentration must be such that it does not adversely affect the production of the target substance. For example, the working concentration is preferably 0.05 or less. Culture conditions can be appropriately selected within a range in which the strain can grow well and produce the substance. For example, the pH of the medium is preferably about 5 to 9, usually around neutrality. The culture temperature is generally maintained at 20 to 40, preferably at 28 to 35. The culturing period is about 2 to 8 days, usually 3 to 5 days. 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 it goes without saying that the optimum conditions can be selected. The bioactive substance EM2487 of the present invention accumulated in the culture solution can be recovered by extracting cells from the cells by a known ordinary solid-liquid separation means such as filtration and centrifugation. .

Separation and purification

 Separation and purification of the bioactive substances EM 2487A, B and C can be performed by selecting and combining various known methods. For example, solvent extraction using methanol, ethyl acetate, acetone, n-butanol, etc., activated carbon, Amberlite XAD (manufactured by Rohm and Haas), Diaion HP-20 (manufactured by Mitsubishi Chemical), etc. Adsorption to water and elution with aqueous alcohol, aqueous acetone, etc., gel filtration with Sephadex LH-20 (Pharmacia), Bio 'Gel P-2 (Bio-Rad), silica gel, alumina, etc. Separation / purification by column method or thin-layer chromatography, high-performance liquid chromatography (preparative HPLC) using normal-phase or reverse-phase columns, alone or in combination as appropriate, and in some cases repeated use. Can do things.

The compound of the present invention can be purified from the culture of the bacteria as follows. After culturing the microorganism of the genus Streptomyces under ordinary appropriate culture conditions, the culture solution is clarified and filtered, and then an organic solvent such as butanol or methyl isobutyl ketone is added for extraction. The organic solvent layer is concentrated under reduced pressure. Then, the mixture is extracted with methanol and treated with petroleum ether (light petroleum) or the like to obtain a crude extract. Next, fractionate using appropriate means such as adsorption chromatography using silica gel, LH20 gel chromatography, distribution chromatography, thin-layer chromatography, paper chromatography, etc., and confirm the active fraction by activity screening. An active substance can be isolated by appropriately combining the above methods. Solvents used for adsorption chromatography include chromatographic form, methanol, acetone, hexane, Using a commonly used organic solvent such as toluene, the concentration can be appropriately selected and used in combination. As a solvent for crystallization, chloroform and hexane, or chloroform and carbon tetrachloride can be used. One method is the method of M. Lumb et al. (Nature. 206, 263, 1965).

 The structural analysis of the isolated compound can be performed by a conventional method such as elemental analysis, GC-MS, NMR, and melting point.

Pharmaceutical applications

 The isolated novel substance inhibits Tat activity, suppresses HIV growth in acute and persistent infection systems, and is useful for treating AIDS caused by the treatment of HIV infection. It may also be used in combination with nucleoside and non-nucleoside reverse transcriptase inhibitors and protease inhibitors to enhance its therapeutic effect or to delay the emergence of resistant viruses.

 It is especially useful for HIV-infected patients who have acquired resistance to nucleoside and non-nucleoside reverse transcriptase inhibitors and protease inhibitors. When the compound is administered as a therapeutic or prophylactic agent for various diseases, it may be administered orally as tablets, powders, granules, capsules, syrups, etc., or as a spray, suppository, injection, external preparation They may be administered parenterally as drops. The dosage varies significantly depending on the severity of symptoms, age, type of liver disease, etc., but usually about 1 mg to 1000 mg per day for adults is administered once or several times a day.

 In the case of formulation, it is manufactured by a usual method using an ordinary formulation carrier. That is, when preparing a solid preparation for oral use, an excipient and, if necessary, a binder, a disintegrant, a lubricant, a coloring agent, a corrective agent, etc. are added to the base drug, and then tablets are prepared in a conventional manner. , Coated tablets, granules, powders, capsules, etc. These tablets and granules may be sugar-coated, gelatin-coated or any other appropriate coating as needed.

