JP2019137640A - Stress reduction agent - Google Patents

Abstract

To provide a stress reduction agent that contains a component derived from deep ocean water, an indole and a salt thereof and a metabolite thereof, as a stress reduction component and is safe and effective.SOLUTION: A stress reduction agent contains an indole represented by the following chemical formula (I), a pharmaceutically acceptable salt thereof, and/or a metabolite thereof, as a stress reduction component. In the stress reduction agent, the indole may have a tryptophan skeleton and its metabolite may have a kynurenine skeleton.SELECTED DRAWING: None

Description

  The present invention relates to a stress-reducing drug that is bred while reducing stress under the breeding conditions of a non-human animal, or that reduces human stress and stabilizes mentally.

  Deep Ocean Water (DOW) is defined as seawater that is more resource-rich than the compensated depth, with superior organic matter decomposition, less vertical mixing and human influence than organic production by photosynthesis. Since such deep ocean water exists at a depth of 200 m or more, (i) it is always kept at a low temperature (low water temperature), (ii) there are few bacteria and pollutants (cleanliness), and (iii) nitrogen and phosphorus -It contains abundant minerals such as silicon and inorganic nutrients (eutrophication), and (iv) is characterized by small seasonal variations and interannual variations in water quality (water quality stability).

  Taking advantage of these features, deep sea water can be used for industrial applications such as mineral sources and refrigerants, as well as for cosmetics and health goods using a healthy and safe image of mineral richness and cleanness. It is used in a variety of fields such as aquaculture where breeding marine products in deep ocean water.

  For example, Patent Document 1 discloses a method of cultivating oysters using deep ocean water, and cultivating oysters in an aquarium by spraying deep ocean water at a seawater temperature of 8 to 18 ° C. for 12 to 72 hours. A method for raising oysters is disclosed.

  Patent Document 2 contains (A) deep ocean water, (B) fresh water having a hardness of 100 mg / L or less, (C) polybasic amino acid, (D) grapefruit seed extract, and has an osmotic pressure of 200 to 350 mOsm. A skin cosmetic composition having a pH of 6.5 to 9.5 is disclosed.

  However, research on the components in the deep ocean water that improves the growth of marine products or their correlation, research on the correlation between the ocean deep water and the ecology or physiological phenomenon or health promotion effect of various animals including humans, There has been little development of new drugs using this.

JP 2006-015947 A JP 2014-234352 A

  The present invention has been made in order to solve the above-mentioned problems, and includes components derived from deep ocean water, indoles, salts thereof and metabolites thereof, containing stress-reducing components, and safe and effective stress. The object is to provide a reducing drug.

（式（Ｉ）中、
Ｒ^１は、水素原子、水酸基、若しくは置換基で置換されていてもよいもので、アルキル基、アルキルオキシ基、アルキルチオエーテル基、アルケニル基、アルケニルオキシ基、アルケニルチオエーテル基、アルキニル基、アルキニルオキシ基、アルキニルチオエーテル基、炭化水素芳香族基、炭化水素芳香族基含有オキシ基、炭化水素芳香族基含有チオエーテル基、飽和複素環基、飽和複素環基含有オキシ基、飽和複素環基含有チオエーテル基、芳香族複素環基、芳香族複素環基含有オキシ基、芳香族複素環基含有チオエーテル基、アラルキル基、アラルキルオキシ基、アラルキルチオエーテル基、アシル基、アシルオキシ基、チオエステル基、チオアミド基、アルキルスルホニル基、アルキルスルホニルオキシ基、アルケニルスルホニル基、アルケニルスルホニルオキシ基、アルキニルスルホニル基、アルキニルスルホニルオキシ基、アリールスルホニル基、アリールスルホニルオキシ基、アラルキルスルホニル基、アラルキルスルホニルオキシ基、又はアミジノ基；
Ｒ^２は、水素原子、ハロゲン原子、又は置換基で置換されていてもよいアルキル基；
ｎ１は、０又は１の数；
Ｒ^３は、水素原子、ハロゲン原子、−ＣＯＯＨ、−ＣＯＯＲ^ａ、−ＣＯＮＨ_２、又は−ＣＯＮＨＲ^ｂ（但し、Ｒ^ａ、Ｒ^ｂは、置換基で置換されていてもよいもので、アルキル基、アルケニル基、アルキニル基、炭化水素芳香族基、飽和複素環基、芳香族複素環基、アラルキル基、アシル基、アルキルスルホニル基、アルケニルスルホニル基、アルキニルスルホニル基、アリールスルホニル基、又はアラルキルスルホニル基）；
Ｒ^４及びＲ^５は、同一又は異なり、水素原子、若しくは置換基で置換されていてもよいもので、アルキル基、アルケニル基、アルキニル基、炭化水素芳香族基、飽和複素環基、芳香族複素環基、アラルキル基、アシル基、アルキルスルホニル基、アルケニルスルホニル基、アルキニルスルホニル基、アリールスルホニル基、アラルキルスルホニル基、アルキルオキシカルボニル基、アリールオキシカルボニル基、アルキルアミノカルボニル基、又はアリールアミノカルボニル基；
ｎ２は、０又は１の数であり、ｎ３は、０又は１の数であって、ｎ２＋ｎ３＞０；
Ｒ^６〜Ｒ^９は、同一又は異なり、水素原子、ハロゲン原子、水酸基、メルカプト基、若しくは置換基で置換されていてもよいもので、アルキル基、アルキルオキシ基、アルキルチオエーテル基、アルケニル基、アルケニルオキシ基、アルケニルチオエーテル基、アルキニル基、アルキニルオキシ基、アルキニルチオエーテル基、炭化水素芳香族基、炭化水素芳香族基含有オキシ基、炭化水素芳香族基含有チオエーテル基、飽和複素環基、飽和複素環基含有オキシ基、飽和複素環基含有チオエーテル基、芳香族複素環基、芳香族複素環基含有オキシ基、芳香族複素環基含有チオエーテル基、アラルキル基、アラルキルオキシ基、アラルキルチオエーテル基、アシル基、アシルオキシ基、アルキルスルホニル基、アルキルスルホニルオキシ基、アルケニルスルホニル基、チオエステル基、チオアミド基、アルケニルスルホニルオキシ基、アルキニルスルホニル基、アルキニルスルホニルオキシ基、アリールスルホニル基、アリールスルホニルオキシ基、アラルキルスルホニル基、アラルキルスルホニルオキシ基、アミジノ基、−ＣＯＯＨ、−ＣＯＯＲ^ｃ、−ＣＯＮＨ_２、又は−ＣＯＮＨＲ^ｄ（但し、Ｒ^ｃ、Ｒ^ｄは、置換基で置換されていてもよいもので、アルキル基、アルケニル基、アルキニル基、炭化水素芳香族基、飽和複素環基、芳香族複素環基、アラルキル基、アシル基、アルキルスルホニル基、アルケニルスルホニル基、アルキニルスルホニル基、アリールスルホニル基、又はアラルキルスルホニル基）、-ＮＨ_２、-ＮＨＲ^ｅ(但しＲ^ｅはアルキル基、アルケニル基、アルキニル基、アリール基、アラルキル基、又はアシル基)、-ＮＲ^ｆＲ^ｆ’(但しＲ^ｆ及びＲ^ｆ’は同一又は異なりアルキル基、アルケニル基、アルキニル基、アリール基、アラルキル基、又はアシル基)、アルキルアミノカルボニルアミド基、又はアリールアミノカルボニルアミド基；
破線／実線平行線は、単結合又は二重結合であり、
前記Ｒ^１〜Ｒ^９中の前記置換基は、夫々単数又は複数であって、水酸基、メルカプト基、ハロゲン原子、アルキル基、アルキルオキシ基、アルキルチオエーテル基、アルケニル基、アルケニルオキシ基、アルケニルチオエーテル基、アルキニル基、アルキニルオキシ基、アルキニルチオエーテル基、炭化水素芳香族基、炭化水素芳香族基含有オキシ基、炭化水素芳香族基含有チオエーテル基、飽和複素環基、飽和複素環基含有オキシ基、飽和複素環基含有チオエーテル基、芳香族複素環基、芳香族複素環基含有オキシ基、芳香族複素環基含有チオエーテル基、アラルキル基、アラルキルオキシ基、アラルキルチオエーテル基、アシル基、チオエステル基、チオアミド基、アシルオキシ基、アルキルスルホニル基、アルキルスルホニルオキシ基、アルケニルスルホニル基、アルケニルスルホニルオキシ基、アルキニルスルホニル基、アルキニルスルホニルオキシ基、アリールスルホニル基、アリールスルホニルオキシ基、アラルキルスルホニル基、アラルキルスルホニルオキシ基、−ＣＯＯＨ、−ＣＯＯＲ^ｇ、−ＣＯＮＨ_２、又は−ＣＯＮＨＲ^ｈ（但し、Ｒ^ｇ、Ｒ^ｈは、アルキル基、アルケニル基、アルキニル基、炭化水素芳香族基、飽和複素環基、芳香族複素環基、アラルキル基、アシル基、アルキルスルホニル基、アルケニルスルホニル基、アルキニルスルホニル基、アリールスルホニル基、又はアラルキルスルホニル基）、-ＮＨ_２、-ＮＨＲ^ｉ(但しＲ^ｉはアルキル基、アルケニル基、アルキニル基、アリール基、アラルキル基、又はアシル基)、-ＮＲ^ｊＲ^ｊ’(但しＲ^ｊ及びＲ^ｊ’は同一又は異なりアルキル基、アルケニル基、アルキニル基、アリール基、アラルキル基、又はアシル基)、アルキルアミノカルボニルアミド基、アリールアミノカルボニルアミド基、又はアミジノ基である。）で表わされるインドール類、それの薬学的に許容される塩、及び／又はそれの代謝物を、ストレス低減化成分として、含有するというものである。 The stress-reducing agent made to achieve the above object is represented by the following chemical formula (I)

