JPS6062989A - Novel naphthalenone compound and its preparation - Google Patents

Abstract

PURPOSE:To prepare a novel naphthalenone compound PD, by cultivating a fungus belonging to the genus specific Penicillium. CONSTITUTION:Penicillium diversum var. aureum (PERM-P 6936) is aerobically cultivated in an ordinary medium at 6.8-7.2pH at 20-25 deg.C for 10-25 days to prepare naphthalenone compound PD shown by the formula. When R1 is H, R2 is OH, and when R1 is OH, R2 is H, the former is PD-2, and the latter is PD-4. PD-2 and PD-4 have improved carcinostatic activity, and low toxicity.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides novel naphthalenone compounds PD covered by the general formula: The present invention relates to a method for producing the same, and an anticancer agent containing the compound as an active ingredient.

Conventionally, cancer chemotherapy agents include alkylating agents (nitrozene mustards, ethyleneimines, sulfonic acid esters), antimetabolites (folate antagonists, purine antagonists,
virimison antagonist), plant pre-fission (colcemid, vinblastine, etc.), antibiotics (dercomycin, cartenophilin, mitomycin, etc.), hormones (adrenal steroids, male hormones, female hormones), and porphyrin complex salts (marphyrin, C0pp). ) etc. are used. However, most of them are cytotoxic substances and exhibit serious side effects, so there is a strong desire to develop substances with low toxicity and excellent anticancer activity.

The present inventor searched for a substance with low toxicity and anticancer activity, and succeeded in isolating a novel naphthalenone compound PD from a culture of Penicillium deversum parietus aureum isolated from a dead pine tree. As a result of intensive research into the physiological activity of this compound, we found that the compound represented by the former has extremely low toxicity and excellent anti-cancer activity, and we have confirmed that this substance can have a remarkable effect on cancer treatment. The present invention has now been completed based on new findings.

The naphthalenone compound PD of the present invention can be an excellent cancer chemotherapeutic agent for warm-blooded animals such as humans, domestic animals, dogs, and cats.

The compound having anticancer activity used in the present invention will be explained below.

The compound of the present invention has the following structural formula.

(1) Hereinafter, the compound (11 will be referred to as “PD-2”, and compound (2) will be referred to as “PD-2”)
It is called "PD-1I-".

The microorganism producing the compound of the present invention is Penicillium (P
Penicillium deipersum paris: r-p
illlLrndiversum var, aureu
m) P-/ (hereinafter referred to as "P-7 strain") is often used. The mycological properties of the P-7 strain are the same as those of the known Penicillium deipersum parietus aureum (Mycologia'+ g+
3'l8fg, see //C/911g).

Strain P-7 was deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology on February 26, 1925, and its accession number is FERM-Pl, No. 6936 (FERM-Pl, 93 Otsu). It is.

The method for producing the 1111t sheath of the compound of the present invention will be described below. That is, the above P-/strain is, for example, 20 to 2,
The culture solution F of the culture obtained by static culture on a flat surface for 3 weeks at t''C is layered with activated carbon, then washed, and desorbed with an organic solvent.The eluates are combined, concentrated, and extracted with an organic solvent.
After washing and drying in a conventional manner, distillation is performed under reduced pressure. The obtained extract was subjected to silica dull column chromatography (elution temperature benzene:acetone = 4t: /), and the commonly used fractions of PD-2 and PD-4 were concentrated and subjected to high performance liquid chromatography. It was separated by chromatography, distilled off under reduced pressure, and crystallized to give PD-
2. Obtain PD-g respectively.

The nutrient source of the culture medium can include carbon sources, nitrogen sources, etc. that are commonly used in microbial culture media, and carbon sources include starch, dextrin, glycerin, glucose, sucrose, galactose, inositol, mannitol, etc. However, yeast extract, begtone, soybean flour, meat extract, rice bran, rice bran, urea, corn steep liquor, ammonium salts, nitrates, and other organic or inorganic compounds are also used as sources.

As a nutrient source, yeast extract or begtone is a suitable ingredient in order to achieve extremely good growth of the microorganisms used. Other inorganic salts, such as table salt,
Phosphates, metal salts such as potassium, calcium, zinc, manganese, iron, etc. may be added as appropriate. Culture conditions such as culture temperature and culture time are determined to be suitable for the growth of the bacteria used and to maximize the production of the target substance. For example, the pII of the medium is Otsu 0g~7.2, 7.2%. The temperature and time of culture should be around 10 to 2 degrees Celsius, 70 degrees centigrade.
About 25 days is good.

