JPH11310530A - Nitrogen monoxide production inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an agent useful as an NO production inhibitor in macrophage and/or endothelial cells by making the agent include, as active ingredient, a compound of specific structure such as chelidonine or protopine. SOLUTION: This agent contains, as active ingredient, a compound (salt) of formula I (R<1> and R<2> are each a lower alkoxy or the like; R<3> is H or the like; R<4> is a lower alkyl; R<5> is hydroxy, a lower alkanoyloxy or the like; R<6> is H or a lower alkyl; dotted line represents a single or double bond) or a compound (salt) of formula II (R<11> and R<12> are each a lower alkoxy or the like; R<16> is joined together with R<13> to represent a single bond, or the like; R<17> is H or the like; R<18> is H or a lower alkyl); wherein the compound of formula I is e.g. (+)-chelidonine, (±)-corynoline, and the compound of formula II is e.g. protopine, (±)-mesocorydaline; these compounds can be prepared by well-known method.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to nitric oxide (N
O) A pharmaceutical composition containing a compound having a production inhibitory action or a salt thereof as an active ingredient. More specifically, the present invention comprises a compound having a cell nitric oxide production inhibitory activity or a pharmaceutically acceptable salt as an active ingredient, septic shock, hypotension, inflammatory tissue disorder, ischemic disease, Allergic diseases, autoimmune diseases,
The present invention relates to a pharmaceutical composition useful for prevention and / or treatment of rheumatoid arthritis, insulin-dependent diabetes, and the like.

[0002]

2. Description of the Related Art Cells are catalyzed by NOS (nitric oxide synthase, NOS) and are converted from L-arginine to nitric oxide.
e, NO). NOS is non-inductive (constituti
ve NOS, cNOS) and inductive (inducible NOS, iNO)
S) isozyme, and cNOS has endothelial type and neural type. Endothelial cNOS is mainly present in vascular endothelial cells, and neuronal cNOS is distributed in central nerve cells and peripheral nerve cells. iNOS is present in macrophages, neutrophils, hepatocytes, chondrocytes, synovial cells, vascular smooth muscle cells, bronchial epithelial cells, glial cells, and the like. The activity of cNOS, which is constantly present in cells, depends on the intracellular calcium concentration and produces NO regardless of stimulation.
On the other hand, iNOS activity is not dependent on intracellular calcium concentration, but is induced only by stimulation with endotoxin, various cytokines, and the like. Calmodulin binds to both NOS and becomes active.

[0003] The action of NO produced by the NOS catalytic reaction is diverse, but it is not clear whether the action is due to cNOS-derived NO or iNOS-derived NO.
The actions of NO include, for example, vasorelaxant action, platelet aggregation inhibitory action, leukocyte adhesion inhibitory action, vascular permeability enhancing action, antigen presenting function lowering function of dendritic cells, lymphocyte division and function lowering action, nerve information transmission Action, central nervous system function regulating action, antimicrobial / antitumor action, and septic shock, septic hypotension, ischemic encephalopathy, autoimmune disease, allergic disease, nephritis, hepatitis, ulcerative colitis, arthritis , Cardiomyopathy and the development and development of insulin-dependent diabetes mellitus and the like.