(In the formula (I),
R ¹ may be a hydrogen atom, a hydroxyl group, or a substituent, and may be an alkyl group, an alkyloxy group, an alkylthioether group, an alkenyl group, an alkenyloxy group, an alkenylthioether group, an alkynyl group, or an alkynyloxy group. , Alkynyl thioether group, hydrocarbon aromatic group, hydrocarbon aromatic group-containing oxy group, hydrocarbon aromatic group-containing thioether group, saturated heterocyclic group, saturated heterocyclic group-containing oxy group, saturated heterocyclic group-containing thioether group, Aromatic heterocyclic group, aromatic heterocyclic group-containing oxy group, aromatic heterocyclic group-containing thioether group, aralkyl group, aralkyloxy group, aralkyl thioether group, acyl group, acyloxy group, thioester group, thioamide group, alkylsulfonyl group , Alkylsulfonyloxy group, alkenylsulfonyl group Alkenylsulfonyloxy group, alkynylsulfonyl group, alkynylsulfonyloxy group, arylsulfonyl group, arylsulfonyloxy group, aralkylsulfonyl group, aralkylsulfonyloxy group, or amidino group;
R ² represents a hydrogen atom, a halogen atom, or an alkyl group which may be substituted with a substituent;
n1 is the number 0 or 1;
R ³ is a hydrogen atom, a halogen atom, —COOH, —COOR ^a , —CONH ₂ , or —CONHR ^b (where R ^a and R ^b are optionally substituted with an alkyl group, Alkenyl group, alkynyl group, hydrocarbon aromatic group, saturated heterocyclic group, aromatic heterocyclic group, aralkyl group, acyl group, alkylsulfonyl group, alkenylsulfonyl group, alkynylsulfonyl group, arylsulfonyl group, or aralkylsulfonyl group) ;
R ⁴ and R ⁵ are the same or different and may be substituted with a hydrogen atom or a substituent, and may be an alkyl group, an alkenyl group, an alkynyl group, a hydrocarbon aromatic group, a saturated heterocyclic group, an aromatic heterocyclic group. A cyclic group, an aralkyl group, an acyl group, an alkylsulfonyl group, an alkenylsulfonyl group, an alkynylsulfonyl group, an arylsulfonyl group, an aralkylsulfonyl group, an alkyloxycarbonyl group, an aryloxycarbonyl group, an alkylaminocarbonyl group, or an arylaminocarbonyl group;
n2 is a number of 0 or 1, n3 is a number of 0 or 1, and n2 + n3>0;
R ^{6 to} R ⁹ are the same or different and may be substituted with a hydrogen atom, a halogen atom, a hydroxyl group, a mercapto group, or a substituent, and an alkyl group, an alkyloxy group, an alkylthioether group, an alkenyl group, an alkenyl group Oxy group, alkenyl thioether group, alkynyl group, alkynyloxy group, alkynyl thioether group, hydrocarbon aromatic group, hydrocarbon aromatic group-containing oxy group, hydrocarbon aromatic group-containing thioether group, saturated heterocyclic group, saturated heterocyclic ring Group-containing oxy group, saturated heterocyclic group-containing thioether group, aromatic heterocyclic group, aromatic heterocyclic group-containing oxy group, aromatic heterocyclic group-containing thioether group, aralkyl group, aralkyloxy group, aralkyl thioether group, acyl group An acyloxy group, an alkylsulfonyl group, an alkylsulfonyloxy group, Lucenylsulfonyl group, thioester group, thioamide group, alkenylsulfonyloxy group, alkynylsulfonyloxy group, alkynylsulfonyloxy group, arylsulfonyl group, arylsulfonyloxy group, aralkylsulfonyloxy group, aralkylsulfonyloxy group, amidino group, -COOH,- COOR ^c , —CONH ₂ , or —CONHR ^d (where R ^c , R ^d may be substituted with a substituent, and an alkyl group, an alkenyl group, an alkynyl group, a hydrocarbon aromatic group, a saturated A cyclic group, an aromatic heterocyclic group, an aralkyl group, an acyl group, an alkylsulfonyl group, an alkenylsulfonyl group, an alkynylsulfonyl group, an arylsulfonyl group, or an aralkylsulfonyl group), —NH ₂ , —NHR ^e (where R ^e is alkyl) Group, alkenyl group, An alkynyl group, an aryl group, an aralkyl group, or an acyl group), —NR ^f R ^{f ′} (wherein R ^f and R ^{f ′} are the same or different, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, an aralkyl group, or an acyl group) ), An alkylaminocarbonylamide group, or an arylaminocarbonylamide group;
Dashed / solid parallel lines are single bonds or double bonds,
The substituents in R ^{1 to} R ⁹ are each singular or plural, and are a hydroxyl group, mercapto group, halogen atom, alkyl group, alkyloxy group, alkylthioether group, alkenyl group, alkenyloxy group, alkenylthioether group. , Alkynyl group, alkynyloxy group, alkynyl thioether group, hydrocarbon aromatic group, hydrocarbon aromatic group-containing oxy group, hydrocarbon aromatic group-containing thioether group, saturated heterocyclic group, saturated heterocyclic group-containing oxy group, saturated Heterocyclic group-containing thioether group, aromatic heterocyclic group, aromatic heterocyclic group-containing oxy group, aromatic heterocyclic group-containing thioether group, aralkyl group, aralkyloxy group, aralkylthioether group, acyl group, thioester group, thioamide group , Acyloxy group, alkylsulfonyl group, alkylsulfonyloxy , Alkenylsulfonyl group, alkenylsulfonyl group, alkynyl-sulfonyl group, alkynylsulfonyl group, an arylsulfonyl group, an arylsulfonyl group, an aralkyl sulfonyl group, aralkyl sulfonyloxy groups, ^-COOH, -COOR g, -CONH _2, or - CONHR ^h (where R ^g and R ^h are an alkyl group, an alkenyl group, an alkynyl group, a hydrocarbon aromatic group, a saturated heterocyclic group, an aromatic heterocyclic group, an aralkyl group, an acyl group, an alkylsulfonyl group, an alkenylsulfonyl group) Group, alkynylsulfonyl group, arylsulfonyl group, or aralkylsulfonyl group), —NH ₂ , —NHR ⁱ (where R ⁱ is an alkyl group, alkenyl group, alkynyl group, aryl group, aralkyl group, or acyl group), —NR ^j R ^{j '(but j} and R ^{j 'are} the same or different alkyl group, an alkenyl group, an alkynyl group, an aryl group, an aralkyl group, or an acyl group), alkylaminocarbonyl amido group, an arylamino carbonyl amide group, or an amidino group. ), A pharmaceutically acceptable salt thereof, and / or a metabolite thereof as a stress reducing component.

  For example, the indole has a tryptophan skeleton, its metabolite has a kynurenine skeleton, or the indole has an indoleacetic acid skeleton.

  This stress-reducing drug is used, for example, for human treatment and / or prevention, or for raising non-human animals.

