WO2004065607A1

WO2004065607A1 - Novel physiologically active substance rs-k3574 and process for producing the same

Info

Publication number: WO2004065607A1
Application number: PCT/JP2003/000532
Authority: WO
Inventors: Tomio Takeuchi; Hironobu Iinuma; Ryuichi Sekizawa; Susumu Matsui
Original assignee: Zaidan Hojin Biseibutsu Kagaku Kenkyu Kai
Priority date: 2003-01-22
Filing date: 2003-01-22
Publication date: 2004-08-05
Also published as: AU2003303775A1; JPWO2004065607A1; JP4376189B2

Abstract

By culturing Pleurotaceae Panus rudis K-3574 strain (FERM BP-8265), a compound RS-K3574 represented by the following formula (I) is produced as a physiologically active substance showing an activity of inhibiting ubiquitin activating enzyme, an activity of inhibiting the ubiquitination of intracellular proteins, an antitumor activity and other various biological activities and having a novel molecular skeleton: (I) wherein the configurations at the 2-, 3-, 4- and 7-positions are respectively S, S, R and R. The substance RS-K3574 is a physiologically active substance which has an activity of inhibiting ubiquitin activating enzyme and an activity of inhibiting the ubiquitination of intracellular proteins as well as an activity of inhibiting the biosynthesis of intracellular proteins, an antitumor activity, an anticancer activity, an anti-inflammatory activity and an antiviral activity.

Description

Specification

New physiologically active substance RS-K3574 and its production method

Surgery g

The present invention has an inhibitory activity on ubiquitin activating enzyme and an inhibitory activity on ubiquitination of intracellular proteins, has an activity of inhibiting biosynthesis of intracellular proteins, and has an antitumor or antitumor activity. The present invention relates to a novel physiologically active substance having cancer activity, RS-K3574. The present invention also relates to a method for producing the above-mentioned physiologically active substance, RS-K3574 substance. Further, the present invention relates to an inhibitor of ubiquitin activating enzyme consisting of a physiologically active substance; RS-K3574, and an inhibitor of ubiquitination of intracellular protein consisting of a physiologically active substance RS-K3574. The present invention relates to an antitumor agent composition, an antiinflammatory agent composition and an antiviral agent composition containing the substance HS-K3574 as an active ingredient. Further, the present invention provides a novel bioactive substance RS-K3574, which is an apoptosis-inducing effect of a cell-killing antitumor agent or anticancer agent for cancer cells or radiation for cancer treatment, and inhibits protein biosynthesis in cancer cells. And an inhibitor of the activity of NF-κΒ transcription factor.

Background branch art

Many various enzyme inhibitors are known, and various many biologically active substances are known. Efforts are being made to apply these enzyme inhibitors or physiologically active substances as therapeutic agents for various diseases. It is also known that one of the extremely important and essential control mechanisms in cell proliferation, differentiation, growth and cell death is the ubiquitination reaction of intracellular proteins. A substance having the activity of inhibiting the ubiquitination reaction of intracellular proteins is expected to be widely applied to the treatment and prevention of diseases, and has been demanded. To date, ubiquitination of the intracellular protein has been requested. No substance having an activity capable of inhibiting the reaction has been found.

Neurological diseases including Alzheimer's disease occur when cell proliferation or cell death occurs in cancer cells or immune cells, when acute or chronic inflammation occurs, or when immune responses such as immune hypersensitivity or immunodeficiency occur. One of the mechanisms that play a crucial and essential role during normal differentiation, such as during infection and propagation of pathogenic viruses, including HIV viruses, and also during fertilization and development of eggs. Ubiquitin It is known that there is a mechanism for ubiquitination of intracellular proteins, which involves the enzyme to be converted.

For example, in cells infected with the HIV-1 virus or HIV-2 virus, it is known that protein ubiquitination is involved in the budding, maturation, and acquisition of infectivity of these HIV viruses. It is known that ubiquitin activating enzyme, an enzyme present in cells, is involved in ubiquitin activation. In addition, by inhibiting the proteasome in cells infected by the HIV virus, the limited degradation of HIV-derived ubiquitinated proteins can be stopped, and the HIV virus can sprout, mature, and acquire infectivity. It is known that the growth can be inhibited [PNAS, Vol. 97, No. 24, 13057-: 13062 (November, 2000)].

Furthermore, it is known that the activation of NF.KB transcription factor by inflammatory site force inevitably occurs when acute or chronic inflammation occurs, and this activation of NF-κΒ transcription factor Upon activation, the gene MAD-3 is expressed [Cell. Vol. 65, pp 1281-1289 H (1991)].

Incidentally, the following formula (A) produced by Panus rudis NREL 3821 and Panus conchatus NRRL 3253 belonging to the genus Panus mushroom (Panus)

OH

Panepoxydone) represented by the following formula is known: [Biochemical and Biophysical Research Communications. 226: 214-221 (1996)]. This document describes that panepoxydone has the activity to inhibit the activation of NF.KB transcription factor involved in inflammation, but panepoxydone produces intracellular protein, RNA or DNA. It is described that it does not show activity inhibiting synthesis. A study of the stereochemical structure of the panepoxydone molecule is described in Helvetica Chimica Acta, 53, Fasc. 7 (1970), No. 186, 1577-, p. 1597; According to the description in Table 2 and 1579 pages pages, Panepokishidon the specific rotation [shed] D ²⁰ - 61 ° (solvent, dichloromethane), a viscous oil of pale yellow indicating the.

