JPS6030289B2 - anticancer drug - Google Patents

Description

[Detailed description of the invention]

The present invention relates to an anticancer agent containing a novel sesquiterbene analog and its salt as an active ingredient. The sesquiterbene analogs used as the active ingredients of the anticancer agent of the present invention can be obtained by, for example, using a compound obtained by purifying the culture product of a microorganism belonging to the genus Stachybotrys (hereinafter referred to as compound [1]) as a raw material compound, and oxidizing it. can. Microorganisms used in the production of compound [1] include:
Examples include the following known microorganisms belonging to the genus Stachybotrys or microorganisms belonging to the genus Stachybotrys newly isolated and obtained from soil. o Stakiboto IJ
Alternance m0 9355 (Snechy or hisal
temansIFO9355) o Stachybotrys cartarum IF05369 (Snechy melon trysChar
tammIF○5369) o Stachybotrys cartarum IF072220 Stachybotrys cylindrospora 8858 (Snechy's tryscyljndros
pora8858) o Stachybotrys echinata 7
525 (S is chy or trysechinata752
5) o Stachybotrys reniformis 7067 (Snechy or trysreniformis 7067) The newly isolated bacteria belonging to the genus Stachybotrys used in the production of compound [1] is a new bacterial species as described below, and the present inventor Stachybotrys
Spy. They are named K-70T-789 and T-791. 1 Collection site 1 Stachybotrys sp. K-176 was isolated from Shiyo, Ishigaki Island, Okinawa Prefecture. 2 Stachybotrys sp. T-1789 isolated from soil in Naruto City, Tokushima Prefecture. 3. Stachybotrys sp. T-1791 was isolated from soil in Tokushima City, Tokushima Prefecture. 0 Properties on various culture media 1 Stachybotrys sp. The characteristics of culturing the K-76 strain on various media based on macroscopic and microscopic observations are shown below. A. Macroscopic observation It shows extremely good growth on various media used for normal filamentous fungus culture, but the settlement of bulwark spores is
Not necessarily good except for oatmeal agar medium. The growth conditions in typical tower sites are described below. (1) Growth on malt extract agar medium is relatively slow.
It shows the huge village on the 5th side. The village grows in a circular shape, with a large jagged edge around the periphery. The surface of the colony is flat, and the central part has a circular mat with thick aerial filaments that grow densely, and the color gradually changes from white at the initial stage of culture to light ivory (LtIvory. Next a). A small number of Enodia spores are formed from around the second week of culture, but they are hardly visible to the naked eye. No sclerotia or other sexual spore organ tubes are formed. The reverse side has a copper pattern due to its colorless color.
, 5ie). The diffusible dye is CopperTan (5i).
e) produced. (2) Growth on potato budu sugar agar is good, reaching 45 to 47 legs after 30 days of culture. The colony grows in a convex shape at the center, and the periphery is jagged, extending from the center of the village to the same circle. From the early stage of culture, the hyphae are thickly covered with white to pearl pink (Peari Pink, 4ca).
As the culture progresses, the color of the bacterial flora becomes black-gray (Gray.i). Enodia spore masses are formed in large numbers. A large amount of droplets can also be seen. The color of the back side is from Rust Tan (51e) to light rose brown (LtRship eBmWn6 back g), and the diffusible pigment is butterscotch (but rScotch, box e), but it is slight.

