JPH1045760A - Therapeutic agent for malignant melanoma - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject agent exhibiting selective toxicity to malignant melanoma cells, excellent in stability and safety, containing a specific compound as active ingredient. SOLUTION: This therapeutic agent for malignant melanoma contains pref. 1-20wt.% of a compound of the formula obtained by isolation and purification from an extract obtained by subjecting a dried product from the leaves of Kepsia tenuis Leenh. & Steenis of the genus Nerium to extraction using an organic solvent such as chloroform or ethanol. It is preferable that the daily dose of this therapeutic agent is 0.1-200mg/m<2> per adult in the case of intravenous administration.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel therapeutic agent for malignant melanoma.

[0002]

2. Description of the Related Art Malignant melanoma is a malignant tumor characterized by having a melanogenic ability, and has a poor prognosis such as high resistance to chemotherapy and difficulty in treatment after metastasis. In recent years, malignant melanoma has been increasing worldwide,
In Japan, nearly 2000 cases are reported annually.
The current treatment for malignant melanoma is mainly surgical treatment, and chemotherapy is given before and after the operation. DTIC (5- (3,3-dimethyl-1-triazenyl)
-1H-imidazole-4-carboxamide: NSC-45388) is the best known (Comis RL et al., Cancer Treat Rep, 60, 1).
65-176, 1976).

[0003]

However, single administration of the above-mentioned chemotherapeutic agent is not sufficient, and there are also problems such as stability and liver damage. Multidrug therapies have also been studied, for example,
With DAV therapy using three drugs, DTIC, ACNU and vincristine, the response rate has reached around 30%. However, it may be ineffective for advanced stages of melanoma, and there is no further chemotherapy, and the development of a new therapeutic agent for melanoma that selectively acts on melanoma cells is desired. . An object of the present invention is to provide a novel therapeutic agent for melanoma which shows selective toxicity to melanoma cells.

[0004]

The object of the present invention is to provide a remedy for malignant melanoma containing a compound represented by the formula (1) as an active ingredient.

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[0005] Accordingly, the therapeutic agent for malignant melanoma of the present invention will be described. The present inventors conducted a screening test using the activity of malignant melanoma cells as an index, and found that lundurine B represented by the chemical formula (1) has the above activity, and completed the present invention. .

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[0006] The therapeutic agent for malignant melanoma of the present invention contains landlin B represented by the above chemical formula (1) as an active ingredient. It is known that Landrin B can be isolated and purified from organic solvent extracts of dried leaves of Kopsia tenuis Leenh. & Steenis of the family Oleander (TSKam et al., Tetrahedron Letters, 36).
(5), 759-762, 1995). The present inventors used Landrin B isolated and purified from Malaysian Coptia tenuis. Landrin B, a compound used in the present invention,
It can be isolated and purified from organic solvent extracts of dried leaves of Coptia tenuis. It is preferable that the raw material Copsia tenuis be dried and further pulverized to prevent spoilage during storage.

[0007] Examples of the hydrocarbon solvent used in the extraction step include hydrocarbons such as pentane, hexane, heptane, octane, petroleum ether, petroleum benzine, ligroin, benzene, toluene, xylene, ethylbenzene, etc .; Alcohols such as methanol, ethanol, butanol and the like, or a mixed solvent thereof. In particular, chloroform,
Ethanol is preferred. Of course, other solvents with similar extraction capabilities besides these solvents may be used.

[0008] The amount of the organic solvent is not particularly limited, but may be any amount that can extract landrin B, and is preferably 100 to 1000 per 50 g of dried leaves of Copsia tenuis.
ml. The extraction temperature is not particularly limited either, but may be any temperature at which landrin B can be stably extracted, and is preferably room temperature to 60 ° C. The extraction time varies depending on the temperature. For example, when the extraction is performed at room temperature, it is 2 to 20 hours. The extraction may be repeated two or more times.

