JPH02233610A - Vascularization inhibitor - Google Patents

Abstract

PURPOSE:To provide a vascularization inhibitor having a remarkable vasculrization-inhibiting activity and useful for preventing or treating diseases caused by the abnormal multiplication of blood vessels by containing WF2015A and/or WF2015B as active ingredients. CONSTITUTION:A vascularization inhibitor having the above-mentioned activity and useful for preventing or treating diabetic retinopathy, rheumatic arthritis, immature infant retinopathy, senile macular degeneration, etc., contains WF2015A (X = formula II) and/or WF2015B (X = formula III) represented by formula I (X is formula II or III) as active ingredients. The active ingredients can be mixed with a carrier acceptable as a drug and orally or parenterally administered in a form of capsules, tablets, granules, powders, oral tablets, sublingual tablets, solutions, etc., preferably in a does of usually 0.01-10mg/kg/ day in the case of injection and 0.5-50mg/kg/day in the case of oral administration.

Description

DETAILED DESCRIPTION OF THE INVENTION This invention relates to a novel angiogenesis inhibitor.
More specifically, the present invention relates to an angiogenesis inhibitor containing WF 2015A and/or WF2015B as an active ingredient. The growth is
A variety of angiogenesis inhibitors have been known in the past, but these did not necessarily have properties that would satisfy them as pharmaceuticals. Therefore, the inventors set out to develop a new type of angiogenesis inhibitor. It is shown that these are the active ingredients of the angiogenesis inhibitor of this invention. (means WF2015B) The compound represented by the above general formula (I) is, for example, disclosed in Japanese Patent Application No. 63
It can be produced by the method described in No.-262044, more specifically by the method shown in the production example below. WF 2015A and B obtained in the production examples described below have the following physical and chemical properties: (1) WF2015A ●》Molecular weight 41G [FAB-MS: s/x 411 (M
+H)] b) Elemental analysis C84. ss: H a. oa: 0 27.
39 (calculated as the difference) C) Specific rotation [α1 -52@O (C-1.0, C}130H) d) UV absorption spectrum terminal absorption (CICI, middle) e) IR absorption spectrum 1170.112G, 1100.1070.1050,
1000. 980, 920, 880.
830 cm-'lf) HNMR absorption spectrum (CDCIs) δ: 8.
95 (IH, dd, J-15.5 and 4.5HZ)
．． 6.20 (IH.dd, J-15.5 and 2Hx
)． 5.70 (IH, wide S》, 5.20 (IH, wide t, J-7}1x), 4.31
(IH 劃》, 3.90 (IH 劃). 3.68 (IH, dd, J-11 and 3Hz) , 3
．． 44 (3Ls). 2.98 (IH, d, J-4.21). 2J2 (IH
．． dd. J-6.5 and l! Hz), 2.58(I
H, d, J-4.2tlz), 2.36 (III, m)
．． 2.18 (1B, +s), 2.10 (1B, +a), 2.00 (IN, m), 1.98 (IH, d, J-11Hx), 1.87 (IH
, m). 1.74 (3H, d, J-1+1z) . 1.86
(3}1, d, J-1+1z) . 1.21 (3H.s
). 1.14 (311, d, J-8.5HZ). 1.07(
1}1. @), g) “C NMR absorption spectrum (CDCli) δ
: 1B5.8(S). 146.6(d). 135.0 ($). 121. ll(d). 118.3 (d). 79. 4 (d), 74.5 (d). 69.9(.d), 66.3(d), 81.1(d). 59.4 (s). 5L9 (S). sa. 7 (q). 50.7 (t), 48.2 (d), 29.2 (t), 27.2 (t), 25.6 (q). 25.6 (t), 17.9 (+1). 17.1 (q), 13.7 (q), h》Solubility Soluble: Chloroform, methanol, acetone, ethanol Insoluble: n-hexane, water》Positive color reaction: Reaction with iodine vapor Negative: Ninhydrin Reaction, Morisch reaction, ferric chloride reaction”) Purchase properties Neutral substances (2) WF2015B a》Molecular weight 447 [F^tMS: m/z 469 (M+Na
)] b) Elemental analysis C 58.03: I% 1.94. Clf. '・3l
: 0 2B. 72 Calculated as C difference) C》Specific optical rotation d) e》 UV absorption spectrum terminal absorption (in CHCIs) IR absorption spectrum CHCIs: 35?0. 3400, ν max×2820. 1?10, 1440, 1400, 1300, 1270, 1080, 1020. 910 cm-' 29150, 1,660. 1380. 1220. 980. 2940, 2870. 1560.14B0, 1360,1340, 1170,1120, 960,940, f) 18 NMR (400Mllz.CDCls)δ
: 6.99 (III, dd, J-5 and ls.!J
z), 6.20 (IL(ld, J-2 and 15.5
Hz), 5.55 (IH)), s. ta (to, haze). 4.36 (IH, fat), 3.98 (IH, dq, J-4 and 6Hz) .3.