[3] It grows well on Beck's agar medium and reaches 270 to 40 to 45 skin after 30 days of culture. It curves in the same circular shape, and the village has a convex bulge in the center. The periphery is wavy. The color of the back side is Lugjitan (Lug saddle geTan, 4ne
). The surface is toasted tan (ToastTan,
Thin, colorless to white hyphae grow in the same D-circular shape from the central part that is not stimulated by the aerial hyphae of 41g). No droplets are observed, but the center is very wet. Almost no Enodia spores are formed. The diffusible pigment is weak and gold (G is d, 21e). {Growth on 4' oatmeal agar medium is extremely rapid, with 6% growth after 30 days of culture at 270°C.
It reaches 0-7. The colony is thin and flat, and colorless to white mycelia are formed on the entire surface from the early stage of culture. Three to four erect hyphae occur only near the center of the colony, but they are not dense. In the second week of culture, the epiphytes of bulwark spores cover the entire village and the color changes to light olive drab (LtOlive Drab, 11i). The back side is beige brown (3)
g) ~Tan (3ie). The diffusible dye is Colonial Y
yellow, next a) is weakly produced. 2 Stachybotrys sp. The culture characteristics of the T-789 Deficient strain T-789 vermilion on various media based on macroscopic and microscopic observations are as described below.
The morphological properties of mochikane observed under a microscope are Stachybotrys sp. It completely matches K-7 Kushu. However, there are differences in their properties on the culture medium. The following two types of media, which are the most different, will be described. A. Macroscopic observation m Malt extract agar medium growth is extremely slow 27q0, 17-2 after 30 days of culture
Sacred ribs. The area around the village grows in waves and is localized. The center of the village shows a convex bulge, and the periphery shows radial sliding. Aerial hyphae are thin and epiphytic over the entire surface, and spore formation gradually occurs from the white color at the initial stage of culture, and grows densely in the center with a mole color (1), but spore formation can be seen in a ring toward the periphery. The back side is Cork Tan (4ie). Diffusible pigments are abundant and amber (Am戊r.$e). {2
) The growth on the Abetsu Agar Plate was good and 270 cells were grown, with 45 cells grown after 30 days of culture. The colony has a convex center and a radial curve, and the periphery shows wavy growth. Thin white aerial mycelium grows outward from the central convex part in the same D-circular shape, and spore formation occurs only in the peripheral part in the same D-circular shape, and the convex part is in a state of dissolved hyphae and is extremely wet. The back side is mustard brown.
, book i). Does not produce diffusible pigments. 3 Stachybotrys sp. The characteristics of culturing T-791 strain T-791 on various media based on macroscopic and microscopic observations are described below. A. Macroscopic Observation 01 Malt Extract Agar The growth on the shore is rapid, showing unfriendly growth, and the color of the underside is tan (Tan, 3ie) to dark brown (DkBrown, 3pn). The settlement is flat and sporulation is good. Mycelium is biscuit (Bi
scuit, next c)~Tan (Tan, 3ie),
Exudate droplets are observed. Does not produce diffusible pigments. (2) Potatoes grow very quickly in glucose medium, reaching 7 acres in 30 days of culture. The peripheral area has a tree-like spread, thin white mycelia are observed, and droplets are also noticeable. Spores There is almost no formation. The back side is light amber (LtAm 戊r, 3ie). There is no diffusible pigment. Glue Poor growth on agar agar base {4} Poor growth on synthetic mucor agar base '5) Very good growth on oatmeal agar base. Cover the Petri dish in 2 weeks. The colony is thin and flat, and mycelium is abundant from the early stage of cultivation, and spore formation is rapid. The color of the bacterial flora changes from white to dark brown (Dk Brown, 3pn) as the culture progresses. A large amount of droplets are produced on the hyphae. Does not produce diffusible pigments. Dish Physiological Properties K-70 T-789 and T-791 are aerobic bacteria and have the following physiological properties. 1 Stachybotrys sp. K-76PH
Temperature conditions for growth 3.51-11.515-35 ○Optimal growth conditions 6.0-9.520-3〆02 Stachybotrys sp. T-789pH Temperature growth conditions 3.5-11.515-383○ Optimum growth conditions 4.5-10.520-3203 Stachybotrys sp. T-791PH Temperature growth conditions 3.5-11.515-3800 Optimal temperature conditions 4.5-9.520-30qON Morphological properties 1
Stachybotrys sp. No sclerotia or other sexual reproductive organs were observed on the various K-76 media, and asexual sporulation of the Enodia spore type was confirmed. Hyphae are formed on a variety of media, are intricately branched, and extend vertically and horizontally with a width of 2 to 4 hyphae. The cyphae is simply branched from the hyphae (there is no branching from one cyphae to another) and has 3 to 4 septa from the podocyte at the base, and is either erect or Stretch out in a gentle curve. The hyphae width of the pedicel is 4.3 to 4.7 degrees at the base and 3 degrees at the center.
．． 6-4.5 Buddha. The tip of the bulrush stalk swells slightly, and from its apex, 3 to 7 stalks of size 7.9 to 9.3 x 3.6 to 4.7 in the shape of a Buddha bucket to a sake bottle are formed upright. Enoki is smooth and colorless to pale yellowish brown. The mycospores are formed by basophilically connecting from the tips of the sycamores and are not formed in a straight chain, but form small semi-moon-shaped clusters of 7 to 26 in number. Enoki pattern is 4.9 to 8.
It is subspherical to ovoid in size, measuring 0 x 3.3 to 4.7 mm, and its surface is rough to warty. Also, the color tone is Dark Ivy (DkIv)
y24po) to exhibit a gray-black color. No coating of viscous material was observed on the Enodia spore mass. The taxonomic position of this bacterium with the above mycological properties can be found in the search guide “The Genera of
High Omycetes From Soil (The Gener
a ○f Hyhomyce ni s From Soi
l, The Wamms & Wilkins
Company Baltimore, G. L. Ban
on, 1968) and “A Manual of
A MANUAL OF SO
ratio FUNGI, The, lowa SPte Uni
venetity press AmeslowaUSA,
J. 〇Oilman, 1971) and ``The Genera of Funjia Sporola.''
ting mPure Cultme, VERAGV○
N.J. CREM old R3301LEHRE, J. A. V
ONARX, 1970) etc., this bacterium was found to be according to Saccardo's classification system.
・It belongs to the genus Phiphomycetes Demachachaeae. In other words, the characteristics of this strain include the fact that it does not have asci or other sexual reproductive organs, produces dark brown sycamore spores from the sycamore, and the produced sycamore spores gather in a half-moon shape at the apex of the sycamore. The characteristics match those of the genus Stachybotrys. The properties of this bacterium are described in the above handbook and by G. Bisbee. R (1943): Tras.
Brit. Mycol. soc. , 26:133-14
3 Zutsuk R. K (1964): Mycologa, 3
8369176, Baron ○. L (1961), Can.
J. B. t. , 39:153-157 and the IFO archived standard strain, it was found that the pulvinar spores of this strain K-76 have a rough surface and wart-like protrusions, and are oval to oval in shape. At the point, Stachybotryslobrata (Snechyb
othis lobula (IF05369) was determined to be the most similar. However, in Stachybotrys lobularis, the pulvinar reaches as far as one side, and in contrast to K-7 mother vermilion, which forms simple branches, Stachybotrys robulae has the property that the pulvinar extends further from one Enoki plant. The points are different. Growth on various media was compared to K-7 mother vermilion, which produced a large amount of diffusible pigment and sporulation was extremely poor on other agar media except potato glycol agar and oatmeal agar. Stachybotrys lopula grows extremely quickly on various media and forms flat colonies without any curvature. In addition, spore formation is so good that it represents the color of the bacterial flora in most media except for Abek Agar Toji, and no diffusible pigment is produced, or even if it is produced, it is very weak. Based on the above differences, this strain K-78 is judged to be a new species different from Stachybotrys lobrata. 2 Stachybotrys sp. T-789 Stachybotrys sp. Although there is a slight size difference with K-76, it is morphologically identical. The above mycological properties were determined by referring to the search guide and literature in the same way as the Stachybotrys sp.
Lobrata m05369 and Stachybotrys sp. Most similar to K-76. This strain is found to form good pulvinar spores on Abek's agar medium, but the former two strains have a cultural difference in that almost no pulvinar spores are formed on Abek's agar medium. Also, the aforementioned Stachybotrys sp. As mentioned in the section on K-7 mother vermilion, there is a clear difference in morphological characteristics from Stachybotrys robulata, and the growth range is pH 4.
．． 5 to 10.5, this strain has a K
-76 is different. Therefore, for the above reasons, this bacterial strain is recognized as a new bacterial species. 3. Stachybotrys sp. T-1791 Enoki stalk is simple and erect without branching. It swells slightly into a club shape at its tip. However, the standard strain Stachybotrys cechinata flower 07525
There is no significant bulk observed in and 8856. The mycelium shows a width of 4.0 to 4.5 mm, which is slightly larger than the Enoko stalk. The pedicel is branched upright from vegetative hyphae or aerial hyphae with podocytes and has 2-3 septa, 40-80 x 3.0-3
．． It exhibits a size of 5〃. No thorn-like protrusions are observed on the cell wall of Enoko stalk, which is smooth. 3 from the apical ampulla of Enoki stalk
Produces ~6 sycamores, and further has 4.3 ~ basilically attached to the tip
Continuously produces spherical to subglobular inchospores with the size of 5.2 x 3.0 to 4.2 Buddhas and 1 cell with spiny projections, 24
Forms ~7 solid spore chains. Enoki has a sake bottle-shaped shape and measures 6.9 to 10.7×
3.5-4.7 Buddha. The ridge stalk and stalks are colorless. Enoki has a color of coffee (Coffee, 3pn) to black. According to the taxonomic position of this bacterium having the above-mentioned mycological properties, the bacterium belongs to the genus Stachybotrys (genus Menmonella) when searched using the search guide given in the section of K-78. That is, it does not have asci or other sexual reproductive organs, and dark brown Enodia spores are continuously produced from the stalk, and the produced spores are the longest chain. The characteristics of the present bacterial strain T-791 are consistent with those of the genus Stachybotrys (genus Menmonella).
The various properties of this bacterium can be found in the above-mentioned search guide and by R.D. K. (
1964, Myc. logia, 38:69-76) and Smith. G(1
962, Trans. Brit. My col. soc.
45:387-394) as well as m○ preservation standard method strains, the property of this strain T-791 to continuously produce basophores from the sycamores is similar to Horner's (H
The genus Menmoniella was named by
a). However, based on the report by Zutsuk and Smith mentioned above, this strain T-
Strain 791 is considered to be a bacterial species related to Stachybotrys cechinata, so Stachybotrys echinata IF0752