When preparing injections, add a pH adjuster, buffer, stabilizer, solubilizer, etc. as necessary to the base drug, and make subcutaneous, intramuscular, or intravenous injections in the usual manner. Example

 Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited thereto. In the examples, percentages (%) refer to weight / volume percentages unless otherwise specified.

Example 1 Separation and purification of EM 2487A, B, C

 From the slope medium (ISP-2) of the Mer-2487 strain, add one platinum loop to a 50πι1 seed culture medium (glycerin 2%, glucose 2%, soybean flour (Essan Meat: Ajinomoto Co.) 2%, yeast extract 0.5%) %, Sodium chloride 0.25%, calcium carbonate 0.32%, copper sulfate 0.0005%, manganese chloride 0.0005%, zinc sulfate 0.0005%, ρΗ7.4) and inoculate a 500 ml triangular flask, and rotate at 28 for 2 days. The seed culture was obtained by culturing on a mill. 150 ml of each of these seed cultures is used for the main culture medium (dextrin 3%, glucose 0.5%, soybean flour (Essan Meat: Ajinomoto Co., Inc.) 1.5%, corn 'Steep' Rica 1 0.5%, calcium carbonate 0.5%, antifoam Ingredients were inoculated into two 30L jar fermenters containing 15L each, and cultured at 28X: for 70 hours with aeration and agitation (aeration rate 7.5L / min, agitation 100-250rpm). . After the completion of the culture, 24 L of the obtained culture solution was centrifuged in a continuous centrifuge to separate the supernatant into bacterial cells. The cells were extracted with 20 L of methanol, and the obtained methanol extract was concentrated under reduced pressure, methanol was distilled off, and the resultant was mixed with the supernatant.

 2.4 L of Diaion HP-20 expanded with water was added to the mixed solution, and after stirring, the resin was collected by filtration, packed in a column, and washed with 6 L of 20% methanol. Subsequently, the mixture was eluted with 41 L of 80% acetone, and the eluate was concentrated under reduced pressure to distill off acetone. The remaining aqueous solution was concentrated to dryness under reduced pressure to obtain a crude extract.

This was dissolved in a small amount of dimethyl sulfoxide, and YMC-GEL 0DS-AM120-S50 (Emushi Co., Ltd.) previously equilibrated with a mixed solution of acetonitrile and 10 mM phosphate buffer (pH 7.0) = 2: 8 (Internal diameter 60 min, length 950 mm). After washing with a mixed solvent of the same composition, elution was carried out with a mixed solvent of a 35:65 composition. The eluate was analyzed by high performance liquid chromatography. 20 mM phosphate buffer (pH7.0) = 35:65, flow rate: 1 mL / min, detection: ultraviolet absorption at 260 nm, retention time: A: 2.5 min, B: 6. lmin, 6.3 min) Was collected to obtain a fraction containing EM 2 487 A, B, C. After the acetonitrile was distilled off under reduced pressure, Diaion HP-20 swollen with water was added, and after stirring, the resin was packed in a column and washed with 20% methanol. Subsequently, elution was carried out with 80% acetone, and the eluate was concentrated under reduced pressure, and acetone was distilled off. The remaining aqueous solution was concentrated to dryness under reduced pressure to obtain a crude extract. This was dissolved in a small amount of dimethylsulfoxide, and fractionated by HPLC (column: Factory sphere ODS-H80, inner diameter 20 mm, length 250 mm, manufactured by YMCS, Inc., moving layer: acetonitrile 20 mM phosphate buffer (pH 7.0 ) = 35:65, flow rate: 10 mL / min, detection: ultraviolet absorption at 260 nm, retention time: A: 12. Omin, B: 22. Omin. C: 26. Omin). This fractionation was repeated, and the eluates showing peaks of EM 2487 A, B, and C were combined, and acetonitrile was distilled off under reduced pressure. Then, Diaion HP-20 expanded with water was added, followed by stirring. Thereafter, the resin was packed in a column and washed with 20% methanol. Subsequently, elution was carried out with 80% acetone, and the eluate was concentrated under reduced pressure, and acetone was distilled off. The remaining aqueous solution was concentrated to dryness under reduced pressure to obtain 432 mg, 44.8 mg and 22.4 mg of pure white powders of EM 2487A, B and C, respectively.