After desorbing the target substance from the culture with acetone in this way, it is treated with an organic solvent to collect a crystalline mixture, and from this mixture ') a method is used that takes advantage of the difference in distribution between K and the organic solvent. Yes. Specifically, activated carbon is first added to the P solution to adsorb it, and then desorbed with acetone. From the resulting hydrous acetone solution, acetone is distilled off, extracted with ethyl acetate, and the resulting residue is removed from the contaminant impurities. The sample is subjected to column chromatography using silica dal in order to remove it.

Benzene-acetone thread is optimal as the solvent system, and the mixing ratio is changed as appropriate to elute while increasing the polarity. This may be changed stepwise or gradiently, and the silica gel column may be adjusted using a commercially available column chromatography agent, or a longitudinally split column may be used. The bath fractions of PO-co and PD-gu are collected and separated by high performance liquid chromatography. The most suitable solvent is hexane-ethyl acetate. After separation, the solvent is distilled off and the residue is recrystallized to obtain PD-λ and PD-4, respectively.

The physicochemical properties of the thus obtained compounds PD-co and PD-1 are as follows.

[Physical and chemical properties of PD-co] (11 Melting point: /da/~/4t3℃ (2) Specific optical rotation: [α]o -7g, Otsu (methanol)
to C=θ, 0g6) (3) Mass spectrum: m/z
/9g (tl1+), KBr −+ (4) IR・Storage, vmaxcm 3'IOθ~
300θ, /63θ, /left gθ (51' H-NMF (spectrum #: (CDC15,
90MHz, δ)λ, θ7(ddd, J-//,'
l, //, 9, /, J'0.2H2XH-3α), 2. g4(dddXJ-11, g, 3.3, //,
9l-IZ 5H-3β) t, J (dd, J=, t, 3, /3..2Hz, H-
2) 11.9g (dcJXJ-'1.g, /81lHz
to H-11) 6.93 (dd, J-co, 0, g, 3H
z, H-7) 7, pII< dd, J-2, 0, za, 3l-IZ, H-5)? , S'7(t, J-3,
,7Hz,H-1,n (6) Toxicity: To mice! ;
Even if 01ng/K9 was administered continuously, no response from 10" was observed.

(Physical and chemical properties of PD-g) (1) Melting point: /3so~/<t/C+21. Optical rotation i: (α)15'-36,3c<, mepnol, C
, -0,062) (3) Mass ξ vector: "η/z
/7g (M”) (41'H-NMR spectrum: (d
-B-Acetone, 9QHz, δ) 2.07Cddd,
J-/θ, g, //, 9, /3. Otsu H2゜1-1-3
α) J, 79 (ddd, J4. Otsu, 5./, / to 9+-1
2゜H-3β) tl, 3g (dd, J-5, /, /3.6Hz, H-;
l) 5.4/(dd, J4.Otsu, 10.gHz, To1
-Hiroshi) 7.09 (dd, J-2,0,7,'7H2XH
-Otsu)ri, 33(tX J-7,7HzXH-7)7,
#? (dd, J-'2.0, '7.7H2, H-etc.) (
5) Toxicity: 5 Qtny/Kg for mice
No maternal effect was observed even after continuous administration.

Next, an anticancer agent containing the naphthalenone compound PD of the present invention as an active ingredient will be described.

The anticancer agent of the present invention can be administered either orally or parenterally; when administered orally, it is administered as a soft/hard capsule, tablet, granule, fine granule, or powder; when administered parenterally, is injectable, infusion and solid form or W! A
It can be administered in the form of a herbal drug, which maintains sustained mucosal absorption as a cloudy viscous liquid.

In order to formulate all the active ingredients of the present invention, the compounds represented by both formulas can be prepared into subcutaneous or intravenous injection preparations, as well as capsules, tablets, fine granules, etc., according to conventional methods. It can be formulated into a dosage form and administered orally.

The formulation of the active ingredient of the present invention includes surfactants, excipients, lubricants, adjuvants, and pharmaceutically acceptable film-forming substances in order to form an enteric formulation in consideration of the properties of the active ingredient. This can be carried out as appropriate using a coating aid, etc., and specific examples thereof are as follows.

In order to improve the disintegration and elution of the composition of the present invention,
Seven or more surfactants such as alcohols, esters, polyethylene glycol derivatives, Sorvita 7Q) MYI fatty acid esters, and sulfated fatty alcohols can be added.