A comparative study was conducted with two strains, 5 and 8856. Morphologically, in the cell wall of this strain T-791, the spiny protrusions that are specific to both standard strains are not observed in the cell wall of the bulonia, and in addition, the tip of the pedicle is 2 to 3 times the width of the hyphae of the pedicle in the standard strain. In contrast, this strain does not show any significant expansion. In addition, the two standard strains showed good growth on soil, especially on potato dextrose soil, forming round, slightly raised colonies, with abundant mycelia, and spore formation. In contrast, this strain shows irregular tree-like growth as described above, and has poor hyphal formation and spore formation. Based on the differences in mycological properties mentioned above, this strain is recognized as a new bacterial species.
The display description of colors can be found in the “Color Harmony Manual (
Coilor Harmony Man Female 1, 1958, Container Corporation of Am
erica)”. The above-mentioned new bacterial species Stachybotrys sp. K-70
Stachybotrys sp. T-789 and Stachybotrys sp. T-791
The microbial accession number is 3801, which is assigned to the Microbiological Research Institute.
No. (FERM-P No. 3801), Microtechnical Laboratory No. 3
No. 802 (FERM-P No. 3802), and FERM-P No. 3803 (FERM-P No. 3803)
It was entrusted as a. The microorganism belonging to the genus Stachybotrys is cultured in a medium containing conventional nutrients and additives. As the culture medium, commonly used nitrogen sources such as soybean flour, soybean oil, cornstarch liquor, yeast extract, dried yeast, oatmeal, meat extract, casein hydrolyzate, ammonium salts, nitrates, etc. and carbonate sources such as glucose, glycerin, Maltose, starch, lactose, sucrose, molasses, etc. can be used. In addition, as additives added to the culture medium, for example, inorganic salts such as calcium carbonate, sodium chloride, magnesium sulfate, and phosphoric acid can be used, and metals such as iron, copper, manganese, and zinc may be added to the soil as necessary. A trace amount of salt can be included. Cultivation can be carried out in a conventional aqueous medium containing the above-mentioned culture medium by either surface culture or deep aerated light bed culture, but deep aeration light bed culture is preferably carried out. The culture conditions are normal aeration conditions, and the liquid pH is 3.
．． 5 to 11.5 preferably PH4.5 to 9.5 and culture temperature and 15 to 35oo preferably 20 to 3〆0 and usually 3
It can be advantageously cultured for ~7 days. Isolation and purification of compound [1] from the culture product produced by the above-mentioned culture can be carried out according to various conventional methods that utilize the physical and chemical properties of compound [1]. For example, methods that utilize differences in solubility with impurities, differences in adsorption affinity for ordinary adsorbents such as activated carbon, XAD-2, silica gel, ion exchange resins, Cephadex, etc., differences in distribution ratio between two liquid phases, etc. It can be carried out by a combination of these methods. More specifically, the culture solution is subjected to oven washing or centrifugation according to a conventional method to remove bacterial cells, then methanol is added to the supernatant solution, left for 2 to 3 hours after drying, and the precipitate is removed by centrifugation again. After extraction with the same amount of ethyl acetate, the solvent was removed, the extract was poured into methanol, and the methanol solution was passed through an activated carbon column. Compound [1] can be isolated and purified by subjecting each fraction obtained by gel incubation at -20° C. to the anti-complement activity test described below, collecting the active portion, and distilling off the solvent. The compound thus obtained [1
The physical and chemical properties of ] are as follows. [1] It is a weakly acidic substance with pale yellow appearance and properties. (2) Soluble methanol Soluble in ethanol, acetone, ethyl acetate, dimethyl sulfoxide, pyridine, acetic acid, ether, and chloroform, slightly soluble in benzene, and immovable in water, hexane, and petroleum ether. {3} Composition The elemental constituent elements are carbon, hydrogen, and oxygen, and do not include nitrogen, sulfur, halogen, etc. '4} Elemental analysis value C: 68.58% H: 7.55% Calculation as C23rd 3o06 Value C: 68.64% H: 7.51% (5- Infrared absorption spectrum (IR) Figure 1 shows the IR analysis diagram of the KBr tablet. The main absorption peaks are as follows: 3400 (s) ), 2
920(m), 1670(s), 1610(s), 15
85(s), 1430(m), 1390(s), 131
0(s), 1240(s), 1195(m), 1115
(m), 1040 (m), 1005 (m), 990 (m)
), 960 (m), 940 (m), 880 (m), 81
5(w), 765(W), 550(W), 515(W)
Kay-1 Tsukuda Ultraviolet absorption spectrum (U.V) 9/64 with ethanol (EtOH) as solvent and sample concentration of 11.9
0 on EtOH, cell length 1c so U. The V analysis is shown in Figure 2. From FIG. 2, it can be seen that maximum absorption is observed at the following wavelengths. 246n skin (Doni 16474) 307n skin (Do = 6659) 35 enemy skin (Doni 5427) [7] Molecular weight 402 (by mass spectrometer) [8} Molecular formula C23 day 3006 '9} TLC board with the following working terms Color reaction 204 above
- Dinitrophenylhydrazine reagent positive/positive phosphomolyptene reagent reaction Positive Tollens reagent reaction
Enteric ferric chloride reaction
Negative {1 Cancer layer chromatography (TLC)
) As a result of TLC analysis using "Silica Gel 6 and 2M" manufactured by Merck, the Rf value is as follows. {i- Ethyl acetate-chloroform-acetic acid (volume ratio 50
:50:2) Rf value = 0.34'ii} Benzene-butanol-acetic acid (volume ratio 60:15:5) Rf value = 0.
58-valent benzene-ethyl acetate-acetic acid (volume ratio 40:5
0:2) Rf value=0.42 (11) Nuclear Magnetic Resonance Spectrum (NMR) An NMR analysis chart measured using pyridine solid state (DSS) as a solvent is shown in FIG. The measurement conditions are as follows. spectral amplitude
4x100 filter
0.1SeCRF output
0.05 skin G sweep time
5mjn sweeping
IQ blood sweep ending
OP style From the above various physicochemical properties and the physicochemical properties of various compounds derived from compound [1], compound [
The results of analyzing the structural formula of 1] are as follows. That is, compound [1] was positive in the 2,4-dinitrophenylhydrazine reagent, phosphomolybdenum reagent reaction, and Tollens reagent reaction, and the NMR spectrum (Figure 3) showed that 6 pyridine = 10.75, 11.02 legs ( For each IH, s) -
Signals of CHO and a Q.6-unsaturated carbonyl band at 1670ka-1 were observed in the IR spectrum (Fig. 1), and the presence of a conjugated aldehyde group was confirmed from these findings. The 2,4-dinitrophenyl hydrazone [la] of compound [1] has a UV result of 395 nm (466 nm).
70) and the NMR result 61MSo-d6=7.
9-9. It can be seen that two molecules of 2,4-dinitrophenylhydrazine are bonded to one molecule of compound [1] from the $ style (spots) and 11.8Qp skin (s spots). The NMR spectrum of compound [1] (Figure 3) showed a songlet signal of 6pyridine-d5 = 7.41 IH, and the tetrahydroform [l] of the oxide [1].
b] From the 13C-NMR results (Fig. 4), 60MSo
-d6=106. Fu 110.3, 110. &141.
8152.1, 160.3 There is a signal of carbon of one benzene ring in the blood, and these show that compound [1] has one pentasubstituted benzene nucleus. Note that FIG. 4 above was measured under the following conditions. Pulse No. 4000° Vless angle 45o Pulse location CS2 Proton DEC. Interval without wide filter Book eC. Rock solv. Sample period 0. In addition, the benzene nucleus has three low-field signals (141.8, 152.1, and 16
0.3 ppm) and three high-field signals (106.5
, 110.3 and 110. The plow m) is shown. The signal with the highest magnetic field is estimated to be tertiary carbon based on the incomplete proton irradiation method. Furthermore, among the carbons that normally constitute the benzene nucleus, carbons that directly bond to oxygen (carbons with oxygen functional groups) are shifted to a lower magnetic field, resulting in 15
The two signals at 2.1 and 160.2 pm are considered to be carbon with an oxygen functional group. Compound [1] NaB
The specific reductant [lc] was negative for 2,4-dinitrophenylhydrazine reagent and phosphomolybdenum reagent, IR spectrum showed no absorption of carbonyl, and NMR showed 6oD3oD=4.62, 4.68 background' Two methylene protons of CH20 bonded to the benzene ring were observed on each day, confirming that compound [1] has two -CHO groups on the benzene ring. The diazomethane methyl ether form [ld] of the reduced form [lc] of NaB day 4 above [ld]
] is the NMR result 6C. 3ooe 3.83 Cape shows a dark blue singlet signal (○-CH3 bonded to the benzene ring). As a result, compound [1] has one -OH on the benzene ring.
It can be seen that it has a group. In addition, the above-mentioned toxylated compound has the molecular peak M/e=
420.2514, and from this result and the elemental analysis results, [ld] is found to have a molecular formula of C24day3606. Also, from the above molecular formula, compound [1] has the molecular formula C23
It is identified as having a solar arc of 06 and a molecular weight of 402. The oxidant [le] obtained by oxidizing compound [1] with A&○ is a positive and
The result of IR spectrum analysis is 1750 skin-1, and the result of NMR spectrum analysis is 8Co3oo
= 6.45, 6.53 skin, and by showing characteristic signals in each IH, it was confirmed that it had a 5-membered lactol structure. The formation of the lactol compound [le] confirms that the two aldehyde groups of compound [1] are present adjacent to each other on the benzene ring. Combining the above results, it can be seen that compound [1] has the following partial structural formula [1-1]. Compound [1] has a molecular formula of C23day3oOG, and in addition to the above partial structural formula (C8 ratio 04) part, it has C, 5days ratio 02 part. This portion is determined to be sesquiterbene based on the NMR spectra of the compound [1] and its various derivatives. More specifically, as a result of NMR spectrum analysis, compound [1] has no double bonds other than the benzene ring, and its degree of unsaturation is 9 from the molecular formula. It can be seen that it has saturated rings. The compound obtained by acetylating the ring element [lc] of compound [1] by a conventional method has the following NMR spectrum analysis results:
CoC13 Sansha = 2.05, 2.09, 2.16, 2.
32 legs (each s): Approximately 5.3 legs (group); 3.49 legs (
IH, d, J = 3HZ), and as a result of IR spectrum analysis, tetraacetate [lf] shows an absorption peak of hydroxyl group at fin turf = 3520 ka-1 and NMR.
Spectrum analysis result 6Cool3=1.97, 2.1
1, 2.12, 2.1fu 2.3 rope plate (each $chi, s):
It showed a signal of about 5.2 legs (dark blue), and IR spectrum analysis confirmed that it was a pentaacetate product [1 g] that did not show an absorption spectrum of hydroxyl groups. From this fact and the fact that the above-mentioned reduced form [lc] has three hydroxyl groups on the benzene ring as mentioned above, the compound [1]
It can be seen that the compound has two hydroxyl groups in a part other than the [1-1] part, that is, in the sesquibene part shown by the C,5 ratio of 602. As a result of IR spectrum analysis, the monoketone [lh] obtained by Jones oxidation of the above tetraacetate [lf] was found to be F1730, 1750, 1770cm-
The absorption peaks of keto group and ester group were observed at 1, and the peak specific to hydroxyl group at 3520 cm-1 disappeared. Also, the [lh] is 6CoC as a result of NMR spectrum analysis.
13 = 5.63 legs with IH (d, d), J = 8.8, 11
．． The signal of 5HZ (=CH-○-Ac) is shown. The signal is a tetraacetate [lf]6ooC13=5
