FI69485B - PROCEDURE FOR THE PHARMACOLOGICAL ACTIVATION OF ACTIVE 6,7-DIHYDROXY-2,5,5,8A-TETRAMETHYL-1,2,3,4,4A, 5,6,7,8,8A-DECAHYDRONAPHTHALEN-1-SPIRO-2 ' - (6 ', 7'-DIFORMYL-4'-hydroxy-2', 3'-DIHYDROBENSOFURAN) - Google Patents

Description

J ANNOUNCEMENT

* · Βί (11) UTLÄGGNINGSSKRIFT 6 9485 C (45) P -. 'Otti 13 y 3 r. ::: 11 y' 'Γ “* · * ·· A t] λ OO 1 ηο / j (51) Kv .lk.> t.Cl. 'c 12 P 17/04, C 07 D 307/94 FINLAND —FINLAND <21) Patent application - Patentansölcning 781517 (22) Filing date - Ansöknlngsdag 12.05.78 (23) Starting date - Glltlghetsdag 12.05.78 (41) Made public - Blivit offentlig j- | 1 2 78

Patent and Registration Office ..-.,. . . . ,. „, 1 (44) Date of publication and hearing publication— τ. i n Qc

Patent and registration authorities in the United States of America have been published in the Official Journal. iu .op (32) (33) (31) Privilege claimed — Begärd priority 30.06.77 »29.12.77, 10.02.78 Japan-Japan (JP) 78641/77, 158957/77, 14674/78 (71) Otsuka Pharmaceutical Co., Ltd., No. 2-9, Kandatsukasa-cho, Chiyoda-ku,

Tokyo, Japan-Japan (jp) (72) Masanao Shinohara, Naruto-shi, Tokushima, Hirotsugu Kaise, Itano-gun, Tokushima, Yoshimasa Nakano, Tokushima-shi, Tokushima, Taketoshi Izawa,

Tokushima-shi, Tokushima, Yasuo Oshiro, Tokushima-shi, Tokushima,

Wasei Miyazaki, Itano-gun, Tokushima, Japan-Japan (JP) (74) Oy Borenius & Co. Ab (54) Method for pharmacologically active 6,7-dihydroxy-2,5,5,8a-tetramethyl--1,2,3,4,4a, 5,6,7,8,8a-decahydronaphthalene-1-spiro For the preparation of -21- (61,71-diformyl-41-hydroxy-21,31-dihydrobenzofuran) - For the preparation of pharmacologically active 6,7-dihydroxy-2,5,5,8a-tetramethyl- 1,2,3,4,4a, 5,6,7,8,8a-Decahydronaphthalene-1-spiro-21- (61,71-diformyl-41-hydroxy-21,31-dihydrobenzofuran )

The invention relates to a process for the preparation of a new sesquiterpene derivative, i.e. 6,7-dihydroxy-2,5,5,8a-tetramethyl-1,2,3,4,4a, 5,6,7,8,8a-decahydronaphthalene-1-spiro For the preparation of -2 '- (6 *, 7'-diformyl-4'-hydroxy-2', 3'-dihydrobenzofuran) of the formula

HO -JJ ^ Y-CHO

0 (I)

HO

BO

OHj CHj and pharmaceutically acceptable salts thereof. The new compound has an inhibitory effect on the animal complement system (anti-complement effect) and an antitumor effect.

According to the invention, the novel compound is prepared by culturing a microorganism belonging to the species Stachybotrys Stachybotrys altermans IFO 9355, Stachybotrys chariarum IFO 5369, Stachybotrys chatrarum IFO 7222, Stachybotrys cylindrospora 8858, Stachybotrys IFotis25, Stachybotrys eco mp. K-76, Stachybotrys complement! nov. T-789 or Stachybotrys (Memmoniella) echinata var. T-791 aerobically in a medium containing assimilable nitrogen sources, carbon sources, inorganic salts and trace elements, at a pH of about 3.5 to 11.5, at a temperature of about 15 to 35 ° C, and recovered as a product 6,7-dihydroxy-2,5,5,8a-tetramethyl-1,2,3,4,4a, 5,6,7,8,8a-decahydronaphthalene-1-spiro-2 '- (6 ', 7'-diformyl-4'-hydroxy-2', 3'-dihydrobenzofuran) from the culture broth, which is optionally converted into a pharmaceutically acceptable salt thereof.

The compound of the invention can be used as an effective component in pharmaceutical compositions which act against autoimmune diseases, nephritis, rheumatism, collagen diseases, allergic diseases, cancers, etc. The compound can also be used as an intermediate to prepare such effective components.

The phrase "complement" refers to a complex group of proteins in body fluids. These proteins interact with antibodies or other factors and play an important role in mediating immune, allergic, immunochemical and / or immunopathological reactions. Reactions in which complement is involved occur in blood serum or other body fluids and are therefore considered humoral or fluid reactions.

