JPH07285957A - New compound a-76341 - Google Patents

Abstract

PURPOSE:To obtain a new compound inhibiting a sphingomyelinase and useful for preventing and treating HIV, diabetes mellitus, arteriosclerosis, osteoporosis, thrombosis, inflammation, respiratory diseases, thyroid diseases, Alzheimer's dementia, hepatitis, nephritis and leukemia, etc. CONSTITUTION:A new compound A-76341 of the formula. The compound is obtained by subjecting A-76341-producing microorganism belonging to the genus Micromonospora consisting of Micromonospora sp. SANK 60294 strain to shake culture using glucose, sucrose, starch, etc., as a carbon source and soybean powder, corn steep liquor, raw yeast, etc., as a nitrogen source, as necessary in the presence of inorganic salts such as calcium carbonate or phosphoric acid under aerobic conditions, preferably at 20-37 deg.C (optimally at about 28 deg.C), normally for 4-8 days, preferably by a deep culture method and then, adding an organic solvent such as acetone or acetonitrile thereto and extracting the cultured product with the solvent.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a novel compound A-763 which inhibits sphingomyelinase and is useful as various medicines.
41 and its manufacturing method.

[0002]

2. Description of the Related Art Interleukin-1β (hereinafter referred to as "IL
-1β ". ) Has various biological actions, and is generally considered to be a biological substance essential for maintaining homeostasis of the living body.
However, an abnormality occurs in the IL-1β production regulation function,
Overproduction of IL-1β causes various diseases. In addition, tumor necrosis factor-α (hereinafter, “TNF-α”)
Say. ) Has the action of killing certain tumor cells or virus-infected cells, or enhancing the antibacterial action of granulocytes, but when TNF-α is also overproduced, it causes some diseases. Is the major etiological factor.

These two cytokines are products of completely different genes and have no similarities in their structures and have unique receptors corresponding to them, but their target cells and biological activities often overlap. For example, both cytokines are major causes of septic shock caused by endotoxin (LPS) that has entered the body [Tracy KJ et al.
Science, 234, 470 (1986), Tracey KJ et al. Natur
e (London), 330, 662 (1987)], others, granuloma (Kobay
ashi K. et al. J. Immunol., 134, 358 (1985)], meningococcal meningitis and malaria infection [Curfs JHA et al. J.
Exp.Med., 172, 1287 (1990)], etc., and is closely related to infectious diseases derived from exogenous microorganisms, parasites, viruses, and the like. Each major stage in such acute phase inflammatory reaction, namely, local infiltration of inflammatory cells [Gamble JR et al. Pro
c.Natl.Acad.Sci.USA, 82, 8667 (1985), Masayuki Miyasaka et al. Annual Review Immune '91, 57 Chugai Medical Co. (199
1)), fever 〔Dinarello CA Lymphokines, 14, 1 (1
987)], induction of acute phase protein [Perimutter DH et al.
J. Clin. Invest., 78,1349 (1986)], for promoting the production of prostanoids, especially PGE2 [Dayer J.-M. et al. JE.
xp.Med., 162, 2163 (1985), Turinsky J. et al. Am.
J. Physiol. 262, E476 (1992), Ballou LR et al.
J. Biol. Chem. 267, 20044 (1992)], both cytokines play an active role.

IL-1β and TNF-α are involved in the development and progression of chronic inflammatory diseases such as rheumatoid arthritis (RA), activation of lymphocyte infiltration in synovial tissue,
It promotes the proliferation of synovial cells, the destruction of chondrocytes, and the activation of osteoclasts to promote bone resorption [Mizel SB et
al.Proc.Natl.Acad.Sci.USA 78, 2474 (1980), Mi
yasaka N. et al. Arthritis Rheum., 31, 480 (198
8), Arend WP and Dayer J.-M. Arthritis Rheumatis
m., 33, 305 (1990)]. In addition, the same rheumatic disease, collagen disease [Tanaka Y. et al. J. Immunol., 143, 1584
(1989)], Kawasaki disease mainly due to systemic vasculitis (Leung
DYM et al. J. Exp.Med., 164, 1958 (1986)], and is known to be involved in granulomas and subsequent chronic inflammation associated with fibrosis (Le J. and Vilcek J. Lab. Invest). ., 56,
234 (1987)]. Glucocorticoids, which are currently used as therapeutic agents for chronic inflammatory diseases, are known to have a part of their action in suppressing the production of these cytokines (Lew W. et al. J. Immunol., 140 , 1895 (1988)
], Glucocorticoid has the disadvantage of inducing various serious side effects due to its various physiological actions.