．． 2. It is observed near the skin that it overlaps with the signal of CH2-CAc that binds to the benzene ring. From the above, it can be seen that the two hydroxyl groups present in the C, 5, 2502 portion are adjacent to each other. In addition, adjacent to this hydroxyl group, the isopropylidene body [li] obtained by treating the above-mentioned reduced body [lc] with dimethoxypropane was found to be 6 pyridine carriers: 1.39, 1.46 ppm (each range) as a result of NMR spectrum analysis. This is also confirmed by the fact that it shows a methyl proton signal. Furthermore, since the signal of the above 8=5.63 blood is dd, it can be seen that a methylene group is adjacent to the =CH-0-Ac group. Furthermore, tetraacetate
As a result of NMR spectrum analysis of the isomer [lf], the proton at the base of the hydroxyl group, which is difficult to be acetylated, is 3.
49 skin exists, and NM of the above monoketone body [lh]
In the R spectrum, only 6=5.63 signals shifted down the magnetic field due to Jones oxidation are observed, indicating that the hydroxyl group is adjacent to a quaternary carbon. Combining the above, C of compound [1], 5 days 0
The sesquiterbene moiety represented by 2 has the following partial structural formula [
1-2]. Furthermore, as a result of NMR spectrum analysis, compound [1]
3 primary methyl groups, 1 secondary methyl group, and 1 methylene sandwiched between quaternary carbons (6 pyridine-d5 = 3.27
It can be seen that the chest has dd). Based on the above, when the above partial structural formulas [1-2] are applied to the skeleton of known sesquiterbenes, Durimane (d
Only the rimane) skeleton satisfied all. Compound [1] has 6 oxygen atoms in the molecule,
From the above, five items were assigned, but the other one cannot be assigned. However, this oxygen atom is recognized to form an enal ring. 6,7-dihydroxy-2,5,5-pine-tetramethyl-1,2,314, represented by structural formula [1]
Umbrella・5・617・8・Shunichi Decahydronaphthalene 1-
It is recognized to be spiro-2'-(6,7-diformyl-4-hydroxy-213-dihydrobenzofuran). Compound [1] obtained above is characterized by having anti-complement activity as described below. This anti-complement activity is shown in “Immunochemistry J, pp. 830-834 (
It is measured and confirmed according to the test method described in the book published by Asakura Shoten, edited by Yuichi Yamamura, 1972). That is, the compound [
1] Aqueous dispersion of 0.5 [, 1 x 1 picel/I sensitized blood cells (EA) 0.5 Sakaki, 5-fold dilution of Veronal buffered saline (GVBH) containing isotonic gelatin 1 Complement serum (g.p.c.) diluted with 15 needles of I and GVB 10 dilutions (g.p.c.) 0
．． After incubating at 37°C for 60 minutes, ice-cold physiological saline was added and centrifuged, and the absorbance of the separated supernatant was measured using an absorbance meter ○D4,3. [1
] is measured by determining the extent to which hemolysis of sensitized blood cells is suppressed. The absorbance and anti-complement activity values measured according to the above method are shown in Table 1 below. Table 1 The blank in Table 1 above is a sample in which only GVB++ and EA were collected without collecting compound [1], and the control* is a sample in which only GVB++ and EA were collected, and the control* is EAO. This is a sample showing 100% hemolysis, taken with 5 ml of water and 2.0 ml of water. And the anti-complement activity value is calculated from sample N from which compound [1] was collected.
O. Blank from the OD side value of 1 (trial flow No. 2 and 3)
The value (OD') obtained by subtracting the OD side average value of
Sample No. 4 and 5), it is obtained by dividing the value (OD') obtained by subtracting the OD4,3 average value of the blank from the OD4,3 average value, that is, the melting plate ratio (y). Therefore, if the port blood ratio (y) is less than 1, it can be considered to have anti-complement activity. It can be said that the smaller the value of the plate ratio (y), the higher the anti-complement activity, and Compound [1] was found to have superior anti-complement activity based on the results of this test. The sesquiterbene analogs used as the active ingredients of the anticancer agent of the present invention are:
It is produced by oxidizing the above compound [1]. The oxidation reaction can be carried out according to a conventional method for obtaining aromatic carboxylic acid from an aromatic aldehyde compound. Such oxidation methods include a method using an oxidizing agent, a non-catalytic light irradiation method, a catalytic oxidation method using air or oxygen in the presence of a catalyst, an electrolytic oxidation method in the presence of a copper compound or sulfuric acid, an oxidation method using oxygen, etc. However, a method using an oxidizing agent and a catalytic oxidation method using air or oxygen are advantageous, especially considering the ease of operation, ease of separation and purification of reaction products, yield, etc. When carrying out the method using an oxidizing agent,
The oxidizing agent is not particularly limited, and both ordinary inorganic and organic oxidizing agents can be used. Representative oxidizing agents are illustrated below. (i) Inorganic oxidizing agents permanganate, manganese oxide, manganese pyrophosphate,
Chromic acid, chromate, silver oxide, nitric acid wire, Tollens reagent, gold oxide, nickel peroxide, selenium dioxide, chlorine, bromine, iodine, hypozinc acid, hypobromous acid, perchloric acid, periodic acid, Salts of the above-mentioned hypohalous acids and perhalogen acids, cobalt salts such as nitric acid, nitric acid, nitrogen tetroxide, nitrogen dioxide, cobalt sulfate, cobalt acetate, cerium sulfate, cerium oxide, cerium perchlorate, etc. Cerium salts, hydrogen peroxide, Izon, etc. Rigid Organic oxidizing agent N-bromosuccinoimide, N-chlorosuccinoimide, sodium-N-chloro-p-toluenesulfonamide, sodium-N-chlorobenzenesulfonamide,
Azo compounds such as ethyl azocarboxylate and 4-phenyl-1,2,4-triazoline-3,5-dione; Carboxylic acids, etc. Among these oxidizing agents, permanganate, silver oxide, hydrogen peroxide, chromic acid, peracetic acid, perbenzoic acid, etc. are preferred, and permanganate and silver oxide are particularly optimal. The oxidation method using the above-mentioned oxidizing agent is usually preferably carried out in a suitable solvent. Examples of solvents include water, alcohols such as methanol and ethanol, ethers such as pyridine, dioxane, tetrahydrofuran and ether, ketones such as acetone and methyl ethyl ketone, carboxylic acids such as acetic acid and propionic acid,
Esters such as ethyl acetate, aromatic hydrocarbons such as benzene and chlorobenzene, halogenated hydrocarbons such as dichloromethane and dichloroethane, and water-containing or anhydrous organic solvents such as hexamethylphosphoamide, dimethylformamide, and dimethyl sulfoxide can be used. . The amount of the oxidizing agent to be used is 1 to 10 equivalents, preferably 1 to 2 equivalents, based on the compound [1] used as the starting material, and the reaction is -10 to 1 equivalent.
00oo, preferably 0 to 5000, for about 30 minutes to 2 hours. In addition, when carrying out the catalytic oxidation method using air or oxygen, oxygen or air is blown into an aqueous solution of sodium hydroxide or potassium hydroxide without a catalyst, or an inorganic material such as manganese acetate, cobalt acetate, cobalt nitrate, Examples include catalytic oxidation in the presence of a salt catalyst, in the presence of a radical generator such as penzoyl peroxide, or under light irradiation. Among the above methods, a catalytic autooxidation method in which air or oxygen is blown into an alkaline aqueous solution without a catalyst can be particularly advantageously carried out. In this case, the reaction is usually room temperature to 10,000, preferably 40 to 5
The process is completed by heating the lamp for about 30 minutes to 2 hours at a temperature of 0.000C. After the above-mentioned oxidation reaction is completed, for example, if an oxidizing agent is used, inorganic substances are removed by decomposition with a reducing agent, filtration, neutralization, vacuum distillation, etc., and in the case of a method of blowing air, etc. is preferably treated with activated carbon, then acidified with hydrochloric acid to precipitate crystals, or extracted with an organic solvent such as ethyl acetate, and then subjected to a conventional separation and purification method such as column chromatography or fractional crystallization to obtain the desired product. Sesquiterbene analogs can be obtained in pure form. The physicochemical properties of the sesquiterbene analog (crystal) thus obtained are shown below. '1} Molecular formula C 23 days 3007 ■ Molecular weight 418 (by mass spectrometer) {3} Melting point Dissolves with color decomposition from around 325 do and does not show a clear melting point. (4) Solubility {i} Insoluble in water and hexane, easily soluble in methanol, ethanol, acetone, chloroform, and ethyl acetate. (ii} Soluble in aqueous sodium hydroxide and potassium hydroxide solutions. ■ Composition There are three elements: C, day, and ○, and N, S, and halogen are not included. [6} Elemental analysis value C: 65.98% H: 7.21% C23 day 3. Calculated value C as 07 : 66.03% H: 7.18% [7} Infrared absorption spectrum (IR) The IR analysis diagram of the KBr tablet is as shown in Figure 6, and shows the following characteristic peaks: 3430 (s), 2975(s), 2950(m), 2
870(s), 1750(s) "1730(shm)
, 1698(m), 1675(shm), 1668(
m), 1640 (sh.s), 1630 (s), 162
0 (sh.s), 1610 (sh.s), 1590 (s.
), 1565 (shm), 1550 (shm), 1
500 (shm), 1480 (shm), 1470
(s), 1460 (sh.s), 1450 (s), 14
30 (sh.m), 1410 (sh.s), 1395 (
s), 1380 (shm), 1370 (shm),
1355(s), 1335(s), 1332(s), 1
305 (sh.s), 1295 (sh.s), 1285
(shm), 1265(s), 1255(s), 12
45 (s), 1210 (m), 1195 (shm),
1185 (m), 1175 (shm), 1165 (s.
h. m), 1160 (s.m.), 1130 (s.m.
), 1098(s), 1045(m), 1035(sh
．． m), 1010 (m), 995 (m), 985 (m)
, 970 (m), 950 (shm), 940 (m),
910 (m), 900 (shm), 890 (m), 8
70 (m), 860 (s.m.), 850 (s.w.)
, 840 (sh.w), 830 (m), 820 (sh.
w), 810 (w), 800 (w), 775 (m), 7
65 (shm), 740 (m), 730 (m), 72
0 (m), 710 (m), 670 (m), (where s is strong absorption, m is medium absorption, w is weak absorption, and sh.
means shoulder) ■ Nuclear magnetic resonance spectrum (
NMR)'i} NM in CD30D (DSS) solution
The R analysis diagram is shown in FIG. {ii} NMR in pyridine-d6 (DSS) solution
An analysis diagram is shown in Figure 8. {iii) Dissolved in dimethyl sulfoxide-d6 solution 2
FIG. 9 shows NMR analysis diagrams after 6 hours and after 6 hours. (The NMR analysis figures after 2 hours and 63 hours are completely consistent.) The measurement conditions in each figure are as follows. Fig. 7 Fig. 8 Fig. 9 Spectrum amplifier 9XIO0 5×100 8×
100 titude filter 0.1sec O.
1sec O. ISeCRF output 0.05m
G.O. 05mG O. 05mG swift collar
5 minutes 5 minutes 5 minute sweep in progress lopp
m loppm loppm swipe end 0
0 0 [9} Thin layer chromatography (T
LC) TL using Merck's "Silica gel hexavalent 2 string"
As a result of C analysis, the Rf values were as follows. River Ethyl acetate-chloroform-acetic acid (volume ratio 50:5
0:2) Rf value = 0.37 valence Benzene-butanol-acetic acid (volume ratio 60:15:5) Rf value = 0.71 side Star color reaction 'i} The color reaction on the TLC slope above is as follows. That's right. Positive for 02,4-dinitrophenylhydrazine reagent]
Positive for phosphomolybdenum reagent reaction, negative for ferric chloride reaction,