The complement system has been suggested to be involved in inflammatory, coagulation, fibrinolytic, antibody-antigen reactions, and other metabolic events (cf. U.S. Patent 4,021,544, Bull. World Health Org., 39, 935-938). (1968), Scientific American, 929 (No. 5) 54-66 (1973), Medical World News, October 11, 53-58, 64-66 (1974), Harvey Lectures, 66 75-104 (1972), The New England Journal of Medicine, 287, 489-495, 3 69485 545-549, 592-596, 642-646 (1972), The Johns Hopkins Med. J. 128, 57-74 (1971), and Federation Proceedings, 32 ^ , 134-137 (1973)).

Many compounds are known to have antimicrobial activity, including ethylenediaminetetraacetic acid (EDTA), Saldox, floricin (Borsos, J. Immunol. 94, (4), 629 (1964)), hydroxybenzene derivatives (Shir Mayer, Biochemistry NY , 1 3003 (1968)), guanidines and phenoxyacetamides (BR, Baker, J. Med. Chem., Ljj, 408 (1968)), phosphonate esters (EL Becker, BBA, 147, 289 (1967)), chlorophyllin, glycyrrhizin- sine, etc. However, these compounds are not suitable for practical purposes because they are highly toxic or have little anti-complement effect. To the best of our knowledge, no anti-complement compound or substance is currently commercially available.

The compound (I) prepared according to the invention and its pharmaceutically acceptable salt can be used in pharmaceutical compositions containing a therapeutically effective amount of these compounds so as to obtain an anti-complement effect in animals. The compound of formula (I) can be used in particular for the treatment of inflammatory diseases of the kidneys in animals by administering a medicament mixture to an animal suffering from such a disease.

The invention will be explained in more detail below on the basis of the accompanying figures.

Figures 1 and 2 show the Stachybotrys complementi Nov of a microorganism producing a compound of formula (I) of the invention. mp. K-76 micrographs.

Figure 3 shows the Stachybotrys complementi Nov. mp. T-789 micrographs.

Figure 4 shows the microorganism Stachybotrys echinata var. T-791 micrographs.

Figure 5 shows the nuclear magnetic resonance spectrum of a compound of formula (I).

4 69485

The compound can be prepared using the following known microorganisms belonging to the genus Stachyhotrvs or microorganisms belonging to the genus Stachybotrys isolated only in the context of the present invention.

Stachybotrys alternans IFO 9355 Stachybotrys chariarum IFO 5369 Stachybotrys chartarum IPO 7222 Stachybotrys cylindrospora IPO 8858 Stachybotrys echinata IFO 7525 and Stachybotrys reniformis IPO 7067

Newly isolated microorganisms are new species belonging to the genus Stachybotrys, as described below, and have been termed Stachybotrys complementi Nov sp. K-76 (hereinafter referred to as Stachybotrys sp. K-76), Stachybotrys complementi Nov sp. T-789 (hereinafter referred to as Stachybotrys sp. T-789) and Stachybotrys (Memmoniella) echinata var. T-791 (hereinafter referred to as Stachybotrys sp. T-79T) ~ (a) Location (1) Stachybotrys sp. K-76 This species was isolated from soil in Ishigaki City, Okinawa, Japan.

(2) Stachybotrys sp. T-789 This species was isolated from soil in Naruto City, Tokushima, Japan.

(3) Stachybotrys sp. T-791 This species was isolated from soil in Tokushima City, Tokushima, Japan.

(B) Properties of different media (1) Stachybotrys sp. K-76 The culture characteristics of this species in many different media are as seen by microscopic observations (Figures 1 and 2) as follows;

II

69485 a) Visual observation

Many good media e 11a, which are commonly used in mold cultures, show very good growth, but the adhesion of fialospores is not always good, except on oatmeal agar. Growth conditions on a typical medium are shown below.

(i) Malt extract agar medium

Growth is relatively slow and after 30 days of cultivation at 27 ° C a large colony with a size of 30 ... 35 mm is formed. The shape of the colony is round and there is a large bump on the perimeter of the colony. The surface of the colony is smooth and the hyphens penetrate the air like a thick round mat in the middle of the colony and grow densely. The color of the colony is white at the beginning of cultivation and changes to a creamy white color (Li Ivory, 2ca). Two weeks after the start of cultivation, phialospores gradually begin to form, but they are barely noticeable to the naked eye. No sclerotia and other sex-forming spore-forming organs are formed. Colony reverse side color colorless to colored copper (Copper Tan, 5ie). Copper Tan (Copper Tan, 5ie) forms a soluble pigment.

(ii) Potato-glucose agar medium. Growth is good and after 30 days of cultivation at 27 ° C the colony size is 45 ... 47 mm. The growth of the colony is convex in the middle and there is a bulge around the perimeter of the colony. The colony has concentric circles or folds starting from the center of the colony. From the beginning of the vijely, the colony is covered by a white or pink (Pearl Pink, 4ca) hyphae. As the rumen is continued, the color of the colony changes to black-gray (Gray, i). Lots of phialospores are formed. A large number of liquid droplets are detectable. Colony reverse side color ranges from rust (Rust Tan, 5ie) as a light red-brown (Li Rose Brown, 6i Ig). The soluble pigment formed is light brown in color (Butter Scotch, 3ne) and does not fade.