 EM 2487A purification and structural analysis data

 1. Color and properties: white powder

2 · Molecular formula: 0!

 3. Mass spectrum (FAB-MS): m / z 828 (M-H) "

4. Specific rotation: ²² -7.9 (c 1.3, MeOH)

 5. Ultraviolet absorption spectrum:

 In neutral methanol: Amax nm (£): 262 (34200)

 In acidic methanol: λ πΐ3χ nm (£): 262 (36100)

 In basic methanol: λ max I (ε): 260 (27100)

 6 · Infrared absorption spectrum:

Measured in KBr powder. Shows major absorption. (Wave number, cm- ¹ )

2920, 2850, 1680, 1470, 1240, 1070, 880, 720, 550 7. Table 1 shows the results of measurements of the MR spectrum, ¹³ C NMR spectrum and HMBC correlations in a heavy methanol solution. The table shows the positions of the carbons of the compounds, their analytical data, ie, the Chemical Shift Table of 2487A of EM 2487A, and the correlation between carbons (HMBC correlat ions) along with the reference value (Reference). Indicated.

Table 1 丽 R spectrum of EM 2487A

Chemical Shift Table of 2487A

position ¹³ C NMR d (mult) Ή NMR d (int, mult, J Hz) HMBC correlations

 2 152.5 (s)

 4 166.2 (s)

 5 103.0 (d) 5.84 (1H, d, 7.9) C4, C6

 6 142.4 (d) 7.91 (1H, d, 7.9) C5, cr

 1 '90.4 (d) 5.98 (1 H, d, 4.6) C2, C6, C2' 2 '75.7 (d) 4.24 (1H, dd, 5.2, 4.6)

 3 '70.3 (d) 4.42 (1H, dd, 5.2, 5.2)

 4 '84.5 (d) 4.22 (1H, ddd, 5.2, 2.7, 2.4)

 5 '69.5 (t) 4.51 (1H, dd, 11.6, 2.7)

 4.27 (1H, dd, 11.6, 2.4)

 1A 155.2 (s)

 2A 81.0 (d) 5.48 (1 H, brm)

 3A 77.0 (d) 4.38 (1H, ddd, 5.8, 4.0,

(V _HP = 2.5)

 4A 71.9 (d) 4.69 (1H, dd, 5.8, 1.0) C1A

 5A 121.4 (s)

 6A 55.4 (t) 4.31 (1H, d, 12.2) C1A, C5A, C4A

4.20 (1H, dd, 12.2, 1.0) 1B 178.7 (s)

2B 51.7 ft) 4.05 (1H, ddd, 17.4, ³ J _PH =: 13.0, 13.0)

3.99 (1H, ddd, 17.4. 3 H = 13.0, 13.0)

 1C 63.2 (t) 2.98 (1 H, brm)

 2.82 (1 H, brm)

 2727 ^ 1.61

 3C-10C 31.1-28.6 1.39-1.28 (16H, brm)

 11C 40.3 (t) 1.21 (2H, brm)

 12C 29.2 (d) 1.56 (1H, sept-t, 6.5, 6.5)

 13C 23.1 (q) 0.91 (3H, d, 6.5) C14C, C12C, C11C

14C 23.1 (q) 0.91 (3H, d, 6.5) C13C, C12C, C11C

N-CH ₃ 46.7 (q) 2.81 (3H, brs) C1C,

Reference ^{, 3} C (500Hz, 20 ° C) CD ₃ OD = 49.0ppm, Ή (500ΜΗζ, 50 C) CD ₂ HOD = 3.35ppm Solubility:

Soluble: neutral water, methanol, dimethyl sulfoxide

Insoluble: Hexane, ethyl ester, black form

High performance liquid chromatography:

Column: J'sphere 0DS-H80, inner diameter 4.6 thigh, length 75 mm) Solvent: acetonitrile-20 mM phosphate buffer (pH 7.0) = 35:65

Flow rate: 1 mL / min

Detection wavelength: 260nm; UV absorption

Holding time: 2.5min

487B purification and structural analysis data

Color and properties: white powder

Molecular formula:. C33H5 ₈ s0 6P ₂

Mass spectrum (FAB-MS): m / z 842 (M-H) —

Ή NMR spectrum

Table 2 shows the results of measurement with the heavy methanol solution. Table 2 EMK 2487B NMK spectrum

position ¹ H NMR d (int, mult, J Hz)

Two

 Four

 5 5.85 (1H, d, 7.9) 6 7.99 (1H, d, 7.9) r 5.99 (1H, d, 4.7)

2 '4.24-4.17 (m)

 3 '4.45 (1 H, dd, 5.0, 5.0) 4' 4.24-4.17 (m)

 5 '4.58 (1H, dd, 11.8, 3.3)

4.24-4.17 (m)

 1A

 2A 5.44 (1 H, brm)

 3A 4.24-4.17 (m)

 4A 4.68 (1H, dd, 6.2, 1.3) 5A

 6A 4.32 (1H, d, 12.3)

 4.20 (1H, dd, 12.3, 1.3)

1B

 2B 4.10-3.92 (2H, m)

 1C 3.27 (1H, brm)

 2.80 (1H, brm)

2C 1.57 (2H, brm) 3C-11 C 1.42-1.28 (16H, brm)

 12C 1.35 (1H, m)

 13C 1.38 (1H, m)

 1.18 (1H, m)

 14C 0.92 (3H, t, 7.0) 15C 0.90 (3H, d, 6.4)

N-CH, 2.82 (3H, brs)

Reference: Ή (400 Ηζ, 30 ° C) CD ₂ HOD = 3.35ppm 5. Solubility:

 Soluble: neutral water, methanol, dimethyl sulfoxide

 Insoluble: Hexane, ethyl ester, black form

 6. High performance liquid chromatography:

 Column: J'sphere 0DS-H80, inner diameter 6 marauder, length 75 marauder (manufactured by YMC) Solvent: acetonitrile 20 mM phosphate buffer (pH 7.0) = 35:65

 Flow rate: 1 mL / min

 Detection wavelength: 260nm; UV absorption

 Retention time: 6. lmin

EM 2487C purification and structural analysis data

1. Color and properties: white powder

2. Molecular formula:, 0 ₁₆ P ₂

 3. Mass spectrum (FAB-MS): m / z 842 (M-H)

 4. Ή NMR spectrum

 Table 3 shows the results measured in a heavy methanol solution.

Table 3 NMK spectrum of EM 2487C

position ¹ H NMR d (int, mult, J Hz)

 Two

 Four

 5 5.85 (1H, d, 7.9)

 6 7.98 (1H, d, 7.9)

 r 5.99 (1H, d, 4.6)

 2, 4.24-4.17 (m)

 3, 4.45 (1H, dd, 5.1, 5.1)

 4, 4.24-4.17 (m)

 5 '4.58 (1H, dd, 11.7, 3.4)

 4.24-4.17 (m)

 1A

 2A 5.44 (1H, brm)

 3A 4.24-4.17 (m)

 4A 4.68 (1H, brd, 6.0)

 5A

 6A 4.31 (1H, d, 12.3)

 4.14 (1H, brd, 12.3)

 1B

 2B 4.10-3.92 (2H, m)

 1C not detected

 2C 1.60 (2H, brm)

 3C-11C 1.42-1.28 (18H, brm)

 12C 1.21 (2H, m)

 13C 1.56 (1H, sept-1, 6.7, 6.7) 14C 0.92 (3H, d, 6.7)

 15C 0.92 (3H, d, 6.7)

 N-CH, 2.83 (3H, brs)