[ii} The star color reaction in a solution dissolved in methanol is as follows. Positive for 02,4-dinitrophenylhydrazine reagent,
o Positive for ferric chloride reaction. From the above physical and chemical properties, sesquiterbene analogs (crystals) have the following formula [n ] 4,6-dihydroxy-8-oxo-2,3,6,8-tetrahydrofuro [3.41 g] Penzofuran-2-
Spiro 1'1 (6, 7-dihydroxy 2, 5, 5,
Shi'-tetramethyl-1', 2, 3, 4', umbrella', 6,
6, 7, 8, decahydronaphthalene). In addition, the sesquiterbene analog represented by the above formula [D] (hereinafter simply referred to as compound [0]), in a solvent, particularly in a basic solvent, is a carboxylic acid represented by the following formula [B]', which is its tautomer. exists as an equilibrium mixture with Tautomer (hereinafter simply compound [
The existence of compound [0] is clear from the results of NMR spectrum analysis of compound [0]. That is, according to NMR spectrum analysis that was performed over time after dissolving compound [2] in dimethyl sulfoxide solvent, a characteristic signal of lactol was observed 20 minutes after dissolution.6.
In addition to the 3-losing side, a slight peak was observed at 9.89 legs, which is a characteristic signal of aldehyde proton (one CHO).
The integral ratio of the two was approximately 73 for the former and approximately 27 for the latter, and after 2 hours, as is clear from Figure 9, the similar peak of the 9.8 $ example increased slightly, reaching an integral ratio of 70:30. . This integral ratio did not change even in the NMR analysis chart after 63 hours. This shows that compound [0]' was produced in the above solution at a ratio of 3:7 to compound [0]. In addition, when methanol and pyridine were used as solvents, as is clear from the NMR spectrum analysis diagrams shown in Figures 7 and 8, respectively, the formation of the tautomer [0]' of the above compound [B] was not observed. Nakatsuta. The anticancer agent of the present invention also includes a basic compound salt of the compound [B] as well as the sesquiterbene analog (compound [B]) represented by the above formula [D] as an active ingredient. The salt of the above-mentioned compound [0] includes a salt in which one or both of the acidic group, ie, the phenolic hydroxyl group and the carboxyl group constituting the lactol ring of the compound [0], reacts with a basic compound. Specifically, the basic compounds used in the production of the above salts include alkali metals or alkali metals such as sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, potassium carbonate, and sodium hydrogen carbonate. Examples include hydroxides and carbonates. Also, for example, methylamine, ethylamine, isopropylamine, morpholine, piperazine, piperidine, 3.4
Organic amines such as -dimethoxyphenethylamine can also be used as the basic compound. The salt-forming reaction of compound [0] with the above-mentioned basic compound can be easily carried out, for example, in a suitable solvent according to a conventional salt-forming reaction. Examples of the solvent used include water, lower alcohols such as methanol, ethanol, and propanol, ethers such as dioxane and tetrahydrofuran, acetone, benzene, ethyl acetate, dimethyl sulfoxide, dimethyl formamide, methylene chloride, and chloroform.
The above reaction is usually carried out at room temperature or under an oxygen-free condition, preferably under an inert gas atmosphere such as nitrogen or argon.
The reaction is carried out at a temperature of 100° C., preferably room temperature to 5,000° C., and the reaction is usually completed in about 5 minutes to 68°C. The amount of the basic compound [B] to be used is not particularly limited, but it is usually an equimole to excess amount, preferably an equimolar to 2 molar amount relative to the compound [B].
It is preferable to use about twice the molar amount. In this way, the desired salt of compound m] can be obtained. The physical and chemical properties of one of the obtained salts are shown below. {1}
Molecular formula C23day2807Na2 (2) Molecular weight 462'3} Solubility (i) Soluble in water. {ii] Soluble in acetone, chloroform, ethyl acetate, and benzene. {4) Consists of constituent elements C, 0, and Na, with no N, S, or halogens. {5) Elemental analysis C: 59.48% H: 5.91% Calculated value as C23 day 2807Na2 C: 59.74% H: 6.10% ■ Infrared absorption spectrum (IR) IR analysis diagram as KBr tablet is shown in Figure 10. The main absorption peaks are as follows. 3400(s), 3280
(sh.s), 2940 (m), 2890 (shm)
, 1750(w), 1720(sh.w), 1705(
sh. w), 1685 (sh.m), 1672 (sh.
m), 1665 (shm), 1655 (shm),
1640 (s), 1575 (sh.s), 1570 (s.
h. s), 1555(s), 1540(s), 1535
(sh.s), 1522 (sh.s), 1517 (sh.s.
．． s), 1490 (sh.m), 1565<Sh-m)
, 1460 (Sh-m>, 1438 (Sh-s), 14
20 (sh.s.), 1400 (s.), 1395 (sh.s.
s), 1365 (shm), 1320 (shm),
1305 (m), 1250 (m), 1215 (sh.w
), 1182 (w), 1165 (sh.w), 1115
(m), 1095 (m), 1046 (m), 1010 (
m), 995 (sh.w), 985 (sh.w), 96
0 (w), 945 (sh.w), 930 (sh.w),
870(w), 840(w), 768(m), 660(
sh. w), 630 (m), 600 (shm), 55
0 (m), 515 (m), 470 (sh.w), (In the above, s indicates strong absorption, m indicates medium absorption, w indicates weak absorption, and sh. indicates shoulder.) [7' Ultraviolet rays Absorption spectrum (UV) sample concentration 6.1
Figure 11 shows a UV analysis diagram of an aqueous solution with a cell length of 1 x 10-6 mol/day. The maximum absorption wavelength is as follows. 25 Shahada (Go: 20
500) 33 Blood (go=49500) [81 Nuclear Magnetic Resonance Spectrum D20 (DSS) NMR analysis diagram in a solution is shown in FIG. The figure was measured under the following conditions. Spectrum amplifier 9x100 filter
0.1SecRF output
0.05mG sweep time
5 minute sweep in progress
IQ ■Swape End 0 [9
} It is positive for the color reaction 2,4-dinitrophenylhydrazine reagent reaction. The salt obtained from the above results has the following structural formula [m]. 8
・Jiichi decahydronaphthalene-1-spiro 2'-(7
'-carboxylate-6'-formyl-4'-oxido-2'-3-dihydrobenzofuran). Compound used as an active ingredient of the anticancer agent of the present invention