  This stress-reducing agent is one that lowers cortisol, raises calcitonin, lowers calcium, and / or activates osteoblasts, for example, in blood and / or urine.

  This stress-reducing agent preferably contains deep ocean water and / or components thereof.

  The stress reducing agent of the present invention contains components derived from deep ocean water, indoles and salts thereof and metabolites thereof as stress reducing components, reduces cortisol, raises calcitonin, and lowers calcium. And / or osteoblasts can be activated.

  Therefore, a stress reducing agent is effective for prevention and treatment of reducing stress in various animals such as humans, fish and shellfish, amphibians, birds, and non-human mammals such as domestic animals, food animals and pets. This stress reducing agent is also effective for a method of transporting fish in a closed space, for example, live fish transport.

It is a graph which shows the cortisol density | concentration at the time of 5 days breeding of a medina in the Example using the stress reduction chemical | medical agent which applies this invention, and the comparative example which does not apply this invention. It is a graph which shows the cortisol density | concentration at the time of 0, 5 and 10-day breeding of a medina in the Example using the stress reduction chemical | medical agent which applies this invention, and the comparative example which does not apply this invention. It is a graph which shows the cortisol density | concentration at the time of 0, 5 and 10 day breeding of a Japanese flounder in the Example using the stress reduction chemical | medical agent to which this invention is applied, and the comparative example which does not apply this invention. It is a graph which shows the calcium density | concentration and inorganic phosphate density | concentration of the blood component of a soleus in the Example using the stress reduction chemical | medical agent to which this invention is applied, and the comparative example which does not apply this invention. It is a graph which shows the blood calcium density | concentration at the time of 0, 5 and 10 day breeding of the Japanese flounder in the Example using the stress reduction chemical | medical agent which applies this invention, and the comparative example which does not apply this invention. It is a graph which shows the blood calcitonin density | concentration at the time of 5 day breeding of the Japanese flounder in the Example using the stress reduction chemical | medical agent which applies this invention, and the comparative example which does not apply this invention. It is a graph which shows the blood cortisol density | concentration at the time of the raising for 5 days of the flounder in the Example using the stress reduction medicine which applies this invention, and the comparative example which does not apply this invention. It is a graph which shows the calcium density | concentration at the time of 5 day breeding of the flounder in the Example using the stress reduction chemical | medical agent which applies this invention, and the comparative example which does not apply this invention. It is a graph which shows the activity of ALP and TRAP of goldfish scales regenerated for 10 days using a stress-reducing drug to which the present invention is applied. It is a graph which shows the activity of ALP and TRAP of goldfish scales regenerated for 14 days using a stress-reducing drug to which the present invention is applied. It is a graph which shows the activity of ALP of goldfish scales cultured for 6, 12, and 24 hours using a stress-reducing drug to which the present invention is applied. The value of the ALP activity of goldfish scales regenerated for 10 days using the stress-reducing agent to which the present invention is applied is shown as a value when the non-added control group is 1, and a DOW value when SSW is 1. It is a graph.

  Hereinafter, although the form for implementing this invention is demonstrated in detail, the scope of the present invention is not limited to these forms.

  The stress-reducing drug contains the indole represented by the chemical formula (I), a pharmaceutically acceptable salt thereof, and / or a metabolite thereof as a stress-reducing component.

In the chemical formula (I), an alkyl group, an alkyloxy group or an alkylthioether group is an alkyl group having 1 to 18 carbon atoms, which is a linear, branched and / or cyclic saturated alkyl such as methyl, ethyl or n-propyl. , Isopropyl, n-butyl, isobutyl, t-butyl, adamantyl;
The alkenyl of the alkenyl group, the alkenyloxy group or the alkenylthioether group is a C2-C18 linear, branched and / or cyclic unsaturated alkenyl such as vinyl and allyl;
The alkynyl of the alkynyl group, alkynyloxy group or alkynylthioether group is a C2-C18 linear, branched and / or cyclic unsaturated alkynyl such as ethynyl, 2-propynyl;
The hydrocarbon aromatic group of the hydrocarbon aromatic group, the hydrocarbon aromatic group-containing oxy group or the hydrocarbon aromatic group-containing thioether group is an aryl such as phenyl, naphthyl, anthracenyl, phenanthryl;
The saturated heterocyclic group of the saturated heterocyclic group, saturated heterocyclic group-containing oxy group or saturated heterocyclic group-containing thioether group is a saturated heterocyclic group containing oxygen and / or nitrogen such as tetrahydrofuranyl, tetrahydropyranyl, pyrrolidinyl, piperidinyl. ;
Aromatic heterocyclic group, aromatic heterocyclic group-containing oxy group or aromatic heterocyclic group-containing thioether group aromatic heterocyclic group is an oxygen and / or nitrogen-containing aromatic heterocyclic ring such as furanyl, pyridyl, indolyl. Group;
Aralkyl of an aralkyl group, an aralkyloxy group or an aralkylthioether group is an arylalkyl such as benzyl or phenethyl;
Acyl of acyl group, acyloxy group and thioester group is a linear, branched and / or cyclic saturated or unsaturated aliphatic acyl group having 1 to 18 carbon atoms (for example, formyl group, acetyl group, propionyl group, (Meth) acrylic group (acrylic group or methacrylic group), 3,3-dimethylacrylic group), aralkyl acyl group (eg phenylacetyl group) or aromatic acyl group (eg benzoyl group, phenolic hydroxyl group-containing benzoyl group) Is a 4-hydroxybenzoyl group, 3,4-dihydroxybenzoyl group or 4-hydroxy-3-methoxybenzoyl group);
The thioketo group of the thioamide group is a thioketo group corresponding to the acyl;
Alkyl of alkylsulfonyl group, alkylsulfonyloxy group and alkylaminocarbonylamide group is alkyl exemplified by the aforementioned alkyl group;
The alkenyl of the alkenylsulfonyl group or the alkenylsulfonyloxy group is the alkenyl exemplified for the alkenyl group;
The alkynyl of the alkynylsulfonyl group or the alkynylsulfonyloxy group is an alkynyl exemplified by the above alkynyl group;
Aryl of arylsulfonyl group, arylsulfonyloxy group and arylaminocarbonylamide group is aryl exemplified in the above-mentioned hydrocarbon aromatic group;
Aralkyl of the aralkylsulfonyl group or the aralkylsulfonyloxy group is arylalkyl exemplified by the above aralkyl group;
The amidino group is C (= NH) -NH ₂ ;
A halogen atom is a fluorine atom, a chlorine atom, an iodine atom;
Is mentioned.

  Among the stress-reducing agents, as the pharmaceutically acceptable salt of the indole represented by the chemical formula (I), an alkali metal salt or an alkaline earth metal salt may be used, and hydrochloride, sulfate, hydrogen bromide may be used. Acid salts, hydroiodide salts, inorganic acid salts such as nitrates, phosphates, metaphosphates, acetates / propionates, fumarate, maleates, citrates / tartaric acid It may be an organic acid salt such as a salt / benzoate, methanesulfonate / toluenesulfonate, naphthalenesulfonate, or a salt with an amino acid.

  This stress-reducing drug contains the indole represented by the chemical formula (I), a pharmaceutically acceptable salt thereof, and / or a metabolite thereof as a stress-reducing component, and if necessary, It may be formulated by mixing with toxic and inert pharmaceutically acceptable excipients such as solid, semi-solid or liquid diluents, dispersants, fillers and carriers. It may be mixed in water or seawater. In addition, stabilizers, preservatives, pH adjusters, binders, disintegrants, surfactants, lubricants, fluidity promoters, corrigents, colorants, fragrance preservatives, media, physiological saline, and other medicinal effects The medicine which it has may be contained as an additive.

  The dosage form of this stress-reducing agent is, for example, elixir, capsule, granule, pill, ointment, suspension, liquid, enteric solvent, emulsion, plaster, suppository, powder, tablet, syrup, injection , Lozenges, ointments, haps, liniments, limonades, lotions. It may be dissolved or suspended in a liquid medium, or may be dispersed in a solid medium. You may mix this stress reduction chemical | medical agent with water or seawater at the time of use, and use it as breeding water or drinking water.

  When used in humans, this stress-reducing drug may be administered orally, or may be administered by intravenous injection or infusion, or may be applied or affixed to the skin for percutaneous absorption.

  The stress reducing agent contains 0.001 to 99% by mass of a stress reducing component in the dosage form. When this stress reducing agent is mixed in water or seawater and used as breeding water, drinking water or feed for non-human animals, it is preferable to use the stress reducing component at a concentration of 0.0001 to 200 μg / kg. Moreover, when using this stress reduction medicine for humans, it is preferable to contain 0.001-100 mg / kg.