Since NO has various physiological actions, NO
Suppression of NO production by cells by S inhibitors or the like is expected to be useful as a treatment method for various diseases,
Development of production inhibitors is underway. Various known documents,
For example, molecular medicine series for new clinicians, “NO radical medicine”, 42-58, Yodosha (1996), “
News and Perspectives "(Drug News
and Perspectives), 204-219 (1996)), `` Expert Opinion on Therapeutic Patents ''
(Expert Opinion on Therapeutic Patents), 7, 717-727
(1996) discloses L-arginine derivatives as substrate competitors for NOS. Examples of such L-arginine derivatives include ^NG -monomethyl-L-arginine (L-NMMA), ^NG -nitro-L-arginine (L-NA), and ^NG -nitro-L-arginine methyl ester. (L-NAME) and the like are known. Also, i
NOS induction inhibitors include corticosteroids, IL
-4, IL-10, IL-13, serine and cysteine protease inhibitors are known. Furthermore, NO
As an inhibitor having a relatively high specificity for each isozyme of S, for example, for iNOS, aminoguanidine, N-iminoethyl-L-lysine, S-methylisothiourea, S- (aminoethyl) isozyme Thiourea, 3
-Amino-4'-hydroxyflavone, 7-propyl-
Homoiminopiperidinium chloride and the like are known. As for cNOS, tannin and quercetin, which are polyphenols, and brefeldin A, which is a product of mold, are known for endothelial cNOS.
7-nitroindazole, 8 for neural cNOS
-Bromo-7-nitroindazole, S-methyl (or ethyl) -L-thiocitrulline and the like are known.

[0005] In addition to these substances, various NOS inhibitors have been reported, for example, "Expert Opinion on Therapeutic Patents", 7, 717-72.
7 (1996) and 7, 1207-1210 (1997) further include amino acid derivatives and their analogs, acyclic amidines and guanidines, cyclic amidines, guanidines, isothioureas, and aminopyridines. , Phenols, aniline derivatives and the like.

On the other hand, compounds similar to the above-mentioned compounds (I) and (II), for example, berberin
e) has an immunosuppressive effect (JP-A-7-316051) and a nerve cell death-suppressing effect (JP-A-7-330623), and a quaternary ammonium tetrahydroprotoberberine compound inhibits arrhythmia and ventricular fibrillation, Protects ischemia (JP-A-7-109276) and corydalin
e) is ACAT (acyl-CoA-cholester)
It is also known that the compound has an inhibitory effect on cholesterol absorption and is useful as a cholesterol absorption inhibitor and a prophylactic / therapeutic agent for arteriosclerosis (Japanese Patent Application Laid-Open No. 8-59469). However, these documents do not suggest that the compounds (I) and (II) of the present invention have an NO production inhibitory action.

[0007]

An object of the present invention is to provide a pharmaceutical composition useful as an NO production inhibitor.

[0008]

Means for Solving the Problems The inventors of the present invention have:
As a result of research to obtain a substance that suppresses NO production from cells, a pharmaceutical composition containing a compound represented by the general formula (I) or (II) or a salt thereof as an active ingredient was converted into a macrophage and / or a macrophage. It has been found that it is useful as an inhibitor of endothelial cell NO production.

The present invention provides a compound represented by the general formula (I):

Embedded image Wherein R ¹ and R ² are lower alkoxy groups,
Or in combination represent -OCH ₂ O-each other, R ³ is bonded to R ⁵ through which, or oxygen atom a hydrogen atom, R ⁴ represents a lower alkyl group, R ⁵ is hydroxy group Or a lower alkanoyloxy group, or a bond to R ³ through an oxygen atom, R ⁶ is a hydrogen atom or a lower alkyl group, and ------ represents a single bond or a double bond. Or a salt thereof, or a compound of the general formula (II)

Embedded image Wherein R ¹¹ and R ¹² are lower alkoxy groups,
Or bonded to each other to represent —OCH ₂ O—, R ¹³ is a lower alkyl group, or together with R ¹⁶ represents a single bond, and R ¹⁴ and R ¹⁵ are a lower alkoxy group, or It represents -OCH ₂ O-bonded to each other,
R ¹⁶ is either a single bond together with R ^13, or together with R ¹⁷ = represents O, R ¹⁷ represents = O together with or is R ^16, a hydrogen atom , R ¹⁸ represents a hydrogen atom or a lower alkyl group, and ------ represents a single bond or a double bond]. The compound (II) or a salt thereof is an active ingredient of a nitric oxide production inhibitor. As a pharmaceutical composition.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The above general formulas (I) and (I)
In I), the lower alkoxy group is a linear or branched alkoxy group having 1 to 4 carbon atoms, and examples thereof include methoxy, ethoxy, propoxy, isopropoxy, butoxy, tert-butoxy and the like. The lower alkanoyloxy group is a linear or branched alkanoyloxy group having 1 to 4 carbon atoms, and includes, for example, acetyloxy, propionyloxy, butyryloxy and the like.