Furthermore, IL-1β and TNF-α adhere to monocytes to vascular endothelial cells and migrate to the subendothelium [Pober JS e
t al. J. Immunol., 137, 1893 (1986), Nelken NA et.
al. J. Clin. Invest. 88, 1121 (1991)], and promote abnormal proliferation of vascular smooth muscle cells in the intimal membrane [Raines EW et
al. Science 243, 393 (1989)], involved in the onset and progression of atherosclerosis.

IL-1β and TNF-α promote the production of platelet activating factor (PAF), induce tissue factor on the endothelial cell membrane surface, decrease thrombomodulin protein C, and produce plasminogen activator inhibitor 1. Suppresses the inhibition and causes platelet aggregation and blood coagulation as a whole, causing thrombus formation [Bevilacqua MP eta
l. Proc.Natl.Acad.Sci.USA, 83, 4533 (1986), Le
J. and Vilcek J. Lab. Invest., 56, 234 (1987), Yasushi Sato Contemporary Medicine, 23, 3163 (1991)].

In insulin-dependent diabetes mellitus (IDDM), latent, chronic, and autoimmune inflammation occurs in pancreatic islets, especially pancreatic β cells in the process leading to its development.
1β and TNF-α are involved in it [Nerup J. et al.
Diabetes Care., 11, 16 (1988). On the other hand, also in non-insulin-dependent diabetes mellitus (NIDDM), TNF-α acts on muscle and hepatocytes through production in adipocytes and is involved in exerting insulin resistance [Spiegelman
BM et al. J. Biol. Chem., 268, 6823 (1993)]. IL-1β and TNF-α are deeply involved in the proliferation of mesangial cells and the growth of substrates, which are the main components of glomerulonephritis development [We.
rber HI et al. J. Immunol., 138, 3207 (1987), Ba
ud L. et al. Kidney Int., 41, 600 (1992)].

IL-1β and TNF-α are I from T cells.
Promotes L-2 production, its secretion, its receptor expression, and
Acts on other immune cells to enhance their function to activate the immune system [Gillis S. and Mizel SB Proc.Na
tl.Acad.Sci.USA 78, 1133 (1981), Scheurich P. e.
t al. J. Immunol., 138, 1786 (1987)]. Due to this action, both cytokines contribute to the development of graft-versus-host disease (GVHD) that occurs during transplantation, for example.

TNF-α suppresses lipoprotein lipase activity of adipocytes in chronic infections and cancer patients to cause anorexia, resulting in extreme weight loss.
It causes depletion (cachexia) and therefore TNF-α is called cachectin [Beutler B.
et al. Nature (London), 316, 552 (1985)]. TNF
-Α activates transcription of HIV (human immunodeficiency virus) -infected cells from the viral genome end: LTR: of HIV inserted into the chromosome through the transcription factor NF-κB, and thus HIV replication is promoted. Advance [Nabel G. e
t al. Nature, 326, 711 (1987), Schreck R. et al. E
MBO Journal, 10, 2247 (1991)]. Others, IL-1
As a disease caused by overproduction of β and TNF-α, fulminant hepatitis [Muto Y. et al. Lancet II, 72 (1988)], asthma,
Idiopathic pulmonary fibrosis (Kelley J. Am. Rev. Respir. Dis., 141
(3), 765 (1990)], ARDS (adult respiratory dis
tress syndrome) (Millar A. et al. Lancet II, 712
(1989)], etc., autoimmune thyroid disease [Katsumi Eguchi et al. Approach from the latest medicine 1 From cytokine, Medical Review, 38 (1991)], Lyme disease [Habicht GS et al. J. Immunol., 134, 3147 (1
985)], Alzheimer's disease [Griffin WST at al. Pr.
oc.Natl.Acad.Sci.USA, 86, 7611 (1989)], Crohn's disease [Ayaguni Yagita, Medical History, 147, 375 (1988)]
], Toxic shock syndrome [Ikejima T. et al. J. Clin. I
nvest., 73, 1312 (1984)], Osteoporosis (Pacifici
R. et al. Proc. Natl. Acad. Sci. USA, 86, 2398 (198
9)], Gout (Di Giovine FS et al. J. Immunol., 13
8, 3213 (1987)], acute myeloid leukemia [Sakai K. et a
lJExp.Med., 166, 1587 (1987)], endometritis (Rome
ro R., et al. Am.J.Obstet.Gynecol. 160, 1117 (198
9)] and the like.