  The dosage and dose of this stress-reducing agent are the effectiveness of the stress-reducing component, the subject of human (patient) or non-human animal administration, the mode and route of administration, the degree of stress, the body type and weight of human and non-human animals・ Select as appropriate according to age, environment, amount and type of food and food. The administration may be carried out 1 to 5 times a day, every 1 to 14 days, or intermittently every 2 to 6 weeks, and breeding water and drinking water such as water and seawater. It may be mixed and added as appropriate.

  The indole represented by the chemical formula (I) in the stress-reducing agent is obtained by halogenating an unsubstituted or partially substituted starting indole compound such as an unsubstituted or partially substituted tryptamine compound or tryptophan compound. Or by substitution such as alkylation, alkenylation, alkynylation, aralkylation, etc., protection by sulfonylation, acylation, etc., or derivatization to another functional group according to conventional methods if necessary can do. For example, Japanese Patent Laid-Open No. 2006-104152 and International Publication No. WO20007 / 010723, M. Somei et al., Heterocycles, 36, p1859 (1993) and M. Somei, et al., Heterocycles, 53, p1725-1736 (2000) and M. Somei, et al., Advances in Heterocyclic Chemistry, Vol. 82, 2002, p101-155. If necessary, it may be a pharmaceutically acceptable salt or hydrate.

で表わされるキヌレニンのようなキヌレニン誘導体であってもよい。 More specifically, the indole has a tryptophan skeleton, such as a tryptophan derivative (specifically, in the formula (I), R ³ is —COOH, —COOR ^a , —CONH ₂ , or —CONHR ^b) . And a derivative represented by n1 = n2 = n3 = 1). As a metabolite, one having a kynurenine skeleton, for example, the following chemical formula (II)

A kynurenine derivative such as kynurenine represented by

Another specific example of the indoles is one having an indole acetic acid skeleton, such as an indole-3-acetic acid derivative (specifically, in the formula (I), R ³ is —COOH, —COOR ^a , —CONH). ₂ or -CONHR ^b , and a derivative represented by n1 = n3 = 0 and n2 = 1).

  Deep sea water such as Noto deep sea water contains kynurenine and indole-3-acetic acid. When fish are raised in such deep ocean water, blood cortisol concentration is decreased, calcitonin is increased, calcium is decreased, and osteoblasts are activated. This suggests that fish stress has been reduced by deep ocean water. Cortisol is known to cause growth-inhibiting effects such as increasing muscle degradation and reducing osteoblast activity. Is expected to improve.

  In addition, as for the effects of deep sea water on fish osteoblasts, the activity of alkaline phosphatase (ALP), an enzyme specific to osteoblasts, was examined using regenerated scales of goldfish (Carassius auratus). When cultivated using the seawater, the ALP activity of the osteoblasts on the scale was significantly higher than that cultivated using the surface seawater.

  The same tendency was observed for the components derived from deep ocean water, the indoles represented by the chemical formula (I), salts thereof and metabolites thereof. Accordingly, deep sea water, components derived from deep sea water, indoles, salts thereof and metabolites thereof have an action of increasing osteoblast activity. Since the growth of the skeleton is indispensable for the growth of fish, the increase in osteoblast activity that promotes it can contribute to aquaculture of fish.

  Hereinafter, the Example which applies this invention and the comparative example which does not apply this invention are demonstrated concretely.

  General materials and methods of Examples and Comparative Examples are as follows.

(Materials and methods)
(1) Test animals As test animals, readily available medina (Girella punctata) and flounder (Paralichthys olivaceus) were used as test fish. After collection or acquisition, habituation treatment for 3 days or more was performed in an outdoor flushing water tank until just before the test.

(2) Breeding water Breeding water for the test is DOW (Deep Ocean Water) from the deep water of 332 m offshore of Ogi Port, Noto Peninsula. Surface seawater pumped at a seaside experimental facility from Kanazawa University Environmental Research Center for the Japan Sea Region from a depth of 2 m in Kujuku Bay as surface water water (SSW) It was used. These seawaters were used as breeding water for testing, which were supplied from the water supply valves of each facility, or were stored in a shaded and dark place by being drawn up in a plastic tank.

(3) Rearing system
(3.1) Breeding system I
It was constructed using a water bath (volume: 120 × 45 × 45 cm ³ ). Two tanks were prepared for the DOW breeding group and the SSW breeding group, respectively. Breeding water was placed in a tank of 200 L and the water was changed 20 L a day. EHEIM FILTER 2260 (manufactured by EHEIM) was used as a biological filtration device for removing ammonia, and cinnabar sand was enclosed as a filter medium. A cooler tower (ZC-200α, manufactured by Zensui Co., Ltd.) and a heater (Protect PRO 500W, manufactured by Nisso Co., Ltd.) were connected to this apparatus, and the water temperature was made constant by thermostat control. In addition, oxygen was supplied to the breeding water by excessive bubbling using an air pump. An LED lamp (flat LED 1200, manufactured by Kotobuki Kogyo Co., Ltd.) was prepared as the light source, and the light was controlled and controlled by a timer from 6:00 to 18:00. Once a day, the feed provided an uneaten amount (about 15 g) of artificial feed for aquaculture in Thailand.

(3.2) Breeding system II
For DOW breeding group and SSW breeding group in the same way as breeding system I, except that one filtration device was connected to two lines of 4 tanks (volume: 60 x 25 x 30 cm ³ ) It was.

(Examples 1-3 and Comparative Examples 1-3)
First, in Examples 1 to 3 and Comparative Examples 1 to 3, blood cortisol concentrations in the DOW breeding group and the SSW breeding group were compared.

(Example 1 and Comparative Example 1: Comparison of blood cortisol concentration between DOW and SSW under medina breeding)
Ten medinas (100 ± 25 g) after acclimation treatment were used and divided into 5 groups of DOW rearing group (Example 1) and SSW rearing group (Comparative Example 1). Breeding at 5 ° C for 5 days. Thereafter, blood was collected under anesthesia.

(Example 2 and Comparative Example 2: Comparison of blood cortisol concentration between DOW and SSW under medina breeding)
Use 16 medina (100 ± 25 g) after acclimation treatment and divide 8 animals into DOW breeding group (Example 2) and SSW breeding group (Comparative Example 2). Breeding at 10 ° C for 10 days. On the 0th, 5th and 10th days of breeding, blood was continuously collected from the same individual under anesthesia.

(Example 3 and Comparative Example 3: Blood cortisol concentration measurement test under DOF and SSW flounder breeding)
16 medinas (350 ± 100 g) after the acclimation treatment were used, divided into 8 animals each for DOW rearing group (Example 3) and SSW rearing group (Comparative Example 3). Breeding at 10 ° C for 10 days. On the 0th, 5th and 10th days of breeding, blood was continuously collected from the same individual under anesthesia.

(Blood collection in Examples 1 to 3 and Comparative Examples 1 to 3)
In order to measure the cortisol concentration in blood, blood was collected from medina or flounder and the plasma was used. Adopting the lower limit concentration at which an anesthetic effect was observed, medina or flounder was anesthetized with an anesthetic solution (0.02% Phenoxyethanol, manufactured by Wako Pure Chemical Industries, Ltd.), and blood was collected from the dorsal aorta with a heparin-treated syringe. The collected blood was immediately centrifuged for 1 minute in a tabletop centrifuge, and the supernatant was separated and stored at −80 ° C. as a plasma sample. The separated plasma sample was passed through 5 times the amount of diethyl ether, and the ether layer was nitrogen-dried to remove proteins. The dried sample is an ELISA assay buffer (50 mM H ₃ BO ₃ ; 0.2% BSAfor ELISA; 0.01% Thimerosal, both manufactured by Wako Pure Chemical Industries, Ltd.) (pH 7.8). And stored at −80 ° C. until immediately before the test.

(Measurement of cortisol concentration in Examples 1 to 3 and Comparative Examples 1 to 3)
<Measurement principle>
For the measurement of cortisol concentration, cortisol in the sample solution and enzyme-labeled cortisol are allowed to undergo an antigen-antibody reaction competitively with the anti-cortisol antibody, and the color of the substrate solution by the subsequent enzyme reaction is changed to a standard curve. A general competitive steroid hormone ELISA method was applied to perform colorimetric quantification.

