JP2003321363A - Vascularization suppressor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a prophylactic and/or treating agent useful for various diseases relating to vascularization such as a malignant tumor including a solid cancer at the pharynx, lung and large bowel, and inflammatory diseases including diabetic retinopathy, rheumatism and psoriasis. <P>SOLUTION: The vascularization suppressor and antineoplastic agent comprise a carboline derivative expressed by formula (a) (R<SP>1</SP>is no substitution or an oxygen atom; R<SP>2</SP>is a hydrogen atom, a hydroxy group, a methoxy group or -OGlc; and Y is a hydrogen atom or a methoxy group) or a quassinoid derivative expressed by formula (b) (R<SP>3</SP>, R<SP>4</SP>and R<SP>5</SP>are the same or different and each represents a hydrogen atom or a hydroxy group) as the effective ingredient. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD [0001] The present invention relates to an angiogenesis inhibitor and an antitumor agent containing a Thai plant Eurycoma harmandiana-derived component as an active ingredient.

[0002]

BACKGROUND OF THE INVENTION Angiogenesis plays an important role in development, wound healing, etc., as well as diabetic retinopathy, macular degeneration,
Retinal diseases such as retinal vein atresia and retinal artery atresia, neovascular glaucoma, rheumatism, rheumatoid arthritis, various inflammatory diseases such as psoriasis, atherosclerosis, solid tumors (stomach cancer, colon cancer, lung cancer, pancreas) It is also known to be related to pathological conditions such as proliferation / metastasis of cancer) and myocardial infarction (see Non-Patent Documents 1 to 5). In recent years, VEGF (Vascul), which is a factor that acts specifically on vascular endothelial cells and promotes angiogenesis
ar Endothelial Growth Factor), angiopoietin, and other ligands and their receptor systems have been found one after another, and the relationship between angiogenesis and the disease has been clarified. Therefore, suppressing angiogenesis is thought to lead to the treatment and prevention of these diseases. For example, for the growth of solid tumors, tumor angiogenesis is required to supply oxygen and nutrients. Therefore, if the formation of such blood vessels is inhibited and the solid tumor is put into a so-called "military attack", As a result, cancer can be expected to shrink. In diabetic retinopathy,
Oxygen deficiency promotes the formation of microcapillaries, which are believed to eventually rupture and bleed to form scar tissue, eventually leading to retinal detachment. The seriousness of can be suppressed.

On the other hand, in recent years, the tendency to attach importance to the quality of life and survival in treating diseases has been increasing socially,
For example, there is a demand for a highly safe drug that can be replaced by a drug having a cytocidal effect that acts not only on cancer cells but also on somatic cells and causes serious side effects like conventional anticancer drugs.

[0004]

[Non-Patent Document 1] Folkman et al., Nature, (1980) Vol. 28.
8, 551-556

[Non-patent document 2] Takagi Hitoshi, Cell Engineering (2000), Vol 19,
No.8, 1160-1165

[Non-Patent Document 3] Hiro Terano, Cell Engineering (1995), Vol 14,
No.4, 426-430

[Non-Patent Document 4] Richard D Connell, Exp. Opin. Ther.
Patents (2000), 10 (6), 767-786

[Non-Patent Document 5] Masafumi Shibuya, Protein Nucleic Acid Enzyme (2000), Vo
l 45, No.6, 1182-1187

[0005]

DISCLOSURE OF THE INVENTION The present invention provides an angiogenesis inhibitor, which is highly safe even when administered for a long time while maintaining its efficacy, particularly a drug effective against solid cancer, diabetic retinopathy and various inflammatory diseases. The purpose is to provide.

[0006]

In view of such circumstances, the present inventors have searched various substances having an angiogenesis-suppressing action, and as a result, have found that the Thai plant Euricoma harmandiana (Eu
Carboline derivative (a) and cacinoid derivative (b), which are components contained in rycoma harmandiana, have excellent angiogenesis inhibitory action, and prevent and / or treat various diseases related to angiogenesis including antitumor agents. They have found that they are useful as agents and have completed the present invention.