87 (1B, d, J-ILHz), 3.49 (IH,
d, J-1182). 3.31 (IH, +s), 3.30 (3H, s). 2.97 (IH, t, JJ. SH, z). 2.56-
2.40 (2B, @) . 2.17 (ILm), 2.05 (LH, s+), 1.90-1.75 (2H, l). 1.73 (3
H,s). 1.61! (3H,s), 1.41 (3Ls), 1.40 (IH, wide d, J-1411z), 1.2
0 (3M, d, J-611z) g) ”C NMR (loOMHx, CDCIs)
δ: 165.7 (s), 14L3 (d), 134.8 (s). 122.4 (d). 118.2(d). 78.7 (d). ? @． Z (a), 74.5 (d). 70.0 (d), 66.1 (d), 64.0 (s), 82.3 (d), 58.7 (d), 50.5 (t), 43.3 (d). 29.1 (t). 27. 5 (t), 25.8 (Q). 23.5 (t). 22.2 (q). 17.9 (q). 17. 6 (q) h) Solubility Soluble: Chloroform, methanol, n, ethanol (N-hexane, water) Positive color reaction: Reaction with iodine vapor Aceto Negative: Ninhydrin reaction, Molissche reaction, chloride chloride From the results of physical and chemical sex trade and separate research, WF
The chemical structures of 2015A and B were determined as described above. Next, the biological contributions of W2015A and B will be explained using the following test examples. 15% fetal bovine serum 100 ug/mI ECG in a 966 microtiter plate coated with fibronectin.
S (Endothelial cell growth
xupple-ment) and MCDB151 medium containing 10 ug/m'1 hebarin, and then a methanol solution of WF2015A or WF2015B at a fixed concentration was added to prepare dilutions of various concentrations. Furthermore, a human mire vein endothelial cell solution was prepared using MCDB151 medium containing the above components, and each well (fi
nal volui+s2 0 0 μ1),
C'O. 37°C in an ink evaporator. Cultured for 5 days. After culturing, MTT method T. MosmanHJ. Ismu
nol. Methods 65.55.1983) to measure the degree of cell proliferation, and WF 2015A or W
The inhibition rate of vascular endothelial cell proliferation against F2015B was calculated. As a result, WF 2015A and WF 2015
B shows a strong inhibitory effect on the proliferation of vascular endothelial cells,
Those ICS. Both values were 2×10” pg/■l. 2.5 μl of the solution and 2.5 μl of the 0.1% methylcellulose aqueous solution.
A sample disk prepared by mixing 5 μl and drying was placed on the chorioallantoic membrane, the window was again closed with a skin closure, and incubation was continued at 37°C. Furthermore, the inhibitory effect of WF 2015A on angiogenesis was observed from the state of development of new blood vessels in the chorioallantoic membrane after 3 days. As a result, in the WF 2015A 10 μg administration group,
Almost complete (approximately 100%) antiangiogenic effect was observed. Approximately 1 l of egg white is aseptically extracted from a 3-day-old fertilized chicken egg using a syringe needle.
Scratch the eggshell with a dental drill, taking care not to damage the chorioallantoic membrane, and break the shell with tweezers for about 1 c++.
”Open the window and cover it with a skin closure 37
Misaki eggs were incubated at ℃. After 24 hours, a certain concentration of W was added in advance.