[0] and its salts have stereoisomers, and these stereoisomers can also be used as active ingredients. The anticancer agent of the present invention containing the above compound [D] and its salt as an active ingredient is usually in the form of a pharmaceutical composition containing the active ingredient together with a pharmaceutical carrier. As carriers, fillers, extenders, binders, wetting agents, disintegrants,
Examples include diluents such as surfactants and lubricants, and excipients, which can be appropriately selected depending on the dosage unit form of the anticancer agent of the present invention. The dosage unit form of the anticancer agent of the present invention can be appropriately selected depending on the therapeutic purpose and the like. Typical examples include tablets, pills, powders, solutions, suspensions, emulsions, granules, capsules, suppositories, and injections (solutions, suspensions, etc.). When forming into a tablet, a wide variety of carriers conventionally known in this field can be used. For example, excipients such as lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid, water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, Binders such as oxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinylpyrrolidone, dried starch, sodium alginate, agar powder, laminaria powder, sodium bicarbonate, calcium carbonate, Win, sodium lauryl sulfate, stearic acid monoglyceride, Disintegrants such as starch and lactose, disintegration inhibitors such as sucrose, stearin, cocoa butter, and hydrogenated oils, absorption enhancers such as quaternary ammonium bases and sodium lauryl sulfate, warming agents such as glycerin and starch, starch, and lactose. , kaolin, bentonite,
Examples include adsorbents such as colloidal silicic acid, purified talc, stearates, boric acid powder, macrogol, and lubricants such as solid polyethylene glycol. When forming into a pill form, a wide variety of carriers conventionally known in this field can be used. Examples include excipients such as glucose, lactose, starch, cacao butter, hydrogenated vegetable oil, kaolin and talc, binders such as gum arabic powder, tragacanth powder, gelatin and ethanol, and disintegrants such as laminaria and agar. Furthermore, the tablets can be made into conventionally coated tablets, such as sugar-coated tablets, gelatin tablets, enteric-coated tablets, film-coated tablets, double tablets, or multilayer tablets, if necessary. When forming into a suppository, a wide variety of known carriers can be used. Examples include polyethylene glycol, cacao butter, higher alcohols, esters of higher alcohols, gelatin, and semisynthetic glycerides. When prepared as injectables, the resulting solutions and suspensions are preferably sterile and isotonic with blood. In forming these solutions and suspensions, any diluent commonly used in this field can be used. Examples include water, ethyl alcohol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitol, sorbitan ester, and the like. In this case, the anticancer agent of the present invention in the form of an injection may contain a sufficient amount of common salt, glucose, or glycerin to prepare an isotonic solution. In addition, the anticancer agent of the present invention includes conventional solubilizing agents, buffering agents,
It may contain a habit-free agent, a preservative, etc., and if necessary, a coloring agent, a preservative, a perfume, a flavoring agent, a sweetener, etc., and other pharmaceuticals. The amount of the active ingredient to be contained in the anticancer agent of the present invention is not particularly limited and can be appropriately selected within a wide range, but is usually 1 to 7% by weight, preferably 5 to 7% by weight based on the total composition.
The content is preferably 50% by weight. The anticancer agent of the present invention can be administered in various ways depending on the form. For example, tablets, pills, solutions, suspensions, emulsions, granules and capsules are administered orally. In the case of an injection, it is administered intravenously alone or mixed with a normal replacement fluid such as glucose or amino acids, and furthermore, if necessary, it is administered alone intramuscularly, subcutaneously, or intraperitoneally. Suppositories are administered rectally. The dosage of the antigasotropic agent of the present invention is appropriately determined depending on the purpose of use, symptoms, etc., but it is usually preferable to use the active ingredient in the range of about 50 9 to 5 ta per person per day. Preferably, the dose is administered in ~4 divided doses. The results of testing the anticancer activity and toxicity of the compound [m], which is the active ingredient of the anticancer agent of the present invention, are shown below. 1 Anticancer effect ICR/JCL male mouse (6 weeks old, weight 30±1
In the evening), 1 × 1 autologous injured cells of Sarcoma 180 grown in the ascites of mice/Pan 9 were inoculated into the lower skin of the left inguinal isthmus of 0.1 mice per mouse, and from 24 hours after inoculation, once a day. Compounds [B] 5-9, 17-claw 9, 50m9 and 150-9 were intraperitoneally administered to groups of 8 mice, respectively, for one week. In addition, a group to which the above compound [m was not administered was used as a control group. Thirty days after the inoculation of tumor cells, cancer cells were excised from each test animal, their weight was measured, and the tumor growth inhibition rate was determined according to the formula below. Taneba member exposure IE rate (%) The obtained results are shown in Table 2 below. The average tumor cell weight and average body weight in the table are the average values of 8 test animals in each group.
The measurements were taken after 1 day. Table 2 From Table 2 above, it can be seen that compound [B] has an extremely excellent anticancer effect, and that this effect is significantly manifested at a dose of only 5 to 9. 2 Acutely toxic compounds