Reference: Ή (400 Ηζ, 30 ° C) CD ₂ HOD = 3.35ppm Solubility:

Soluble: neutral water, methanol, dimethyl sulfoxide

Insoluble: Hexane, ethyl acetate, black form

High performance liquid chromatography:

Column: J'sphere ODS-H80, inner diameter 4.6 mm, length 75ππη (manufactured by IMC) Solvent: acetonitrile- 20 mM phosphate buffer (PH7.0) = 35:65

Flow rate: lmL / min

Detection wavelength: UV absorption at 260im

Retention time: 6.3min 99/03428 Example 2 Tat inhibitory activity

 (1) Construction of reporter gene

 To screen for compounds that inhibit Tat activity, we constructed a repo all-in-one gene whose transcription from the HIV LTR containing the TAR region could be measured. Goto et al. Reported a TNF-α promoter followed by a secreted alkaline phosphatase PLAP, followed by a TNF-α repo overnight plasmid containing a Neo resistance gene.TNF-a-PLAP-PGK-neo (Goto M. et al., Mol. Pharmacol. 49: 860 (1996)), in the Xbal-Hinddlll region containing the TNF-α promoter, pUC-BENN- obtained from the National Insiitute of Allergy and Infectious Disease AIDS Research and Reference Reagent Program. The repo overnight gene PPGKF was constructed by inserting the Xbal-Hindlll site containing the HIV LTR of CAT and sequences derived from infected cells.

 (2) Construction of Tat-expressing gene

 It has been reported that the 2nd exon of Tat is required for transcriptional activation from the HIV LTR integrated into the chromosome of cells (Jeang. TJ of Biol. Chem. 268 (33): 24940 (1993)). Tat expression vector obtained from National Institute of Allergy and Infectious Disease AIDS Research and Reference Reagent Program pSV2 Tat 2nd.exon sequence (Collman R. et al., J. of Virol. 66 (12) : 7517 (1992)). After annealing the synthetic oligonucleotides of SEQ ID NO: 1 and SEQ ID NO: 2, an extension reaction was performed using Vent polymerase (manufactured by New England Biochemistry), digested with BamHI and Bglll, and inserted into the BamHI-Bglll site of pSV2 Tat to insert pSV2 Tat- Constructed a long.

 (3) Gene transfer by electoral portation method

10 to 40 ^ g of DNA containing the gene sequence to be transferred is mixed with 3 x 10 ⁶ to 1 x 10 ⁷ human leukemia cell CEM suspended in 1 ml of serum-free RPMI medium, and 4: 10 minutes After standing, the power was supplied to the product using a Gene Pulser (manufactured by Biorad) under the conditions of 300 V and 1000 ¥. The suspension was immediately diluted in RPMI medium (10% FCS / RPMI medium) containing 10¾ FCS and cultured in a C0: incubator.

(4) Measurement of alkaline phosphatase activity The solution was treated at 65 ° C for 20 to 30 minutes in order to inactivate the alkaline phosphatase contained in the FCS in the test solution. To 96 play Bok for chemiluminescence measurement, test solution 10 ii, carbonate buffer one _{(0.28 M Ma 2 C0 3 buifer} pH 10.0, 8 mM MgS0 4) (manufactured by Sumitomo Metal Co., Ltd.) 50 1 and Rumisute in 50 ^ 1 After left for 1 hour at room temperature, the chemiluminescence was measured using a microplate luminometer LB96P (Berthold).

 (5) Establishment of repo overnight cell line

To screen for compounds that inhibit Tat activity, a reporter cell line expressing a reporter gene was established. The reporter gene pPGKF was introduced into human leukemia cell CEM cells by the electrophoresis method. After culturing overnight in C0 ₂ Inkyube Isseki were cultured in suspension for 24 well plates in 10% FCS / RPMI culture locations containing 0.8 mg / ral geneticin (Sigma Co.). Two weeks later, the AL-rephosphatase activity in the culture supernatant of the cells in which the cells had proliferated was measured, and PLAP-producing cells were cloned from the active wells by the ultradilution method.