<Operation 1: Preparation of secondary antibody-immobilized plate>
First, an anti-rabbit antibody goat antibody was immobilized on a plate. A commercially available anti-rabbit antibody goat antibody (CPL55641, manufactured by Cosmo Bio Co., Ltd.) was added to a carbonate buffer (15 mM Na ₂ CO ₃ ; 35 mM NaHCO ₃ ; 3 mM NaN ₃ ; both manufactured by Wako Pure Chemical Industries, Ltd.) (pH 9.6). A secondary antibody solution was prepared by diluting to 15 μg / mL. 100 μL of this solution was poured into a 96-well plate (C8 MAXISORP, Nunc-Imnomodule), sealed with cellophane tape, and incubated at room temperature for 48 hours to solidify. The plate was then blocked with sugar. The secondary antibody solution was discarded and washed 4 times with 250 μL of physiological saline. Pour 200 μL of blocking buffer (50 mM H ₃ BO ₃ ; 0.1% BSA; 3% Sucrose, Wako Pure Chemical Industries, Ltd.) (pH 7.8), seal with cellophane tape, and incubate at room temperature for 48 hours. And blocked. Thereafter, the blocking buffer was discarded, sealed with cellophane tape, and stored at 4 ° C.

<Operation 2: Preparation of dilution series of cortisol solution (standard solution) of known concentration>
A solution in which unlabeled cortisol was dissolved in ethanol to 1 mg / mL was prepared. Dilute serially with assay buffer to 100 ng / mL, 50 ng / mL, 25 ng / mL, 12.5 ng / mL, 6.25 ng / mL, 3.23 ng / mL, 1.56 ng / mL, 0.78 ng / mL each time, Each sample was incubated at the same time as the standard solution.

<Operation 3: Preparation of horseradish peroxidase (HRP) -labeled cortisol>
HRP-labeled cortisol (FKA 403, manufactured by Cosmo Bio Co., Ltd.) was stored at 4 ° C. and diluted 1/50 times with assay buffer for each measurement.

<Operation 4: Preparation of primary antibody solution>
Using anti-cortisol rabbit antibody (FKA 404-E, Cosmo Bio), dissolve 20 μL of the inner solution in 9.98 mL of assay buffer, dilute 1/500, and dispense 440 μL into Eppendorf tubes. Stored at -80 ° C. Immediately before the experiment, it was used after being diluted 1/10 times with assay buffer.

<Operation 5: Antigen-antibody reaction>
50 μL of the solution prepared as described above was added to each well of the secondary antibody-immobilized plate in the order of sample or standard solution, HRP-labeled cortisol solution, and primary antibody solution. Thereafter, it was sealed with cellophane tape, shaken gently, and incubated at room temperature for 24 hours.

<Operation 6: Preparation of HRP substrate solution and color reaction>
As a substrate for the enzyme reaction by HRP, o-phenylenediamine dihydrochloride (o-Phenylenediamine: OPD, manufactured by Wako Pure Chemical Industries, Ltd.) was used. OPD is oxidatively cleaved by a reaction catalyzed by HRP and exhibits an absorption wavelength of 492 nm. Immediately before the color reaction, OPD and hydrogen peroxide (Hydrogen peroxide: H ₂ O ₂ , manufactured by Wako Pure Chemical Industries, Ltd.), citrate buffer (0.2 M Citric acid, manufactured by Wako Pure Chemical Industries, Ltd.) (pH 4.5) Was used to prepare a substrate solution (0.08% OPD, 0.02% H ₂ O ₂ in Citricacic buffer). After completion of the antigen-antibody reaction procedure, the plate was washed with 250 μL of plate washing solution (131 mM NaCl; 9 mM Na ₂ HPO ₄ ; 1.1 mM NaH ₂ PO ₄ , Wako Pure Chemical Industries, Ltd .; 1.5 mM KH ₂ PO ₄ , All of the above were washed 4 times with Wako Pure Chemical Industries, Ltd .; 0.05% Tween20) (pH 7.4). Thereafter, 100 μL of the substrate solution was poured, incubated in the dark for about 20 minutes with light shaking. After confirming that the substrate solution was sufficiently colored, the enzyme reaction was stopped by adding 50 μL of normality 3 sulfuric acid. The plate was converted into data by measuring the absorbance with a plate reader set to an absorption wavelength of 492 nm, and the absorbance was compared with that of the standard solution to determine the cortisol concentration in the sample solution.

(Statistical treatment of cortisol concentration in Examples 1 to 3 and Comparative Examples 1 to 3)
When the data of Example 1 and Comparative Example 1 are processed statistically, first, an outlier test (Grubbs' outlier t-test) is applied to each group of DOW breeding group and SSW breeding group, and p <0.05. Was found as an outlier and then excluded from the average calculation. Next, a one-sided t-test with two samples assuming equal variance was performed. For the resulting p-value, p <0.05 was the statistical significance level, and p <0.10 was the statistical significance trend. Elements that were outliers were inserted with x marks in the graph.
On the other hand, when the data of Examples 2 to 3 and Comparative Examples 2 to 3 are statistically processed, the blood cortisol concentration value of each individual bred for 5 days and 10 days is first expressed as blood on the 0th day. The rate of change was calculated by dividing by the value of medium cortisol. Using this value, Sidak's multiple comparison test was performed for each time point on the 5th and 10th days. For the p-values as the results, p <0.05 was defined as the statistical significance level, and p <0.10 was defined as the statistical significance trend.

(Results of cortisol concentration in medina in Example 1 and Comparative Example 1)
FIG. 1 shows the results of the cortisol plasma concentration of mezzina in the DOW breeding group (Example 1) and the SSW breeding group (Comparative Example 1), each of which was fed with 5 Medina 5 days at 20 ° C. When the average value was obtained and subjected to statistical tests, as is clear from FIG. 1, the plasma cortisol concentration in the DOW group was significantly lower than that in the SSW group.

(Results of cortisol concentration in medina in Example 2 and Comparative Example 2)
FIG. 2 shows the results of the cortisol plasma concentration of medina in the DOW breeding group (Example 2) and the SSW breeding group (Comparative Example 2) that were raised at 20 ° C. for 5 and 10 days. The change rate of plasma cortisol on the 5th and 10th day relative to the 0th day (initial) of each individual showed that the plasma cortisol concentration increased as the breeding period increased in the SSW breeding group, whereas the DOW breeding group However, the plasma cortisol concentration hardly changed even when the breeding period increased.

(Results of cortisol concentration in flounder in Example 3 and Comparative Example 3)
FIG. 3 shows the results of cortisol plasma concentrations in Japanese flounder in the DOW breeding group (Example 3) and the SSW breeding group (Comparative Example 3) that were raised at 20 ° C. for 5 and 10 days. The change rate of plasma cortisol on the 5th and 10th day relative to the 0th day (initial) of each individual is increased in the SSW breeding group as the breeding period increases, whereas in the DOW breeding group, the plasma cortisol concentration increases. The plasma cortisol concentration remained almost unchanged even as the breeding period increased.

  Cortisol is known as one of the stress hormones and is known to be an indicator of chronic stress because it is secreted from the adrenal cortex (interrenal gland in fish) and increases in blood concentration when the organism is stressed. Yes. As compared with the results of Examples 1 to 3 and Comparative Examples 1 to 3, the blood cortisol concentration increased with time due to the SSW breeding, so that the breeding in the aquarium itself is a stress for the fish. In contrast, fish such as medina or flounder raised in deep ocean water (DOW) were found to have little decrease in blood cortisol levels. DOW breeding has been found to reduce the chronic stress associated with breeding in narrow aquariums. Cortisol is known to cause growth-inhibiting effects such as enhancing muscle breakdown, and lowering this is thought to free it from growth inhibition and improve growth.

(Example 4 and Comparative Example 4)
Next, in Example 4 and Comparative Example 4, blood components under flounder breeding in the DOW breeding group and the SSW breeding group were examined.