F 2015A Methanol 1 Harvest New Year: The angiogenic effect within the rabbit cornea is due to M. ^, Gimbron
e et al. method (Journal National Ca
ncer Institute52, 413.1974
) was investigated. Specifically, using a rabbit (New Zealand White breed, female, 9 weeks old), under Nembutal anesthesia, an approximately 2 mm incision was made in the central part of the cornea with a scalpel, and a pocket-like structure was made in the corneal part through the incision using an ophthalmological grade iris subatel. Had made. At the bottom of the pocket, Elvax (+
Insert a pellet (1 mm square), and then
Flbroblast Growth Factor,
An Elvax pellet containing 1 μg of comb brain origin was then inserted, and new blood vessels extending from the corneal ring toward the FGF pellet were observed over time (2 weeks). As a result, W F 2GISA completely inhibited (approximately 100%) blood vessels generated by FGF in the 200 μg administration group. A physiological saline solution of WF 2015A or B was intraperitoneally administered to five ICR mice (female, 5 weeks old) once a day for 5 days (one dose: 100 μg/kg). As a result, no abnormal symptoms were observed in the mice. As is clear from the above test examples, the angiogenesis inhibitor of the present invention has a remarkable angiogenesis inhibitory effect and is effective against diseases caused by abnormal proliferation of blood vessels, such as diabetic retinopathy, rheumatoid arthritis, etc. It is used to prevent or treat retinopathy of prematurity, senile macular degeneration, etc. The active ingredient of the angiogenesis inhibitor of the present invention is mixed with a pharmaceutically acceptable carrier and administered to humans in the form of a formulation such as a capsule, tablet, granule, powder, oral tablet, sublingual tablet, liquid, etc. It can be administered orally or parenterally to suckling animals. Pharmaceutically acceptable carriers include various organic or inorganic carriers customary for pharmaceutical purposes, such as excipients such as sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, etc. , calcium carbonate, etc.), binders (cellulose, methyl cellulose, hydroxybrobyl cellulose, polybrobyl pyrrolidone, ze)tin, gum arabic,
Polyethylene glycol, sucrose, starch, etc.], disintegrants (e.g., starch, carboxymethyl cellulose, carboxymethyl cellulose calcium, hydroxybrobyl starch, sodium glycol starch, sodium bicarbonate, calcium phosphate, calcium citrate, etc.), Thickeners (e.g. magnesium stearate, Aerosil, talc, sodium lauryl sulfate, etc.)
, flavoring agents (e.g. citric acid, menthol, glycine, orange powder, etc.), preservatives (sodium benzoate, sodium bisulfite, methylbarben, probylbarben, etc.), stabilizers (citric acid, sodium citrate, acetic acid, etc.) ), suspending agents (e.g., methylcellulose, polyvinylpyrrolidone, aluminum stearate, etc.), dispersing agents (e.g., surfactants, etc.), aqueous diluents (e.g.,
water), oils (e.g., sesame oil, etc.), base waxes (e.g., cocoa butter, polyethylene glycol, white petrolatum, etc.), etc. The dosage of the active ingredient depends on the type of disease, the patient's weight and/or or age, and should be modified depending on various factors such as the route of administration. The preferred dose of WF 2015A or B is usually
0 for injections. In the case of oral administration, the dosage is appropriately selected from the range of 0.5 to sogeg/kg/day. The following production example is intended to explain a specific method for producing the active ingredient of the angiogenesis inhibitor of the present invention. Soluble starch 2%, corn starch 1%, glucose 1%, cottonseed flour 1%, dry yeast 1%, pebtone 0.5%
, an aqueous medium containing 0.5% corn stew liquor and 0.2% calcium carbonate (pl 16.0) (
160ml) in a 500μl Erlenmeyer flask19
The solution was dispensed into books and sterilized at 120°C for 20 minutes. Scolecoba-sidium Arenarium Scolecoba-sid
Inoculate 1 platinum loop of slant culture of F-2015 Chokoken Joyori No. 1520 into each medium and culture with shaking at 25°C for 3 days! ! I did. The resulting culture was incubated at 200°C, which had been previously sterilized at 120°C for 20 minutes.