As mentioned above, ubiquitination of proteins in cells is an important biochemical reaction, but substances with the activity to directly inhibit ubiquitination of proteins in cells are currently not available. There is still a need for new substances with such activity, as they have not yet been discovered. If new substances with the ability to inhibit the mechanism of ubiquitination of intracellular proteins could be provided, such new substances would be known anti-tumor tumors known or used in the past. Compounds, anti-inflammatory and analgesic compounds, anti-rheumatic drugs, anti-paralytics, immunosuppressants or immunostimulants, antivirals, anti-dementia drugs, anti-cardiomyopathy, anti-obesity drugs, anti-glucoseuria It is expected that it can be used as a compound having a different action point and a new chemical structure from drugs and organ failure drugs. Research is being conducted to find new substances with the above-mentioned inhibitory activities.

Launch announcement

DISCLOSURE OF THE INVENTION The present inventors have a novel activity having an inhibitory activity on intracellular ubiquitin activating enzyme and an inhibitory activity on intracellular protein ubiquitination that can meet the above-mentioned demands, and also have an antitumor activity and an antiviral activity. For the purpose of providing substances, research on the development and practical application of useful bioactive substances has been promoted. As a result of the research, they found that a strain of the genus Aratake mushroom, which belongs to the genus Pleurotus sp., Produces bioactive substances with a new structural skeleton. Successful isolation of this novel physiologically active substance, confirming that it has the following physicochemical properties and the steric chemical structure shown by the following formula (I), the physiologically active substance RS-K3574 It was named. Furthermore, this novel physiologically active substance RS-K3574 has an inhibitory activity on ubiquitin activating enzyme and ubiquitination of intracellular protein, as well as antitumor, anti-inflammatory and antiviral activities. I found it to have.

That is, in the first present invention, this RS-K3574 substance is represented by the following formula (I)

Wherein the configuration at the 2-, 3-, 4- and 7-positions is S, S, R and R, respectively, and the specific rotation [α] D ²⁶ — 62.3 ° (c 1.0, dichloromethane) is a colorless and viscous oily substance, which is measured with a thin-layer silica gel chromatography developed with a developing solvent consisting of chloroform-methanol (10: 1). The RS-K3574 substance shows an Rf value of 0.39, and the main peak in the ultraviolet absorption spectrum of the RS-K3574 substance measured in a methanol solution was _max nm (ε); 209 (8300) and 241 (5700); main absorption band in infrared absorption spectrum (KBr tablet method) is _max (cmi); 3300-3500, 2979, 2911, 1681, 1446, 1379, 1043, 995, 850, 817, 570 There; measured at 125MHz in heavy black port Holm CDC1 ₃ solution (using tetramethylsilane as internal standard) (5 values (ppm) were 3.47 (dd, J = 3.9 Hz, J = 1.0 Hz) and 3.81 (ddd, J = 3.9) in the proton nuclear magnetic resonance (Ή-MR) spectrum of the RS-K3574 material. Hz, J = 1.2Hz, J = 2.5Hz), 4.69 (br s, J = 1.2Hz, J = 5.0Hz, J = 1.0Hz), 6.71 (ddd, J = 5.0Hz, J = 1.2Hz, J = 2.5Hz), 5.29 (br d, J = 9.0Hz, J = 1.2Hz), 5.02 (dt, 9.0Hz, J = 1.2Hz, J = 1.2Hz), 1.72 (d, J = 1.2Hz), 1.72 ( d, J = a 1.2 Hz), also in heavy chloroform CDC1 ₃ solution (internal standard using tetramethylsilane as) in carbon 13 nuclear magnetic resonance RS-K3574 material measured at 125MHz in (@ 13 C-NMR) spectrum , 5 values (ppm) are 194.6 (s), 53.9 (d), 57.7 (d), 63.2 (d), 137.8 (d), 139.0 (s), 65.3 (s), 123.6 (d), 138.4 ( s), wherein 25.8 (q) ヽ 18.4 (q), wherein the physiologically active substance HS-K3574 is provided.

Next, the physicochemical properties of the first physiologically active substance of the present invention; RS-K3574 will be described.

A) Appearance and properties: colorless viscous oily substance

B) Specific rotation [D] D ²⁶ —62.3 ° (cl.0, dichloromethane)

C) 1 £ value of 1 ^ 〇: 0.39 When measured by thin-layer chromatography on silica gel (Art.l05715, Merck) using a developing solvent consisting of chloroform-methanol (10: 1).

D) FAB mass spectrum (m / z): 233 (M + Na) ⁺

Note that M + was not observed, and (M-18) + was observed.

E) molecular formula: C _u H I4_rei_4

F) UV absorption spectrum

(i) The ultraviolet absorption spectrum measured in the methanol solution is shown in Fig. 1 of the attached drawings. The main peaks are as follows.

Amax nm (ε): 209 (8300), 241 (5700)

(ii) The ultraviolet absorption spectrum measured in a methanol-HC1 solution is shown in Fig. 2 of the accompanying drawings. The main peaks are as follows.

Input nm (ε): 205 (14200), 240 (sh 5500)

(ii) UV absorption spectrum measured in methanol-NaOH solution is shown in Fig. 3 of the attached drawings. The main peaks are as follows.

Amax nm (ε): 206 (16400), 240 (sh 5300), 304 (2400)

G) The infrared absorption spectrum (KBr tablet method) is shown in Fig. 4 of the accompanying drawings. The main absorption bands are as follows.

