JPH07258132A - Novel hydroquinone compound - Google Patents

Abstract

PURPOSE:To obtain the compound inhibiting sphingomyelinase, and useful as a medicine among various ones. CONSTITUTION:The novel compounds, i.e., F-11334A1 of the formula I, F-11334A2 of the formula II, F-11334A3 of the formula III, F-11334B1 of the formula IV, and F-11334B2 of the formula V. This novel compound can be obtained by culturing Acremonium bacteria capable of producing the compound [e.g. Acremonium sp. SANK 20793 strain (FERM BP-4539)] and then isolation from the cultured product. This compound can be used as an anti-HIV agent, antidiabetic agent, anti-arteriosclerotic agent, anti-osteoporotic agent, anti- thrombotic agent, anti-inflammatory agent, immunosuppressive agent, diuretic agent, and as a preventive or therapeutic agent for respiratory diseases, thyropathy, Alzheimer's disease, hepatities, nephritis, leukemia and cachexia.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a novel compound F-113 which inhibits sphingomyelinase and is useful as various medicines.
34A1, F-11334A2, F-11334A3,
It relates to F-11334B1 and F-11334B2 and a method for producing the same.

[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 PGE ₂ [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 cause of glomerulonephritis [Werber HI et al. J. Immunol., 138, 32]
07 (1987), Baud L. et al. Kidney Int., 41, 600 (1
992)].

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.

[0010] 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)].

[0011] TNF-α is HIV inserted into the chromosome in cells infected with HIV (human immunodeficiency virus).
Transcription from the viral genome end: LTR: is activated via the transcription factor NF-κB to promote HIV proliferation [Nabel G. et al. Nature, 326, 711 (1987),
Schreck R. et al. EMBO Journal, 10, 2247 (199
1)].

In addition, as a disease caused by overproduction of IL-1β and TNF-α, fulminant hepatitis [Muto Y. et al. Lan
cet II, 72 (1988)], asthma, idiopathic pulmonary fibrosis (Kelley
J. Am. Rev. Respir. Dis., 141 (3), 765 (1990)], AR
DS (adult respiratory distress syndrome) [Mill
ar A. et al. Lancet II, 712 (1989)] and other respiratory diseases, autoimmune thyroid disease [Kaguchi Eguchi et al. Approach from the latest medicine 1 From cytokines, Medical View, 38 (1991)], Lyme disease (Habicht GS et al. J.
Immunol., 134, 3147 (1985)], Alzheimer's disease [Gr
iffin WST at al. Proc.Natl.Acad.Sci.USA, 86,
7611 (1989)], Crohn's disease [Ayaguni Yagita, Ayumi Kyoku, 147, 375 (1988)], shock shock syndrome [Ikejim
a T. et al. J. Clin. Invest., 73, 1312 (1984)], osteoporosis (Pacifici R. et al. Proc. Natl. Acad. Sci.
USA, 86, 2398 (1989)], gout (Di Giovine FSe
t al. J. Immunol., 138, 3213 (1987)], acute myeloid leukemia (Sakai K. et al. J. Exp. Med., 166, 1587 (198).
7)], Endometritis [Romero R., et al. Am. J. Obstet. Gy
nercol. 160, 1117 (1989)] and the like.

Sphingomyelinase is an enzyme which 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 is then transduced intracellularly []. Dressler KA et al. Science, 25
5, 1715 (1992), Mathias et al. Science, 259, 519 (1
993)].

Therefore, it is possible to block the signal transduction of these TNFα and IL1β by the inhibitor of sphingomyelinase activity, and it is possible to improve the pathological condition in which these cytokines are involved.

On the other hand, it has been shown that the reaction product of sphingomyelinase activates cyclooxygenase and promotes PGE ₂ production via this (Ballou LR et.
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. Acta., 1126, 291 (199
2), Chatterjee SJBiol.Chem., 268, 3401 (199
3)], it is expected to be involved in the progression of this disease state.