Sphingomyelinase is an enzyme that decomposes this into ceramide and phosphorylcholine using sphingomyelin, which is one of the choline-containing lipids contained in the cell membrane system and nucleus of the body, as a substrate. This enzyme is
Initially, it was found as one of the lysosomal hydrolases that have an optimum pH in the acidic range, but recently it has been found to be optimal in the neutral range.
The same enzyme activity having H is also found in the microsomal fraction and plasma membrane [Allan D. et al. Biochim. Biophys.
Acta., 693, 53 (1982), T-Koizumi K. and Kojima K.
J. Biochem., 99, 1803 (1986), it is considered that these enzymes are actually involved in the metabolism of sphingomyelin in vivo.

Ceramide, one of the products of the above reaction, is further hydrolyzed by ceramidase to produce fatty acid and sphingosine. It has been confirmed by in vivo experiments that sphingomyelin is metabolized in the mammalian body to form ceramide and sphingosine [Schneide
r PB and Kennedy EPJLipid Res., 9, 58 (196
8)]. Ceramide and sphingosine, which are degradation products of sphingomyelin, have been shown to be involved in cell proliferation and differentiation, and the signaling mechanisms closely related to them [Kijima Kojima and Keiko Koizumi Protein Nucleic Acid
Enzyme, 36, 629 (1991)], this reaction pathway is called the sphingomyelin pathway.

It has been shown that this sphingomyelin pathway is involved when IL-1β or TNF-α binds to the receptor of the target cell, and then intracellular signal transduction is carried out [ Dressler KA et al. Science, 25
5, 1715 (1992), Mathias et al. Science, 259, 519 (1
993)].

Therefore, the signal transduction of these TNFα and IL1β can be blocked by the inhibitor of sphingomyelinase activity, and the pathological condition involving these cytokines can be improved.

On the other hand, it has been shown that a sphingomyelinase reaction product activates cyclooxygenase and promotes PGE2 production via this (Ballou LR et al.
al. J. Biol. Chem. 267, 20044, (1992)).

Further, the sphingomyelinase reaction itself is the LDL or degenerated LDL involved in the development of atherosclerosis.
Promotes uptake into the peripheral cells,
Increases ester synthesis and its intracellular accumulation [Stein O.
et al. Biochim. Biochim. Ata., 1126, 291 (199
2), Chatterjee SJBiol.Chem., 268, 3401 (199
3)], it is expected to be involved in the progression of this disease state.

Furthermore, activation of sphingomyelinase reduces the content of sphingomyelin in the apical membrane of the sinusoidal side in the proximal tubule of the kidney and reduces the uptake of phosphate and sugar which function in a Na-dependent manner. (Vrtovsnik
F. et al. Kidney International., 41, 983 (199
2)].

It was also shown that the amount of ceramide was increased in CEM cells infected with HIV [Veldhoven PP
V. et al. Biochem. Biophys. Res. Comm., 187, 209
(1992)], it has been shown that sphingomyelinase is activated in HIV-infected cells in vivo.

From the above facts, specific inhibitors for sphingomyelinase are anti-HIV agents, anti-diabetic agents, anti-arteriosclerotic agents, anti-osteoporosis agents, anti-thrombotic agents, anti-inflammatory agents, immunosuppressive agents, It can be used as a diuretic, and as a preventive or therapeutic drug for respiratory system diseases, thyroid diseases, Alzheimer's disease, hepatitis, nephritis, leukemia, and mosquitoes.

However, no specific and potent inhibitory substance for sphingomyelinase has been found to date.

Soluble IL-1 receptor and IL-1 receptor antagonist have hitherto been found as substances which specifically inhibit the action of IL-1β, and the use of these substances improves symptoms in septic shock patients and RA patients. Is seen.