S maxfcnT ¹ ): 3300-3500 ヽ 2979, 2911 ヽ 1681, 1446, 1379, 1043, 995,

850, 817, 570

H) The Ή-NMR spectrum (in the form of heavy chloroform / internal standard tetramethylsilane) is shown in FIG. 5 of the accompanying drawings.

In the heavy black port Holm CDC1 ₃ solution (as an internal standard using tetramethylsilane) RS-K3574 proton nuclear magnetic resonance of the substance measured in 500MHz in (Ή-NMR) scan Bae transfected Le, straight (ppm) is 3.47 ( dd, J = 3.9Hz, J = 1.0Hz), 3.81 (ddd, J = 3.9Hz, J = 1.2Hz, J = 2.5Hz), 4.69 (br s, J = 1.2Hz, J = 5.0Hz, J = 1.0Hz), 6.71 (ddd, J = 5.0Hz, J = 1.2Hz, J = 2.5Hz), 5.29 (br d, J = 9.0Hz, J = 1.2Hz), 5.02 (dt J = 9.0Hz, J = 1.2 Hz, J = 1.2 Hz), 1.72 (d, J = 1.2 Hz), and 1.72 (d, J = 1.2 Hz).

I) ¹³ C-NMR spectrum (in the form of double-mouthed form / internal standard tetramethylsilane) is shown in FIG. In the heavy black port Holm CDC1 ₃ solution (internal standard using tetramethylsilane as) in carbon 13 nuclear magnetic resonance RS-K3574 material measured at 125MHz in ⁽¹³ ONMR) scan Bae transfected le, 5 value (ppm) is 194.6 (s), 53.9 (d), 63.2 (d), 137.8 (d), 139.0 (s), 65.3 (s), 123.6 (d), 138.4 (s), 25.8 (q), 18.4 (q) is there. The fact that the stereochemical structure of the RS-K3574 substance of the present invention is as shown in the above formula (I) means that a crystal of a certain derivative from the RS-K3574 substance was analyzed by powder X-ray diffraction. I was able to confirm by doing.

Equation (A) above

OH

Compared with the stereochemical structure of panepoxydone, the bioactive substance RS-K3574 of the present invention is recognized as a new stereoisomer (7-epimani) of the known substance panepoxydone.

Further, the biological properties of the physiologically active substance RS-K3574 of the present invention are described below.

A) Measurement of inhibitory activity of RS-K3574 substance on ubiquitin activating enzyme It has the ability to completely inhibit ubiquitin activating enzyme.

The inhibitory activity of the RS-K3574 substance on ubiquitin activity was measured according to the method described in J. Nat. Prod., Vol. 65, pp. 1491-1493 (2002). That is, human-derived ubiquitin activating enzyme was expressed in Escherichia coli by recombinant genetic engineering, and a ubiquitin activating enzyme sample was prepared by collecting and purifying the ubiquitin activating enzyme from the E. coli. Piotinylated ubiquitin prepared by biotinylation of ubiquitin derived from pest was used as a substrate. The ubiquitin activating enzyme and the substrate were reacted with ATP (adenosine-1,3-phosphate) for 15 minutes at 3TC in the presence or absence of the test RS-K3574 substance. The resulting reaction solution was subjected to polyacrylamide gel electrophoresis, and the fractionated protein in the gel, which was bound to the ubiquitin bitionate, was adsorbed on the polyvinylidene difluoride membrane by elect opening plot. . The amount of the enzyme protein bound to the biotinylated ubiquitin on the membrane was assayed using the ECL method (see “Clin. Chem.j, Vol. 25, pp. 1531 to 1546 (1979).) In the presence of the RS-K3574 substance The amount of enzyme protein bound to piotinylated ubiquitin was detected in the test group where the above enzyme reaction was performed, and the amount was detected in the control test group where the enzyme reaction was performed in the absence of RS-K3574. By comparing with the amount of the enzyme protein bound to the biotinylated ubiquitin, the extent to which the amount of the bound product of the ubiquitin-activating enzyme bound to the biotinylated ubiquitin was suppressed by the RS-K3574 substance was determined.

B) Measurement of inhibitory activity of RS-K3574 substance on ubiquitination of intracellular protein

The inhibitory activity of the physiologically active substance RS-K3574 according to the present invention on protein ubiquitination in cells completely inhibits the production of ubiquitinated proteins induced in cells by RS-K3574 substance at a concentration of 2 μg / ml or more. It has the strength to do.

The production of ubiquitinated protein in the cells was detected as follows. That is, RS-K3574 substance was added in various concentrations to a medium containing human breast cancer cells MCF7 in advance. Thirty minutes after the addition, MG-132, an inhibitor of proteasomes in cancer cells, was further added to the medium at a concentration of 1 M. (The proteasome was completely added with MG-132 added to the medium.) Inhibition, which allows the accumulation of ubiquitinated proteins in the cells to be degraded by the proteasome). Then, after culturing the cancer cells in the medium for 3 hours, the cancer cells were dissolved. The obtained cell soluble fraction was subjected to polyacrylamide gel electrophoresis. The fractionated protein in the gel is adsorbed onto a polyvinylidene difluoride membrane by electroprotocol, and the adsorbed ubiquitinated protein on this membrane is reacted with an anti-ubiquitin antibody. Selectively detected. For detection, the ECL method (see "Clin. Chem.j, Vol. 25, 1531-: p. 1546 (1979)") was used.