The acute toxicity test of [0] was carried out in 1. This was done on V rats. As a result, the compound

[0] LD5. The value is 9 of 500
/k9. This value is considerably different from the above-mentioned effective dose for inhibiting tumor growth, indicating that the anticancer agent of the present invention is excellent. In order to further clarify the present invention, the production example of compound [1] will be used as a reference example, and the compound

Examples of the production of [0] and its salts and examples of the anticancer agent of the present invention are listed below. Reference example 1 Stachybotrys sp. Pour 100ml of K-76 into a Sakaguchi flask with the following composition and heat at 28oC.
Reciprocal shaking culture was performed for 4 days at OPH=6. Glycerin 0.5% Starch 1.0% Lactose 0.2% Soy flour 0.5% Yeast extract 0.1% Malt extract 0.2% CaC03 0.3%MgS04
0.05% 1 bottle of the seed culture obtained above was used for 30 minutes, and 2 hours of culture medium with the above composition was placed in a Jarfamenter, and the culture temperature was 2800, the air flow rate was 1/min, and the number of revolutions was 300. Centrifuge at 1/2 min to remove bacterial cells, add methanol to this supernatant plate, stir and leave for 3 hours, centrifuge to remove precipitate, and extract with an equal volume of ethyl acetate. . After the solvent in the acetate ethyl ester layer was distilled off under reduced pressure, the remaining groove was dissolved in methanol and passed through an activated carbon column.
The eluate was concentrated to dryness under reduced pressure, then dissolved in chloroform:ethyl acetate = 1:1 (V/V), and Sephadex LH
-20 column, remove the anti-complement activity peak, and distill off the solvent to obtain a pale yellow weakly acidic substance (compound [1]) having anti-complement activity. (The physical and chemical properties of this substance are as described above). Reference example
2. The known Stachybotrys cartarum IF05369 was cultured in a 500-liter Sakaguchi flask with 100 volumes of a medium having the following composition, and cultured with reciprocating shaking at 2800 and pH=6 for 4 days. Glycerin 0.5% starch
1.0% lactose
0.2% soy flour
0.5% yeast extract 0.1%
Malt extract 0.2%CaC03
0.3%MgS04
0.05% 2 bottles of the seed culture obtained above were placed in a jar fermenter for 30 minutes, and the culture medium with the above composition was placed in a jar fermenter at a culture temperature of 2800 degrees, aeration rate of 1 minute, and a number of flies of 300 revolutions/min. Culture was carried out for 5 days. The obtained culture solution was centrifuged at 8,000 rpm to remove the bacterial cells, and methanol was added to the supernatant solution and allowed to stand for 3 hours.Then, the precipitate was removed by centrifugation, and an equal amount Extract with ethyl acetate. After the solvent in the ethyl acetate layer was concentrated to dryness under reduced pressure, the residue was dissolved in methanol and passed through an activated carbon column. The eluate was concentrated to dryness under reduced pressure, and then chloroform:ethyl acetate = 1:1 ( V/V), gel filtered through a Sephadex LH-20 column to extract the active peak, and the solvent was removed to obtain a pale yellow weakly acidic substance (compound [1]) with anti-complement activity. ) 2.2 days were obtained (the physical and chemical properties of this substance were consistent with those obtained in Reference Example 1). Reference example 3 Stachybotrys sp. T-789 was cultured and purified in the same manner as in Reference Example 1 in a medium with the following composition adjusted to pH = 7.5 at 3.0 to obtain a pale yellow weakly acidic substance (compound [1 ]) was obtained (the physical and chemical properties of this substance were consistent with those obtained in Reference Example 1). Glycerin 0.5% glucose
1.2% corn stew liquor
0.5% dry nose yeast
0.1% malt extract 0.2%
MgS04 0.05%N
aCI O. 3% Reference Example 4 Stachybotrys sp. A pale yellow weakly acidic substance (compound [1]) having anti-complement activity was obtained by culturing and purifying T-791 in a medium with the following composition to pH = 5.5 at 25 qo in the same manner as in Reference Example 1. The physicochemical properties of this substance were consistent with those obtained in Reference Example 1). Glycerin 0.5% starch
1.0% sucrose
0.2% soy flour
0.5% Bepton 0.1%
Malt extract 0.2%MgS04
0.3%HCI
O. Example of 05% production Dissolve 2.1 liters of silver nitrate in 11' of water, add 3.5 liters of a 5.8 molar aqueous sodium hydroxide solution, and stir for 20 minutes at room temperature.
Add a solution obtained by dissolving compound [1] overnight in 3 parts of ethanol. After the reaction medium was heated at room temperature for 1.5 hours, the pH was adjusted to about 2 with 2N hydrochloric acid. The reaction solution was extracted with the same amount of ethyl acetate,
The extract and solvent were removed under reduced pressure, and the resulting silica gel column chromatography [Silica gel "Wakou C-200"]
Wako Pure Chemical Industries, Ltd. product, eluent: chloroform-ethyl acetate-acetic acid (volume ratio 100:50:2)].
By collecting the fractions corresponding to the above TLC and distilling off the solvent, 9 of 700 of a sesquiterbene analog (compound [B]) in the form of pale yellow amorphous crystals is obtained (physical and chemical properties of this compound [B]). is as described above.Manufacturing example Silver dioxide 0
．． 65% of the suspension was suspended in an IN-sodium hydroxide aqueous solution, and 1.0% of the compound [1] was added while stirring at room temperature. After soaking at the same temperature for 1 hour, filter the precipitate and add 10w of water.
Wash 5 times. The washing liquid and furnace liquid were combined, concentrated hydrochloric acid was added to adjust the HZ to about 2 to 3, and the mixture was allowed to cool. The precipitated crystals are collected in an oven, washed 5 times with 10 g of ice-cold water, and then dried. The dried powder crystals are dissolved in 50 ml of ethyl acetate, the unmelted portion is separated by a furnace, and the furnace solution is concentrated under reduced pressure to 50 ml of ethyl acetate. Add 50 pieces of ligroin to the concentrated solution, stir well, and let cool.
After washing twice with 20 mg of ligroin and drying, the compound becomes a white amorphous crystal.