(iii) Czapek agar medium

Growth is good and after 30 days of cultivation at 27 ° C the colony size is 40 ... 45 mm. The colony has a concentric fold and there is a bulge in the middle of the colony. The perimeter of the colony is wavy. Colony reverse side color is dark brown (Luggage Tan, 4ne). The edges of the colony adhere to the colorless or white hyphae in a thinly concentric shape and its toast-colored (Toast Tan, 41g) central part has no air hyphen. No liquid droplets are detected, but the center of the colony is hygroscopic. Phialospores are hardly formed at all. The soluble pigment is light and golden in color (Gold, 21e).

6 69485 (iv) Oatmeal agar medium

Growth is very fast and after 30 days of cultivation at 27 ° C the colony size is 60 ... 70 mm. The colony is thin and flat and from the beginning of cultivation a colorless or white hyphae forms on the entire surface of the colony. A 3 to 4 mm high, upright hyphae is formed only in the middle of the colony, but it is not dense. Two weeks from the start of culture, the formation of phialospores spreads over the entire surface of the colony, and the color of the colony changes to a light olive color (Li Olive Drab, 1 li). Colony reverse side color varies from light tan (Brown Beige, 3ig) of a tan (Tan, 3ie). The soluble pigment is opaque yellow (Colonial Yellow, 2ca).

(2) Stachybotrys sp. T-789

The culture characteristics of the T-789 species belonging to Fungi-Inperfect on many different culture media were determined visually and microscopically (as in Figure 3), and the results are shown below. The microscopically observed morphological characteristics of T-789 are well suited to Stachybotrys sp. To the corresponding characteristics of the K-76 species. However, different results are achieved on different media. On the next two platforms, the features differ the most.

a) Visual observation (i) Malt extract agar medium

Growth is very slow and after 30 days of culture at 27 ° C the colony size is 17 ... 20 mm. The peripheral part of the colony grows in a wavy manner, the growth is limited in the middle part. The colony has a bulge and radial folds appear in the perimeter of the colony. Few air hybrids grow on the entire surface of the colony. In the early stages of cultivation, the hyphal is white and conidia are gradually formed. The middle part of the colony turns roar-colored (Mole 1) and the conidia grow densely. Conidia formation is annular near the perimeter of the colony. Colony reverse side color is brown cap (Cork Tan, 4ie). Amber-colored (Amber, 41e) soluble pigment is abundant.

(ii) Czapek agar medium

Growth is good and after 30 days of culture at 27 ° C the colony size is 45 mm. The central part of the colony is convex and the colony forms radial folds. The peripheral part of the colony shows undulating growth. The light air sac grows sparsely in concentric circular shapes outward from the convex portion of the central portion. Conidia are formed concentrically only in the periphery of the colony. The hyphae is dissolved in a convex part and is very hygroscopic. Colony reverse side color is brown mustard (Mustard 3rown; 2pi). no soluble pigment is formed.

(3) Stachybotrys sp. T-791

The culture characteristics of the T-791 species belonging to Fungi-Imperfect on many different culture media were determined visually and microscopically (as in Figure 4), and the results are shown below.

a) Visual observation (i) Malt extract agar medium

The growth is opaque and irregular. Colony reverse side color ranging from tan (Tan, 3ie) to dark brown (Dk Brown, two pn). The colony is flat and conidia formation is good. The color of the hyphal varies from light brown (Biscuit, 2ec) to skin brown (Tan, 3ie) and oozing liquid droplets can be observed. No soluble pigment is formed.

(ii) Potato-glucose medium

Growth is very fast and after 30 days of culture at 27 ° C the colony size is 70 mm. The peripheral part of the colony spreads branchedly and a hyphae rising from the menus can be detected. Lots of liquid droplets are also noticeable. Colony reverse side color is light amber-colored (Li Amber, 3ie). No soluble pigment is formed.

(iii) Czapek-Dox agar medium Growth is weak.

(iv) Synthetic Home Agar Medium (Mucor Brush Home)

Growth is weak.

(v) Oatmeal agar medium

The growth is so good that the petri dish is covered after two weeks. The colony is thin and flat. Hypha formation is abundant from the beginning of cultivation, and conidia formation is also rapid. The color of the colony changes from white to dark brown (Dk Brown, 3pn) as cultivation continues. A lot of liquid droplets are formed by the hyphae. No soluble pigment is formed.

8 69485 (C) .Physiological properties

Stachybotrys K-76, T-789 and T-791 are all aerobic species and have the following physiological properties.

pH Temperature (1) Stachybotrys sp. K-76

Growth conditions 3.5 ... 11.5 15 ... 35 ° C

Optimal growth conditions 6.0 ... 9.5 20 ... 32 ° G

(2) Stachybotrys sp. T-789

Growth conditions 3.5 ... 11.5 15 ... 38 ° C

Optimal growth conditions 4-, 5 ... 10.5 20 ... 32 ° C

(3) Stachybotrys sp. T-791

Growth conditions 3.5 ... 11.5 15 ... 38 ° C

Optimal growth conditions 4.5 ... 9.5 20 ... 30 ° C

(D) Morphological characteristics (1) Stachybotrys sp. K-76

Figures 1 and 2 show the following. No sclerotia or other sexual reproductive organs are observed in the various media, but phialospore-type asexual spores appear to form.