The salt of compound (I) and compound (II) is not particularly limited as long as it is a pharmaceutically acceptable salt. Specifically, for example, hydrochloride, hydrobromide, sulfate or phosphate Inorganic salts such as salts, maleates, tartrate,
Organic acid salts such as citrate, succinate or methanesulfonate are mentioned. Compound (I) or compound (I
When I) is a quaternary ammonium salt, examples of the counter ion include a halogen anion, and among them, a chloride anion is preferable.

The compound (I) and the compound (II) are both known and known methods, for example, Helvetica Chimica Acta, 66, 473 (1983),
Archiv der Pharmazi
e), 317,223 (1984), Tetrahedron 35,1
099 (1979), 35, 1977 (1979), Tetrahedron Letters, 1979, 495, Annalen der Pharmcie, 29, 113 (1839),
Canadian Journal of Chemistry
ian Journal of Chemistry), 47,3701 (1969), Chemical & Pharmaceutical Bulletin (Chemical & Phar
maceutical Bulletin), 21,1020 (1993), 21,1096 (197
3) and the like, or can be obtained from a compound obtained by these known methods according to a conventional method.

Representative examples of compound (I) and compound (II) are shown below, but the present invention is not limited to these examples.

(A) (+)-Keridonin (B) (+)-O-Acetylkeridonine (C) (±) -Corinoline (D) (±) -Corinololoxin (E) Sanginaline chloride (F) (+ ) -Homokeridonin (G) protopin (H) (±) -mesocoridaline (I) 13-methyl berberine chloride (J) 13-ethyl berberine chloride

Each of the above compounds is represented by the following structural formula.

Embedded image

Compound (I) and compound (II) have various stereoisomers and tautomers, and these isomers are all included in the present invention. When the molecule contains an asymmetric center, the configuration of the asymmetric center may be either R or S, or a mixture thereof.
These isomers can be obtained, for example, by using an optically active compound as a starting material, or by purifying the obtained isomer mixture by column chromatography or recrystallization.

Compound (I) and compound (II)
Can be produced and purified by a usual salt-forming reaction.

Compounds (I) and (II) and their pharmaceutically acceptable salts significantly inhibit NO production by cells and have low toxicity as shown in the test examples described below. And is useful as a NO production inhibitor for mammals. Specifically, various diseases caused by NO production of cells, such as septic shock, hypotension, inflammatory tissue disorder, ischemic disease, allergic disease, autoimmune disease, rheumatoid arthritis, insulin-dependent diabetes, etc. Useful as a prophylactic or therapeutic agent.

The pharmaceutical composition of the present invention can be prepared in various dosage forms depending on the purpose, for example, oral preparations such as tablets, pills, powders, granules, fine granules, capsules, liquids, suspensions and emulsions. It is used as a parenteral preparation such as injections, drops, suppositories, ointments, plasters, patches and aerosols. These preparations can be manufactured by a conventional method generally known in the art.

The pharmaceutical composition of the present invention may contain, if desired, ordinary additives such as coloring agents, preservatives, flavors, flavors, sweeteners, and other pharmaceuticals, as long as the effects are not impaired. It can also be blended.

In the case of molding into a tablet form, it is produced by a conventional method using a conventional excipient such as lactose, starch, crystalline cellulose or the like as a carrier. In this case, if desired, a starch solution, a binder such as carboxymethyl cellulose, a disintegrant such as dried starch and calcium carbonate, a disintegration inhibitor such as sucrose, stearic acid, cocoa butter, hydrogenated oil, a quaternary ammonium base, lauryl Absorption promoters such as sodium sulfate, humectants such as glycerin and starch, adsorbents such as starch, lactose, kaolin, bentonite and colloidal silicic acid, purified talc, stearate,
Lubricants such as boric acid powder and polyethylene glycol can be used.