In addition, excipients include, for example, sucrose, lactose, starch,
Seven types or a combination of two or more types such as crystalline cellulose, mannitol, light anhydrous silicic acid, magnesium aluminate, magnesium metasilicate aluminate, synthetic aluminum silicate, calcium carbonate, sodium hydrogen carbonate, calcium hydrogen phosphate, calcium carboxymethyl cellulose, etc. can be added.

Examples of lubricants include magnesium stearate,
Seven or more types of talc, hydrogenated oil, etc. can be added, and salt, saccharin,
Sweeteners such as sugar, mannitol, orange oil, licorice extract, citric acid, glucose, menthol, eucalyptus oil, and malic acid, fragrances, colorants, preservatives, and the like may be included.

Adjuvants such as suspending agents and wetting agents include, for example, coconut oil, olive oil, sesame oil, fallen beef oil, calcium lactate,
Safflower oil, soybean phospholipids, etc. can be contained.

In addition, cellulose acetate phthalate (CAP) is a carbohydrate derivative such as cellulose and saccharides as a membrane-forming substance.
In addition, examples of polyvinyl derivatives such as acrylic acid copolymers and dibasic acid monoesters include meboul acrylate/methacrylic acid copolymers and methyl methacrylate/methacrylic acid copolymers.

In addition, when coating the above-mentioned film-forming substances, in addition to commonly used coating aids such as plasticizers, it is possible to add various additives to prevent the chemicals from adhering to each other during coating operations. It can improve properties and make coating operations easier. Note that the active ingredient may be encapsulated using a film-forming substance and then mixed with excipients and the like to form a dosage form.

The compounding ratio in a typical dosage form of qiVC is as follows.

Particularly preferred range Active ingredient θ, /~90 M-m% 0.3~/! ;
1iii: % excipient /θ-'? 9. g z g left~de, ql lubricant θ~Sθ 0-2°I surfactant θ~30 0-co. l Film-forming substance θ, / size o 〃 θ, 3-20 I Particularly preferred excipients are lactose, crystalline cellulose, and Calcium. It is oxymethylcellulose calcium.

In addition, the expected dose is insufficient to effectively treat the target tumor, and it depends on the symptoms of the tumor, administration route, dosage form, etc., but in general, when administered orally, for adults, it is approximately 7 days per day. θ, 0/~100g/edge weight (for dwarfs, θ, θ/
~6oπy/1 (y body weight), the upper limit is preferably about sθη/edge weight, more preferably about 10 kg/
~ 1 in the body, and in the case of parenteral administration, the upper limit is approximately 1
0rrry/Lg body is about 1, preferably S/K
y body weight, preferably 2 kg/9 body weight is appropriate.

Next, the anticancer activity testing method for confirming the anticancer activity of the above compound will be described.

Yoshida jm cells (Yoshldasa jm cells) were extracted from the ascites of rat 1.
rcoma cells) f and cultured in DM-/6θ medium containing 20% fetal bovine serum (a pure synthetic medium for tissue culture that does not contain proteins or lipids and contains essential amino acids as well as dispensable amino acids). / 1 in tttl
Dilute to 0 to 5 x/0' cells. When 20 ml of this cell suspension is transferred to a TO4° flask and cultured at 37C for 3 to 3 days, the cells proliferate to 10 to 30 x 10 cells per liter of waste.

This proliferated cell suspension is again diluted with siJ DM-/O θ medium to reach a cell count of 7S to θ × 104 cells in /d. The total TD of this diluted cell suspension
Transfer to a 4o flask and culture at 37° C. for 3 to q days to proliferate the cells. In this way, the D
Tissue cultures of Yoshikawa sarcoma cells are maintained through passage by repeating the growth, dilution, and growth sequence using M-/Otsu theta medium.

One Yoshida sarcoma cell transferred to tissue culture and grown in a TO4o flask was suspended in the above DM-/Otsu0 medium to reach a number of 2θ~22X/0' cells in the DM-/O medium. Adjust to. Dispense 1 volume of this cell suspension into each vial, dissolve the test compound in 10 μt of methanol so that the final reduction is at a predetermined value, add it to each vial, and culture at 37°C for 1 day. . After culturing, take a portion of the cell suspension in the vial and count the number of cells under a microscopic microscope using a collapsing ball it jE m.

The present invention will be explained in detail below using production examples, formulation examples, and test examples.

For production example culture, Czapek-Yeast extra with the following composition was used.
ct) medium was used.