That is, the present invention has the following general formula (a):

[0008]

[Chemical 5]

[In the formula, R ¹ represents an unsubstituted or oxygen atom, and R ² represents a hydrogen atom, a hydroxy group, a methoxy group or-
Represents OGlc, and Y represents a hydrogen atom or a methoxy group. ]
The present invention provides an angiogenesis inhibitor and an antitumor agent containing the carboline derivative represented by

The present invention also provides the following general formula (b):

[0011]

[Chemical 6]

[In the formula, R ³ , R ⁴ and R ⁵ represent the same or different hydrogen atom or hydroxy group. ]
The present invention provides an angiogenesis inhibitor and an antitumor agent, which comprises as an active ingredient a carcinoid derivative represented by

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION The carboline derivative of the general formula (a) and the carcinoid derivative of the general formula (b) of the present invention are both Euricoma harmandiana, which is a plant of the Simaroubaceae family native to Thailand.
It is a component isolated from Thai name Ian-don (Phytochem
istry 56, (2001), 383-386, Phytochemistry 57, (2001),
1205-1208). The root of Eurycoma harmandiana is used in Thailand as a remedy for aphrodisiacs, tumors or malaria, but no pharmacological studies have been conducted on the above ingredients.

In the carboline derivative represented by the formula (a) of the present invention, -OGlc represented by R ¹ means O-glucoside, preferably O-β-glucopyranoside.

The carbophosphate derivative (a) of the present invention
Is added with strong acids such as hydrochloric acid, sulfuric acid, nitric acid,
It is possible to form pharmaceutically acceptable salts, and such salts are also included in the present invention. In addition, the carcinoid derivative (b) of the present invention includes, for example, acetic acid, butyric acid, propionic acid,
Lower fatty acids such as lactic acid can be added to form a pharmacologically acceptable acetate, which is also included in the present invention.

Preferred specific examples of the carboline derivative (a) and the carcinoid derivative (b) of the present invention are shown below.

[0017]

[Chemical 7]

The carboline derivative (a) and the carcinoid derivative (b) of the present invention can be obtained by extraction, fractionation and purification from plants such as Euricoma harmandiana (Phytochemistry 56, (2001), 383). -386, Phyt
ochemistry 57, (2001), 1205-1208, AD Kinghorn et
al., J. Nat. Prod., 54 (5), 1360-1367 (1991)., K. Mit
sunaga et al., Phytochemistry, 35 (3), 799-802 (199
Four).). In addition, commonly used synthetic methods and semi-synthetic methods,
Alternatively, it can be obtained by a combination of known synthesis methods.

The method for obtaining the carboline derivative (a) and the carcinoid derivative (b) by the extraction method will be described below. The extraction is not particularly limited, and Euricoma
Method of heating Harmandiana root, wood, branch, leaf, or seed with water, a water-soluble organic solvent such as lower alcohol such as methanol or ethanol, or acetone at room temperature or with heating, and water and these water-soluble organic solvents A method of extracting with a mixture, further chloroform, dichloromethane, acetates, toluene, a method of extracting with a mixture of a hydrophobic organic solvent such as a supercritical fluid such as carbon dioxide gas and a water-soluble organic solvent such as methanol can be used, but in particular, Cut or crush the roots, water, lower alcohols such as methanol, ethanol,
It is preferable to chill soak in preferably ethanol, more preferably 95% ethanol for about 12 hours (three times).

The extract thus obtained is concentrated and then distributed with a suitable organic solvent such as dichloromethane, ethyl acetate, n-butanol, etc., followed by silica gel, chemically bonded silica gel,
The carboline derivative (a) and the carcinoid derivative (b) can be isolated by fractionating and purifying using column chromatography with alumina, celluloses, and porous polymers in an appropriate combination (see Production Examples 1 to 1 below).
8).

The carboline derivative (a) and the carcinoid derivative (b) of the present invention thus obtained have a vascular endothelial cell growth inhibitory activity and a vascular cell capillary network formation inhibitory activity, as will be shown in the Examples below, and therefore have a vascular property. It is useful as an anti-neoplastic agent and also as an anti-tumor agent because it has a cytotoxic activity against tumor cells.