Further, activation of sphingomyelinase reduces the content of sphingomyelin in the apical membrane of the sinusoidal side in the renal proximal tubule 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 include 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 inhibitor of sphingomyelinase has been found to date.

Conventionally, soluble IL-1 receptor and IL-1 receptor antagonist have 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.

[0026]

The present inventors searched for a substance having a sphingomyelinase inhibitory action from secondary microbial metabolites and isolated from soil, Acremonium sp. SANK20793 strain belonging to the genus Acremonium (FERM BP- 4539) A novel compound, F-11334A1, F-11334A2, having a sphingomyelinase inhibitory action in the culture solution of the strain,
The present invention has been completed by finding that F-11334A3, F-11334B1 and / or F-11334B2 are produced.

[0027]

Means for Solving the Problems That is, the present invention provides (1) a novel compound F-1133 represented by the general formula (I).
4A1:

[0028]

[Chemical 6]

(2) Novel compound F-11334A2 represented by the general formula (II):

[0030]

[Chemical 7]

(3) Novel compound F-11334A3 represented by the general formula (III):

[0032]

[Chemical 8]

(4) Novel compound F-11334B1: represented by the general formula (IV)

[0034]

[Chemical 9]

(5) Novel compound F-11334B2 represented by the general formula (V):

[0036]

[Chemical 10]

(6) F-11334A1, F-11334A2, F belonging to the genus Acremonium
-11334A3, F-11334B1 and / or F
-11334B2 producing bacterium was cultivated, and F-
11334A1, F-11334A2, F-11334
A3, F-11334B1 and / or F-11334
F-11334A1, characterized by collecting B2,
F-11334A2, F-11334A3, F-113
34B1 and / or a method for producing F-11334B2, (7) F- belonging to the genus Acremonium (Acremonium)
11334A1, F-11334A2, F-11334
A3, F-11334B1 and / or F-11334
The B2-producing bacterium is Acremonium sp.
The production method according to (6), which is SANK20793 strain, (8) Acremonium sp. SANK20793 strain.

F-11334A1 and F-113 of the present invention
34A2, F-11334A3, F-11334B1 and / or F-11334B2 have the following physicochemical properties.

F-11334A1 1) Properties; white substance, 2) Solubility; soluble in organic solvents such as methanol and ethyl acetate 3) Color test: 50% sulfuric acid, iodine positive.