Sucrose 5oy KCtO, 5t MSO4θ, left? 2HPO4/f NaN05.21 yeast extract 3f sterilized water 101C pH7, Co) Dispense the above medium into 30 flat culture flasks (33θWLl per flask), cover with aluminum foil, and heat in an autoclave at 720°C. ．． /, sterilize for minutes.

Sterile bales, the above P-/bacteria [Feikoken Bacteria No. 6936 (FE
RM-PA'136)) inoculated with 10-2 SC
We held a period of quiet cultivation. Activated carbon 10θ2 was added to the culture F solution 101 collected by suction filtration from the flask, and then degassed to adsorb the product onto the activated carbon. This I-occupied charcoal was filtered out by suction and washed with about 1/l of distilled water.

Next, this activated carbon was immersed in about 2 tons of acetone to remove the absorbed material. Furthermore, this operation was repeated to completely desorb the remaining adsorbed matter. The aqueous solution obtained by combining the acetone eluates and concentrating under reduced pressure was diluted with about 2 liters of ethyl acetate.
Extracted twice. The obtained extract was washed with four portions of saturated brine and dried by adding anhydrous sodium sulfate. After drying, it was distilled under reduced pressure to obtain an ethyl acetate extract/jF. The extract was dissolved in a small amount of acetone, adsorbed onto a small amount of silica gel, the acetone gold was evaporated and dried, and this was placed on a silica gel column (Silica Gluco 00?).

The elution solvent for the column was benzene:acetone (4j=/
) was used. By this column chromatography, t)P
The part containing D-3 was separated, and Rf/, 4-core a was detected on TLC.
Fractions of j (hexane: ethyl acetate =/: l) were collected, concentrated, and subjected to high performance liquid chromatography (hexane: ethyl acetate =/:/, Nucleosil 30-!; g
f! j x 300tn) for a holding time of 79.2 minutes (
The peaks of PI)-1J) and 2hiro and partial (PD-2) were collected, concentrated, and recrystallized from the above solvent to obtain colorless needle crystals, respectively. The fit is 15-. It was 20q.

Formulation example/(injection/infusion) Powdered glucose 52 was added to contain compound P[)-U(lO■), dispensed aseptically into vials, sealed, and inert gas such as nitrogen, helium, etc. Seal and store in a cool, dark place. Before use, add physiological saline lθ0-
Added to make an intravenous injection, 1 day, 10 ~ / 00
Administer m77 by intravenous injection or drip depending on the symptoms.

Formulation Example 2 (Injection/Drop) Compound PD-IIC2mf) was prepared as an intravenous injection for mild symptoms in the same manner as in Formulation Example/.
Administer 00ml by intravenous injection or drip depending on the symptoms.

Formulation Example 3 (Enteric Coated Capsules) Compound po-a (sr), lactose λ, 9 was added to Otsu 1 and hydroxyprobil cellulose 0.0 Da 2 were mixed well, and then granulated according to the "rjC method". This is thoroughly dried and sieved to produce granules suitable for bottles, heat-seal packaging, etc. Next, cellulose acetate phthalate 0
, 3? and hydroxyglobil methylcellulose phthalate 0. , tf2 dissolves 7 and a, package 2! /! The granules are suspended and flowed, and the base material is coated to form enteric-coated granules.

This composition is filled into capsules to produce enteric-coated cassette-wrapping agent/θθ pieces.

Test Example Using the above compounds PO-U and P[)-Hiro, Yoshikawa sarcoma cells (Yoshlda sarcoma cells) were isolated by the above test method.
The growth inhibitory activity against (Acells) Ic was investigated. All compounds showed growth inhibitory activity at 20-50 μm741. From this result, it is clear that the above test compound exhibits an excellent effect on suppressing the proliferation of cancer cells.

Patent applicant: Physical and Chemical Research B [

Claims (1)

[Claims] (11) A naphthalenone compound PD represented by the general formula (when R,=H, R2=OH%; when R,=OH, R2 represents H). (2J R,= The compound according to claim 1, where HXR2=OH. The compound according to claim 11, where 31R,=OHSR2=1-1. (4) A!=silium (Penlcllllum)
A method for producing a naphthalenone compound f'D, which has a lFr characteristic, by culturing a naphthalenone compound PD-producing bacterium belonging to FA, and separating and collecting the naphthalene compound f''D from the culture. (5) Naphthalenone compound belonging to the genus Henicillicum. P.D.
The producing bacteria are Penicillium deipersum parietus.
Aureum (Peniclliundlversum)
var, aurem) P −/
93 No. O)), the manufacturing method according to claim (4).