When formed into pills, the carrier may be, for example, excipients such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, kaolin, talc, binders such as gum arabic powder, tragacanth powder, gelatin, etc. Laminaran,
Disintegrators such as agar can be used.

Tablets, pills, granules and the like may be in the form of a usual coating, if necessary, such as sugar-coated tablets, gelatin-encapsulating agents, enteric-encapsulating agents, film-coating agents and the like. The tablet may be a multilayer tablet such as a double tablet.

Capsules are prepared by mixing a compound of the present invention or a salt thereof with any of the above-listed carriers and filling into hardened gelatin capsules, soft capsules, and the like, according to conventional methods.

When preparing a liquid preparation for oral use, a carrier such as a flavoring agent, a buffering agent, a stabilizer and the like are added to the active ingredient of the present invention, if necessary, and a liquid solution for internal use, syrup,
An elixir or the like can be produced. In this case, sucrose, orange peel, citric acid, tartaric acid and the like can be used as a flavoring agent, sodium citrate and the like can be used as a buffer, and tragacanth, gum arabic, gelatin and the like can be used as a stabilizer.

When preparing an injection or infusion, a compound of the present invention or a salt thereof may be added, if necessary, with a pH adjuster,
Add carriers or diluents such as buffers, stabilizers, local anesthetics, and isotonic agents, and intravenously, intramuscularly, subcutaneously,
Intradermal or intraperitoneal injections or drops can be prepared.

Examples of pH adjusters and buffers include sodium citrate, sodium acetate, sodium phosphate and the like, and stabilizers include sodium pyrosulfite, ethylenediaminetetraacetic acid, thioglycolic acid, thiolactic acid and the like. Examples thereof include procaine hydrochloride, lidocaine hydrochloride, and the like, and examples of the tonicity agent include salt, glucose, glycerin, and the like. Examples of the diluent include water, ethyl alcohol, macrogol, and propylene glycol.

The injection or infusion is preferably sterilized and isotonic with blood.

Suppositories can be prepared in a conventional manner after adding a base and, if necessary, a surfactant to the compound of the present invention or a salt thereof. As the base, for example, oily bases such as macrogol, lanolin, cocoa oil, fatty acid triglyceride, and Witepsol (manufactured by Dynamite Nobels) can be used.

When preparing an ointment, a carrier such as a base, a stabilizer, a wetting agent, a preservative and the like which are usually used for the compound of the present invention or a salt thereof are blended if necessary, and mixed by a conventional method. Is formulated. Liquid paraffin,
White petrolatum, beeswax, octyldodecyl alcohol, paraffin and the like. As a preservative, methyl paraoxybenzoate, ethyl paraoxybenzoate,
And propyl paraoxybenzoate.

A patch can be prepared by applying the above-mentioned ointment, paste, cream, gel or the like to a usual support in a conventional manner. Examples of the support include a woven or non-woven fabric made of cotton, cloth, and synthetic fibers, a film of soft vinyl chloride, polyethylene, polyurethane, or the like, or a foam sheet.

The pharmaceutical composition of the present invention can be administered orally, intravenously, subcutaneously / intradermally, intramuscularly, intraarticularly, rectally, depending on the dosage form, age of the patient, degree of disease, etc. Although it can be used by parenteral administration such as transmucosal administration, oral administration, intravenous administration and intraarticular administration are preferred.

The dose of the compound of the present invention for therapeutic use depends on the type of disease, the symptom / severity of the patient, weight, age, sex, tolerability of the drug, dosage form of the drug and other conditions. The dose may be adjusted as appropriate, but usually 1 to 2000 mg, preferably 10 to 500 mg per day for an adult.

[0034]

The present invention will be described below with reference to Reference Examples, Test Examples and Examples, but the present invention is not limited to these Reference Examples, Test Examples and Examples.