 After growing the cloned cells, clones with increased PLAP production due to introduction of the Tat-expressing gene and TNF were selected. The selected repo overnight cells were subcultured and used to measure Tat inhibitory activity.

 (6) Measurement of living cells by MTT method

The cells cultured in a 96-well plate, 2 hours were cultured in 7.5 mg / inl of MTT solution 20 w l was added C0 ₂ in Kyubeta. The 150 1 supernatant was removed without sucking cells, and 100 ίΐ 1 of 10% TritonX-100 in isopropanol was added to solubilize the dye (formazan) formed by reducing MTT. The absorbance at 540 nm of each well was measured using a plate reader (BioRad Model 3550) and used as an indicator of living cells.

(7) EM 2487A, B, C inhibitory activity against Tat expressed in cells

Tat expressed by electroporation in reporter cell gene pSV2 Tat - after introduction of the long gene were cultured in C0 ₂ incubator. Cells into which the Tat expression gene was not introduced were also cultured. The next day, the cultured cells were collected by centrifugation at 1000 rpm for 5 minutes, adjusted to a concentration of 1.25xlOVml in 10% FCS / RPMI medium to obtain a cell suspension. The test sample was dissolved in DMSO and diluted 100-fold with 10% FCS / RPMI to prepare a sample diluent.

In a 96-well plate, 10¾ FCS / RPMI medium 20 1 not containing or containing sample diluent 20 1, 20 ng / nil of TNF, was added the cell suspension 160 n 1, and cultured for 2 days in C0 ₂ incubator. The culture supernatant lOi was collected and the alkaline phosphatase activity was measured. The cytotoxicity of the sample was measured by the MTT method.

 Table 4 shows the lipophilic phosphatase activity in the culture supernatant and the absorbance of the MTT method (the percentage in parentheses is the percentage when the control without drug was set to 100%). EM 2487A, B, and C did not affect the production of al force phosphatase in cells not transfected with a Tat-expressing gene at a concentration of 10 g / ml, and did not show cytotoxicity. The introduction of the Tat-expressing gene enhanced the production of alkaline phosphatase by about 20-fold, but EM 2487A, B, and C suppressed only the production of alkaline phosphatase promoted by Tat. In addition, in the presence of TNF, alkaline phosphatase production in cells into which Tat-expressing genes had been introduced and in which cells had not been introduced was further promoted by about 3 times. In this case as well, EM 2487A, B, and C did not affect the production of alkaline phosphatase in cells not transfected with the Tat-expressing gene, and did not affect the production of alkaline phosphatase promoted by the introduction of the Tat-expressing gene. Only production was suppressed.

 (8) Inhibitory activity of EM 2487A on extracellular Tat

Incubate the reporter cells for 2 hours in the presence of 2 / xg / ml Tat (Immuno Diagnostics), ImM Dit iothratol, oth / ol ChloroQuin in serum-free medium, and centrifuge at 1000 rpm for 5 minutes. % were suspended in FCS / RPMI medium were cultured in C0 ₂ incubator. Cells treated similarly in the absence of Tat were also cultured. The next day, the cultured cells were collected by centrifugation at 1000 rpm for 5 minutes, and adjusted to a concentration of 1.25 × 10 ⁵ / ml in 10¾ FCS / RPMI medium to prepare a cell suspension.

A 96-well plate, sample diluent 20μ1, 20 ng / ml of or without including TNF 1 0¾ FCS / RPMI medium 20 1, the cell suspension 160 1 was added and cultured for 2 days in a C0 ₂ incubator. 10 1 of the culture supernatant was collected and measured for alkaline phosphatase activity. In addition, the cytotoxicity of the specimen was measured by the ΜΤΤ method. 3428 Alkaline phosphatase production was increased about 30-fold by supplying Tat from outside the cell. In the presence of TNF, alkaline phosphatase production was further promoted about three-fold. Table 4 shows the alkaline phosphatase activity in the culture supernatant and the absorbance measured by the MTT method (the values in parentheses are the percentages when the control without drug was set to 100%). Regardless of the presence or absence of TNF, EM 2487A did not affect the production of alkaline phosphatase in cells that did not supply Tat at a concentration of 10 g / ml, and did not affect the production of Tat-expressing genes. Only phosphatase production was suppressed. Table 4 shows the results together with no treatment (None Treat).