In addition, the test was performed in the same manner as described above, without adding the RS-K3574 substance to the medium containing human breast cancer cells MCF7. As in this latter control study, the absence of RS-K3574 When the test was conducted under the above conditions, the ubiquitinated protein was accumulated, but when the test was performed in the presence of the RS-K3574 substance added to the above medium, the presence of the RS-K3574 substance was observed. It was confirmed that the accumulation of ubiquitinated protein was suppressed depending on the concentration. As is evident from this, HS-K3574 is effective in suppressing the production of functional intracellular proteins whose properties are directly or indirectly controlled by being ubiquitinated in cells. .

C) Inhibitory activity of RS-K3574 substance on cancer or tumor cell proliferation

The physiologically active substance RS-K3574 according to the present invention has an activity of suppressing the growth of cancer or tumor cells. The concentration of the RS-K3574 substance that inhibits the growth of various cancer cells by 50% (IC ₅₀ value) was measured by the MTT method (see Journal of Immunological Methods, Vol. 65, pp. 55-60 (1983)). Are shown in Table 1 below.

As is clear from the results in Table 1, the physiologically active substance RS-K3574 according to the present invention has an antitumor activity that suppresses the growth of various cancer cells, and is therefore useful as an antitumor agent. .

D) Anti-cancer activity of RS-K3574 substance

The physiologically active substance RS-K3574 according to the present invention has an anticancer activity. The RS-K3574 substance has a life-prolonging effect on mice transplanted with, for example, Ehrlich ascites cancer. When RS-K3574 substance was administered to these tumor-bearing mice at a dose of 250 μg / mouse per day for 9 consecutive days, the life-span effect was longer than when ES-K3574 substance was not administered. Reaches 200%. This life extension effect was determined as follows.

That is, 2 × 10 ⁶ Ekdicli cancer cells were transplanted intraperitoneally into 4-week-old ICR mice, and from the next day, the physiologically active substance RS-K3574 was administered intraperitoneally at a dose of 250 or 62.5 g / animal per day for 9 days. Dosing continued daily. In the untreated group, ascites caused by Ehrlich cancer accumulated in the abdominal cavity, and all patients died about 2 weeks later. Therefore, the survival effect was evaluated by the value of the quotient obtained by dividing the average survival days of the treatment group to which the RS-K3574 substance was administered by the average survival days of the non-administration group. In the group treated with the RS-K3574 substance, the accumulation of ascites was significantly suppressed, and as a result, a life-promoting effect of 200% was obtained at a dose of 250 g / mouse per day for 9 days, and 62.5 g / mouse Even when administered at the same dose for 9 days, a survival effect of 160% or more was observed.

As is clear from these results, the physiologically active substance RS-K3574 has an inhibitory effect on the malignant transformation of cancer or tumor in a living body. Useful as

E) Enhancement effect of RS-K3574 substance on apoptosis susceptibility of cancer cells by cytocidal antitumor agent, anticancer agent or radiation for cancer treatment

The physiologically active substance RS-K3574 according to the present invention enhances the apoptosis-inducing effect of the antitumor agent, the anticancer agent, or the radiation for cancer treatment on cancer cells, and is irradiated with the antitumor agent, the anticancer agent, or the radiation for cancer treatment. For example, it has an activity of increasing the antitumor activity or anticancer activity of γ-rays, X-rays, and α-rays.

When human leukemia cells U937 are treated in vitro with 10 ng / ml of a cell-killing anticancer agent, TNP tmnor necrosis factor, for 4 hours, apoptosis of U937 cells is not observed. On the other hand, when U937 cells were previously treated with 5 g / ml of the physiologically active substance RS-K3574 and then treated with 10 ng / ml of TNF, remarkable apoptosis was observed after 4 hours. Observed in U937 cells.

This apoptosis was confirmed by measuring the ladder formation of DNA, which is characteristic of apoptosis, by the method used in BBRC. Vol. 209, pp. 907-915 (1995). The potentiating activity of RS-K3574 on TNF-induced cancer cell apoptosis is exactly the same when tested using a TNF-like apoptosis-inducing ligand (TRAIL) instead of TNF. Is recognized.

In addition, when HeLa cells derived from human uterine cancer are treated with adriamicin, which is used as a cell-killing anticancer agent, to induce apoptosis in vitro, the number of viable HeLa cells can be reduced by simultaneously adding the KS-K3574 substance. Can be significantly reduced. That is, while the number of viable cells when HeLa cells were treated with 1 j glml of adriamicin for 24 hours was 100%, 5 g / ml of the RS-K3574 substance and 1 μg / ml of adriamicin were simultaneously used. When HeLa cells are treated, the number of surviving cells is reduced to 50%.

As is evident from these results, the physiologically active substance RS-K3574 has an activity of enhancing the apoptosis-inducing action of a cell-killing antitumor agent or an anticancer agent on cancer cells. Alternatively, the antitumor effect of the anticancer agent is increased. It also has the effect of similarly enhancing cancer cell apoptosis due to radiation irradiated for cancer treatment.

It can be expected that RS-K3574 substance has. Therefore, it can be used as an anti-tumor agent or anti-cancer agent or as an enhancer of cancer cell apoptosis-inducing action of radiation irradiated for cancer treatment, and as a concomitant agent for enhancing the therapeutic effect of anti-tumor agent or anti-cancer agent in cancer treatment. The K3574 substance is useful.

F) Inhibitory activity of HS-K3574 on protein biosynthesis

As described in Biochemical and Biopnysical Research Communications Vol. 226, 214-221 Sada (1996), the known substance panepoxydone does not inhibit the biosynthesis of any of the proteins: NA and DNA. The RS-K3574 substance of the present invention has an activity of remarkably and specifically inhibiting intracellular protein biosynthesis. Its inhibitory activity is such that the 50% inhibitory concentration of the RS-K3574 substance is 4.5 μg / ml. The inhibitory activity was measured as follows.