Synthesis Example 1 (+)-chelidoni (chelidoni)
ne) Production of (A)

Embedded image A methanol extract was prepared from Shirokuna (Daiwa Shokai kk, dry weight 30 kg) by a conventional method to obtain a tertiary non-phenolic alkaloid fraction. Then, separation by silica gel column chromatography (benzene / ether) yielded 7 g of an oily substance as the main base, which was recrystallized from methanol / ether to give m.p. p. The title compound at 136 ° C. was obtained as colorless prisms.

Synthesis Example 2 Production of (+)-O-acetylchelidonine (B)

Embedded image 70 mg of (+)-keridonine was dissolved in 0.5 ml of acetic anhydride, several drops of pyridine were added, and the mixture was allowed to stand at room temperature for 4 hours. After the reaction, a small amount of ice chips was added to the reaction solution to decompose acetic anhydride, and the mixture was made alkaline by adding aqueous ammonia, and then extracted with ether. Then, the extract was dried over anhydrous potassium carbonate, ether was distilled off, and the obtained colorless oily residue was recrystallized with an acetone / ether mixed solvent to give mp16.
70.5 mg of (+)-O- as plate crystals at 5-166 ° C.
Acetylkeridonine was obtained.

Synthesis Example 3 (±) -corinoline
Production of (C)

Embedded image A methanol extract of 3.7 kg dry weight of purple mushroom (Fukui Prefecture) was extracted with 3% tartaric acid. The tartaric acid layer was subjected to ether extraction to remove neutral and acidic substances, made alkaline with aqueous ammonia, and then subjected to ether extraction. After the ether layer was dried over anhydrous potassium carbonate, the ether was distilled off to obtain 14.1 g of a red-brown oily residue. The residue was recrystallized with a mixed solvent of chloroform / methanol, and mp21
Colorless prism (±) -corinoline 3.5 at 6-217 ° C
g was obtained.

Synthesis Example 4 (+)-Corinoloxine (corynol
oxine) (D)

Embedded image Dissolve 100 mg of (+)-corinoline in 5% acetic acid and add Hg
It was heated for 4 hours at ₂ (OAc) onto the ₂ 210 mg added water bath. After the reaction, Hg (OAc) ₂ precipitated was separated by filtration, the filtrate was made alkaline with aqueous ammonia, and extracted with ether. After the ether layer was dried over anhydrous potassium carbonate, the solvent was distilled off to obtain a yellowish oily substance. This was purified by alumina column chromatography (benzene) and recrystallized from methanol to obtain 55 mg of (+)-corinoloxin as colorless prism crystals having an mp of 147 to 148 ° C.

Synthesis Example 5 Sanguina Link Chloride (sangu
inarine chloride) (E)

Embedded image

5 g of (+)-O-acetylchelidonine was added to 5
% Acetic acid dissolved in 1000 ml, added with 15 g of mercury oxide,
The mixture was heated under reflux for 4 hours. After cooling, the solids were removed by filtration, and the filtrate was made alkaline with 28% aqueous ammonia to give ether 3
Extracted twice with 00 ml. The ether layer was dried over anhydrous potassium carbonate and concentrated under reduced pressure to give 2.7 g of 5,6-dihydrosanginalin. p. Obtained as prism crystals at 190-192 ° C (chloroform / methanol). 2 g of this 5,6-dihydrosanginalin is mixed with 80 ml of benzene.
, And 36 ml of a 5% aqueous sodium hydroxide solution was added thereto.
1.96 g of 5,6-dicyano-para-benzoquinone was added dropwise little by little. After completion of the dropwise addition, the mixture was stirred at room temperature for 1 hour. After the reaction, a 10% aqueous sodium hydroxide solution 20
After adding 00 ml, the mixture was extracted twice with 300 ml of ethyl acetate. The ethyl acetate layer was washed with saturated saline, dried over anhydrous potassium carbonate, and the solvent was distilled off under reduced pressure to obtain 2 g of an oily residue. 10 ml of this oily substance in methanol
Was suspended and acidified by adding 35% hydrochloric acid to obtain 2.1 g of sanguinarine chloride as red-orange needles.
Decomposition point 287-289C.