The angiogenesis inhibitor and antitumor agent of the present invention are:
Within a range that does not reduce the effect, it is mixed with a carrier such as a dispersion aid, an excipient, etc., which is usually used for formulation, and powders, liquids, capsules, suspensions, emulsions, syrups, elixirs, It can be used in the form of internal preparation such as granules, pills, tablets, troches, limonades, or injections.

Examples of such a carrier include water-soluble monosaccharides such as mannitol, lactose and dextran, oligosaccharides or polysaccharides; gel-forming or water-soluble celluloses such as hydroxypropyl cellulose, hydroxypropylmethyl cellulose and methyl cellulose. Water-absorbent and sparingly water-soluble celluloses such as crystalline cellulose, α-cellulose, cross-linked sodium carboxymethyl cellulose and derivatives thereof; for example, hydroxypropyl starch, carboxymethyl starch, cross-linked starch, amylose, amylopectin, pectin and Water-absorbing and poorly water-soluble polysaccharides such as derivatives thereof; for example, water-absorbing and poorly water-soluble gums such as gum arabic, tragacanth gum, glycomannan and their derivatives; for example polyvinylpyrrolidone Crosslinked polyacrylic acid and its salts,
Examples thereof include crosslinked vinyl polymers such as crosslinked polyvinyl alcohol, polyhydroxyethylmethacrylate and their derivatives; and lipid sugars that form molecular aggregates such as liposomes such as phospholipids and cholesterol.

The angiogenesis inhibitor or antitumor agent of the present invention is
The carboline derivative (a) or the carcinoid derivative (b) may be used alone, or two or more kinds may be used in combination.

The dose of the angiogenesis inhibitor or antitumor agent of the present invention may be appropriately adjusted according to the patient's symptoms and the like. For example, as the carboline derivative (a) or the carcinoid derivative (b), 0.01 ~ 2000 mg / day, preferably 0.1-500 mg / day, especially 0.5-200 mg
It is preferably administered daily.

[0026]

EXAMPLES The present invention will be described in more detail with reference to examples. Production Example 1 Production of Eurycoma halmandiana extract Dried bittern family Euricoma halmandiana (Eu
7 kg of roots of rycoma harmandiana) were extracted with 18 L of 95% ethanol at 90 ° C. for 3 hours. After cooling to room temperature and filtering with filter paper, the solvent was distilled off under reduced pressure with a water-jet pump.
0 g of extract concentrate was obtained.

Production Example 2 Production of compound (a1)

[0028]

[Chemical 8]

The concentrate of the extract was extracted with dichloromethane, ethyl acetate and n-butanol (2 L each) in this order and concentrated to dryness to obtain a dichloromethane fraction, an ethyl acetate fraction, a butanol fraction and a residue. Next, the butanol fraction (45
g) was subjected to column chromatography packed with a copolymer (porous polymer) consisting of divinylbenzene and styrene, and eluted with water, methanol, and acetone in this order. The fraction eluted with methanol was further subjected to silica gel column chromatography to obtain a mixture of ethyl acetate-methanol (9: 1) and ethyl acetate-methanol-water (4: 1: 0.1, 7 :).
A mixture of 3: 0.3 or 6: 4: 1) was used as a mobile phase for elution to obtain 9 fractions (A, B, C, D, E, F, G, H and I). Of these, fractions E and F were subjected to column chromatography using octadecyl silylated silica gel as a packing material and eluted with 70-50% hydrous methanol to give compound (a
75 mg of 1) was isolated.