Further, a clinical test was conducted using soluble TNF receptor and anti-TNF antibody which specifically inhibit the action of TNF-α for endotoxin shock and acute GHVD, and its effectiveness was observed. (Vincen
t J.-L. et al. Chest, 101,810 (1992), Herve P. et
al. Blood, 79, 3362 (1992)].

However, since all of these are peptidic or high molecular weight substances, they have drawbacks in terms of drug absorbability into the body, stability in blood, and the like. From this point of view, a low molecular weight physiologically active substance having a specific inhibitory activity against sphingomyelinase has been desired.

[0023]

DISCLOSURE OF THE INVENTION The present inventors searched for substances having a sphingomyelinase inhibitory action from secondary microbial metabolites, and isolated from soil SANK60294 strain belonging to the genus Micromonospora (FERM BP-4603) The present invention was completed by finding that a novel compound, A-76341, having a sphingomyelinase inhibitory action is produced in the culture solution of the strain).

[0024]

That is, the present invention is
(1) Novel compound A-7634 represented by general formula (I)
1:

[0025]

[Chemical 2]

(2) Micromonospor
a) A-76341-producing bacterium belonging to the genus is cultured, and A-76341 is collected from the culture.
-76341 manufacturing method, (3) Micro Monospora (Micr
A-76341-producing bacterium belonging to the genus omonospora is a Micromonospora sp. SAN.
The production method according to (2), which is K60294 strain, and (4) Micromonospora sp. S.
Regarding the ANK60294 strain.

A-76341 of the present invention has the following physicochemical properties.

1) Properties; white substance 2) Solubility; soluble in organic solvents such as methanol, ethyl acetate, chloroform 3) Color test: 50% sulfuric acid, iodine positive.

[0029] 4) molecular _{formula; C 19 H 22 O 5 5} ) molecular weight: the 330 (high resolution FAB-MS method [M ⁺ H] + 33
1.1539 (Measured value)) (Calculated value: 331.1545) 6) Specific optical rotation; [α] +285.6 (c 0.5 in MeOH) 7) Ultraviolet absorption spectrum; 244 nm (ε5940) 8) Infrared absorption spectrum; 3460, 2960, 2930, 1740,
1700, 1620, 1220cm ^-1 9) ¹ H-nuclear magnetic resonance spectrum; (in deuterated chloroform: PP
M, TMS standard) 6.78 (d, J = 12Hz, 1H), 5.92 (dt, J = 10Hz, 1H), 5.62 (d
t, J = 10Hz, 1H), 4.92 (m, 1H), 3.63 (dd, J = 111, 6.5Hz,
1H), 3.44 (ddd, J = 12, 6.5, 2Hz, 1H), 3.15 (dd, J = 18,
7Hz, 1H), 3.14 (m, 1H), 2.88 (br, s, 1H), 2.65 (m, 1H),
2.65 (d, J = 18Hz, 1H), 2.49 (ddd, J = 15, 11, 4Hz, 1
H), 2.15 ~ 2.40 (m, 2H), 2.02 (m, 1H), 1.70 ~ 1.90 (m,
2H), 1.48 (ddd, J = 13,10.5, 10.5Hz, 1H), 1.10 (d, J =
6.5Hz, 3H) 10) ¹³ C-nuclear magnetic resonance spectrum; (in deuterated chloroform: P
PM, deuterated chloroform standard) 217.4 (s), 194.2 (s), 165.7 (s), 155.1 (d), 136.4 (s), 127.8
(d), 123.5 (d), 72.9 (d), 66.7 (d), 45.8 (d), 45.7 (t), 43.9
(d), 41.2 (t), 40.7 (t), 38.6 (d), 35.3 (d), 34.7 (d), 33.2
(t), 13.9 (q) 11) High performance liquid chromatography Retention time: 5.6 minutes Column: Senshu Pak PEGASIL, 4.6 φ × 150 mm Solvent: Methanol-0.2% TEAP (pH3.3) 1: 1 Flow rate: 1 ml / ml Detection: UV 210 nm producing bacterium Actinomycete used in the present invention Micromonospora Espi-
The morphological characteristics, physiological properties and chemotaxonomic properties of (Micromonospora sp.) SANK 60294 strain are as shown below.