Synthesis Example 6 (+)-Homokeridonine (homocheli
donine) (F) production

Embedded image A methanol extract was prepared from Shirokuna (Daiwa Shokai kk, dry weight 30 kg) by a conventional method to obtain a tertiary non-phenolic alkaloid fraction. This fraction was separated by silica gel column chromatography (benzene / ether), and (+) −
The fraction eluting next to kelidonin was isolated. When recrystallized from the fraction from acetone / ether mixed solvent, mp18
80 mg of (+)-homokeridonin was obtained as prism crystals at 1-182 ° C.

Synthesis Example 7 Protopine (G)
Manufacturing of

Embedded image

A methanol extract was prepared from Shirokuna (Daiwa Shokai kk, dry weight 30 kg) by a conventional method to obtain a tertiary non-phenolic alkaloid fraction. Protopin was separated from this fraction by silica gel column chromatography (benzene / ether = 1/1, then methanol) and recrystallized from chloroform / methanol to give m.p. p. 2
1.5 g of the title compound at 05-206 ° C was obtained as colorless prisms.

Synthesis Example 8 (±) -mesocoridaline (meso-co
rydaline) (H)

Embedded image

In a separating funnel, palmatin chloride 10 was added.
g, acetone (200 ml) and a 10% aqueous sodium hydroxide solution (100 ml) were added, and the mixture was shaken at room temperature for 30 minutes. The precipitated crystals were filtered to obtain a palmatin acetone adduct. The palmate acetone adduct (2 g) was dissolved in acetone in a pressurized bottle, 5 ml of methyl iodide was added, and the mixture was reacted at 50 ° C. for 4.5 hours. After the reaction, the precipitated crystals were separated by filtration to obtain 13-methylpalmatine iodide. 2.0 g of 13-methylpalmatine iodide, 300 ml of methanol, 2 g of Zn powder and 6 ml of concentrated hydrochloric acid were placed in an eggplant-shaped flask, and heated at 60 ° C. for 6 hours in a water bath. After the reaction, the Zn powder was filtered off, 20 ml of water was added, and methanol was distilled off under reduced pressure. Water was added to the obtained residue to make the total amount about 200 ml, then 25% aqueous ammonia was added to make the mixture alkaline, and extracted twice with 300 ml of chloroform. The extract was dried over anhydrous magnesium sulfate, and then the solvent was distilled off to remove oily (±) -coridaline.
A (±) -mesocoridaline reaction mixture was obtained. This was separated by silica gel column chromatography (benzene / ether / methanol) and recrystallized from chloroform to give m.p. p. 180 mg of (±) -mesocoridaline at 155.5-156.7 ° C was obtained.

Synthesis Example 9 Preparation of 13-methylberberine chloride (I)

Embedded image

Berberine chloride 10 was added to the separating funnel.
g, acetone 200 ml and a 10% aqueous sodium hydroxide solution (100 ml) were added and shaken at room temperature for 30 minutes, and the precipitated crystals were filtered to obtain a berberine acetone adduct. Berberine acetone adduct 2 in a pressurized bottle
g was dissolved in 80 ml of acetone, 5 ml of methyl iodide was added, and the mixture was reacted at 50 ° C. for 4.5 hours. After the reaction, the precipitated crystals were separated by filtration to obtain 13-methyl-berberine iodide. Then, 1.5 g of Amberlite CG-4
Was suspended in water, packed in a column, and a solution of 50 mg of 13-methyl-berberine iodide in 20 ml of methanol was injected. The eluate eluted with water / methanol is concentrated to dryness under reduced pressure to give 13
38 mg of -methyl-berberine chloride were obtained as a yellow powder. Decomposition point 162.3-165.8C.