(Example 4 and Comparative Example 4: Measurement of blood components under DOF and SSW flounder breeding)
Examination of blood measurement in human health checkup for each of the test items of plasma of 8 flounder each in DOW rearing group and SSW rearing group that were reared for 10 days at 20 ° C in Example 3 and Comparative Example 3 above In the same way as the item, total protein (TP) (g / dL), albumin (ALB) (g / dL), urea nitrogen (BUN) (mg / dL), creatinine (CRE) (mg / dL), sodium ( Na) (mEq / L), potassium (K) (mEq / L), chlor (Cl) (mEq / L), calcium (Ca) (mg / dL), inorganic phosphorus (IP) (mg / dL), asparagine Acid aminotransferase (AST) (IU / L), alanine aminotransferase (ALT) (IU / L), lactate dehydrogenase (LDH) (IU / L), amylase (AMY) (IU / L), γ-glutamyl Transferase (rG) (IU / L), Total cholesterol (T-CHO) (mg / dL), Neutral fat (TG) (mg / dL), High density lipoprotein-cholesterol (HDL-C) (mg / dL ), Total bilirubin (T-BIL) (mg / dL), and glucose (GLU) (mg / dL).

(Statistical treatment of blood components in Example 4 and Comparative Example 4)
For statistical processing of the analyzed data, two-sided t-test (student's t-test) with two samples assuming equal variance for each test item in the DOW breeding group (Example 4) and the SSW breeding group (Comparative Example 4) Went. Regarding p-values, p <0.05 was regarded as a statistical significance level, and p <0.10 as a statistical significance trend.

(Results of each inspection item with flounder in Example 4 and Comparative Example 4)
As a result of the blood component screening of Japanese flounder in the DOW rearing group (Example 4) and the SSW rearing group (Comparative Example 4) that were reared for 5 and 10 days, as shown in FIG. 4, the blood was higher in the DOW rearing group than in the SSW rearing group. Medium calcium concentration was significantly lower and inorganic phosphorus concentration was significantly higher.

(Example 5 and Comparative Example 5)
Therefore, next, in Example 5 and Comparative Example 5, the blood calcium concentration under flounder breeding in the DOW breeding group and the SSW breeding group was measured at each time point.

(Example 5 and Comparative Example 5: Comparison of blood calcium concentration under flounder breeding between DOW and SSW)
About 8 flounder (0th, 5th, 10th day) each in the DOW rearing group and SSW rearing group reared for 10 days at 20 ° C. in Example 3 and Comparative Example 3 in the blood The calcium concentration was measured.

(Measurement of calcium concentration in Example 5 and Comparative Example 5)
<Measurement principle>
Since arsenazo III, a chelating agent in the color solution, absorbs light at 660 nm when chelated to calcium in the sample, the absorbance increases in proportion to the calcium concentration. By creating a standard curve, the calcium concentration is colorimetrically determined.

<Calculation procedure of calcium concentration>
The calcium concentration was measured using a kit of Aqua Auto Kinos Ca Reagent (manufactured by Kinos Co., Ltd.). A calcium solution (standard solution) with a known concentration and 4 μL of sample plasma were placed in a 1.5 mL Eppendorf tube, 360 μL of reagent was added, and the mixture was mixed by inversion. Thereafter, each solution was placed in a predetermined position of a 96-well plate (Code No. 269620, manufactured by Thermo Fisher Scentific) at a rate of 100 μL per well for triplication. The plate was converted into data by measuring the absorbance with a plate reader whose absorption wavelength was set to 660 nm, and the absorbance was compared with that of the standard solution to determine the calcium concentration in the sample solution.

(Statistical treatment of calcium concentration in Example 5 and Comparative Example 5)
When the data of Example 5 and Comparative Example 5 are processed statistically, first, an outlier test (Grubbs' outlier t-test) is performed for each DOW breeding group and SSW breeding group, and elements that satisfy p <0.05 are excluded. The value was excluded from the average calculation. Thereafter, a two-sided t-test (student's t-test) was performed with two samples assuming equal variance for each time point. Regarding p-values, p <0.05 was regarded as a statistical significance level, and p <0.10 as a statistical significance trend.

(Results of calcium concentration in Japanese flounder in Example 5 and Comparative Example 5)
Results of blood calcium concentration on day 0, 5 and 10 of 8 flounder in DOW rearing group (Example 5) and SSW rearing group (Comparative Example 5) reared at 20 ° C for 5 and 10 days Is shown in FIG. The average value was obtained and subjected to a statistical test. As is clear from FIG. 5, the blood calcium concentration in the DOW group was significantly lower in the sample on the 10th day than in the SSW group as shown in FIG.

(Example 6 and Comparative Example 6)
Then, next, in Example 6 and Comparative Example 6, the blood calcitonin concentration in the flounder breeding in the DOW breeding group and the SSW breeding group was measured at each time point.

(Example 6 and Comparative Example 6: Comparison of blood calcitonin concentrations in DOW and SSW under flounder breeding)
In the above Example 3 and Comparative Example 3, the plasma of each flounder in the DOW breeding group and the SSW breeding group that were raised for 10 days at 20 ° C. for 10 days (Day 0, Day 5) was diluted 5-fold and added to the blood. Calcitonin concentration was measured.

(Measurement of calcitonin concentration in Example 6 and Comparative Example 6)
<Measurement principle>
Calcitonin competition ELISA was used. In this competitive ELISA method, the pre-incubated sample and primary antibody solution are poured onto a calcitonin-immobilized plate so that the calcitonin on the solid phase and the calcitonin in the sample compete with each other for the antigen antibody. Then, the coloration of the substrate solution by the enzymatic reaction is colorimetrically determined in accordance with the standard curve.

<Operation 1: Preincubation>
A preincubation operation was performed in which the calcitonin in the sample solution was reacted with an anti-salmon calcitonin rabbit antibody, which is a primary antibody, as an antigen-antibody. First, a primary antibody solution was prepared. PBS (0.13 M NaCl, 9 mM Na ₂ HPO ₄ , 1.1 mM NaH ₂ PO ₄ , 1.5 mM KH ₂ PO ₄ , 0.1% BSA, 0.1% so that the anti-salmon calcitonin rabbit antibody is 1/320000 of serum It was diluted with NaN ₃ ) (pH 7.4) and used as a primary antibody solution.
Next, a dilution series of a calcitonin solution (standard solution) with a known concentration was prepared. First, a solution in which unlabeled calcitonin (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in distilled water (DW) so as to be 500 ng / μL was prepared, dispensed into an Eppendorf tube, and stored at −80 ° C. For each experiment, dilute with PBS to 1600 pg / mL, 800 pg / mL, 400 pg / mL, 200 pg / mL, 100 pg / mL, 50 pg / mL, 25 pg / mL, 12.5 pg / mL, and use them as standard solutions. Used for preincubation at the same time as the sample. Thereafter, the primary antibody solution and an equal amount of blood sample or standard solution were placed in a 5 mL glass test tube. The tube was capped and preincubated for 72 hours at 4 ° C.

<Operation 2: Preparation of calcitonin-immobilized plate>
First, calcitonin was immobilized on a plate. Prepare a salmon calcitonin solution diluted to 0.1 ng / μL with carbonate buffer (15 mM Na ₂ CO ₃ , 35 mM NaHCO ₃ , 3 mM NaN ₃ ) (pH 9.6), and add all wells to a 96-well plate (Falcon). 100 μL each was sealed with cellophane tape and incubated at 25 ° C. for 24 hours to immobilize. The plate was then blocked with skim milk. After incubation, wash 4 times with 250 μL of plate wash (131 mM NaCl, 9 mM Na ₂ HPO ₄ , 1.1 mM NaH ₂ PO ₄ , 1.5 mM KH ₂ PO ₄ , 0.05% Tween20) (pH 7.4) for blocking Buffer (5% skim milk in carbonate buffer) was poured in 250 μL aliquots, incubated at 25 ° C. for 1 hour, and blocked. This plate was further washed and used as a calcitonin immobilized plate.

<Operation 3: Antigen-antibody reaction>
100 μL each of the pre-incubated sample solution and standard solution was poured into a predetermined position on the calcitonin immobilized plate. Thereafter, it was sealed with cellophane tape, shaken gently, and incubated at 4 ° C. for 24 hours.

<Operation 4: Reaction of secondary antibody>
A biotin-labeled anti-rabbit antibody goat antibody (Polyclonal Goat Anti-Rabbit Immunoglobulins / Biotinylated, manufactured by Dako) was diluted 5000 times with PBS to prepare a secondary antibody solution. 100 μL of the solution was poured into all wells of the washed plate, sealed, and incubated at 25 ° C. for 2 hours.

<Operation 5: Reaction of horseradish peroxidase (HRP) -labeled streptavidin>
HRP-labeled streptavidin (Streptavidin / HRP, manufactured by Dako) was diluted 10,000 times with PBS to prepare an HRP-labeled streptavidin solution. 100 μL of the solution was poured into all wells of the washed plate, sealed, and incubated at 25 ° C. for 1 hour.