1) Morphological characteristics SANK60294 strain is ISP "International
Streptomyces Project (International St
The following morphological characteristics were exhibited by culturing at 28 ° C. for 14 days on a medium defined by “reptomyces Project”). That is, the basal hyphae are well elongated and branched, and no aerial hyphae are observed. One spore is formed on the basal hyphae, and its mode is monopodial type. In addition, the surface has a protrusion shape (Warty). The spores are spherical or subspherical and the size is 0.7-0.8 x 0.7-1.1 µm. Other special organs such as sporangia, hyphal axle branch, and sclerotium are not found.

2) Culture properties on various culture media Table 1 shows the culture properties of the SANK60294 strain on an agar medium after culturing at 28 ° C for 14 days.

[0032]

[Table 1] Type of medium Item Properties of SANK60294 strain Yeast extract / G ^{* 1} : Very good, wrinkled, light brown (7.5YR 7/6) ^{* 2} Malt extract agar R: Gray taste olive (5Y 4/4) (ISP 2) SP: Not produced Oatmeal agar G: Good, flat, grayish yellow tea (2.5Y 5/4) (ISP 3) R: Grayish yellow tea (2.5YR 5/4) SP: Produce Starch / inorganic salt agar G: Very good, flat, dark brown (10YR 3 /
2) (ISP 4) R: Dark brown (10YR 3/2) SP: Not produced Glycerin / G: Poor, flat, light yellowish tea (2.5Y 8/4) Asparagine agar R: Light yellowish tea (2.5Y 8 /) 4) (ISP 5) SP: Not produced Peptone yeast G: Good, flat, light yellowish tea (10YR 8/6) Extract / Iron agar R: Light yellowish tea (10YR 8/6) (ISP 6) SP: Produce No Tyrosine agar G: Not good, flat, grayish olive (5Y 5/4) (ISP 7) R: Grayish olive (5Y 5/4) SP: Not produced Sucrose G: Not very good, flat , Light yellowish tea (10YR 8/4) Nitrate agar R: Light yellowish tea (10YR 8/4) SP: Not produced Glucose / G: Poor, flat, brownish white (7.5YR 9/2) Asparagine agar R: Brown white (7.5YR 9/2) SP: Not produced Nutrient agar G: Good, flat, yellowish tea (10YR 7/6) (DIFCO) R: Yellowish tea (10YR 7/6) SP: Not produced Potato extract G: Not very good No, flat, yellowish tea (2.5Y 6/4) Ginseng extract agar R: Tea taste ash (2.5Y 6/2) SP: Not produced, water agar G: Poor, flat, light brown (2.5Y 8/2) R: Light brown (2.5Y 8/2) SP: Not produced * 1: G: Growth, R: Back side, SP: Soluble pigment * 2: Color tone display according to Munsell method 3) Physiological properties After culturing at 28 ° C, SANK observed during 2 to 21 days
The physiological properties of strain 60294 are as shown in Table 2.

[0033]

[Table 2] Physiological properties SANK60294 strain Hydrolysis of starch Positive liquefaction of gelatin Negative nitrate reduction Negative nitrate coagulation Positive milk peptone of milk Positive melanin-like pigment productivity (medium 1, 2, 3) Anionic Substrate degradability: Casein positive Tyrosine negative xanthine negative growth temperature range (medium 4) 10-37 ° C appropriate growth temperature (medium 4) 20-35 ° C growth in the presence of salt (medium 4) 3% medium 1: Tryptone, yeast extract, broth (ISP 1) Medium 2: Peptone, yeast extract, iron agar (ISP 6) Medium 3: Tyrosine agar (ISP 7) Medium 4: Yeast extract, malt extract agar (ISP 2) SANK60 observed after culturing at 28 ° C for 14 days using GOTRYB agar (ISP 9)
The carbon source assimilation of 294 strains is shown in Table 3.