[0040] 4) Molecular _{formula; C 11 H 16 O 4 5} ) molecular weight: the 212 (high resolution EI-MS method [M] + 212.
1051 (measured value)) (calculated value: 212.1049) 6) Specific optical rotation; [α] +36.0 (c 0.2 in MeOH) 7) UV absorption spectrum (methanol); 216 nm sh
(ε4580), 226 nm sh (ε3690), 293 nm (ε3050) 8) Infrared absorption spectrum (KBr); 3340, 1650, 16
10, 1500, 1460 cm-1 9) ¹ H-nuclear magnetic resonance spectrum; (in deuterated methanol: PPM,
TMS standard) 6.63 (d, J = 8.5Hz, 1H), 6.62 (d, J = 3.0Hz, 1H), 6.50 (d
d, J = 8.5, 3.0Hz, 1H), 3.60 (dd, J = 10.0, 2.0Hz, 1H),
2.80 (dd, J = 13.5, 2.0Hz, 1H), 2.60 (dd, J = 13.5, 10.0
Hz, 1H), 1.23 (s, 6H) 10) ¹³ C-nuclear magnetic resonance spectrum; (in deuterated methanol: PP
M, heavy methanol standard) 151.8 (s), 150.1 (s), 129.6 (s), 119.3 (d), 117.7 (d), 115.2
(d), 81.2 (d), 74.3 (s), 34.8 (t), 26.2 (q), 25.4 (q) 11) High Performance Liquid Chromatography Retention time: 2.2 minutes Column: Senshu Pak PEGASIL, 4.6φ × 150mm (Sensu Kagaku solvent: methanol-water 1: 1 flow rate: 1 ml / ml detection: UV 210 nm F-11334A2 1) Properties; white substance 2) Solubility; soluble in organic solvents such as methanol and ethyl acetate 3) Color study; 50% sulfuric acid, positive 4) molecular formula _{iodine; C 11 H 14 O 3 5} ) molecular weight: the 194 (high resolution EI-MS method [M] + 194.
0955 (measured value)) (calculated value: 194.0942) 6) Specific optical rotation; [α] +49.8 (c 1.0 in MeOH) 7) UV absorption spectrum (methanol); 216 nm sh
(ε4020), 229 nm (ε4310), 303 nm (ε3670) 8) Infrared absorption spectrum (KBr); 3230, 3160, 14
90 cm-1 9) ¹ H-nuclear magnetic resonance spectrum; (in deuterated methanol: PPM,
TMS standard) 6.62 (br, 1H), 6.52 (br d, J = 8.5Hz, 1H), 6.48 (br dd,
J = 8.5, 2.5Hz, 1H), 4.50 (dd, J = 9.5, 8.5Hz, 1H), 3.
12 (dd, J = 15.5, 8.5Hz, 1H), 3.06 (dd, J = 15.5,9.5Hz,
1H) 1.22 (s, 3H), 1.20 (s, 3H) 10) ¹³ C-nuclear magnetic resonance spectrum; (in deuterated methanol: PP
M, heavy methanol standard) 155.0 (s), 152.6 (s), 129.7 (s), 115.3 (d), 113.5 (d), 110.2
(d), 90.7 (d), 73.0 (s), 32.5 (t), 25.9 (q), 25.6 (q) 11) High performance liquid chromatography Retention time: 14.5 minutes Column: Senshu Pak Silica-4251-N, 10φ x 250mm
(Sensu Kagaku solvent: hexane-ethyl acetate 2: 1 Flow rate: 5 ml / ml Detection: differential refractometer F-11334A3 1) Properties; white substance 2) Solubility; soluble in organic solvents such as methanol and ethyl acetate 3) color test; 50% sulfuric acid, positive 4) molecular formula _{iodine; C 11 H 14 O 3 5} ) molecular weight: the 194 (high resolution EI-MS method [M] + 194.
0941 (measured value)) (calculated value: 194.0942) 6) Specific rotation: [α] -20.4 (c 1.0 in MeOH) 7) UV absorption spectrum (methanol); 218 nm sh
(ε5590), 227 nm (ε5240), 297 nm (ε3670) 8) Infrared absorption spectrum (KBr); 3410, 3200, 15
00 cm-1 9) ¹ H-nuclear magnetic resonance spectrum; (in deuterated methanol: PPM,
TMS standard) 6.56 (d, J = 8.5Hz, 1H), 6.52 (dd, J = 8.5, 2.5Hz, 1H),
6.50 (br, 1H), 3.70 (dd, J = 7.5, 5.5Hz, 1H), 2.92 (dd,
J = 16.5, 5.5Hz, 1H), 2.63 (dd, J = 16.5, 7.5Hz, 1H) 1.29 (s, 3H), 1.20 (s, 3H) 10) ¹³ C-nuclear magnetic resonance spectrum; (in deuterated methanol : PP
M, deuterated methanol standard) 152.2 (s), 147.8 (s), 122.4 (s), 11
8.9 (d), 116.9 (d), 116.1 (d), 78.0 (s), 71.2 (d), 32.9 (t), 2
6.3 (q), 21.4 (q) 11) High performance liquid chromatography Retention time: 15.5 minutes Column: Senshu Pak Silica-4251-N, 10 φ × 250mm
(Sensu Kagaku solvent: hexane-ethyl acetate 2: 1 Flow rate: 5 ml / ml Detection: differential refractometer F-11334B1 1) Properties; pale yellow substance 2) Solubility; soluble in organic solvents such as methanol and ethyl acetate 3 ) color test: 50% sulfuric acid, positive 4) molecular formula _{iodine; C 11 H 14 O 3 5} ) molecular weight: 194 (by high resolution EIMS method [MH ₂ O] + 17
6.0844 (measured value)) (calculated value: 176.0837) 6) UV absorption spectrum (methanol); 248 nm sh
(ε8500), 322 nm (ε3840) 7) Infrared absorption spectrum (KBr); 3330, 1660, 16
10, 1500, 1460 cm-1 8) ¹ H-nuclear magnetic resonance spectrum; (in deuterated methanol: PPM,
TMS standard) 6.83 (d, J = 3Hz, 1H), 6.82 (d, J = 16Hz, 1H), 6.61 (dd,
J = 8.5Hz, 1H), 6.51 (dd, J = 8.5, 3Hz, 1H), 6.28 (d, J = 1
6Hz, 1H), 1.37 (s, 6H) -producing bacterium Acremonium (Acremoniu) used in the present invention
Examples of strains belonging to the genus m) include Acremonium sp.
SANK 20793 strain (FERM BP-4539) can be mentioned, and the mycological properties of this strain are as follows.