Properties: Yellow amorphous powder MS (HR-FAB-MS): m / z 399.1205 [M + H] ⁺ (C ₂₀ H ₁₉ N ₂ O ₇ 39
9.1192); ¹ H-NMR (DMSO-d ₆ ) δ _ppm : 8.79 (1H, d , J = 5Hz), 8.29
(1H, d , J = 9Hz), 8.211 (1H, d , J = 5Hz), 8.17 (1H, d ,
J = 2Hz), 8.12 (1H, d , J = 10Hz), 7.30 (1H, dd , J = 9,
2Hz), 6.97 (1H, d , J = 10Hz), 5.45 (1H, d , J = 3Hz),
5.03 (1H, dd , J = 1, 3Hz), 4.57 (1H, t , J = 3Hz), 4.08
(1H, q , J = 3Hz), 3.72 (1H, m ), 3.54 (1H, m ), 3.4-
3.2 ( m ), 3.16 (1H, d , J = 3Hz); ¹³ C-NMR (DMSO-d ₆ ) δ _ppm : 159.7, 158.8, 145.9, 139.
9, 139.8, 135.2, 131.7, 129.2, 128.1, 124.3, 118.
1, 116.4, 114.2, 104.4, 101.0, 77.2, 76,5,73.3, 6
9.5, 60.6

Production Example 3 Production of compound (a2)

[0032]

[Chemical 9]

Dichloromethane fraction of Preparation Example 2 (90 g)
Was subjected to column chromatography packed with silica gel, and a mixture of dichloromethane and a mixture of dichloromethane-methanol (1
9: 1, 9: 1, 4: 1, 3: 2) and methanol were eluted in this order to obtain 6 fractions (D-1 to D-6). Of these, Fraction D-2 was eluted by silica gel chromatography with a linear gradient from dichloromethane to dichloromethane containing 5% methanol. Furthermore, reverse phase chromatography using octadecyl silylated silica gel (solvent system: 60% to 0
% Water-containing methanol) to give compound (a2) 367m
g was obtained.

Properties: Yellow needle crystals MS (HR-MS): m / z 236.0590 [M] ⁺ (C ₁₄ H ₈ N ₂ O ₂ 236.058
6); ¹ H-NMR (CDCl ₃ ) δ _ppm : 8.76 (1H, d , J = 5Hz), 8.17 (1
H, d , J = 9Hz), 8.14 (1H, d , J = 5Hz), 8.10 (1H, d , J =
10Hz), 8.00 (1H, d , J = 3Hz), 7.00 (1H, dd , J = 9, 3H
z), 6.96 (1H, d , J = 10Hz); ¹³ C-NMR (CDCl ₃ ) δ _ppm : 160.5, 158.9, 146.0, 140.5,
140.0, 135.60, 131.7, 129.9, 128.0, 124.7, 116.0,
115.6, 114.0, 103.0

Production Example 4 Production of compound (a3)

[0036]

[Chemical 10]

From the fraction D-2 of Production Example 3, the compound (a
At the same time as 2), 900 mg of the compound (a3) as a separate fraction
Got

Properties: Yellow crystals Positive HR-FAB-MS: m / z 251.2660 (C ₁₅ H ₁₁ N ₂ O ₂ requi
res 251.2668); ¹ H-NMR (DMSO-d ₆ ) δ _ppm : 8.74 (1H, d , J = 5Hz), 8.14
(1H, d , J = 2Hz), 7.98 (1H, d , J = 10Hz), 7.89 (1H, d ,
J = 9Hz), 7.80 (1H, d , J = 5Hz), 7.03 (1H, dd , J = 9, 2H
z), 6.93 (1H, d , J = 2Hz), 3.98 (1H, s ); ¹³ C-NMR (DMSO-d ₆ ) δ _ppm : 162.5, 159.7, 146.0, 141.
2, 139.9, 135.6, 132.3, 130.4, 128.5, 123.3, 117.
2, 115.5, 114.2, 101.2, 56.0

Production Example 5 Production of compound (a4)

[0040]

[Chemical 11]

From the fraction D-2 of Production Example 3, the compound (a
At the same time as 2), 28 mg of compound (a4) was obtained as a separate fraction.