That is, 2 × 10 ⁵ human breast cancer-derived MCF7 cells were seeded on a 24-well plate containing a serum-containing medium and cultured for 24 hours. The medium was replaced with a serum-free medium, and various concentrations of RS-K3574 were added. Here, tritium-labeled leucine was added at a concentration of lCi / ml. After an additional hour of culture, leucine is taken up by the cells during this time and protein biosynthesis proceeds within the cells. Thereafter, the medium was removed, and an ice-cooled 10% aqueous solution of trifluoroacetic acid was added to the cultured MCF7 cells. Culture play The sample was allowed to stand on ice for 30 minutes, and then washed twice with an ice-cooled 10% aqueous trifluoroacetic acid solution. This operation destroyed the cells. After this washing, fractions containing the insoluble protein were collected, dissolved in 0.1N aqueous sodium hydroxide, and the radioactivity of the resulting solution was measured.

The measured radioactivity of the resulting solution indicates the amount of protein biosynthesized in the cell. Of added RS-K3574 substance

When the above test in which MGF7 cells were cultured in the presence of the above concentration, the measured value of radioactivity was 180 cpm. In comparison, when MCF7 cells were cultured in the absence of the RS-K3574 substance and a control test was performed, the measured radioactivity was 700 cpm. From these results, the concentration of RS-K3574 substance that inhibits protein biosynthesis in MCP cells 50% (IC ₅₀ value) was calculated to be δμ ^ / ιηΐ.

G) Inhibitory effect of RS-K3574 on activation of NF-κΒ transcription factor

The physiologically active substance RS-K3574 has an activity to completely inhibit the activity of NF-κΒ transcription factor at a concentration of 4 Ug / ml. This activity was determined as follows.

That is, when TNF acting as an inflammatory cytokine at a concentration of 10 ng / ml or Interleukin-l (IL-1) at a concentration of 2 ng / ml is added to the culture medium of human breast cancer cells MCF7, about 1 hour later Activation of the NF.KB transcription factor is observed. Activation of this NF.KB transcription factor is caused by a specific increase in the mRNA of MAD-3 (Cell, vol. 65, pp. 1281-1289, 1991), a gene that is expressed with the activation. Can be confirmed by measuring with the RT-PCII method. At this time, if the RS-K3574 substance was added in advance to the culture medium at a concentration of 4 μg / ml, the expression of the gene MAD-3 was not observed at all, and the activation of the NF.KB transcription factor could be completely inhibited. . From these results, it has been confirmed that the RS-K3574 substance has an activity of inhibiting the activation of NF.KB transcription factor necessary for inducing acute and chronic inflammation, and is useful as an anti-inflammatory agent. Furthermore, according to the second present invention, a P. mushroom (Panus rudis) producing the physiologically active substance RS-K3574 represented by the above formula (I) is cultured in a nutrient medium, and the physiologically active substance HS-K3574 is obtained from the culture. And a method for producing a physiologically active substance RS-K3574.

As an example of a bacterium producing RS-K3574 which can be used in the second method of the present invention, There is also an oyster mushroom genus Aragueka (Emm§mdk) strain K-3574. In addition, this strain was obtained from a fungus hypha obtained from the Fermentation Research Institute (IFO) more than 10 years ago from July 2001, and the potato 'dextrose and agar was used for more than 10 years from that time. It is a strain that has been subcultured on a culture medium at a laboratory of Calagari Co., Ltd. The strain K-3574 is mutated during the passage, and the production amount of the physiologically active substance RS-K3574 is higher than that of the Agarage moss (Panus rudis) IFO 8994 strain deposited at the Institute of Fermentation (IFO). And the composition of other metabolites is also different. From this, this strain K-3574 is a different strain from Panus rudis IFO 8994 strain.

June 2001, Allageka mushroom (Panus rudis) K-3574 strain was deposited at the National Institute of Advanced Industrial Science and Technology, National Institute of Advanced Industrial Science and Technology at 1-1 1-1 Tsukuba-Higashi, Ibaraki, Japan June 2001 An application for deposit was made on the 26th, and the deposit was received under deposit number FERM P-18397. Also, on the date of the transfer acceptance on December 25, 2002, under the provisions of the Budapest Treaty, the Aragageka mushroom K-3574 strain was deposited at the above depository under the accession number of FEEM BP-8265.

In addition, Aratakeka mushrooms are known mushrooms (basidiomycetes) and are distributed almost all over the world, but they occur relatively commonly in beech trees in Japan. The mushroom mycological characteristics of Kiagega are as follows. The umbrella of the fruiting body is 1.5-5cm in diameter, initially bun-shaped, later opened to form a slightly funnel-shaped, tough fleshy-slightly leathery, with coarse hair on the surface, initially brownish purple, and sometimes purple. The edges are almost flat, the handle is generally short (0.5-2 cm), eccentric to central, rarely lateral, and the surface is almost the same as an umbrella. The spore print is white. The spores are 4.5 ~ 5.5 Χ2 ~ 2.5μηι, spheroidal, and the basidium is four spores. Thick-membrane cystizia are 53-70 X 9.5-; 14 μιη, club-column-shaped, or spindle-shaped. There is no hyphal peg. The hypha composition of the meat tissue is dimitic ("Primary Color Japanese New Fungi Guidebook (1)"), p. 32, Rokuya Imaseki, Tsuguo Hongo, Nursery, June 30, 1987.