The strain SANK 20793 was isolated from sea sand using sterile balsa as a bait.

The mycological characteristics of this strain are as follows.

Growth on PDA medium is 12 days, 45 mm at 23 ° C.
Reach Initially, it secretes a white fluffy, reddish-brown soluble pigment, but by further culturing, conidia are formed, and accordingly, a black powdery substance is formed from the central part. The whole is almost flat, but a part of the colony is fluffy due to the formation of hyphae bundles. In addition, conidia are actively formed and sparse parts are fan-shaped. Vegetative hyphae are colorless and several hyphae form a bundle on which a large number of conidial cells are formed. Conidia formation is fiaro type. Fear Ride is a cut type, tapering toward the tip, its size is 18
-39x2.5-3.5 μm, with some particles attached, partially rough surface, and unclear collar. Conidia gather on the phialide to form a pseudo-head-like mucus. Conidia are oval, single-celled, dark brown, and their size is 5-6.5x2-3.5 μm.

The above features are described by Gams, W. (1971).
Cephalosporium-artige Schimmelpilze, 262p.G.Fis
Glioma from the system of cher, Stuttgart.
This is consistent with the characteristics of the Acremonium spp. However, the genus Acremonium contains a number of species, including Gams, W. (1
971) By Cephalosporium-artige Schimmelpilze, 262p.
Due to the large number of species other than G. Fischer, Stuttgart., It is difficult at present to cover all of them and perform species-level identification, which may lead to lack of accuracy. . Therefore, the identification of the species level of this bacterium was suspended and it was identified as SANK20793 Acremonium sp. Acremonium sp.

This bacterium was produced on January 20, 1994.
It was internationally deposited on the day with the Institute of Biotechnology, Institute of Biotechnology, Ministry of International Trade and Industry, and was given the deposit number FERM BP-4539.

Culturing Method and Purification Method The separation medium used for separating the new strain of the present invention is a medium containing appropriately selected from carbon source, nitrogen source, inorganic ion, organic nutrient source and the like. Either a synthetic or natural medium can be used. F-1133
4A1, F-11334A2, F-11334A3, F
-11334B1 and / or F-11334B2 is S
It is obtained by culturing the ANK20793 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 flour, corn steep liquor, raw yeast, potato, ammonium sulfate, sodium nitrate and the like can be used. In addition to this, inorganic salts such as calcium carbonate and phosphate are added as necessary, and the growth of strains is assisted by F-11334A1 and F-11334.
A2, F-11334A3, F-11334B1 and /
Alternatively, organic and inorganic substances that promote the production of F-11334B2 can be appropriately added.

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 culturing is carried out under aerobic conditions, and the suitable temperature for culturing is 20-30 C, but in most cases 23 C
Incubate in the vicinity. F-11334A1, F-11334
A2, F-11334A3, F-11334B1 and F
The production of -11334B2 usually peaks in 5-8 days with shaking culture.