Hypophilic, branched and cross-crossed hyphae are formed with many different media. The width of the hyphen is 2 .., 4 yu.

Phialophores simply branch from the hyphal but not from the phialophore to another. There are 3 .., 4 partitions between the foot cell and the phialophore. The phialophores are vertical or slightly bent. The hyphal width of the phialophores is 4.3 ... 4.7 in the lower part and 3.6 ... 4.5 in the middle part. The tips of the phialophores are slightly bulging, and the extreme phialophores form 3 to 7 ellipsoidal or malleable vertical fialides measuring 7.9 to 9.3 x 3.6 to 4.7 yu. The fialides are smooth and vary in color from colorless to pale yellowish brown. Fialospores are formed from the ends of the fialides. Fialospores do not form straight chains, but form mucus-down-hanging small semicircular compositions numbering from 7 to 26. The shape of the phialophores varies from circular to right and their size is 4.9 ... 8.0 x 3.3 ... 4.7 yu. The surface of the phialophores is rough and bumpy, and their color varies from dark ivy (Dk Ivy 24po) to gray-black. The compositions of the fialospores do not show the membrane from which the mucus would come.

il 9 69485

The taxonomic status of a species with the above microbiological characteristics has been sought in the following works: G.L. Barron, The Genera of Hyphomycetes from Soil, The Williams & Wilkins Company, Baltimore (1968), J.C. Gilman, A Manual of Soil Fungi. The Iowa State University Press, Ames, Iowa (1971), and J.A. von Arx, The Genera of Fungi Spore-lating in Pure Culture, Vergang von J. Cremer 3301 Lehre (1970).

According to the Saccardo taxonomic system, the species in question belongs to the class Hyphomycetes. To the family Dematlaceae, genus Stachybotrys. The characteristics of this species are described by the absence of ascocarp and other sexual reproductive organs, the formation of dark brown fialos pores from fialides, and the accumulation of formed fialospores in a circular shape at the ends of fialides, the characteristics of which match well with those of the genus Stachybotrys.

The various characteristics of the species in question are identified in the references in the above-mentioned manuals, and in the literature references such as G.R. Bisby, Trans. Brit. Mycol. Soc., 26, 133, 143 (1943); R.K. Zuck, fcycologia, 38, 69, 76 (1946); GL Barron, Can. J. Bot .. 39, 153 ...

157 (1961), and have been compared to type species stored at an institution called the Institute for Fermentation, Osaka, Japan (IFO).

It has been concluded that this K-76 species is mainly similar to Stachybotrys lobulata IFO 5369, as the Fialospores of the K-76 species have a rough and bumpy protrusion and the Fialospores are oval or elliptical. However, there are differences, for example, the size of the phialophore of Stachybotrys lobulata may be 1 mm, and simple branching occurs in K-76, whereas Stachybotrys lobulata has the property that one phialophore branches and spreads from another phialophore. Considering the growth with different medium, K-76 species forms a large amount of soluble pigment, and conidia formation on agar medium is very weak, except on potato-glucose agar medium and oatmeal agar medium. Stachybotrys lobulata, on the other hand, grows very rapidly on many different media and an unfolded flat colony is formed. Conidia formation is good on almost all media except Czapek's Dox agar medium, where colony color is typical, no or very little soluble pigment is formed.

10 69485

Due to the above differences, it has been concluded that this species K-76 is a new species, different from Stachybotrvs lobulata, and this species has been given the name Stachybotrys sp. K-76, (2) Stachybotrys sp. T-789

The following can be seen from Figure 3. The morphology of this species is exactly the same as that of Stachybotrys sp. K-76 species, although there are differences in colony size.

The microbiological properties mentioned above are, in the same way as Stachybotrys sp. K-76, compared to the above-mentioned manuals and literature references and Ii’0 type species. In conclusion, this species is mainly similar to Stachybotrys lobulata IFO 5369 and Stachybotrys sp. K-76 species. The formation of fialospores of this species on Czapek agar medium is good. However, the formation of phialospores of the first two species on Czapek's Dox agar medium is hardly observed. In this respect, the cultures differ.

In addition, Stachybotrys- sp. K-76 that the morphological characteristics are clearly different from Stachybotrys lobulata. in addition, the pH range for optimal growth of the species in question is 4.5 to 10.5. Here, too, this species differs. K-76 species.

For the above reasons, this species has been considered a new species and has been named Stachybotrys sp. T-789.

The following can be seen from Figure 4. The phialospores are simply branched and upright. The ends of the phialophores are slightly bulging into rod-shaped. However, the bulges are not as large as those observed for Stachybotrys echinata IFO 7525 and 8856. The hyphal width of this species is 4.0 ... 4.5, which is slightly larger than the width of the phialophores. Phialophores, which branch upright with their leg cells from the vegetative hyphae or air hyphae, have two or three partitions and are 40 ... 80 x 3.0 ... 3.5 yu in size. The cell wall of the Fialo traffic light is not bumpy but smooth.

Three to six fialides consist of a rod-like part at the ends of the phialophores, in addition to round or almost round unicellular phialospores with a bumpy protrusion and a size of 4.3 --- 5.2 x 11 69485 3.0. ..4.2 yu, is continuously formed from the ends of fialides and chains with 24 to 70 conidia are formed. Fialid has a mallet shape and is 6.9 ... 10.7 x 3.5 ... 4.7 yu in size. The phialophores and fialide are colorless, and the color of the fialide varies from coffee (Coffee, 3pn) to black.