Table 4 Tat inhibitory activity of EM 2487A, B, C

 1) Inhibitory activity of EM 2487A, B, C against Tat expressed in cells

 None treat TNF Tat TNF + Tat-MTT P and luminosity No drug added 376 (100) 1240 (100) 8989 (100) 22367 (100) 0.474 (100) EM 2487 A (10 / ig / ml) 400 (106) 1285 (104) 5825 (65) 11420 (51) 0.477 (101)

EM 2487B (10 / ^ g / ml) 437 (116) 1617 (131) 5521 (61) 11248 (50) 0.495 (104)

EM 2487C (10 / g / ml) 390 (104) 1362 (110) 5576 (62) 11455 (51) 0.483 (102)

 2) Inhibitory activity of EM 2487A against extracellular Tat

 None treat ― TNF Tat TNF + Tat — MTT absorbance No drug added 357 (100) 1046 (100) 11058 (100) 36354 (100) 1.090 (100)

_EM 2487A (10A <g / ml) _369 (104) —1134 (108) —7630 (69) — 19124 (53) — 1.122 (103) —Example 3 EM 2487A, B, C in Molt-4 acute infection system Anti-HIV activity

Human leukemia cells Molt-4 were infected with the HIV-1 HTLV-IIIB strain. HIV-infected cells and uninfected cells, lxi0 ⁵ cells / ml, were cultured at 37 with various concentrations of the drug, and after 4 days, diluted 1: 5 with a culture solution containing the same concentration of the drug. 7 days after the start of culture

The number of living cells was measured by the MTT method, and the cytotoxicity inhibitory activity (anti-HIV activity) and cytotoxicity of EM 2487A, B, C were determined.

 A drug that exhibits 50% cytotoxicity-inhibiting activity against HIY-infected cells, where the number of live cells in non-drug-free HIY-infected cells is 100% and the number of live cells in drug-free HIV-infected cells is 0%. The concentration EC ^ was determined.

The number of viable cells in non-infected cell culture without drug was set to 100%, and viable cells in medium alone Drug concentration CC _5, shown as a number, 50% of the cytotoxic activity against non-infected cells. Was asked.

As shown in Table 5, £ M 2487A, B, and C showed anti-HIV activity in HIV proliferation in the Molt-4 acute infection system. Table 50% cytotoxicity inhibitory concentration (EC5. (^ M)) , 50% cytotoxicity concentration _{(CC 5. (M))} , showed selectivity coefficient (selectivity index).

Table 5 Anti-HIV activity of EM 2487A, B, C in Molt-4 acute infection

EC ₅₀ M) CC ₅₀ (M) selectivity index

 EM 2487A 0.27 4.84 18

 EM 2487B 0.28 4.69 17

 EM 2487C 1.28> 10> 7.8

Example 4 Anti-HIV Activity of EM 2487A in MT-4 Acute Infection System

Human leukemia cells MT- were infected with the HIV-1 HTLV-IIIB strain. And cultured at 37 with HIV-infected cells and uninfected cells IX 10 ⁵ cei is / ml of various concentrations of the drug. On the 4th day from the start of culture, live cells were measured by the MTT method, and cytotoxicity-suppressing activity (anti-HIV activity) and cytotoxicity were determined by drug addition.

 As shown in Table 6, Ro 24-7429 had no effect in the MT-4 acute infection system, but EM 2487A showed anti-HIV activity.