In practicing the second method of the present invention, the above-mentioned Aratake Kazukitake K-3574 strain was inoculated into a nutrient medium as a strain producing a physiologically active substance RS-K3574 belonging to the genus Pleurotus edulis, and cultured. I do. The nutrient medium used here is assimilated by the aforementioned production strain. It contains carbon and nitrogen sources as nutrients.

As the nutrient source of the medium, those commonly used as nutrient sources for basidiomycetes strains, for example, assimilable nutrient sources such as carbon source, nitrogen source and inorganic salts can be used. For example, carbohydrates such as glucose, maltose, molasses, dextrin, glycerin, and starch; and carbon sources such as soybean oil, peanut oil, and other fats, as well as peptone, meat extract, cottonseed powder, soybean powder, yeast extract, and casein. Nitrogen sources such as corn 'steep' liqueur, NZ-amine, ammonium sulfate, ammonium nitrate, ammonium chloride, etc., as well as potassium nitrate, sodium phosphate, salt, calcium carbonate, magnesium sulfate, manganese chloride, etc. Inorganic salts can be blended. If necessary, trace metals such as cobalt and iron can be added. As a nutrient source, any known nutrient source can be used as long as the RS-K3574 producing strain used for producing the physiologically active substance RS-K3574 can be used.

The mixing ratio of the above-mentioned nutrients in the medium is not particularly limited, and can be varied over a wide range. Depending on the RS-K3574 substance producing strain used, the optimum composition of the nutrients and the mixing ratio are as follows. However, it can be easily determined by a person concerned with a simple small-scale experiment. The nutrient medium consisting of the above-mentioned nutrients can be sterilized prior to cultivation, and before or after this sterilization, the pH of the medium is in the range of 5 to 7, especially in the range of pH 5.5 to 6.5. It is advantageous to adjust

Cultivation of the RS-K3574 substance-producing strain in such a nutrient medium can be carried out according to a method generally used in the production of a physiologically active substance using common mushrooms. Usually, it is preferable to culture under aerobic conditions. Usually, the culture can be performed with stirring and / or aeration. As the culture method, any of stationary culture, shaking culture, and liquid culture with aeration and stirring can be used, but liquid culture is suitable for mass production of RS-K3574 substance.

The culture temperature that can be used is not particularly limited as long as the growth of the RS-K3574 substance producing strain is not substantially inhibited and the physiologically active substance RS-K3574 can be produced. The culture temperature in the range of 25 to 30 ° C. is particularly preferred. Culture can usually be continued until the RS-K3574 substance has sufficiently accumulated. The culture time depends on the composition of the medium, the culture temperature, The target RS-K3574 substance can usually be obtained by culturing for 14 to 30 days, depending on the temperature used, the production strain used, etc.

The accumulated amount of the physiologically active substance RS-K3574 during the culture can be quantified by the above-described method for measuring the inhibitory activity of ubiquitin activating enzyme.

The RS-K3574 substance accumulated in the culture obtained by the culture is collected from the culture. After the culture, if necessary, the bacterial cells are removed by a known separation method such as filtration or centrifugation, and the culture filtrate is subjected to solvent extraction using an organic solvent, particularly butyl acetate, etc., adsorption, The RS-K3574 substance can be isolated, further purified, and collected by treating the ion-exchange chromatography, gel filtration, or chromatography using countercurrent distribution alone or in combination. . Activated carbon, silica gel, porous polystyrene-divinyl benzene resin or various ion exchange resins can be used as a carrier used for adsorption or a carrier used for mouth chromatography having ion exchange ability. Thus, a novel physiologically active substance RS-K3574 having the aforementioned properties can be obtained.

Further, the third present invention provides an inhibitor of a ubiquitin activating enzyme, comprising the physiologically active substance represented by the above formula (I): RS-K3574.

In a fourth aspect of the present invention, there is provided an inhibitor of ubiquitination of an intracellular protein, comprising the physiologically active substance RS-K3574.

Further, in the fifth invention, there is provided an antitumor agent composition comprising the above-mentioned physiologically active substance HS-K3574 as an active ingredient, and being mixed therewith and containing a pharmaceutically acceptable carrier.

Further, in the sixth aspect of the present invention, there is provided an anti-inflammatory composition comprising the above-mentioned physiologically active substance RS-K3574 as an active ingredient, and a pharmaceutically acceptable carrier mixed therewith.

In a seventh aspect of the present invention, there is provided an antiviral composition comprising the above-mentioned physiologically active substance RS-K3574 as an active ingredient, and a pharmaceutically acceptable carrier mixed therewith. You.

The third inhibitor of the ubiquitin activating enzyme according to the present invention and the fourth inhibitor of the ubiquitination of the intracellular protein according to the present invention are each prepared from the RS-K3574 substance alone. Is what can be. Further, the antitumor agent composition according to the fifth aspect of the present invention, the anti-inflammatory agent composition according to the sixth aspect of the present invention and the antiviral agent composition according to the seventh aspect of the present invention each comprise the RS-K3574 substance as an active ingredient thereof. In the form of a composition mixed with a conventional pharmaceutically acceptable solid or liquid carrier. Further, the first physiologically active substance RS-K3574 of the present invention is used as an anti-rheumatic drug, an anti-periodical drug, an immunosuppressive or immunostimulating substance, an anti-dementia drug, an anti-cardiomyopathy, an anti-obesity drug or an anti-diabetic drug. Is also expected to be available.