The taxonomic status of a species with the above-mentioned microbiological characteristics has been sought in the manuals mentioned in the section dealing with the X-76 species. As a result, it was found that this species belongs to the genus Stachybotrys (genus Hemmoliella). It is characteristic that this species has no ascocarps or other genital organs, and dark brown phialospores are continuously formed from fialid. Long spore chains are formed. The characteristics of this T-799 species fit well with those of the genus Stachybotrys (genus Memmoniella).

The characteristics of the species in question have been sought in the above-mentioned manuals and literature references, such as R.K. Zuck, Mycologia, 38, 69 ... 76 (1964) and G. Smith, Trans. Brit. Mycol. Soc., 45, 387-394 (1962) and have been compared to the types stored in the IFO.

It has been concluded that since fialospores are continuously formed from the ends of fialides, this species belongs to the species Memmoniella echinata called Höhnel. However, this type of T-791 was R.K. According to Zuck and G. Smith, considered the same species as Stachybotrys echinata. It was compared to Stachybotrys echinata 1F0 7525 and IFO 8856.

Cystic protrusions, which are morphologically typical of both species, are not detectable in the cell walls of the phialophores of this T-791 species. In addition, the ends of the phialophores of the type species bulge 2 to 3 times the width of the hyphal of the phialophores, whereas in the species in question no bulge is observed. In addition, both Type species show good growth on many different media, especially potato-glucose-agar medium, and form round, slightly elevated colonies, and the hyphae is strongly adherent. In addition, considerable conidia formation is observed. The growth of the species in question, on the contrary, is branchedly irregular, as shown above, and the observed hypha formation and conidia formation are poor. Due to the microbiological differences mentioned above, this species has been considered as a new species and has been named Stachybotrys sp. T-791.

12 69485

The color references above and below follow the method set forth in the Color Harmony Manual, Container Corporation of America (1958).

New species, Stachybotrys sp. K-76, Stachybotrys sp. T-789 and Stachybotrys sp. T-791 »samples have been collected at the following plant; Fermentation Research Institute, Agency of Industrial Science and Technology, Japan, under deposit numbers FERM-P No. 5801, FERM-P No. 3082, and FERM-P No. 3803.

Production of a substance of general formula (I) by microorganisms belonging to the genus Stachybotrys

The preparation of the substance of the general formula (I) by means of the above-mentioned microorganisms belonging to the genus Stachybotrys is carried out as follows.

The microorganisms are first cultured on a medium containing common food sources and additives. Nitrogen-based, commonly used media include, for example, soybean meal, soybean oil, corn liquor, yeast extract, dry yeast, oatmeal, meat extract, hydrolyzed casein, ammonium salts, and nitrate salts. Suitable carbon sources include, for example, glucose, glycerol, maltose, starch, lactose, sucrose and molasses. Culture medium additives include, for example, inorganic salts such as axiurocarbonate, sodium chloride, magnesium sulfate and phosphoric acid. In addition, small amounts of metal salts such as iron, copper, manganese and zinc salts may be added to the culture medium as needed. The culture can be carried out in a conventional aqueous medium containing the above-mentioned substrates, using either a surface culture technique or a liquid culture technique with aeration and mixing. Liquid culture with aeration and mixing is recommended. Cultivation can be advantageously carried out at a temperature of 15 to 35 ° C, preferably 20 to 32 ° C, for approximately 3 to 7 days under normal aeration conditions, maintaining the pH of the culture medium at about 3.5 to 11.5, the best has a pH of 4.5 to 9.5.

13 69485

The material formed after culturing is obtained from the culture broth. In particular, the recovery method is not limited, and many different methods can be applied using the physicochemical properties of the recovered substances. For example, recovery can be accomplished by a method that utilizes differences in solubility between products and impurities, or by utilizing differences in adsorption strength and affinity of common adsorbents such as activated carbon, silica gel, ion exchange resins, or Sephadex (Pharmacia Pine Chemistry). differences in partition coefficients between the two liquid phases, and combinations of such methods.

More specifically, the culture broth is filtered or centrifuged in the usual manner to allow the cells to evaporate. Methanol is then added to the rising liquid, and the mixture is stirred and allowed to stand for 2-3 hours. The precipitate is removed by a second centrifugation. The residue is extracted with the same volume of ethyl acetate, and the solvent is distilled off. The extract is mixed with methanol, and the methanol solution is passed through a column of activated carbon, after which the solvent is distilled off from the eluate. The residue is gel filtered using Sephadex 1H-20. Each fraction obtained is examined by the anti-complement activity test described below. The active fractions are collected and the solvent is distilled off. Thus, pure 6,7-cLihydroxy-2,5,5,8a-tetramethyl-1,2,3,4,4a, 5,6,7,8,8a-decahydronaphthalene-1 of the above structural formula can be isolated. spiro-2 '- (6 *, 7, -diformyyli-4-hydroxy-2', 3'-dihydrobenzofuran.