Property: Light yellow needle crystal Positive HR-FAB-MS: m / z 221.0718 (C ₁₄ H ₉ N ₂ O ₂ requir
es 221.0714); ¹ H-NMR (DMSO-d ₆ ) δ _ppm : 8.61 (1H, d , J = 12Hz), 8.37
(1H, d , J = 11Hz), 8.32 (1H, d , J = 6 Hz), 7.96 (1H,
d , J = 12 Hz), 7.78 (1H, d , J = 6Hz), 7.42-7.72 (2H,
m ), 6.92 (1H, d , J = 11Hz);

Production Example 6 Production of compound (a5)

[0044]

[Chemical 12]

Dichloromethane fraction of Production Example 2 (90 g)
Was subjected to column chromatography packed with silica gel, and a mixture of dichloromethane and dichloromethane-methanol (1
Elution in the order of 9: 1, 9: 1, 4: 1) and 6 fractions (E-
1 to E-6) were obtained. Of these, the fraction E-2 was eluted with a linear gradient from dichloromethane to dichloromethane containing 5% methanol by silica gel chromatography.
Further, it was fractionated by reverse phase chromatography using octadecylsilylated silica gel to obtain 8.0 mg of compound (a5).

Positive HR-FAB-MS: m / z : 281.0931 (C ₁₆ H
₁₃ N ₂ O ₃ requires 281.0926) ¹³ C-NMR (CD ₃ Cl, 100MHz) 159.6, 152.3, 148.0, 145.
7, 139.6, 135.7, 134.6, 131.9, 130.6, 128.6, 116.
4, 115.1, 104.0, 100.3, 56.4, 54.6,

Production Example 7 Production of compound (b1)

[0048]

[Chemical 13]

The butanol fraction of Production Example 2 was subjected to the same operation to elute Fraction B obtained by silica gel chromatography using a linear gradient of dichloromethane-5% methanol-containing dichloromethane to fractionate into 6 fractions. (Fraction 2-1
To 2-6). Fractions 2-4 were subjected to column chromatography using octadecyl silylated silica gel as a packing material, and 50-
Elution with 0% hydrous methanol gave 268 of compound (b1).
mg was obtained.

Properties: colorless amorphous Negative HR-FAB-MS: m / z 377.1609 (C ₂₀ H ₂₅ O ₇ requires
377.1600); ¹ H-NMR (C ₅ D ₅ N) δ _ppm : 6.08 (1H, br.s ), 4.54 (1H, b
rs ), 4.35 (1H, s ), 4.11 (1H, d , J = 9Hz), 3.95 (1H,
d , J = 4Hz), 3.73 (1H, d , J = 9Hz), 3.35 (1H, dd , J = 19,
14Hz), 3.29 (1H, s ), 3.00 (1H, br.d , J = 12Hz ), 2.8
6 (1H, dd , J = 19, 5Hz), 2.42 (1H, m ), 2.16 (1H, dt ,
J = 12Hz), 1.96 (1H, m ), 1.71 (3H, s ), 1.52 (3H, s ),
1.09 (3H, d , J = 7Hz); ¹³ C-NMR (C ₅ D ₅ N) δ _ppm : 197.5, 170.5, 162.3, 126.1,
110.6, 84.4, 79.6,78.6, 71.4, 46.2, 45.4, 44.5, 4
2.7, 42.5, 31.6, 30.6, 26.1, 22.4, 13.3, 10.3

Production Example 8 Production of compound (b2)

[0052]

[Chemical 14]

Six fractions of Production Example 7 (fractions 2-1 to 2-
Of 6), Fraction 2-6 was subjected to silica gel column chromatography and eluted with a mixed solution of ethyl acetate-methanol (9: 1) to obtain 24 mg of compound (b2).

Properties: colorless amorphous Negative HR-FAB-MS: m / z 393.1557 (C ₂₀ H ₂₅ O ₇ require
s 377.1600); ¹ H-NMR (C ₅ D ₅ N) δ _ppm : 6.07 (1H, br.s ), 5.44 (1H,
d , J = 11Hz), 4.67 (1H, t , J = 2Hz), 4.30 (1H, s ), 4.11
(1H, d , J = 9Hz), 4.08 (1H, d , J = 2Hz), 3.85 (1H, d ,
J = 9Hz), 3.29 (1H, s ), 3.09 (1H, br.d , J = 12Hz ), 2.6
9 (1H, m ), 2.33 (1H, dd , J = 11, 6Hz), 2.15 (1H, dt ,
J = 15, 3Hz), 2.00 (1H, td , J = 15, 2Hz), 1.71 (3H,
s ), 1.70 (3H, d , J = 7Hz), 1.55 (3H, s ); ¹³ C-NMR (C ₅ D ₅ N) δ _ppm : 197.4, 174.1, 162.5, 126.1,
110.8, 84.5, 80.4,78.2, 71.5, 49.6, 47.5, 45.8, 4
5.5, 42.3, 38.6, 33.0, 26.2, 22.3, 16.3, 10.6

