JP2001097855A - Antifungal agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new antifungal agent containing a compound having hydronaphthalene ring as an active component. SOLUTION: This agent is obtained by including a compound comprising a hydronaphthalene ring having a methyl group and a (protective group-having or nontoxic salt-forming) lower alkyl or (protective group-having) hydroxyl group at 4-position, a methylene group at 8-position, an alkyl group having two unsaturated bonds at 9-position and a methyl group at 10-position.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an antifungal agent containing a compound having a hydronaphthalene ring structure as an active ingredient, and belongs to a pharmaceutical production technique.

[0002]

2. Description of the Related Art With the remarkable development of antimicrobial drugs, mainly antibiotics, antifungal agents are not always in a satisfactory state, judging from their type and effectiveness. Things. Fungal infections to humans include candidiasis, aspergillosis, cryptococcosis, mucormycosis, and other imported mycosis, including actinomycosis, nocardiosis, chromoblast mycosis, histoplasmosis, coccidioidomycosis,
Geotrichum disease, penicillium disease and the like are known.
There are also superficial mycosis such as athlete's foot and bugs. In particular,
In recent years, general use of anticancer drugs, immunosuppressants, steroid hormones, etc.
Opportunistic infections caused by fungal infections such as Candida, Aspergillus, and Cryptococcus have become one medical problem, and the development of therapeutic agents for them is desired. At present, chemotherapeutic agents such as amphoterin B, fluconazole, itraconazole, miconazole, and 5-fluorocytosine are used as therapeutic agents for these fungal infections. However, these chemotherapeutic agents are not always satisfactory in terms of toxicity and therapeutic effect, and the emergence of resistant bacteria is also a problem. In order to solve this problem, a clinically useful antifungal drug excellent in selectivity is desired. The present inventors have conducted intensive studies in order to respond to this demand, and have previously found that cryptoporic acid derivatives are excellent as antifungal agents, and have made one proposal (International Publication WO96 / 25385).

[0003]

SUMMARY OF THE INVENTION The present inventors have paid attention to the fact that cryptoporic acid derivatives are excellent as antifungal agents, studied the mechanism of action of cryptoporic acid derivatives, and further studied their functions and structures. From these results, intensive studies are underway to find compounds other than cryptopolonic acid derivatives that have strong antibacterial activity against the above-mentioned fungi that cause opportunistic infections, that is, compounds that can be effectively used as antifungal agents. They continued. That is, the present invention seeks to provide a novel antifungal agent.

[0004]

Means for Solving the Problems The present inventors have made intensive studies in order to achieve the above-mentioned object, and have found that, among the structures of cryptoporic acid derivatives, the hydronaphthalene ring structure has a great effect on the antifungal activity. That is, any of sclareol, sclareolide, manol, labdanolic acid having the structure thereof, those obtained by adding various substituents thereto, and derivatives thereof, and bicyclic diterpenes obtained from a copearl resin are all anti-oxidants. The inventors have found that they have a fungal action and completed the present invention.

That is, the present invention relates to an antifungal agent comprising a compound represented by the following structural formula as an active ingredient.

[0006]

Embedded image

In the formula, A is a carboxyl group which may have a protecting group or may form a non-toxic salt or a lower alkyl group having a hydroxyl group which may have a protecting group.

[0008]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail.
The compound of the present invention has a hydronaphthalene ring at the center of its structure. More specifically, as shown in the above structural formula, a non-toxic salt having a methyl group and a protecting group at the 4-position is used. A carboxyl group which may be formed or a lower alkyl group having a hydroxyl group which may have a protecting group, particularly a methyl group, a methylene group at the 8-position, a specific alkyl group having two unsaturated bonds at the 9-position, 10 It consists of a hydronaphthalene ring with a methyl group added at the position. The protecting group is a methoxymethyl group (MOM), methylthiomethyl (MTM), tetrahydropyranyl (THP), t-butyl (t-Bu), trityl (Trt), benzyl (Bzl), p- Ether-type protecting groups such as methoxybenzyl (MP), acyl-type protecting groups such as acetyl groups, trimethylsilyl (TMS), silyl-type protecting groups such as t-butyldimethylsilyl (TBDMS), etc. Protecting groups generally used in organic synthesis and the like can be used, such as an ester-type protecting group such as a benzoyl group and a silyl ester-type protecting group such as a trimethylsilyl group. Examples of the non-toxic salt include salts of sodium, potassium, calcium, choline, meglumine, diethanolamine and the like. The compound in the present invention can be easily obtained from commercially available products such as sclareol, manol, labdanolic acid and the like by using conventional methods. In addition, it can be isolated and synthesized from naturally occurring copearl resin or leaves of Konotegashiwa.