The sesquiterene derivatives of formula (I) and their salts prepared according to the present invention are useful as a treatment agent for nephritis, and when used in this way they have been added to pharmaceutical compositions together with customary pharmaceutically acceptable carriers.

There is no particular restriction on the use of the therapeutic agent as a treatment agent for nephritis, and the therapeutic agent may be administered by suitable means depending on the particular form of the therapeutic agent. For example, tablets, liquid preparations, suspensions, emulsions, granules and capsules are administered orally. Syringe preparations are administered into a blood vessel either alone or with a common excipient such as glucose and amino acids. In addition, if required, the therapeutic agent may be administered intramuscularly, intradermally, subcutaneously, or intraperitoneally. A suppository is administered rectally.

The dose of the nephritis treatment agent is appropriately selected according to the intended use, the symptoms, etc. Generally, a suitable dose of the compound of this invention is about 0.5 to 20 mg / kg of body weight per day in single or multiple doses.

The compound of formula I has anti-complementary activity and is also useful as a therapeutic agent in the treatment of autoimmune diseases, collagen diseases and rheumatic diseases, each of which includes a complement system.

The results of experiments on the pharmacological efficacy of the compound of formula (I) and its pharmaceutically acceptable salts are shown below.

(1) Anti-complement effect

The anti-complement effect was measured and determined according to the method described in Meneki Kagaku (Immuno-Chemis-try), Yuichi Yamamura et al., Ed. pages 830 ... 834, Asakura Shoten, Tokyo, Japan (1973). Specifically, 0.5 ml of an aqueous dispersion of test compound, 0.5 ml of photosensitive red blood cells was added to the test tube as a solution containing 1 x 10® cells / ml, 1 ml of a five-fold diluted solution of Veronal buffer containing gelatin, Ca ++ and Mg ++ (GVB ++), and 0.5 ml complement serum (guinea pig complement) diluted 150-fold with GVB ++. The mixture was treated hot at 37 ° C for 60 minutes. 5 ml of ice-cold physiological saline was then added, and the mixture was centrifuged. The absorbance of the obtained supernatant was measured in OD413, and the extent to which the test compound inhibited the hemolysis of photosensitive red blood cells was determined. The value of the effect of 50% hemolysis inhibition (y / ml) measured from the test compound according to the above method is shown in Table 1 below.

(2) Acute toxicity

The LDjq values (mg / kg) of the test compounds when administered intravenously to mice were measured, and these results are also shown in Table 1 below.

table 1

Compound Anti-complement LD50 value Activity value (mg / kg) (y / ml)

Compound of formula I

Chlorophyllin 40 (3) Therapeutic effect on nephrotoxin-type nephritis

Rat nephrotoxin (abbreviated "NT") was obtained as follows. The rat renal cortex was homogenized with the same amount of physiological saline. The homogenized mixture was mixed with "Freund's complete excipient" (product of Difco Company) in a volume ratio of 1: 1. Two milliliters of this solution was injected into the rabbit muscle (body weight 3,100 g) to render the rabbit immune. After one and a half months, blood was taken from the heart of the rabbit to obtain serum. This serum was inactivated at 56 ° C for 30 minutes, then desalted with 40% saturated aqueous ammonium sulfate, and fractionated, the γ-globulin fraction (IgG) was collected to obtain NT. .

Evaluation was performed using male Wistar rats weighing 150-160 g using three replicate experiments for each test compound. The test compound was administered intraperitoneally once every 24 hours for seven days. One hour after administration of the test compound, NT was administered on the third day. 1 ml of NT was injected into the tail vein of each rat. Chlorophyllin (CP) was used as a reference solution and physiological saline was used as a control.

The amount of protein urea (total amount of urine excreted over 24 hours) was measured using nephelometry and, as a control, serum albumin with sulfosalicylic acid.

The results obtained are shown in Table 2 below.

Table 2

Compound Dose Parallel (mg rat sample No. Daily reading *) 1 4 7 10

1 2.5 ** 1.5 0.8 8 kainen combined 3 2 4.3 2.1 1.8 10 diste 3 1.7 1.1 0.9 7 average 2.8 1, 6 1.2 8 1 2.4 1.5 0.9 9

Chlorophylline 5 2 4.4 2.6 1.7 8 3 3, 2 2.1 3, 5 6 average 3.4 2.1 2.0 8 1 13 16 21 29 (control) - 2 19 23 25 35 3 11 18 24 40 average 14 19 23 35 * The daily reading in Table 2 is calculated from the time the test compound was administered, which was 1 hour before NT administration ** The amount of protein urea is expressed in mg / day 17,694 8 5

The protein urea level in a healthy rat is 0.5 to 5 mg / day.

When the protein urea level exceeds this limit, especially when the protein urea level is higher than 10 mg / day, it can be said with certainty that nephritis occurs. From the results in Table 2, it is noted that nephritis was present in the control batch and that in the batches of the compound of formula I and chlorophyllin, the amount of protein urea was approximately the same as in the healthy rat 10 days after NT administration. Thus, administration of a compound of formula I may be considered to inhibit primary and secondary immune responses.