Synthesis Example 10 Preparation of 13-ethyl berberine chloride (J)

Embedded image

Berberine acetone adduct 1 in a pressure bottle
g was dissolved in 30 ml of acetone, 8 ml of ethyl iodide was added, and the mixture was reacted at 60 ° C. for 5 hours. After the reaction, the precipitated crystals were separated by filtration and 13-ethylberberine iodide 350
mg were obtained as yellow needles. Decomposition point 207.8-21
0.7 ° C. Next, 28.6 mg of 13-ethyl berberine chloride was obtained as a yellow powder by the same operation as in Reference Example 9. Decomposition point 174.8-177.4 ° C.

Test Example 1 Inhibitory effect on NO production through inducible NOS (iNOS) A macrophage-derived cell line RAW264 was implanted in a 48-well plate at 4 × 10 ⁵ cells / cm ^{2 and} modified by Dulbecco's method containing 10% fetal bovine serum. Eagle culture (Dulbecco '
1 ng / ml lipopolysaccharide (LPS) (E. Coli Sero) in modified Eagle's medium)
The cells were stimulated at 37 ° C. for 20 hours with type 0111: B4). Test compounds were added to the cells simultaneously with LPS.
After stimulation, the amount of NO produced in the culture was determined by the grease method (Green LC et al., Analytical Biochemistry, 126, 131-138 (1982)).
Was measured as the amount of nitrite ion (NO ₂ ⁻ ). That is, 50 μl of a grease reagent (containing 0.1% naphthylethylenediamine, 1% sulfanilamide, and 5% phosphoric acid) was added to 75 μl of the culture supernatant, and 10 minutes later, 54 μl of the grease reagent was added.
The absorbance from 0 nm to 650 nm was measured. The inhibitory activity was Δ (NO production of LPS-stimulated cells without addition of compound) −
(NO production of unstimulated cells without addition of compound) = 100
It was calculated as%｝. This value is shown in Table 1. As comparative drugs, dexamethasone (Dex), which is an iNOS induction inhibitor, and non-inducible NOS (c
^NG -nitro-L-arginine methyl ester (L-NAME), which is said to inhibit NOS) relatively strongly, was used.

[0051]

[Table 1]

Test Example 2 Inhibitory effect on NO production through non-induced NOS (cNOS) Bovine pulmonary artery endothelial cells (CPAE) cultured to confluence in a 24-well plate were subjected to Dulbecco's modified Eagle culture containing 10% fetal bovine serum. Liquid (Dulbecco's modified Eagl
In e's medium), the cells were stimulated with 10 μM ionomycin (calcium ionophore) at 37 ° C. for 40 hours.
Test compounds were added to the cells simultaneously with ionomycin.
The NO production amount in the culture solution was measured by the same operation as in Test Example 1. The inhibitory activity was calculated as {(NO production of ionomycin-stimulated cells without addition of compound) − (NO production of unstimulated cells without addition of compound) = 100%}. This value is shown in Table 2. As a comparative drug, as in Test Example 1,
Dexamethasone (Dex) and ^NG -nitro-L-arginine methyl ester (L-NAME) were used.

[0053]

[Table 2]

Test Example 3 The compounds described in Tables 1 and 2 were subjected to a cytotoxicity test by the following method, and it was confirmed that no toxicity was observed at the concentrations described. That is, after culturing the cells by adding the test compound under the same conditions as in Test Examples 1 and 2, the culture solution was removed, and 0.3 mg / ml WS was added to the cells.
T-1 (2- (4-Iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl)-
2H-tetrazolium) and 6.4 μg / ml 1-
PBS (phosphate-buf) containing methoxy PMS (1-methoxy-5-methylphenazinium methyl sulfate)
fered saline). After incubating this at 37 ° C. for 1 hour, the formed formazan was removed at 450 nm-6.
It measured by the absorbance of 50 nm, and compared with the case where a test compound was not added. As a result, the amount of formazan generated by the action of living cells did not change even when the test compound was added.