[0034]

Table 3 Properties of carbon source SANK60294 strain D-glucose + L-arabinose ++ D-xylose + inositol-D-mannitol-D-fructose ± L-rhamnose-sucrose ++ raffinose-control-++: Strongly assimilated, +: Assimilate, ±: Weakly assimilate,-: Not assimilate 4) Chemotaxonomy The cell wall of the SANK60294 strain is the method of Hasegawa et al., “Hasegawa et al., The Journal of General and Applied Micro”. Biology (The Journal of Genera
l and Applied Microbiology) 29, 319-32.
Page 2, 1983 ”, mesodiaminopimelic acid was detected. In addition, the major sugar component in the whole cells of the SANK60294 strain was determined by the method of MP Rechevalier, “MP Lechevalier, Journal of Laboratory and Clinical Medicine (Journal of
Laboratory and Clinical Medicine) 71, 934
-944, 1968 ”, arabinose and xylose were detected. Furthermore, SANK602
When 94 strains of menaquinone were examined by the method described in "Experimental method for identifying actinomycetes: edited by the Japanese Society of Actinomycetes, (1985)", the major menaquinone was MK-10 (H4).
Regarding the acyl group of cell wall peptidoglycan, the method of Uchida et al., “Uchida et al., The Journal of General and Applied Microbiology (The Journal
Volume of General and Applied Microbiology 25, 169
Pp. 183, 1979 ”, it was found to be glycolyl type. When the G / C content of DNA was examined by the method described in “Experimental method for identifying actinomycetes: edited by Japan Society of Actinomycetes, (1985)”, it was 72.4 mol%.

From the above, it is clear that the SANK60294 strain belongs to the genus Micromono-spora among actinomycetes. Therefore, SANK6029
4 strains are Micromonospora
sp.). SANK60294 strain
It has been internationally deposited as FERM BP-4603 at the Institute of Biotechnology, Institute of Biotechnology, Ministry of International Trade and Industry on March 15, 2014.

Culturing Method and Purification Method As the separation medium used for separating the new strain of the present invention, a medium appropriately containing a carbon source, a nitrogen source, an inorganic ion, an organic nutrient source and the like can be used. Either a synthetic or natural medium can be used. A-7634
1 is obtained by culturing the SANK60294 strain in an appropriate medium and then collecting it. As the nutrient source, known ones conventionally used for culturing fungal strains can be used. For example, glucose, sucrose, starch, glycerin, starch syrup, molasses, soybean oil and the like can be used as the carbon source. As the nitrogen source, soybean powder, corn steep liquor, raw yeast, potato, ammonium sulfate,
Sodium nitrate or the like may be used. In addition, if necessary, inorganic salts such as calcium carbonate and phosphate may be added, as well as organic and inorganic substances that assist the growth of the strain and promote the production of A-76341.

As the culturing method, the liquid culturing method, particularly the submerged culturing method, is most suitable as in the method for producing general antibiotics. The culture is carried out under aerobic conditions, and the suitable temperature for the culture is 20-37 ° C, but in most cases, the culture is performed at around 28 ° C. A-76341 production usually peaks in 4-8 days in shake culture.

After the completion of the culture, A-76341 present in the bacterial cells or the filtrate in the culture solution is extracted by adding an organic solvent such as acetone or acetonitrile in a volume of the culture solution and mixing them. A-76 present in the filtrate or the supernatant is separated by filtering or centrifuging the solid part present in the extract with diatomaceous earth as a filter aid.
341 is extracted and purified by using its physicochemical properties, using the sphingomyelinase inhibitory activity as an index. For example, A-76341 present in this extract is an organic solvent immiscible with water under acidic conditions of about pH 3, such as n-butanol, methyl ethyl ketone, ethyl acetate, after first removing the organic solvent mixed in the concentration operation. Chloroform, ethylene chloride, methylene chloride, etc. alone or
It is possible to extract and purify by a combination thereof.
Alternatively, as an adsorbent, for example, activated carbon or an adsorbent resin such as Amberlite XAD-2, XAD-4 (manufactured by Rohm and Haas Co.) or the like, or Diaion HP-1
0, HP-20, CHP-20P, HP-50 (manufactured by Mitsubishi Kasei Co., Ltd.) and the like can be used. A-76341
The solution containing a is passed through a layer of the adsorbent as described above to adsorb and remove impurities, or after adsorbing A-76341, elution is performed using methanol water, acetone water, n-butanol water, or the like. Is obtained by A-76341 thus obtained was further treated with silica gel,
Adsorption column chromatography using a carrier such as Florisil, partition column chromatography using Sephadex LH-20 (manufactured by Pharmacia), gel filtration chromatography using Sephadex G-25 (manufactured by Pharmacia), and the like. It can be purified by high performance liquid chromatography or the like using a phase or reverse phase column.