(4) Therapeutic effects on Heymann-type nephritis

Male Wistar rats weighing 180-200 g were used in the experiment. The rat renal cortex was extracted and homogenized with the same volume of physiological saline. The homogenized solution was centrifuged at 1500 G for 1 hour. The supernatant solution was purified according to the method of T.S. Edgington et al., Journal of Experimental Medicine, 127, 555 (1968), and mixed with Freund's complete excipient 37 Ra (product of Difco Company) in a volume ratio of 0.4: 1. The resulting mixture was injected intraperitoneally into isological rats, 0.5 ml per rat. Thereafter, the same amount of excipient was administered every other week until the protein urea level exceeded 100 mg / day. (This period was about 6 to 8 weeks).

Each test compound in Table 3 was administered intraperitoneally to rats suffering from Heymann-type nephritis (weighing 300-350 g) once daily for 7 days, after which the amount of protein urea (mg / day) was measured by the above-mentioned bile. CP was used as a reference compound and physiological saline was used as a control. Three parallel experiments were performed on each co-compound. The results obtained are shown in Table 3 below.

18 69485

Table 3 ______ Daily reading__

Dose Parallel

Compound no (mg / rat) band- _____ sample no of 1 4__7_14 21

A compound of the formula I 3 1 95 90 47 17 9 10 2 132 125 65 35 15 12 3 121 120 70 27 10 9

average 116 111 61 26 11 1C

Chlorophyllin 5 1 123 116 72 32 16 10 (comparison) 2 117 108 63 29 12 8 3 129 121 52 22 15 13 ski value 123 115 62 28 14 10 (control) - 1 135 127 132 135 114 126 2 121 105 121 103 105 109 3 137 117 135 121 109 132 mean 131 116 129 119 109 122 2 .. .3 weeks after the start of the experiment, the body weight of the rats increased to 400 .. .500 g and normal protein urea levels were likely to be 5-15 mg /day. As can be seen from the results in Table 3, the compound of formula I can ameliorate Heymann-type kidney disease.

To illustrate this invention in more detail, the preparation of a compound of formula (I) of this invention is shown in the following examples. Unless otherwise stated, all parts, percentages, ratios, etc. are by weight and the term room temperature as used herein means 15 ... 25 °.

11 x9 69485

Example 1 A 500 ml Sakaguchi flask was charged with 100 ml of medium according to the following composition, and Stachybotrys sp. K-76 was cultured at 28 ° G and pH 6 for 4 days with shaking.

Medium composition%

Glycerol 0.5 Starch 1.0

Lactose 0.2

Soy flour 0.5

Yeast extract 0.1

Malia mouth 0.2

CaCO 3 0.3

MgSO 4 0.05 A 30 liter fermentation flask was charged with 20 liters of medium according to the above composition, and one flask of the obtained inoculum was cultured in medium at 28 ° C for 5 days with stirring at 4-00 rpm and aeration of 1 liter of medium per liter per minute. The resulting culture broth was centrifuged at 8,000 rpm to remove microbial cells. To the supernatant solution was added 5 liters of methanol, and the mixture was stirred, after which the mixture was allowed to stand for 3 hours. The mixture was centrifuged and the solid removed. The supernatant was extracted with an equal volume of ethyl acetate. The solvent of the ethyl acetate layer was distilled off under reduced pressure. The precipitate was dissolved in methanol, and passed through a column of activated carbon. The eluate was concentrated to dryness under reduced pressure. The dry mass was dissolved in a mixture of chloroform and ethyl acetate (1: 1 v / v) and gel filtered through a column of Sephadex LH-20. The filtrate was subjected to thin layer chromatography (using a mixture of ethyl acetate, chloroform and acetic acid (50: 50: 2 by volume) as the developing solution), and the fraction having an anti-complement effect corresponding to Rf = 0.34 was collected. Alternatively, the filtrate was subjected to thin layer chromatography. (using a mixture of benzene, butanol and acetic acid (in a volume ratio of 60:15:15) as the developing solution) and a fraction having an anti-complement effect corresponding to Rf * 0.58 was recovered, and 2.0 g of the solvent was evaporated. 7-dihydroxy-2,5,5,8a-tetramethyl-1,2,3,4,4a, 5,6,7,8,8a-decahydronaphthalene-1-spiro-2 * - (6 *, 7'- diformyl-4'-hydroxy-2 ', 3'-dihydrobenzofuran) as a pale yellow, slightly acidic substance with anti-complement activity, the formation of which was confirmed by the following physicochemical properties.

69485 20 (1) 1 ~ CxJq ° - -4-8 ° (C <2.5, grain ethanol) (2) Elemental analysis values for C23H30 ° 6

Calculated (%): C 68.84, H 7.51

Found (%): C 68.58, H 7.55 (5) Ultraviolet absorption spectral analysis (UV) ethanol »246 nm (ε * 16474) λ max = 307 nm (ε» 6659) (4) Nuclear Magnetic Resonance Spectrum Analysis (NKR) The analysis was performed using pyridine-N, N-sodium 2,2-dimethyl-2-silapentane-5-sulfonate (DSS) as an internal standard. The NMR result obtained is shown in Fig. 5. The conditions of the NMR measurement were as follows:

Spektraalilaajuus; 4 x 100 filter 0.1 sec

RF power 0.5 mG

sweep time 5 minutes sweep width 10 ppm sweep limit 0 ppm

Example 2 A 500 ml Sakaguchi flask was charged with 100 ml of medium according to the following composition, and a known species of Stachybotrys chartarum IFO 5369 was cultured in medium at 28 ° G for four days at pH 6 with shaking.