Production Example 9 Production of compound (b3)

[0056]

[Chemical 15]

Ethyl acetate fraction of Preparation Example 2 (27.5 g)
Was subjected to column chromatography using octadecyl silylated silica gel as a packing material, eluted with 70-50% hydrous methanol, and further purified with 60% -0% hydrous methanol to obtain 84 mg of compound (b3).

Properties: colorless amorphous Negative HR-FAB-MS: m / z 409.1501 (C ₂₀ H ₂₅ O ₉ require
s 409.1498); ¹ H-NMR (C ₅ D ₅ N) δ _ppm : 6.13 (1H, br.s ), 5.62 (1H,
s ), 4.64 (1H, d , J = 9Hz), 4.39 (1H, s ), 4.14 (1H,
d , J = 4Hz), 4.12 (1H, d , J = 3Hz), 4.04 (1H, d , J = 9H
z), 3.51 (1H, s ), 3.17 (1H, br.d , J = 14Hz ), 2.84 (1
H, m ), 2.27 (1H, dd, J = 14, 3Hz), 2.03 (1H, dt , J = 1
4, 3Hz), 1.85 (3H, d , J = 7Hz), 1.77 (3H, s ), 1.62 (3H, s ); ¹³ C-NMR (C ₅ D ₅ N) δ _ppm : 197.5, 17
4.5, 162.7, 126.0, 110.3, 84.5, 79.6,76.5, 75.1, 7
1.8, 67.3, 52.7, 46.9, 45.6, 42.2, 25.7, 22.4, 14.
0, 10.6

Example 1 As a test sample for the vascular endothelial cell growth inhibitory activity, the compound (a) obtained in the above Production Example was used.
1) to (a5) and (b1) to (b3) were used.
Human umbilical cord endothelial cells (HUVE cells; purchased from Dainippon Pharmaceutical Co., Ltd.) were used as cells.

1) Cells and Culture HUVE cells were cultured in CS-C medium (Cell Systems Corpora).
culture) and 10% under the condition of 5% CO ₂ and 37 ° C.
It was cultured in a 0 mm collagen type I coating dish (manufactured by Iwaki Glass). When used in the experiment, the cells were washed with PBS (−) before reaching confluence, trypsinized, and centrifuged at 1000 × g for 8 minutes to dilute the cell suspension to an appropriate concentration.

2) Growth inhibition assay HUVE cells were seeded on a 96-well collagen type I coated plate at 5 × 10 ³ cells / 50 μL / well, and cultured under conditions of 5% CO ₂ and 37 ° C. After 4 hours, 50 μL of Dulbecco's modified Eagle's medium containing the test substance was added to each well and cultured for 4 days (5% C
O ₂ , 37 ° C.). After culturing, 10 μL of WST-
8 solution (Tetra Color One Cell proliferation assay s
ystem, manufactured by Seikagaku Corporation) was added to each hole. As it is 2
After incubating for 5 hours (5% CO ₂ , 37 ° C), a microplate reader (SPECTRA MAX 250, Molecular Devices Ca, U
The absorbance at 490 nm was measured by SA). Assuming that the absorbance of the control group (DMSO only added) is 100%, D
50% growth inhibitory concentration of the test substance dissolved in MSO (IC
₅₀ ) asked. The results obtained are listed in Table 1.

[0062]

[Table 1]

As shown in Table 1, all the compounds have H
Suppresses the growth of UVEC, especially the compounds (a2) and (b)
A high effect was recognized in 1).

Example 2 As a test sample for the activity of inhibiting the formation of capillary networks of vascular cells, the compound (a) obtained in the above Production Example was used.
1) to (a3) and (b1) to (b2) were used.
In addition, bovine aortic endothelial cells (BAE cells; purchased from Dainippon Pharmaceutical Co., Ltd.) were used as cells, and 10% FCS-containing Dulbecco's modified Eagle medium (DM
EM: Nissui Pharmaceutical Co., Ltd. was used.