The sphingomyelinase inhibitory activity can be measured by the following method.

That is, first, as a substrate solution, 10 μl of [N-methyl- ¹⁴ C] sphingomyelin (cow, 52 m
Ci / mmol, 25 μCi / ml; Amersham) and 200 μl of sphingomyelin (cow, 20 mM, Sigma) were dried with nitrogen gas, and then 200 μl of 1M
Tris-hydrochloric acid (pH 7.5), 40 μl of 10% (v /
v) Triton X-100, 20 μl of 0.5M MgC.
L ₂ and 1.24 ml of H ₂ were added, incubation was carried out at 48 ° C. for 30 minutes, and ultrasonication was performed twice for 15 seconds under the output condition of 20 W by a probe type ultrasonic disruption device. A mixed micelle system containing ¹⁴ C] sphingomyelin was prepared.

In the sphingomyelinase reaction, 150 μl of the substrate solution thus prepared was mixed with 10 μl of the sample solution, and the microsome fraction (25,000 × g-100,000 × g, of Wistar Imamiti male rat brain) was mixed. 40 μl of protein concentration 3-4 mg / ml) was added as an enzyme solution and incubated at 37 ° C. for 40 minutes. After completion of the reaction, 500 μl of chloroform: methanol (2: 1, v / v) was added to perform extraction operation, and 150 μl of the obtained aqueous layer was added to 3 ml of Picoflow ™ 4.
The amount of [ ¹⁴ C] phosphorylcholine as a reaction product was measured by mixing with 0, using a liquid scintillation counter. The sphingomyelinase activity is calculated as a value obtained by subtracting the measurement value when MgCl ₂ necessary for the sphingomyelinase reaction is removed from this measurement value.

[0042]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto.

Example 1. Purification of A-76341 substance (1) Culture The SANK60294 strain was aseptically inoculated into a branch test tube of 50 ml containing 20 ml of sterilized seed medium having the composition described below. Then, this was precultured at 28 ° C. for 5 days on a rotary shaker at 120 rpm.

[0044] Seed Medium Composition Glycerol 25g glucose 25g raw yeast 10g soybean meal _{10g K 2 HPO 4 0.5g MgSO 4} · 7H 2 O 0.5g CaCO 3 5g Nissan Disconnect Form CB-442 0.005% (v / v) Tap water 1000 L pH 7.0 (before sterilization) Main culture was performed as follows. Put 1 ml of each seed culture solution into a 500 ml Erlenmeyer flask with a velvet containing 80 ml of sterilized culture medium of the following composition, and keep at 28 ° C for 6 days.
The culture was performed on a rotary shaker at 00 rpm.

Glucose 20 g Soluble starch 10 g Raw yeast 9 g Meat extract 5 g Polypeptone 5 g CaCO _{3 3} g NaCl 5 g Nissan Disfoam CB-442 0.005% (v / v) Tap water 1000 L pH 7.4 (before sterilization) (2) Sphingomyelii Method for Measuring Nase Inhibitory Activity Using rat brain as an enzyme source of sphingomyelinase, first, its microsome fraction was prepared as follows. Ten Wistar Imamiti male rats (9 weeks old) were exsanguinated from the carotid artery, and the whole brain was extracted. Immediately after removing the cerebellum, buffer A (0.25M
Sucrose, 1 mM EDTA, 1 mM PMSF, 0.1
mM leupeptin, 5 mM Tris-HCl buffer, p
H7.4) (130 ml) was added, and the brain cells were disrupted using a Potter homogenizer at 4 ° C. next,
The resulting cell lysate was centrifuged at 700 xg for 10 minutes at 4 ° C, and the supernatant was further subjected to 2 ° C under 2 ° C.
Centrifugation was performed at 5,000 xg for 10 minutes. Finally, the obtained supernatant was subjected to ultracentrifugation at 100,000 xg for 60 minutes at 4 ° C, and the precipitate was used as a microsome fraction. This fraction was cryopreserved under liquid nitrogen until the activity of sphingomyelinase was measured, and buffer A was used to prepare a protein concentration of about 3 to 4 mg / ml.