The antifungal agent containing the compound of the present invention as an active ingredient is particularly useful as a remedy for mycosis involving fungi such as molds and yeasts which are included in humans, and tablets for oral administration. In the case of parenteral administration, it can be used as a solution, suspension, ointment and the like. The dose varies depending on the severity of the disease, the weight of the patient, and the like, but is generally preferably about 1 to 500 mg, and is preferably administered once to several times a day. . In the case of an injection solution, it can be administered by putting it in a drip. In the case of formulation, commonly used vehicles, carriers, swelling agents, excipients, binders, lubricants, buffers, antioxidants, preservatives, stabilizers, flavoring agents, etc. are required for the formulation. The dose is mixed and formulated.

[0010]

EXAMPLES Examples of the present invention will be described below, but these examples do not limit the present invention in any way. In addition, the antifungal activity measurement method and the infection treatment test method in each Example are as follows. ○ Anti-Candida albicans (Candida albicans) activity measurement method Candida albicans (Candid albicans)
a albicans) as a test bacterium, i.
Tested in vitro to confirm its activity. Antifungal susceptibility test, Journal of the Japanese Society for Medical Mycology, Vol. 36 (1), 62-64 (1995)
Performed in accordance with the method proposed in The strains used in the experiments used were strains TIMM1768 and TIMM3318, which were provided by Teikyo University Medical Mycology Research Center. Among them, the TIMM3318 strain has resistance to an azole antifungal agent. The culture medium for sensitivity measurement is RP
MI1640 (Irvine Scientific Cat. # 9512) for 10.
4 g and 2.0 g of sodium bicarbonate in 900 m of sterile distilled water
After dissolving in l, 34.53 g of MOPS was added as a buffer,
Stir well until dissolved, then p with sodium hydroxide
After adjusting to H7.0, the volume was adjusted to 1 liter by adding sterile distilled water, and the solution was sterilized by filtration and stored at 4 ° C. Test bacteria are YM
Using an agar medium (Difco), the cells were subcultured by culturing at 35 ° C. for 24 to 48 hours twice or more, and then suspended in 5 ml of sterile physiological saline. The absorbance of this suspension was measured at 600 nm, the amount of bacteria was determined from a calibration curve created in advance, and 2 × 1
It was diluted with a culture medium for sensitivity measurement so as to have a cell count of 0 ³ cells / ml to prepare a test bacterial solution. The test sample was dissolved using a methanol solution containing 20% DMSO, and a test assay solution was prepared by 2-fold serial dilution. The test for antifungal susceptibility was 9
Dispense 90 μl of sensitivity measurement medium into a 6-well flat bottom microplate, add 10 μl of test assay solution and 100 μl
Was added to each well to prepare a predetermined concentration.
The microplate was placed in a container keeping humidity and cultured at 35 ° C. for a maximum of 72 hours. Observation was made every 24 hours, and when the turbidity of the growth control reached 0.2, the turbidity of each well was determined. It was measured. To determine the 80% growth inhibitory concentration (IC ₈₀ ), a microplate was stirred with a mixer, 40 μl of a growth control culture was taken, and 160 μl of a culture medium for sensitivity measurement was added thereto to prepare a well equivalent to IC ₈₀ . The wells exhibiting a turbidity equal to or less than the turbidity of this well were defined as the end points. ○ Anti-Aspergillus fumigatus
igatus) Activity measurement method 1. Drug solution preparation method The test drug was weighed, and a drug dilution step was prepared with methanol containing 20% DMSO. 2. Sensitivity measurement medium and preparation method RPM11640 (Irvine Scientific Cat. # 9512) 10.4
g, 2.0 g of NaHCO _{3 in} 900 ml of sterile distilled water
34.53 g of MOPS was added as a buffer solution, and the mixture was stirred.
After adjusting the pH to 7.0 with H, the volume was adjusted to 1 liter and sterilized by filtration before use. 3. Preparation of Inoculated Bacterial Solution Test bacteria were cultured on a potato dextrose agar medium (Difco) at 30 ° C. for 1 week, and then 0.05% Tween80 was added.
A sterile physiological saline solution was added to obtain a conidia suspension. The number of conidia was counted under a microscope using a hemocytometer and adjusted to 2.5 × 10 ⁵ cells / ml in a measurement medium. 4. Culture 90 μl of medium was dispensed into a 96-well flat bottom microplate, 10 μl of the drug solution prepared in 1 and 80 μl of the inoculum were added.
20 μl of alamar blue solution was added to each well (final 1% DMSO, 4% methanol, alamar blue
Is 10%, and the growth control is the same medium without drug). Incubate at 30 ° C in a wet container to prevent drying, 2
Observed every 4 hours, OD _{570 of} the growth control was 0.6.
The end point was judged when reaching. 5. Judgment of results 80% growth inhibitory concentration (IC ₈₀ ) relative to growth control
This was defined as the minimum inhibitory concentration with the endpoint as the endpoint. More specifically, the drug concentration was determined to be equal to or less than 20% of the measured value of the growth control, but the drug concentration was calculated as I
C ₈₀ (MIC).治療 Infection treatment test Healthy growing mice (ICR, 4 weeks old, female, 19-2)
2g, Charles River Japan) 0.1% Tween8
0.2 ml of a bacterial solution of Aspergillus fumigatus (1.0 × 10 ⁶ CFU / mouse) suspended in physiological saline to which 0 was added.
Inoculation was performed through the tail vein to establish infection. Treatment is performed by dissolving or suspending the specimen in a 5% gum arabic aqueous solution, 0.2 m each time.
l was orally administered using a gastric probe (10 mg / kg and 2 mg / kg).
kg) . The first time is 1 hour after inoculation of the bacteria, and once a day thereafter 2 times
Six doses were administered every 4 hours (once / day, for a total of 7 days). The control group received 0.2 ml of the vehicle alone. The efficacy was determined as follows from the number of surviving mice in the sample administration group at the time when all the control animal groups died. Effective 60% or more survival rate Slightly effective 40% survival rate Ineffective 20% or less survival rate