Further, in the eighth aspect of the present invention, there is provided a potentiator comprising a physiologically active substance RS-K3574, which enhances a cancer cell apoptosis-inducing effect of a cell-killing antitumor agent or anticancer agent or radiation for cancer treatment on cancer cells. . The cell-killing antitumor agent or anticancer agent according to the eighth aspect of the present invention refers to TNF, a TNF-like apoptosis inducer, adriamycin, and other clinical anticancer agents.

In the ninth aspect of the present invention, there is provided an inhibitor of protein biosynthesis in cancer cells, comprising the physiologically active substance RS-K3574.

Further, in the present invention of a 10 consists bioactive substance RS-K3574, NF- _K B inhibitors on activation of the transcription factor are provided.

An aqueous solution of the RS-K3574 substance dissolved in water containing 10% dimethyl sulfoxide (DMS0) and 0.5% Tween 80 (surfactant) was administered at a dose of 2 mg or more per mouse to the RS-K3574 substance. (Four weeks old, female, average body weight 21.6 g, 5 animals per group) were injected intravenously via the tail vein, but in 2 weeks after administration, all cases survived and no abnormalities were observed. Intravenous administration of RS-K3574 substance to adult humans (body weight 60 kg) at doses from l mg to 5000 mg is not expected to show any acute toxicity. The dose of the S-K357 substance to humans can be adjusted to an appropriate level by a specialist through preliminary tests, depending on the type of disease to be treated, symptoms, and other factors.

In a pharmaceutical composition such as an antitumor agent composition, an anti-inflammatory agent composition or an antiviral agent composition according to the present invention, a RS-K3574 substance as an active ingredient is converted into a pharmaceutically acceptable conventional liquid carrier, For example, it can be mixed with ethanol, aqueous ethanol, water, physiological saline, or a solid carrier such as a crystal cell mouth, starch and the like to form a composition containing the RS-K3574 substance. The RS-K3574 substance, which is an active ingredient used in a pharmaceutical composition according to the present invention, can be administered orally. Alternatively, it can be administered parenterally by intravenous or intramuscular or subcutaneous injection, or intraperitoneal or rectal administration.

For oral administration, in the pharmaceutical composition according to the present invention, the RS-K3574 substance is mixed as an active ingredient with a conventional pharmaceutically acceptable solid or liquid carrier, and the resulting mixture is powdered. , Tablets, capsules, suspensions, syrups and the like.

When the pharmaceutical composition of the present invention is formulated for injection, a desirable form of the injection preparation is a sterile aqueous solution or a lyophilized agent containing the RS-K3574 substance as an active ingredient. Liquid carriers used in injectable preparations include, for example, water, saline,

Phenol, hydrated ethanol, glycerol, propylene glycol, vegetable oil and the like.

The ratio of the RS-K3574 substance as an active ingredient to be incorporated in the pharmaceutical composition as described above according to the present invention may vary depending on the dosage form. For example, the content ratio of the active ingredient may be about 1 to 1% by weight of the dosage unit. Can be in the range of 95%.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows an ultraviolet absorption spectrum of a RS-K3574 substance in a methanol solution. Figure 2 shows the UV absorption spectrum of the RS-K3574 substance in methanol-HC1 solution.

FIG. 3 is an ultraviolet absorption spectrum of NaOH in a methanol solution of RS-K3574 substance.

Figure 4 shows the infrared absorption spectrum of the RS-K3574 substance measured by the KBr tablet method.

FIG. 5 is a proton nuclear magnetic resonance spectrum measured with a heavy chloroform solution of RS-K3574 substance (internal standard: tetramethylsilane) at 500 MHz.

FIG. 6 is a 13C nuclear magnetic resonance spectrum measured at 125 MHz using a heavy chloroform solution of RS-K3574 substance (internal standard: tetramethylsilane).

Form of the turtle

Next, production examples of the physiologically active substance HS-K3574 of the present invention will be described in more detail by way of Examples. As will be described, the present invention is not limited to the following examples.

Example 1 Production of bioactive substance RS-K3574

1% glucose, 0.5% of polypeptone, 0.3% yeast _{_{extract, KH 2 P0 4 0.3%,}} MgS0 4 - 7H 2 0 0.1%, the liquid containing medium (pH unadjusted) shake flask (500ml volume) each 200ml dispensing Then, it was sterilized at 120 ° C for 20 minutes by an ordinary method. The sterilized liquid medium was inoculated with a mycelium of Pleurotus edulis (Panus dis) K-3574 strain (FERM BP-8265) cultured on an agar slant medium. Thereafter, the cells were cultured in a liquid medium at 27 ° C for 3 days. Thereafter, the cells were cultured at 27 ° C. for 2 days under rotary stirring. The culture obtained here was used as a seed culture.

1% glucose, 0.5% of polypeptone, 0.3% yeast _{_{extract, KH 2 P0 4 0.3%,}} MgS0 4 '7H 2 0 0.1%, by 200ml liquid medium (pH unadjusted) in Fuban flask (500ml volume) containing minute The solution was poured and sterilized at 120 ° C for 20 minutes by an ordinary method. Thereafter, 7 ml of each of the seed cultures obtained above was inoculated into a sterilized liquid medium, and cultured at 27 ° C for 15 days.