<Operation 6: Preparation of HRP substrate solution and color reaction>
O-Phenylenediamine dihydrochloride (OPD) was used as a substrate for the enzymatic reaction by HRP. A substrate solution (0.08% OPD, 0.02% H ₂ O ₂ in citrate buffer) was prepared using OPD, hydrogen peroxide, and citrate buffer (0.2 M Citric acid, pH 4.5) immediately before the color reaction. After completion of the HRP-labeled streptavidin incubation procedure, the plate was washed 4 times with 250 μL of plate wash. Thereafter, 100 μL of the substrate solution was poured in, protected from light, and incubated for about 30 minutes with gentle shaking. After confirming that the substrate solution was sufficiently colored, the enzyme reaction was stopped by adding 50 μL of normality 3 sulfuric acid. The plate was converted into data by measuring the absorbance with a plate reader whose absorbance wavelength was set to 492 nm, and the absorbance was compared with a calcitonin standard to determine the calcitonin concentration in the sample solution.

(Statistical treatment of calcitonin concentration in Example 6 and Comparative Example 6)
When the data of Example 6 and Comparative Example 6 are processed statistically, the outlier test (Grubbs' outlier t-test) is applied to each of the DOW breeding group and the SSW breeding group to remove the element that gives p <0.05. Excluded after finding the value. Next, a two-sided t-test with two samples assuming equal variance was performed. Regarding p-values, p <0.05 was regarded as the statistical significance level, and p <0.10 as statistical significance.

(Results of calcitonin concentration in Japanese flounder in Example 6 and Comparative Example 6)
Fig. 6 shows the results of the statistical test by calculating the mean value of the blood calcitonin concentration on the 5th day of breeding for 8 flounder each in the SSW breeding group and the DOW breeding group raised at 20 ° C for 10 days. Show. As is apparent from FIG. 6, on the fifth day, the mean value of blood calcitonin concentration in the DOW group was higher than that in the SSW group (p = 0.10).

  As shown in the comparison of the results of Examples 5-6 and Comparative Examples 5-6, the blood calcium concentration was significant by raising fish in deep sea water (DOW) compared to fish raised in surface seawater (SSW). Declined. Calcium exists in the body in the form of hydroxyapatite mainly in bones and teeth. Calcium ions act as an activator for maintaining muscle and nerve functions, blood coagulation, and enzymes, and also act as regulators of intracellular signal transduction along with cAMP. Also, cadherin, a molecule responsible for cell-cell adhesion, adheres adjacent cells in a calcium-dependent manner.

  The reason for the change in blood calcium concentration is estimated as follows. Cortisol present in the blood as shown in the results of Example 1 and Comparative Example 1 seems to be involved in changes in the calcium concentration in the blood. Cortisol is a steroid hormone secreted from the fish interrenal gland (adrenal cortex in mammals) and acts as a stress hormone, but seems to affect calcium metabolism. Osteoblasts have the function of forming bone (bone formation) and deposit calcium in bone tissue. Osteoclasts also enzymatically break down bone (bone resorption) and release calcium. Cortisol increases osteoclast differentiation promoting factor RANKL (receptor activator of nuclear factor κ B ligand) and suppresses OPG (osteoclastogenesis) expression at its decoy receptor. Promotes and increases bone resorption. It also suppresses osteoblast differentiation and proliferation and increases apoptosis of mature osteoblasts. As a result, the deposition of calcium on the bone matrix is delayed, and it is presumed that the blood calcium concentration tends to increase. As the results of Examples 1 to 3 and Comparative Examples 1 to 3, the blood cortisol concentration was kept low by DOW breeding compared to SSW breeding. The change in calcium concentration is considered to correspond to this change in cortisol concentration.

  In most teleosts, it is known that osteoblasts and osteoclasts on scales are involved in the regulation of blood calcium levels. The teleost scales have a bone matrix composed of a fibrous layer and a bone layer similar to mammalian bones, and bone metabolism is regulated by osteoblasts and osteoclasts. Since the spine is important for swimming in fish, it is thought that the scale's bone matrix is used as a reservoir for calcium to be taken in and out in order to maintain blood calcium homeostasis. In fact, it is known to use mainly the osseous bone matrix for calcium regulation, not the spine, in situations such as sexual maturity and starvation where calcium is deficient. It is presumed that DOW affected the regulation of bone metabolism by osteoblasts and osteoclasts on fish scales, and that the blood calcium concentration changed.

  Furthermore, it seems that the hormone calcitonin, which has the function of lowering the blood calcium level, is also involved. Calcitonin binds to a receptor on osteoclasts and contracts osteoclasts through the destruction of the osteoclast skeleton (actin ring) to suppress bone resorption. It is known that calcitonin has a role of maintaining the plasma calcium concentration at a physiological concentration in marine fish.

  Calcitonin is known to have analgesic effects, and there are various factors regarding its mechanism (inhibition of prostaglandin synthesis, activation of endogenous opioid system, sensory C fiber involved in serotonin receptor fluctuation, suppression of CGRP release) Etc.). In mice, calcitonin suppresses proteolysis due to stress through suppression of the activity of a proteolytic enzyme called calpain to alleviate shock. Thus, calcitonin is involved in maintaining the health of the living body by acting suppressively on several targets. DOW may have affected fish scales and increased calcitonin expression, which may be released into the blood and affect the nervous system.

(Example 7 and Comparative Example 7)
Next, in Example 7 and Comparative Example 7, stress reducing components in DOW and SSW used as breeding water were identified.

<Filtering seawater samples>
Install the glass fiber filter (GC-50, manufactured by Advantec) and C-18 filter (2215-C18, manufactured by Empore) in this order on the filter holder unit, and fix it with a clamp. Connected. 50 mL of methanol (for HPLC, manufactured by Wako Pure Chemical Industries, Ltd.) and 100 mL of milli-Q water prepared with an ultrapure water production apparatus manufactured by Millipore were poured into the filter unit in this order to hydrophilize the silicon filter. 3 L of seawater sample was poured into the filter unit, the inside of the filter bottle was made negative pressure with a suction pump, and the seawater was passed through the filter. At this time, when the glass fiber filter was clogged and the flow rate dropped significantly, the clamp was removed and the glass fiber filter was replaced. After passing 3 L of the seawater sample, 50 mL of milli-Q water was poured and the filter was lightly washed. Thereafter, air was aspirated for 20 minutes to dry the filter.

<Elution of components from the filter>
A C-18 filter through which a seawater sample was passed was installed in the filter holder unit and fixed with a clamp. Pour 10 mL of ethanol and 30 mL of benzene, and pass through the filter for 10 minutes to elute the compound. This liquid was once collected in a conical beaker. The eluted filter was discarded and the filter holder unit was rinsed with a small amount of ethanol. This ethanol was also collected in a conical beaker. The solution collected in the conical beaker was nitrogen-dried to about 10 mL. This solution was transferred with a Komagome pipette into a 20 mL volume brown bottle with a lid. Further, the conical beaker was washed twice with about 3 mL of ethanol, and the solution was also transferred to a brown bottle. All solutions collected in the brown bottle were dried to nitrogen. Immediately before the measurement, the dried sample was redissolved in 500 μL of DW, and the solution was analyzed by LC-MS / MS. In the analysis, the concentration of each compound was analyzed using 10 types of indole compounds as standard (melatonin: MEL, N-acetyl-5-methoxyquinuramine: AMK, N-acetyl-N-formyl-5 -Methoxyquinuramine: AFMK, serotonin: 5HT, N-acetyl serotonin: NAS, hydroxymelatonin: HaMT, quinuramine, kynurenine, 5-methoxytryptamine: 5MTP, indoleacetic acid: IAA). The results of the detected components are shown in Table 1.

  As is clear from Table 1, Noto Ocean Deep Water (DOW) contained more kynurenine than surface seawater (SSW). Indole-3-acetic acid was also contained. This indicates that the effect of DOW on fish physiology may be due to the action of kynurenine and indoleacetic acid.