1) Cells and Culture BAE cells were cultured in Dulbecco's modified Eagle medium (Nissui Pharmaceutical) containing 10% fetal bovine serum and antibiotics. BAE cells at 10% under the condition of 5% CO ₂ and 37 ° C.
When cultured in 0 mm collagen type I coated dish and used in the experiment, it was washed with PBS (-) before reaching confluence, trypsinized, and 1000
What was centrifuged at xg for 8 minutes was diluted to a cell suspension having an appropriate concentration.

2) Capillary network (lumen) formation in collagen type I gel Montesano et al. (Montesano, R.et.al, J. Cell Bi
ol. 97: 1648-1652, 1983) with a partial modification. Collagen type I solution was prepared from adult rat tail and diluted with 0.1% (v / v) acetic acid solution. 10
1 volume of double concentration DMEM, 4 volumes of collagen type I solution and 5 volumes of 11.76 mg / mL sodium hydrogen carbonate solution were mixed on ice to prepare a collagen mixture. 50 μL of the ice-cold collagen mixture was dispensed into each well of the 96-well culture plate and gelled at 37 ° C. About 2 × 10 ⁴ BAE cells were seeded in each well and cultured for 1-2 days so that the gel surface was confluent. The medium on the gel was removed, 50 μL of ice-cold collagen mixture was newly added, and the mixture was left at 37 ° C. for 20 minutes to solidify the gel. DM
50 μL of Dulbecco's modified Eagle medium containing the test substance dissolved in SO was added to each well and cultured. 72 hours later, the formation of the capillary network (lumen) was observed under a microscope. The criteria for the effect of the test substance are negative (ineffective) when the network is formed, positive (effective) when the network is not formed at all or is fragmented even if formed, and apparently no network due to cytotoxicity. When it was not formed, it was regarded as toxic. In addition,
If the test result is positive, the effective minimum concentration of the test substance in the test concentration range is described. The results obtained are listed in Table 2.

[0067]

[Table 2]

As shown in Table 2, each compound was found to have a capillary network formation inhibitory activity.

Example 3 As a test sample for cytotoxic activity against tumor cells, the compound (a) obtained in the above Production Example was used.
1) to (a4) and (b1) to (b3) were used.
Moreover, as a tumor cell, a pharyngeal cancer cell (KB cell; Amer
ican Type Culture Collection (ATCC)), lung cancer cells (A549 cells; purchased from ATCC),
Colorectal cancer cells (HT29 cells; purchased from ATCC) were used. In the case of A549 cells and HT29 cells, 10% heat-inactivated fetal calf serum (FCS), 10 mg /
A cell suspension prepared in F-12 medium (manufactured by Sigma-Aldrich) containing ml gentamicin was dispensed at 240 ml per well of a 96-well plate, so that the number of cells per well was 4 × 10 ³ cells. Sowed. In the case of KB cells, the cell suspension prepared in Dulbecco's modified Eagle medium containing 10% FCS was dispensed at 50 ml per well of a 96-well plate on day 0 of the experiment. Was sowed to be 2 × 10 ³ cells.
These cells were cultured under the conditions of 5% CO ₂ and 37 ° C. On the first day of the experiment, a medium containing a constant concentration of the test sample was added to the cell culture system by serial dilution. On the 3rd day of the experiment, the cytotoxic activity of the test sample was measured using Tetra Color One cell proliferation (manufactured by Seikagaku Corporation) for A549 cells and HT29 cells, and CellTiter 96 ^{R for} KB cells.
It was measured using AQueous One Solution Cell Proliferation (manufactured by Promega). The cytotoxic activity measurement method used this time is to detect and quantify the activity of dehydrogenase in intracellular mitochondria spectroscopically using a synthetic chromogenic substrate (WST-8 and MTS). Microplate absorptiometer Was used to measure the absorbance at a wavelength of 490 nm. The concentration of the test sample when the number of cells was reduced to 50% was determined by setting the number of cells when the various cells were cultured in a medium not containing the test sample as 100%, and this was calculated as IC _50.
(Mg / ml). The strength of the activity of the test sample is IC _50.
It was evaluated by. The results obtained are listed in Table 3.