Sphingomyelinase activity was measured as follows using a mixed micelle system. That is, first, 10 μl of mixed [N-methyl- ¹⁴ C] sphingomyelin (cow, 52 mCi / mmol, 25 μCi / ml; Amersham) and 200 μl of sphingomyelin (cow, 2
0 mM, Sigma) was dried with nitrogen gas, and then 200
μl of 1 M Tris-HCl (pH 7.5), 40 μl of 1
0% (v / v) Triton X-100, 20 μl of 0.5
Add M MgCl ₂ and 1.24 ml H ₂ O to add 4
Incubation was carried out at 8 ° C for 30 minutes. And
With a probe type ultrasonic crusher, under output conditions of 20 W, 1
Sonication was performed twice for 5 seconds to prepare a mixed micelle system containing [ ¹⁴ C] sphingomyelin.

In the sphingomyelinase reaction, 150 μl of the substrate solution thus prepared is mixed with 10 μl of the sample solution, and 40 μl of the enzyme solution described above is added to the reaction solution.
The incubation was carried out at 40 ° C. for 40 minutes.
After the reaction was completed, chloroform: methanol (2: 1, v /
v) was added in an amount of 500 μl, extraction was performed, 150 μl of the obtained aqueous layer was mixed with 3 ml of Picoflow ™ 40, and the amount of [ ¹⁴ C] phosphorylcholine as a reaction product was measured with a liquid scintillation counter. The sphingomyelinase activity is calculated as a value obtained by subtracting the measurement value when MgCl ₂ necessary for the sphingomyelinase reaction is removed from this measurement value.

The concentration of A-76341 required to inhibit the sphingomyelinase reaction by 50% measured by this method is 74 μg / ml.

(3) Isolation The culture solution for 20 Erlenmeyer flasks was rotated at 3000 rpm for 10 minutes,
After centrifugation, the resulting supernatant was adjusted to pH 3 and extracted 3 times with an equal volume of ethyl acetate. The ethyl acetate layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give a brown oily substance. This was dissolved in a small amount of methylene chloride-methanol, 20: 1 solvent and charged to a 200 ml silica gel column equilibrated with the same solvent. This column was eluted with 2 L of methylene chloride-methanol, 20: 1 solvent and fractionated. Fractions showing enzyme inhibition activity were concentrated to obtain 70 mg of a pale yellow oily substance. This was dissolved in a small amount of methanol and purified by preparative HPLC as follows. That is, about 15 mg each, methanol-0.2%
HPLC column equilibrated with TEAP (pH 3.3) 1: 1 solution (Nacalai Tesque Cosmosil 5C18-AR, 20 φ × 250
mm) and developed with the same solvent at a flow rate of 9 ml / min. Absorption at 210 nm was detected, and the peak eluting at about 11 minutes was collected. The separated solution was concentrated under reduced pressure to remove methanol, an appropriate amount of water was added to adjust the pH to 3, extracted with ethyl acetate and concentrated to obtain 14 mg of this substance.

[0050]

The novel compound A-76341 of the present invention is
Anti-HIV agents, anti-diabetic agents, anti-arteriosclerotic agents, anti-osteoporosis agents, anti-thrombotic agents, anti-inflammatory agents, immunosuppressants, diuretics, and respiratory diseases, thyroid diseases, Alzheimer's disease, hepatitis, nephritis It can be used as a preventive or therapeutic drug for leukemia, leukemia, and cabbage.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location A61K 31/365 ACB ACD ACX ADD ADP ADX ADY C12P 17/06 7432-4B // (C12P 17/06 C72R 1:29) (72) Inventor Wazawa, 1-58, Hiromachi, Shinagawa-ku, Tokyo 1-258 Sankyo Co., Ltd. (72) Inventor Ryuzo Enota 33 Miyukigaoka, Tsukuba, Ibaraki Sankyo Co., Ltd. (72) ) Inventor Nao Okazaki 33 Miyukigaoka, Tsukuba City, Ibaraki Sankyo Co., Ltd.

Claims (4)

[Claims] 1. A novel compound A-7 represented by the general formula (I).
6341. : [Chemical 1] 2. A-763, which comprises culturing an A-76341-producing bacterium belonging to the genus Micromonospora and collecting A-76341 from the culture.
41 manufacturing method. 3. The process according to claim 2, wherein the A-76341-producing bacterium belonging to the genus Micromonospora is Micromonospora sp. SANK60294 strain. 4. Micromonspora SP
ospora sp.) SANK60294 strain.