[0009] Landrin B is purified from the obtained extract. For purification, for example, once the extract is concentrated,
It can be performed by distribution using a base or chromatography. Chromatography is preferably column chromatography, gel filtration chromatography, high performance liquid chromatography, liquid-liquid countercurrent distribution chromatography, thin layer chromatography, centrifugal chromatography and the like. Further, a combination of a plurality of the above-mentioned chromatographys may be used. Particularly preferred is column chromatography. As the column chromatography, one using Merck silica gel 60 (Merck) as a carrier and a combination of chloroform and methanol as a solvent is preferable.

[0010] The therapeutic agent for malignant melanoma of the present invention only needs to use Landrin B as an active ingredient, and Landlin B to be used is not limited to the extract from Copsia tenuis described above.

The therapeutic agent for malignant melanoma of the present invention contains landrin B in an amount of 0.01 to 50% by weight, preferably 1 to 50% by weight, in the composition.
20% by weight is blended.

Pharmaceutically acceptable carriers used in the therapeutic agent for malignant melanoma of the present invention include excipients such as natural or synthetic aluminum silicate, microcrystalline cellulose, talc, dextrin, starch, lactose, and the like. Diluents such as vegetable oil and propylene glycol are mentioned. In addition to this, other optional ingredients include pharmaceutically acceptable stabilizers, coating agents, suspending agents, emulsifiers, solubilizing agents,
Preservatives, buffers, sweeteners and the like can also be present. Known additives can be used in appropriate combination as these additives. The dosage form may be any form such as powders, granules, tablets, capsules, and injections.

The therapeutic agent for malignant melanoma of the present invention is mainly administered intravenously. However, the method of administration of the therapeutic agent of the present invention,
It is not limited to this. The daily dose for an adult varies depending on the administration method, disease state and the like. In the case of intravenous administration, which is the main administration method, 0.1 to 200 mg / m ²
Is the usual dose. However, the dose is not limited to this.

(Examples) The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the examples.

(Separation and Purification of Landrin B) 1 kg of dried leaves of Coptia tenuis was ground with a grinder, and extracted repeatedly with 95% ethanol at room temperature until no color appeared. The extracts were combined, and the solvent was distilled off under reduced pressure to obtain an extract. To this extract was added an excess of 5% hydrochloric acid,
The alkaloid in the extract was dissolved, and the non-basic substance in the insoluble portion was removed with a filter. A concentrated ammonia solution (about pH 10) was added to the alkaloid solution to make the solution basic, and then chloroform was added for partition. The chloroform layer was recovered, and the solvent was distilled off under reduced pressure to obtain a crude alkaloid mixture. Next, the crude alkaloid mixture was subjected to silica gel column chromatography.
Using a mixed solvent obtained by gradually adding methanol to chloroform as an elution solvent, the polarity was gradually increased to obtain 89 fractions. Among them, fractions 42 to 51 containing Landrin B
The fractions were collected and the solvent was distilled off under reduced pressure to obtain a crude landrin B fraction (2.6 g). This is again subjected to silica gel column chromatography (1% methanol-chloroform mixed solvent) for purification, and then centrifugal chromatography (adsorbent; silica gel, elution solvent; diethyl ether:
Chloroform = 1: 1) and Landrin B (16 m
g) was obtained.

[Physicochemical properties of Landrin B] On silica gel TLC (trade name: Merck 5715, manufactured by Merck)
Rf values of Landrin B were 0.11 and 0.31, respectively, in a developing system: diethyl ether: ethyl acetate = 20: 1 and 3% methanol-chloroform solution. Landlin B EI mass spectrum and UV
The spectrum measurement results were as follows.

[Α] D = −34 ° (CHCl ₃ , c = 0.15)
8). EIMS, m / z (rel.int.): 352 [M +, C21H24N2O3] (100), 33
7 (12), 324 (15), 323 (45), 309 (10) and 293 (15) UV (EtOH), λmax (log ε) 211 (4.43), 252 (4.97)
and 299 (3.92)

The measurement results of ¹ H-NMR and ¹³ C-NMR spectra are as shown in Tables 1 and 2.