After the completion of the culture, F-11334A1, F-11334A2 existing in the bacterial cells or the culture solution in the culture medium,
F-11334A3, F-11334B1 and / or F-11334B2 is extracted by adding and mixing an organic solvent such as acetone or acetonitrile in a volume of the culture solution. The solid part present in the extract is separated by filtration or centrifugation using diatomaceous earth as a filter operation aid, and F-11334 present in the filtrate or supernatant is separated.
A1, F-11334A2, F-11334A3, F-
11334B1 and / or F-11334B2 are extracted and purified by using their physicochemical properties with the sphingomyelinase inhibitory activity as an index. For example, F-11334A1, F-11334A present in this extract
2, F-11334A3, F-11334B1 and / or F-11334B2 is an organic solvent immiscible with water under acidic conditions of about pH 3 after first removing the organic solvent mixed in the concentration operation, for example, n-butanol, methyl ethyl ketone, Extraction and purification can be performed using ethyl acetate, chloroform, ethylene chloride, methylene chloride, etc. alone or in combination. 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, Diaion HP-10, HP-20, C
HP-20P, HP-50 (manufactured by Mitsubishi Kasei Co., Ltd.) and the like can be used. F-11334A1, F-1133
A liquid containing 4A2, F-11334A3, F-11334B1 and / or F-11334B2 is passed through a layer of the adsorbent as described above to adsorb and remove impurities;
Or F-11334A1, F-11334A2, F-
11334A3, F-11334B1 and / or F-
It is obtained by adsorbing 11334B2 and then eluting it with methanol water, acetone water, n-butanol water or the like. F-11334 thus obtained
A1, F-11334A2, F-11334A3, F-
11334B1 and / or F-11334B2 is further subjected to adsorption column chromatography using a carrier such as silica gel or Florisil, Sephadex LH-20.
Partition column chromatography using (Pharmacia), Sephadex G-25 (Pharmacia)
Gel filtration chromatography using, and normal phase,
It can be purified by high performance liquid chromatography using a 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, protein concentration 3˜5) of the Wistar Imamiti male rat brain 40 μl of 4 mg / ml) was added as an enzyme solution and the mixture was 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.

[0052]

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. F-11334A1, F-11
334A2, F-11334A3, F-11334B1
And purification of F-11334B2 (1) Culture Acremonium sp. SANK 20793 strain was aseptically and sterilized, and 500 m containing 100 ml of culture medium having the composition described below
The Erlenmeyer flask (seed flask) of 1 was inoculated. Then, this was precultured for 5 days on a rotary shaker at 200 rpm.

Medium composition Glycerol 50 g Potato 50 g East extract 5 g Malt extract 5 g Defoamer (CB-442) 0.005% (v / v) Ion-exchanged water 1000 ml pH unadjusted Main culture is as follows went. 500 ml Erlenmeyer flask containing 100 ml of sterilized culture medium of the above composition 1
Add 5 ml of seed culture solution to each of 5 tubes, and keep at 23 ° C for 7 days.
The culture was performed on a rotary shaker at 200 rpm.

(2) Method for measuring sphingomyelinase inhibitory activity Using rat brain as the 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 obtained 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 was mixed with 10 μl of the sample solution, and 40 μl of the enzyme solution described above was added to obtain 37
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.

F-11334A required to inhibit the sphingomyelinase reaction by 50% measured by this method
The concentrations of 1, F-11334B1 and F-11334B2 are 38.8, 9.9 and 7.3 μg / ml, respectively.