Example 1 Preparation of compound 300 g of ethyl acetate was added to 50 g of soybean syrup, and extracted at room temperature for 24 hours. After filtration, 300 ml of ethyl acetate was added to the residue.
And extracted again at room temperature for 24 hours. Both extracts were combined and the solvent was distilled off to obtain 2.14 g of extract. This extract was subjected to activated carbon chromatography (5.0 g, 2.5
cmID × 8cm, developing solvent: methanol / chloroform = 1
0/0 to 4/1) and silica gel column chromatography (30 g, 2 cm ID × 22 cm, developing solvent: hexane / ethyl acetate = 9/1 to 7/3) to give a compound represented by the following structural formula (hereinafter referred to as COP-). 343 mg (referred to as f) (mixture of cis and trans) was obtained. The ¹ H-NMR (400 MHz, CDCl ₃ ) spectrum chart is as shown in FIG. Communi COP-f
c acid. The Rf value of COP-f silica gel thin-layer chromatography (silica gel 60 F254 (Merck)) is 0.1.
43 (hexane / ethyl acetate = 4/1), the color reaction was positive for anisaldehyde reagent (purple). 30 of the above COP-f
0 mg (0.99 mmol) is dissolved in 5 ml of diethyl ether,
Diazomethane is added little by little to give the methyl ester 272
mg was obtained. This methyl ester is treated with diethyl ether 5
Dissolve in water and stir with ice-cooled lithium aluminum hydride 3
6 mg was added little by little, and the mixture was stirred at room temperature for 3 hours. After extracting the reaction solution with ethyl acetate, a saturated aqueous solution of sodium hydrogen carbonate was used,
After washing with a saturated saline solution, the solution was dried over anhydrous magnesium sulfate, and the solvent was distilled off. The residue was subjected to silica gel column chromatography (5.0 g, 1 cm ID × 16 cm, developing solvent: hexane / ethyl acetate = 9/1 to 7/3) to give a compound represented by the following structural formula (hereinafter referred to as COP-f-OH). 114 mg (mix of cis and trans, yield 40%) were obtained. Its ¹ H-NMR (400 MH
z, CDCl ₃ ) is as shown in FIG.
Silica gel thin layer chromatography (silica gel 60 F25
4 (Merck)) has an Rf value of 0.32 (hexane / ethyl acetate = 4
/ 1), the color reaction is positive for anisaldehyde reagent (purple).

[0012]

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[0013]

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測定 Measurement of compound properties Tables 1 and 2 show the results of the antifungal activity of the prepared compound and the results of an infection treatment test.

[0015]

[Table 1]

[0016]

[Table 2]

[0017]

From the above results, it can be seen that the compound of the present invention is a pathogenic fungus infecting humans, Candida albicans (Candida albicans).
dida albicans) and Aspergillus fumigatus (Asper
gillus fumigatus), indicating that these compounds are effective as chemotherapeutic agents against fungal infections such as candidiasis and aspergillosis.

[Brief description of the drawings]

FIG. 1 is a ¹ H-NMR chart of COP-f.

FIG. 2 is a ¹ H-NMR chart of COP-f-OH.

 ──────────────────────────────────────────────────の Continuing from the front page (72) Muneaki Kano, 2nd Okubo, Tsukuba, Ibaraki Prefecture, Toagosei Co., Ltd. (72) Inventor Toshiaki Segawa, 2nd Okubo, Tsukuba, Ibaraki, Toagosei Co., Ltd. 72) Inventor Shigeo Nozoe 1-10-4 Yagiyama Honcho, Taishiro-ku, Sendai-shi, Miyagi F-term (reference) 4C206 AA01 AA02 CA09 CA14 CA24 DA13 DB11 KA04 MA01 MA04 MA37 MA43 MA48 MA55 MA57 MA63 MA72 MA75 ZB35 4H006 AA03 AB29 BJ30 BS20 FC32 FC78 FE11

Claims (1)

[Claims] 1. An antifungal agent comprising a compound represented by the following structural formula as an active ingredient. Embedded image However, in the formula, A is a carboxyl group which may have a protecting group or may form a non-toxic salt or a lower alkyl group having a hydroxyl group which may have a protecting group.