1.01 of [0] was obtained (the physicochemical properties of the obtained compound [0] are as described above). Production Example 3 1.0 ml of compound [1] was dissolved in 10% aqueous sodium hydroxide solution for 50 ml, and concentrated for 3 minutes while introducing air at 50 qo. Thereafter, the mixture is made hydrochloric acidic (pH 2 to 3), and the precipitated crystals are collected in a furnace, washed 5 times with 10 drops of cold water, and then dried. The crystals are dissolved on 50 ml of ethyl acetate, the unfiltered portion is filtered out, treated with activated carbon, and concentrated to 50 ml under reduced pressure. The concentrated solution was added to 50 g of ligroin while stirring vigorously, and the precipitated crystals were collected in a furnace, washed with ligroin, and dried to obtain a pale yellow amorphous crystal compound [0.72 m of the obtained compound]. The physical and chemical properties are as described above). Production Example 4 1.0 μl of compound [1] is dissolved on 25 μl of an IN-potassium hydroxide aqueous solution, and 50 μl of an aqueous solution in which 0.578 μl of potassium permanganate is dissolved is added little by little while pouring vigorously. Stop the fly pestle when the color of potassium permanganate disappears,
The precipitate is separated in a furnace using Celite as a furnace aid. The furnace solution is made acidic with hydrochloric acid (pH approximately 2 to 3), and the precipitated crystals are collected from the furnace, washed three times with 10 drops of water, and then dried. Dried Sun Qing crystal was dissolved in 30 ml of ethyl acetate, unmelted matter was removed by a furnace, decolorized, and concentrated under reduced pressure. Add 50m' of ligroin to the concentrate and pour it over. After removing the precipitated crystals from the furnace and washing them with ligroin,
A pale yellow amorphous crystal compound was obtained by drying under reduced pressure at

0.91 ta of [0] is obtained (the physicochemical properties of the obtained compound are as described above). Manufacturing example 5 0. Sodium hydroxide aqueous solution 5' and ethanol 5
'to the compound

Add 9 of 418 of [0] and stir for 30 minutes at 30 to 40 oo of nitrogen flow. After the reaction is completed, the solvent is distilled off under reduced pressure and dried. To the residue, 10 parts of acetone is added and the dissolved portion is removed in an oven. The obtained crude crystals were recrystallized from water-acetone to give the compound as pale yellow amorphous crystals.

Disodium salt of [0] (compound [m])
9 of 342 was obtained (the physicochemical properties of the obtained compound [m] are as described above). Production Example 6 Compound [D] 418-9 was dissolved in 10 parts of ethyl acetate, 0.33 mol of an aqueous sodium hydroxide solution was added under stirring, and the mixture was stirred at room temperature for 10 minutes under a nitrogen stream. The precipitated crystals were collected in a furnace, washed three times with 10 ethyl acetate, and dried to obtain 9 of 390 of the disodium salt [m] of the compound [B] in the form of pale yellow amorphous crystals. Compound〔
The physical and chemical properties of [m] are as described above). Example 1 Disodium salt [m] of compound [b] and glucose are dissolved in distilled water for injection and then injected into ampoule 5'. After purging with nitrogen, autoclaving is performed at 121° C. for 15 minutes to obtain the anticancer agent of the present invention in the form of an injection. Example 2 According to Example 1, an anticancer agent of the present invention in the form of an injection was obtained. Example 3 Compound of the present invention in semi-synthetic glyceride base

Add [0],
After mixing and suspending the mixture at a temperature of 5,000 °C, the mixture was poured into a mold, allowed to cool naturally, and taken out to obtain the anticancer agent of the present invention in the form of a suppository. Example 4 According to Example 3, an anticancer agent of the present invention in the form of a suppository is obtained. Example 5 Compound [m, Apichell, cornstarch and magnesium stearate were taken and mixed, polished and sugar-coated with RIO.
Compress the tablets with a rib kine. The obtained tablets were mixed with TC-5 and polyethylene glycol-6.
000, coated with a film coating agent consisting of castor oil and methanol to produce the anticancer agent of the present invention in the form of a film-coated tablet. Example 6 Compound [2], Abishel, cornstarch, and magnesium stearate were mixed, polished, and then compressed into tablets using a sugar-coated RI powder. The obtained tablets are coated with a film coating agent consisting of methyl acrylate-methacrylic acid copolymer, triacetin, and ethanol to produce the anticancer agent of the present invention in the form of enteric-coated tablets. Example 7. [B] Mix citric acid, lactose, monocalcium phosphate, Puron F-68 and sodium lawalyl sulfate. The above mixture was added to NO. 60 screen sieve, polyvinylpyrrolidone, carbovac 1500
Wet granulation with an alcoholic solution consisting of Add alcohol if necessary to make the powder into a pasty mass. Add cornstarch and continue mixing until uniform particles are formed. No. Pass through 10 screens, put in a tray and dry in a 100q0 oven for 12 to 1 hour. Dry particles are NO. Add and mix the sieved dry sodium lauryl sulfate and dry magnesium stearate on a 16 screen and compress into the desired shape on a tablet press. The core is treated with varnish and sprinkled with talc to prevent moisture absorption. A subbing layer is applied around the core. Apply varnish enough times for internal use. Further subbing layers and smooth coatings are applied to make the tablet perfectly round and smooth. Pigmented coatings are applied until the desired shade is obtained. After drying,
Polish the coated tablet to give it a uniform luster. In this way, the anticancer agent of the present invention in the form of a tablet is obtained.

[Brief explanation of the drawing]

Figure 1 is an infrared absorption spectrum analysis diagram of compound [1],
Figure 2 is an ultraviolet absorption spectrum analysis diagram of the same compound [1], Figure 3 is a nuclear magnetic resonance spectrum analysis diagram of the same compound [1], and Figure 4 is a 1C nuclear magnetic resonance spectrum of the same compound [1]. The analysis diagram and FIG. 5 show high-resolution mass vector analysis diagrams of the same compound [1]. Figure 6 is an infrared absorption spectrum analysis diagram of compound [D].
Figures 7 to 9 are nuclear magnetic resonance spectroscopy charts of the same compound [B], Figure 10 is the compound

Basic compound salt of [0] [m
11 is an infrared absorption spectrum analysis diagram of the salt [m
] and FIG. 12 are the ultraviolet absorption spectrum analysis diagram of the salt [
This figure shows a nuclear magnetic resonance spectrum analysis diagram of the town. Soup 2 Diagram * Diagram M Bo Dimension * Diagram Bottom Diagram Wooden Box N * Diagram To ☆ Unillustrated Bottom Diagram ★ Diagram 〇★ O 1 Diagram

Claims (1)

[Claims] 1 (a) has a molecular formula of C_2_3H_3_0O_7, (b) has an elemental analysis value of C, 66.03; H, 7.18, (c) has a molecular weight of 418, (d) water, Insoluble in hexane, easily soluble in methanol, ethanol, acetone, chloroform, and ethyl acetate, soluble in aqueous sodium hydroxide and potassium hydroxide, and has the following infrared absorption spectrum as a (e) KBr tablet, 3430 ( s),
2975(s), 2950(s), 2870(m), 1
750(s), 1730(shm), 1698(m)
, 1675 (shm), 1668 (m), 1640 (
sh. s), 1630 (s), 1620 (sh.s),
1610 (sh.s), 1590 (s), 1565 (s.
h. m), 1550 (shm), 1500 (shm)
), 1480 (shm), 1470 (s), 1460
(sh.s), 1450(s), 1430(shm)
, 1410 (sh.s), 1395 (s), 1380 (
sh. m), 1370 (shm), 1355 (s),
1335(s), 1320(s), 1305(sh.s.
), 1295 (sh.s), 1285 (shm), 1
265(s), 1255(s), 1245(s), 12
10 (m), 1195 (shm), 1185 (m),
1175 (s.m.), 1165 (s.m.), 116
0 (s.m.), 1130 (s.m.), 1098 (s.m.)
), 1045 (m), 1035 (s.m.), 1010
(m), 995 (m), 985 (m), 970 (m),
950 (shm), 940 (m), 910 (m), 9
00 (shm), 890 (m), 870 (m), 86
0 (sh.m), 850 (sh.w), 840 (sh.
w), 830 (m), 820 (sh.w), 810 (w
), 800 (w), 775 (m), 765 (shm)
, 740 (m), 730 (m), 720 (m), 710
(m), 670 (m), (where s means strong absorption, m means medium absorption, w means weak absorption, and sh. means shoulder) (f) The following color reaction on a silica gel plate. Contains as an active ingredient at least one sesquiterpene analog and its salt that is positive to 2,4-dinitrophenylhydrazine reagent, positive to phosphomolybdenum reagent reaction, and negative to ferric chloride reaction. An anticancer agent characterized by