[0019]

[Table 1] Position dC dH −−−−−−−−−−−−−−−−−−−−−−−−−−−−−− 2 48.1-3a 64.6 3.45 dt (15, 2) 3b -4.00 br d (15) 5a 50.2 2.89 dt (12.7, 4.4) 5b-3.13 td (12.7, 2.4) 6a 28.5 2.30-2.42 m 6b-2.56 ddd (15.8, 4.4, 2.4) 7 33.4-8 139.0-9 109.7 6.81 d (2,4) 10 155.8-11 111.3 6.65 dd (8.8, 2.4) 12 116.2 7.50 br s 13 136.2-14a 124.6 5.61 dt (6, 2) 14b--15 137.5 5.40 br d (6) 15-- −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− a CDCl ₃ , 270 MHz; assignments based on COSY, HETCOR and HMBC.

[0020]

Table 2 Position dC dH −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− 16 28.2 1.04 d (5.4) 17 27.6 1.94 dd (14.6 , 5.4) 17-2.34 d (14.6) 18a 19.8 2.26 dd (14.7, 6) 18b-2.48-2.62 m 19a 34.5 1.45 br dd (13, 6) 19b-1.68-1.84 m 20 67.7-ArOMe 55.7 3.78 s NCO _{2 Me 52.7 3.86 s NCO 2 Me 154.9} - ------------------------------ a CDCl 3, 270 MHz; assignments based on COSY , HETCOR and HMBC.

From these physicochemical properties, EI mass spectrum, UV spectrum and NMR spectrum, it was found that the purified product was landrin B represented by the above chemical formula (1).

[Measurement of Melanocidal Melanoma Cell Activity] Mouse melanoma B-16 cells (10% in Eagle's minimum essential medium)
(Fetal calf serum added) was plated on a 96-well flat bottom microplate (4 × 10 ³ cells / 200 μl / well) and cultured for 1 day. Various concentrations of Landrin B were added to each well and cultured for 3 days. After the culture, the cells were stained with Giemsa's solution, and morphological changes were observed with a microscope. In addition, Landlin B
Lundurine A and lundurine C (lundurine C) isolated and purified from Copsia tenuis
C) was used as a control drug.

The results are as shown in Table 3. From these results, it was confirmed that only landlin B had excellent melanocidal melanoma cell activity.

[0024]

[Table 3] ++++: all cells killed, + to ++: morphological change,-: invalid

[Acute Toxicity of Landrin B] The LD50 value of intravenous toxicity to mice was as follows, and it was confirmed that the toxicity was lower than that of existing plant alkaloid antitumor agents. Landrin B 27.6 mg / ml vincristine sulfate 2.5 mg / ml vindesine sulfate 9.1 mg / ml vinblastine sulfate 15.2 mg / ml

[0026]

The therapeutic agent for malignant melanoma according to the present invention contains Landrin B represented by the following chemical formula (1) as an active ingredient. The therapeutic agent for malignant melanoma of the present invention comprises Landrin B
Can effectively treat malignant melanoma.

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 ──────────────────────────────────────────────────続 き Continuation of the front page (71) Applicant 596054364 Toseok Kam TOH-SEOK KAM Petaling Jaya, Daman Salautama, Jalan SS 21/21, 3 (72) Inventor Hiroaki Takahashi 1500 Inoguchi, Nakaicho, Ashigara-gun, Kanagawa Prefecture. Inside Terumo Corporation (72) Inventor Takashi Koyano 1500 Inoguchi, Nakai-machi, Ashigara-gun, Kanagawa Prefecture Inside Terumo Corporation (72) Inventor Hiroki Komiyama 5-9-1 Shirokane 5-chome, Minato-ku, Tokyo Inside the Kitasato Institute (72) Inventor Tosseok Kham Malaysia, Petaling Jaya, Daman Salautama, Jalan SS 21/21, 3 (72) Inventor Kanagasunderam Yoga Nathan Malaysia, 59100 Kuala Lumpur Lemba Pantai (no address), Sea Oh Uni Vel City Malaya

Claims (1)

[Claims] 1. A therapeutic agent for malignant melanoma, comprising a compound represented by the chemical formula (1) as an active ingredient. Embedded image