Test Example 4 Acute toxic effect by intraperitoneal administration of mouse 5% polyethylene glycol 400 and 1% Tween
Test compounds (A, B, C, D, H) suspended in phosphate buffered saline containing n80 were intraperitoneally injected into BALB / c male mice (20 to 25 g, 4 mice per group). Two doses of 100 mg / kg were administered at 6-hour intervals, but no deaths were observed 24 hours after the first dose.

Example 1 20 g of (±) -corinoline was taken, and 90 g of lactose, 59.5 g of corn starch, 20 g of crystalline cellulose, 10 g of calcium carboxymethylcellulose and 0.5 g of magnesium stearate were added to make a total amount of 200 g of a powder. After mixing well, a tablet of 200 mg per tablet was produced. Example 2 40 g of (±) -corinoloxin was taken, 78 g of lactose, 51.5 g of corn starch, 20 g of crystalline cellulose, 10 g of calcium carboxymethylcellulose and 0.5 g of magnesium stearate were added to make a total amount of 200 g of a powder, which was mixed well. Thereafter, a tablet of 200 mg per tablet was manufactured.

[0057]

The compounds (I) and (II) of the present invention have an inhibitory effect on NO production from cells, and cause septic shock, hypotension, inflammatory tissue disorders, ischemic diseases, allergic diseases. It is useful for the prevention and / or treatment of autoimmune diseases, rheumatoid arthritis or insulin-dependent diabetes.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI A61K 31/435 ABN A61K 31/435 ABN ABV ABV ADP ADP ADP 31/395 ADZ 31/395 ADZ // A61K 35/78 35/78 C C07D 455/03 C07D 455/03 491/056 491/056 491/147 491/147 491/153 491/153 (72) Inventor Miyoko Uegakiuchi 3-8-6 Shinsenri-Kitamachi, Toyonaka-shi

Claims (4)

[Claims] 1. A compound of the general formula (I) Wherein R ¹ and R ² are lower alkoxy groups,
Or in combination represent -OCH ₂ O-each other, R ³ is bonded to R ⁵ through which, or oxygen atom a hydrogen atom, R ⁴ represents a lower alkyl group, R ⁵ is hydroxy group Or a lower alkanoyloxy group, or a bond to R ³ through an oxygen atom, R ⁶ is a hydrogen atom or a lower alkyl group, and ------ represents a single bond or a double bond. Or a salt thereof, or a compound of the general formula (II): Wherein R ¹¹ and R ¹² are lower alkoxy groups,
Or bonded to each other to represent —OCH ₂ O—, R ¹³ is a lower alkyl group, or together with R ¹⁶ represents a single bond, and R ¹⁴ and R ¹⁵ are a lower alkoxy group, or It represents -OCH ₂ O-bonded to each other,
R ¹⁶ is either a single bond together with R ^13, or together with R ¹⁷ = represents O, R ¹⁷ represents = O together with or is R ^16, a hydrogen atom , R ¹⁸ is a hydrogen atom or a lower alkyl group, and ------ represents a single bond or a double bond.] Compound (II) or a salt thereof is an active ingredient of a nitric oxide production inhibitor. A pharmaceutical composition containing 2. Compound (I) is (+)-keridonine,
The pharmaceutical composition according to claim 1, which is (+)-O-acetylchelidonine, (±) -corinoline, (±) -colinoloxine, sanguinarine chloride or (+)-homokeridonin. 3. The pharmaceutical composition according to claim 1, wherein the compound (II) is protopin, (±) -mesocoridaline, 13-methylberberine chloride or 13-ethylberberine chloride. 4. A preventive / therapeutic agent for a nitric oxide production inhibitor for septic shock, hypotension, inflammatory tissue disorder, ischemic disease, allergic disease, autoimmune disease, rheumatoid arthritis or insulin-dependent diabetes mellitus. The pharmaceutical composition according to claim 1, which is