10 L (liter) of the culture thus obtained was filtered, and the culture filtrate was separated. The culture filtrate was extracted with ethyl acetate, and the ethyl acetate layer was concentrated to dryness under reduced pressure. A solution obtained by dissolving the obtained residue in black-mouthed form was subjected to silica gel gel chromatography (80 g), and black-mouthed form-methanol (100: 0 to 50: 1, volume ratio), Eluted step by step. HS-K3574 was contained in the fraction eluted with black form-methanol (200: 1 to 100 ··· 1). Fractions containing the RS-K3574 substance were collected and concentrated to dryness to obtain 1.17 g of a crude product. The crude product was further subjected to liquid-centrifugal partition chromatography (250 mL) by an ascending method using a liquid phase consisting of chloroform-methanol-water (5: 6: 4). Fractions were collected to separate unwanted and required components. The fraction containing the RS-K3574 substance was obtained as the 24th to 27th fractions when fractionated by 10 ml. These active fractions were concentrated to dryness under reduced pressure to obtain 716 mg of a purified RS-K3574 substance of the present invention.

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As described above, the present invention provides a novel physiologically active substance, RS-K3574 substance. This RS-K3574 substance is used for cell proliferation of cancer cells or immune cells or It not only has the activity of inhibiting the intracellular enzyme ubiquitin activating enzyme, which is involved in the generation of apoptosis or inflammation, but also has antitumor, anticancer and antiinflammatory activities. The activity of inhibiting the biosynthesis of proteins in cells. The RS-K3574 substance according to the present invention is useful as an antitumor agent, an anticancer agent, an antiinflammatory agent or an antiviral agent.

Claims

, The scope of the claims

1. This HS-K3574 substance has the following formula (I)

(

[Wherein the configuration at the 2-, 3-, 4-, and 7-positions is S, S, R, and R, respectively], and the specific rotation [H] D ²⁶ — 62.3 ° (c 1.0, dichloromethane) is a colorless and viscous oily substance, which is measured by developing it with a developing solvent consisting of chloroform-form-methanol (10: 1) by thin-layer silica gel chromatography. this RS-K3574 material shows a Rf value of 0.39, a main peak in still present RS-K3574 main material Yunoichiru solution UV absorption scan Bae spectrum measured in the example _m ax nm _(ε) in; 209 (8300) and is in the 241 (5700); main absorption bands in infrared absorption scan Bae spectrum (KBr tablet method) is _{m ax (cm-; 3300~3500, 2979} , 2911, 1681, 1446, 1379, 1043, 995, 850, 817, located at 570; measured at 500MHz in heavy black port Holm CDC1 ₃ solution (using tetramethylsilane as internal standard) 5 values (ppm) of 3.47 (dd, J = 3.9Hz, J = 1.0Hz) and 3.81 (ddd, J = 3.9Hz) in the proton nuclear magnetic resonance (Ή-ΝΜΙΙ) spectrum of the RS-K3574 material , J = 1.2Hz, J = 2.5Hz), 4.69 (br s, J = 1.2Hz, J = 5.0Hz, J = 1.0Hz), 6.71 (ddd, J = 5.0Hz, J = 1.2Hz, J = 2.5 Hz), 5.29 (br d, J = 9.0Hz, J = 1.2Hz), 5.02 (dt, J = 9.0Hz, J = 1.2Hz, J = 1.2Hz), 1.72 (d, J = 1.2Hz), 1.72 (d, J = 1.2 Hz), and carbon-13 nuclear magnetic resonance (i ³ C-NMR) of RS-K3574 substance measured at 125 MHz in deuterated chloroform CDCls solution (tetramethylsilane used as internal standard). In the spectrum, the d value (ppm) is 194.6 (s), 53.9 (d), 57.7 (d), 63.2 (d), 137.8 (d), 139.0 (s), 65.3 (s), 123.6 (d), Physiologically active substance Κ ^ · Κ3574, characterized by 138.4 (s) and 25.8 (qX 18.4 (q).

2. A protein producing the physiologically active substance RS-K3574 according to claim 1. The physiologically active substance RS-K3574 according to claim 1, characterized in that the gekazuki yuga (Fanus rudis) is cultured in a nutrient medium, and the physiologically active substance RS-K3574 is collected from the obtained culture. Manufacturing method.

3. As a bacterium producing the physiologically active substance RS-K3574, the National Institute of Advanced Industrial Science and Technology

2. The method according to claim 1, wherein a strain of P. mushroom (Panus rudis) · K-3574 deposited under a deposit number of BP-8265 is cultured.

4. An inhibitor of ubiquitin activating enzyme, comprising the physiologically active substance HS-K3574 according to claim 1.

5. An inhibitor of ubiquitination of an intracellular protein, comprising the physiologically active substance HS-K3574 according to claim 1.

6. An antitumor agent composition comprising the physiologically active substance RS-K3574 according to claim 1 as an active ingredient, and a pharmaceutically acceptable carrier mixed therewith.

7. An anti-inflammatory composition comprising the physiologically active substance RS-K3574 according to claim 1 as an active ingredient, and a pharmaceutically acceptable carrier mixed therewith.

8-An antiviral composition comprising the physiologically active substance RS-K3574 according to claim 1 as an active ingredient, and a pharmaceutically acceptable carrier mixed therewith.

9. An enhancer for a cancer cell apoptosis-inducing effect of a cell-killing antitumor agent or anticancer agent or cancer therapeutic radiation against cancer cells, comprising the physiologically active substance HS-K3574 according to claim 1.

10. An inhibitor of protein biosynthesis in cancer cells, comprising the physiologically active substance RS-K3574 according to claim 1.

11. consisting physiologically active substance HS-K3574 according to claim 1, wherein, NF-? _K B inhibitors on activation of the transcription factor.