Aqueous medium containing 3% soluble starch, 1% glucose, 1% wheat germ, 0.5% cottonseed flour and 0.2% calcium carbonate in a 120f jar fermenter
T) and cultured at 25°C for 3 days. The culture solution thus obtained was filtered using diatom ± (20 kg). The filtrate (954!) was diluted with ethyl acetate (95!
Extracted using 1). This extraction operation was performed twice and the extracts were combined. After dehydration over anhydrous magnesium sulfate, the ethyl acetate layer was concentrated under reduced pressure. The concentrate was applied to a silica gel chromatography column (11). The column was diluted with n-hexane (
3A) and n-hexane-ethyl acetate mixture (1:1,
3IL). The active fraction was eluted with ethyl acetate (4A) and then concentrated under reduced pressure. This was further applied to a silica gel column (400 ml). Chloroform (1.
24! ) and chloroform-methanol mixture (10
0: 1, 1.2 Jl! ．． ) After washing the column with chloroform-methanol) mixture (75:1, 50:
1, and 25:1). The active fraction was concentrated under reduced pressure, and the residue was subjected to reverse phase silica gel ODS reverse column chromatography. The column was washed with a warm methanol/water solution (1:1, 300 ml), and then developed stepwise with a methanol/water mixture (3:2 and 7:3, 300 ml each). The active fraction was evaporated to dryness under reduced pressure to obtain purified colorless oil WF 2015A (92
mg) was obtained. ■Yuskoreko Basidium Arenarium F - 2015
1 platinum loop of slant culture (Shikoken Joyose IJ No. 1520) was added to 2% soluble starch, 1% corn starch, 1% glycose, 1% cottonseed powder, 1% dry yeast, and 0 pebtone.
．． 5%, cornstarch 0.5%, NaCl
3% and CaCO. ．． A mild medium containing 0.2% (p
ns. o) (1 6 0 ■ 1) 20 pieces of 50
Dispense the mixture into 0.01-volume Erlenmeyer flasks, sterilize them in the usual manner, and inoculate them using a rotary shaker at 250 rpi+.
The cells were cultured at 25°C for 72 hours. The obtained seed culture solution was
160 JL of the same sterile medium as above was inoculated into a stainless steel 2001 jar fermenter, and the fermenter was stirred at 20 rpm and 25° C. for 48 hours. The thus obtained seed culture 901 was further added with 3% soluble starch, 1% glucose, 0.5% cottonseed flour, and wheat embryo #. 1% NaC
400 containing 3% I and 0.2% CaC
Sterile production medium 300 in Oflili fermenter
Inoculated at 0ft. 3ooofL/min ventilation and 13
Culture was carried out at 25° C. for 72 hours under stirring at 0 rpm. The culture solution <<285 of L>> was filtered using diatomaceous earth (askg). The filtrate was passed through a column of activated carbon (6001). The column was washed with 180 ft of deionized water and eluted with 80% acetone water (TAOG).
It was concentrated to 71. The concentrate was extracted with 181 ml of ethyl acetate. The ethyl acetate layer was separated and dried under reduced pressure to give a powder (61
．． 3g) was obtained. This crude powder was mixed with 0.3 silica gel.
Mixed with 1. The mixture was subjected to column chromatography using silica gel (3'j!). Fill the column with hexane tojl, then hexane-ethyl acetate (1:1).
It was washed with IOjL and eluted with ethyl acetate 101. The active fractions were collected and evaporated to dryness to obtain a powder, which was mixed with silica gel. The mixture was subjected to column chromatography using silica gel (11). The column was filled with 3 IL of chloroform, then chloroform-methanol (10
0:1) 31 and chloroform-methanol (7:1).
5:1) 3J! It was eluted. The eluate was dried under reduced pressure to obtain a powder (16.4 g), and this powder was purified by reverse phase silica gel OD.
Mixed with 30ml of S. Mixture on reversed phase silica gel ODS
(600+wl) column, methanol-water (1:
1) Washed with 2.41. Fill the column with methanol-water (3
: It was eluted in 2). The active fractions were collected and concentrated under reduced pressure to obtain oily WF2015B (305ag).

Claims (1)

[Claims] An angiogenesis inhibitor containing WF2015A and/or WF2015B as an active ingredient.