Table 6 Anti-HIV activity of EM 2487A in MT-4 acute infection

EC ₅₀ (^) L / C ₅ o (β Μ) selectivity index

 EM 2487A 0.97 4.8 5

 Ro 24-7429> 2.8 2.8 <1 Example 5 Anti-HIV activity of EM 2487A in acutely infected human peripheral monocytes (PBMC)

HIV-1 HTLV-IIIB strain was infected to PBMC IX 10 ⁵ cells / well stimulated with PHA, and cultured at 37 with various concentrations of drugs. On the 6th day from the start of the culture, the P24 antigen was measured by ELISA to determine the total amount of T cells, and the viable cells were measured by the ΜΤΤ method. Was determined.

The p24 S original amount of drug additive-free and 10M, drug concentration EC ₅ showing 50% of HIV proliferation inhibitory activity. I asked.

As shown in Table 7, 2487A exhibited anti-HIV activity in acute PBMC infection. Example 6 OM10. Anti-HIV activity in Keji infection system

HIV-1 infected promyelocytic OM10.1 cells, Ixi0 ⁵ cells / well, were pretreated with various concentrations of the drug for 2 hours, and then stimulated with lng / ml of TNF-α, and then in the presence of the drug. The culture was continued. On the third day from the start of the culture, the amount of HIV is quantified by measuring the ρ24 antigen by ELISA, and the viable cells are measured by the MTT method. The activity of suppressing the HIV production (anti-HIV activity) and The judgment was made.

 As shown in Table 7, EM 2487A showed anti-HIV activity in the OM10.1 persistent infection system.

Table 7 Anti-HIV activity of EM 2487A in PBM acute infection system and OM10.1 persistent infection system

EC ₅₀ (μ M) CG ₅₀ M) selectivity index

 PBMC acute infection 1.6 55 35

 OM10.1 Persistent infection system 0.17 9.3 55 According to the present invention, in addition to the nucleoside and non-nucleoside reverse transcriptase inhibitors and protease inhibitors that have been put to practical use, it is possible to provide a third means of treating AIDS Becomes

 When used in combination with a reverse transcriptase inhibitor or a protease inhibitor, it is expected that HIV growth will be further suppressed, so that it can be used to delay the emergence of resistant viruses. Particularly useful for HIV-infected patients who have acquired resistance to nucleoside and non-nucleoside reverse transcriptase inhibitors and protease inhibitors because of different action points and no crossover of resistance It is.

Claims

The scope of the claims 1. The following general formula (I)

(In the formula, including those with a branch in § alkyl group having from c ₁₂ c ₁₈ and the higher alkyl. This indicated a higher A kill group.) Or a salt or hydrate thereof represented by 2. R _t is the following general formula (II):

(Wherein, R ₂ is a methyl group or an ethyl group) The compound according to claim 1, which is a group represented by the formula: 3. R [is the following formula (III):

2. The compound according to claim 1, which is a group represented by the formula: or a salt or hydrate thereof. 4. Culturing Streptomyces sp. SM-2487 (Streptomyces sp. Mer-2487, FERM BP-6T62) bacteria in a nutrient medium, and using the culture solution as claimed in claim 1 or 3 4. The method for producing a compound, a salt thereof, or a hydrate thereof according to claim 1, wherein the compound, a salt thereof, or a hydrate thereof is collected. 5. A medicament comprising the compound according to any one of claims 1 to 3, a salt thereof, or a hydrate thereof as an active ingredient.  6. A human immunodeficiency virus (HIV) growth inhibitor comprising the compound according to any one of claims 1 to 3, a salt thereof, or a hydrate thereof as an active ingredient.  7. A pharmaceutical composition comprising an effective amount of the compound according to any one of claims 1 to 3, a salt or a hydrate thereof, and a pharmacologically acceptable carrier.  8. Use of the compound according to any one of claims 1 to 3, a salt thereof, or a hydrate thereof for the manufacture of a human immunodeficiency virus (HIV) growth inhibitor.  9. A method for inhibiting the growth of human immunodeficiency virus (HIV) by administering to a human an effective amount of the compound according to any one of claims 1 to 3, a salt thereof, or a hydrate thereof.