[0070]

[Table 3]

As shown in Table 3, compounds (b1) to
(B3) exhibited cytotoxic activity against all cells of KB cells, A549 cells, and HT29 cells, and in particular, high activity was observed with compound (b1). In addition, compound (a2) exhibited cytotoxic activity against KB cells and A549 cells. On the other hand, the compounds (a1), (a3) and (a4) showed cytotoxic activity on KB cells and were found to have high selectivity.

Example 4 As a test sample for cytotoxic activity against tumor cells, the compound (a) obtained in the above Production Example was used.
1) was used. Moreover, lung cancer cells (A
549 cells; purchased from ATCC) were used. The test was carried out in the same manner as in Example 3, the cytotoxic activity of the test sample was measured on the fifth day of the experiment, and the strength of the activity of the test sample was evaluated by IC ₅₀ . The results obtained are listed in Table 4.

[0073]

[Table 4]

As shown in Table 4, the compound (a1) was A
Cytotoxic activity was observed on 549 cells.

[0075]

INDUSTRIAL APPLICABILITY Since the carboline derivative (a) and the carcinoid derivative (b) have an excellent anti-angiogenic effect and an antitumor effect, malignant cancers such as pharyngeal cancer, lung cancer and colon cancer It is useful as a preventive and / or therapeutic agent for various diseases associated with angiogenesis such as tumors, diabetic retinopathy, inflammatory diseases such as rheumatism and psoriasis.

Front page continuation (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 29/00 A61P 29/00 35/00 35/00 // C07D 471/16 C07D 471/16 493/08 493/08 B (72) Inventor Tripetch Kanchanaphum 2-3, Hikarigaoka, Higashi-ku, Hiroshima-shi, Hiroshima Asian Cultural Center (72) Inventor Shusuke Hashimoto 1-1-1 Higashishimbashi, Minato-ku, Tokyo Yakult Head Office (72) Inventor Ritsuo Aiyama 1-1-19 Higashishimbashi, Minato-ku, Tokyo Yakult Honsha Co., Ltd. (72) Inventor Ken Matsuzaki 1-1-19 Higashishimbashi, Minato-ku, Tokyo Yakult Honsha Co., Ltd. In-house F-term (reference) 4C065 AA07 AA18 BB09 CC03 DD02 EE03 HH01 JJ01 KK01 LL04 PP01 4C071 AA03 AA08 BB01 BB07 CC12 EE05 FF17 HH05 HH09 KK17 LL01 4C0 86A16 A08 CA26 CB01 BA32 B11 ZABA CB01 ZA14B14 ZA14 B11 ZA33 B11 ZA14 B11 ZA14 B11 ZA14 B11 ZA14 B11 ZA33 B11 B03 ZA14 ZA36 ZB11 ZB26

Claims (4)

[Claims] 1. The following general formula (a): [In the formula, R ¹ represents an unsubstituted or oxygen atom, R ² represents a hydrogen atom, a hydroxy group, a methoxy group or —OGlc,
Y represents a hydrogen atom or a methoxy group. ] An angiogenesis inhibitor containing a carboline derivative represented by the following as an active ingredient. 2. The following general formula (a): [In the formula, R ¹ represents an unsubstituted or oxygen atom, R ² represents a hydrogen atom, a hydroxy group, a methoxy group or —OGlc,
Y represents a hydrogen atom or a methoxy group. ] An antitumor agent comprising a carboline derivative represented by the following as an active ingredient. 3. The following general formula (b): [In the formula, R ³ , R ⁴ and R ⁵ represent the same or different hydrogen atoms or hydroxy groups. ] An angiogenesis inhibitor, which comprises a cacinoid derivative represented by the following as an active ingredient. 4. The following general formula (b): [In the formula, R ³ , R ⁴ and R ⁵ represent the same or different hydrogen atoms or hydroxy groups. ] The antitumor agent which uses the cacinoid derivative represented by these as an active ingredient.