(3) Isolation The culture solution for 7 Erlenmeyer flasks was rotated at 3000 rpm for 10 minutes,
Centrifugation was performed. 80% acetone was added to the obtained bacterial cells 1
L was added and extracted for 5 hours. The mixture is suction filtered,
The filtrate was concentrated under reduced pressure to remove acetone. It was then 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 1.6 g of a brown powder. This powder was dissolved in a small amount of methylene chloride-methanol, 15: 1 solvent and charged on a 150 ml silica gel column equilibrated with the same solvent. This column is packed with 1.5 L of methylene chloride-methanol,
It was eluted with a 15: 1 solvent and fractionated. Fractions showing enzyme inhibitory activity were concentrated to obtain 200 mg of a pale yellow substance. These were dissolved in a small amount of methanol and purified by preparative HPLC as follows. That is, an HPLC column (Cosmocil 5C18-AR manufactured by Nacalai Tesque, 20φ × 250 m
m) and developed with the same solvent at a flow rate of 9 ml / min. Absorption at 210 nm was detected, and peaks eluted at about 7, 8, 10, and 12 minutes were collected. The collected solution is concentrated under reduced pressure, and 19 mg of F-113 is extracted from the peak eluted at about 7 minutes.
34A1, 34 mg of F-11334 from the 10 minute peak
A2 and F-11334A3 mixture, from the 8 min peak
4.5 mg of F-11334B1, 10m from the peak at 12 minutes
g of F-11334B2 material was obtained. F-11334A
The 2 and F-11334A3 mixture was further separated as follows. That is, about 5 mg each was charged to an HPLC column (Senshu Pak-Silica-4251-N, 10φ × 250 mm) equilibrated with a solvent of hexane-methanol, 2: 1 and developed with the same solvent at a flow rate of 5 ml / min. did. Detection was performed with a differential refractometer, and peaks eluted at about 14 and 15 minutes were collected. The separated solution was concentrated under reduced pressure to obtain 7 mg of F-11334A2 from the peak at 14 minutes and 23 mg of F-11334A3 from the peak at 15 minutes.

[0060]

The novel compound F-11334A of the present invention
1, F-11334A2, F-11334A3, F-1
1334B1 and / or F-11334B2 are anti-H
IV agents, anti-diabetic agents, anti-arteriosclerotic agents, anti-osteoporosis agents, anti-thrombotic agents, anti-inflammatory agents, immunosuppressive agents, diuretics, and respiratory diseases, thyroid diseases, Alzheimer's disease, hepatitis, nephritis, It can be used as a preventive or therapeutic drug for leukemia and mosquitoes.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location C12N 1/14 A 8828-4B C12P 7/22 8114-4B 17/04 7432-4B 17/06 7432 −4B // A61K 31/34 AED (C12P 7/22 C12R 1: 645) (C12P 17/04 C12R 1: 645) (C12P 17/06 C12R 1: 645) (72) Inventor Go Hosoya Tsukuba City, Ibaraki Prefecture Miyukigaoka 33 Sankyo Co., Ltd. (72) Inventor Kohei Furuya 33 Miyukigaoka Tsukuba, Ibaraki Sanko Co., Ltd.

Claims (8)

[Claims] 1. A novel compound F-1 represented by the general formula (I).
1334A1. : [Chemical 1] 2. A novel compound F- represented by the general formula (II)
11334A2. : [Chemical 2] 3. A novel compound F represented by the general formula (III)
-11334A3. : [Chemical 3] 4. A novel compound F- represented by general formula (IV)
11334B1. : [Chemical 4] 5. A novel compound F-1 represented by general formula (V)
1334B2. : [Chemical 5] 6. F-11334A1, F-11334A2, F-11 belonging to the genus Acremonium
334A3, F-11334B1 and / or F-11
A 334B2-producing bacterium was cultured, and F-113 was obtained from the culture.
34A1, F-11334A2, F-11334A3,
F-11334A1, F-1 characterized by collecting F-11334B1 and / or F-11334B2
1334A2, F-11334A3, F-11334B
1 and / or a method for producing F-11334B2. 7. F-11334A1, F-11334A2, F-11 belonging to the genus Acremonium.
334A3, F-11334B1 and / or F-11
334B2 producing bacteria are Acremoniums
p. The production method according to claim 6, which is SANK20793 strain. 8. Acremonium sp. SANK2079
3 shares.