Medium composition% glycerol 0.5 starch 1.0 lactose 0.2 soy flour 0.5 yeast extract 0.1 crude extract 0.2

CaCO 3 0.3

MgSO 4 0.05 A 30 liter fermentation flask was charged with 20 liters of medium according to the above composition, and two flasks of the obtained inoculum were cultured in medium at 28 ° C for five days with stirring at 2 rpm and 1 liter of aeration per liter of medium per minute. . The resulting broth was centrifuged at 8,000 rpm to remove microbial cells. To the supernatant solution was added 5 liters of raethanol, and the mixture was stirred and allowed to stand for 3 hours. The mixture was centrifuged and the solid removed. The supernatant was extracted with an equal volume of ethyl acetate. The solvent of the ethyl acetate layer was removed under reduced pressure and concentrated to dryness.

The precipitate was dissolved in methanol and passed through a column of activated carbon. The eluate was concentrated to dryness under reduced pressure, dissolved in a mixture of chloroform and ethyl acetate (1: 1, v / v), and gel filtered through a column of Sephadex LH-20. Fractions with peaks corresponding to the Rf values of the thin layer chromatography described in Example 1 were collected, and 2.2 g of (6,7-dihydroxy-2,5,5,8a-tetramethyl-1,2,3,4,4a) was obtained after evaporation of the solvent. , 5,6,7,8,8a-decahydronaphthalene-1-spiro-2 '- (6', 7'-diformyl-4'-hydroxy-21,3'-dihydrobenzofuran), as a pale yellow, slightly acidic substance with This compound had the same physicochemical properties as shown in Example 1, and these properties confirmed the formation of this compound.

Example 3

Stachybotrys sp. T-789 was cultured and purified in the same manner as described in Example 1, except that the composition of the medium was as follows and the pH was 7.5 and the culture temperature was 32 ° C.

Media composition% glycerol 0.5 glucose 1.2 maize leachate 0.5 dried yeast 0.1 malt extract 0.2

MgSO 4 0.05

NaCl 0.3 There was thus obtained 6,7-dihydroxy-2,5,5 * 8a-tetramethyl-1,2,3,4,4a, 5,6,7,8,8a-decahydronaphthalene-1-spiro-2 ' - (6 ', 71-diformyl-4'-hydroxy-2', 3'-dihydrobenzofuran), as a pale yellow slightly acidic substance with anti-complement activity. The physicochemical properties of this compound correspond to the values of the compound isolated and purified in Example 1.

22 6 9 4 8 5

Example 4

Stachybotrys sp. T-791 was cultured and purified in the same manner as in Example 1 except that the composition of the medium was as follows, the pH was 5.5, and the culture temperature used was 25 ° C.

Composition of the medium /> glycerol 0.5 starch 1.0 sucrose 0.2 soy flour 0.5 peptone 0.1 malt extract 0.2> -g304 0.3 HOI 0.05 There was thus obtained 6,7-dihydroxy-2.5, 5,8a-tetramethyl- ", 2,3,4,4a, 5,6,7,8,8a-decahydroraphthalene-1-spiro-21- (6 ', 7'-difformyl-4 * -hydroxy- 2 ', 31-dihydrobenzofuran), as a pale yellow slightly acidic substance with anti-complement activity, the physical and chemical properties of this compound correspond to those of the compound isolated in Example 1.

Claims (1)

23 69485 Claim A method of 6,7-dihydroxy-2,5,5,8a-tetramethyl-1,2,3,4,4a, 5,6,7,8,8a-decahydronaphthalene-1 having anti-complement activity. to prepare -spiro-2 "- (6 ', 7" -diformyl-4 * -hydroxy-2', 3'-dihydrobenzofuran) of formula (I): H0 (i) HO OHj OHj or its for the preparation of pharmaceutically acceptable salts, characterized in that the microorganism Stachybotrys altermans IFO 9355, Stachybotrys chariarum IFO 5369, Stachybotrys chatrarum IFO 7222, Stachybotrys cylindrosi 7525, Stachybotrys cylindrospora 8858, Stachybotrys cylindrosi 8858, Stachybotrys cylindrosi . mp. K-76, Stachybotrys complementi Nov. T-789 or Stachybotrys (Memmoniella) echinata var. T-791 aerobically in a medium containing assimilable nitrogen sources, carbon sources, inorganic salts and trace elements, at a pH of about 3.5 to 11.5, at a temperature of about 15 to 35 ° C, and recovering the product 6.7 dihydroxy-2,5,5,8a-tetramethyl-1,2,3,4,4a, 5,6,7,8,8a-decahydronaphthalene-l-spiro-2 '- (6', 7'-diformyl -4'-hydroxy-2 ', 3'-dihydrobenzofuran) from a culture broth which, if desired, is converted into a pharmaceutically acceptable salt thereof.