  Kynurenin is one of the intermediate metabolites of the kynurenine pathway, a type of tryptophan metabolic pathway. In mammals, tryptophan is catalyzed by tryptophan oxygenase (Tryptophan 2,3-dioxygenase, TDO) present in the liver, and then enters kynurenine pathway via N'-formylkynurenine. Tryptophan that reaches the brain without entering the kynurenine pathway is metabolized by tryptophan 5-hydroxylase to become serotonin in the brain. Quinolic acid, 3-hydroxykynurenine and kynurenic acid, which are intermediate metabolites of the kynurenine pathway, are neuroactive substances, such as human Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, AIDS dementia complex, It is known to be involved in many diseases and disorders including malaria, cancer, depression and schizophrenia. Indoleamine 2,3-dioxygenase 1: IDO1, which is strongly expressed in antigen-presenting cells, epithelial cells and tumor cells, is another enzyme that converts tryptophan to kynurenine. Tryptophan depletion and production of kynurenine metabolites play important regulatory roles in T cell-mediated immune responses. Kynurenin itself, as an endogenous ligand of the aryl hydrocarbon receptor, has the function of controlling the innate immune response by suppressing the induction of type I interferon production.

  On the other hand, regarding fish physiology, kynurenine itself has been identified as a sex pheromone of salmon salmon. In relation to the kynurenine pathway, allylformamidases extracted from cattle and rainbow trout liver (catalyzing the reaction from N'-formylkynurenine to kynurenine) were compared, and the properties such as heat resistance and feedback mechanism were compared. I know there is a big difference. As for knowledge related to antistress and health, in the zebrafish tank behavior test, exposure to kynurenic acid (KYNA) produced from kynurenine is known to have an anxiolytic-like effect (similar to the knowledge of mice and rats). Is the result). In vitro, the growth of rainbow trout immune cells was observed after exposure to certain concentrations of KYNA, suggesting that KYNA may activate fish lymphocytes.

(Example 8 and Comparative Example 8)
Therefore, next, in Example 8 and Comparative Example 8, changes in the blood cortisol and calcium concentrations of flounder by artificial seawater breeding containing kynurenine or indoleacetic acid were measured, and the effects of kynurenine and indoleacetic acid were examined.

<Creation III>
It was constructed using a plastic case water tank (300 × 195 × 205 mm ³ ). 6 tanks are prepared, 2 of which are used for breeding the untreated group (control), 2 are used for breeding the indole-3-acetic acid (IAA) treated group, and 2 are used for the kynurenine treated group. did. As breeding water, 3% artificial seawater (SEALIFE, manufactured by Nippon Seawater Co., Ltd.) was used as a solvent, and the following three kinds of seawater were prepared and used.
(i) Untreated artificial seawater: Prepared by dissolving artificial seawater in tap water to 3% practical salinity.
(ii) IAA-containing artificial seawater: Prepared by adding IAA (manufactured by Wako Pure Chemical Industries, Ltd.) to untreated artificial seawater so as to be 10 ⁻⁶ M.
(iii) Artificial seawater containing kynurenine: Kynurenin (manufactured by Sigma) was added to untreated artificial seawater to a ^{concentration} of 10 ⁻⁶ M.
Breeding water was placed in a case of 5 L and the water was changed 2 L a day. Here, IAA and kynurenine lost due to water replacement are replaced with 1 mL of ethanol (for HPLC, manufactured by Wako Pure Chemical Industries, Ltd.) and 19 mL of DW. Every time, the amount that was reduced in the calculation was supplemented with fresh artificial seawater. In addition, oxygen was supplied to the breeding water by bubbling using an air pump. It was controlled by a timer from 6:00 to 18:00, and the light was adjusted. No food was given during the experiment.

<Raising and blood collection>
Use 18 flounder (15 ± 6 g) after acclimation treatment, and divide 6 each into an untreated group (Comparative Example 8), an IAA-treated group, and a kynurenine-treated group (Example 8). I put it in the aquarium. The water temperature was kept at 20 ° C for 5 days. On day 5, flounder was anesthetized with an anesthetic solution to obtain a sample for measuring blood cortisol and calcium concentrations, and blood was collected from the dorsal aorta with a syringe not subjected to heparin treatment. The collected blood was allowed to clot for 2 hours at room temperature and 24 hours at 4 ° C. Thereafter, the mixture was centrifuged at 5,000 rpm for 15 minutes, and the supernatant was separated and stored as a serum sample at −80 ° C.

<Measurement of blood cortisol and calcium concentration>
In the measurement of the cortisol concentration, a cortisol measurement system by a competitive ELISA method was used in the same manner as in Examples 1 to 3 and Comparative Examples 1 to 3. Further, the calcium concentration was measured in the same manner as in Example 5 and Comparative Example 5 using a kit of Aqua Auto Kinos Ca reagent (manufactured by Kinos Co., Ltd.).

(Statistical treatment of calcium concentration in Example 8 and Comparative Example 8)
When statistically processing these data, the outlier test (Grubbs' outlier t-test) is applied to each group for the cortisol concentration or calcium concentration of the untreated group, IAA-treated group, and kynurenine-treated group, and p < Elements with 0.05 were found as outliers and excluded from the average calculation. Thereafter, the untreated group was used as a control, and a multiple comparison test by Dunnett's method was performed with the null hypothesis that there was no difference from the control. For the resulting p-value, p <0.05 was the statistical significance level, and p <0.10 was the statistical significance trend. Elements that were outliers were inserted with x marks in the graph.

(Results of calcium concentration in Japanese flounder in Example 8 and Comparative Example 8)
Serum cortisol and calcium concentrations were measured for 6 flounder each in the untreated group, IAA-treated group, and kynurenine-treated group that were raised at 20 ° C for 5 days. The results are shown in FIGS. 7A and 7B. As is clear from FIG. 7, the blood cortisol concentrations of the IAA treatment group and the kynurenine treatment group tended to be lower than the blood cortisol concentration of the untreated group (p = 0.09). In addition, the blood calcium concentration in the IAA treatment group tended to be lower than the blood calcium concentration in the untreated group (p = 0.06), and the blood calcium concentration in the kynurenine treatment group also showed a similar tendency (p = 0.18).

(Example 9 and Comparative Example 9)
Next, in Example 9 and Comparative Example 9, an experiment was conducted using scales that are tissues deeply involved in calcium metabolism in fish.

(Example 9 and Comparative Example 9: Enzymatic activity of osteoblasts and osteoclasts using goldfish scales)
The teleost scale has a bone matrix composed of a fibrous layer and a bone layer, as well as osteoblasts and osteoclasts, and performs bone metabolism similar to that of mammalian membranous bone. Using a bioassay system with goldfish scales, we investigated the effects of DOW and kynurenine on bone metabolism in fish, and examined the mechanisms of the effects of DOW and kynurenine on fish physiology.

<Test fish>
Goldfish (Carassius auratus) was used as the test fish. In the week before the test, goldfish were aliquoted and placed in a small aquarium, the water temperature was kept constant at 26 ° C. by thermostat control, and food was fed twice a day for about 2 minutes (about 1-2 g).

<Process for obtaining recycled scales>
When conducting experiments using the goldfish scale, the phosphatase activity of osteoblasts and osteoclasts, and the level of gene expression in scales, regenerated scales were used rather than regular scales. It is known that the measurement sensitivity is higher. Moreover, it is known that in the scales in the corresponding positions on the left and right sides of the body side, the marker enzyme activities of osteoblasts and osteoclasts are almost the same. The marker enzyme for osteoblasts is alkaline phosphatase (ALP), and the marker enzyme for osteoclasts is tartrate resistant acid phosphatase (TRAP). In the assay system of goldfish scales, this was used to analyze the scales on one body side as the control group and the scales on the other side as the experimental group. Anesthetize goldfish with an anesthetic solution (0.03% MS-222, manufactured by Sigma; 0.03% NaHCO ₃ , manufactured by Wako Pure Chemical Industries, Ltd.), select the necessary number of scales at the corresponding positions on the left and right body sides, Removed with tweezers. After removing the scales, the goldfish are transferred to a water tank with a constant temperature of 26 ° C, and the food is fed twice a day for about 2 minutes (about 1-2 g) and kept for 10 or 14 days. Uruko renewed.

<Preparation of medium for culturing scales>
In culturing scales, L-15 liquid medium containing antibiotics was used (1% Penicillin-Streptomycin-Amphotericin, ICN Biomedicals; Leibovitz's L-15, manufactured by Life Technologies). For the test, the following various media were prepared, and an experiment was set up by appropriately combining them.

<Influence of DOW addition>
A medium containing SSW at a certain ratio was used as a control group, and a medium containing DOW at the same ratio was used as an experimental group.
(i) 10% SSW L-15 vs 10% DOW L-15
(ii) 20% SSW L-15 vs 20% DOW L-15

<Influence of kynurenine addition>
A medium without solute added was used as a control group, and a medium containing a certain percentage of kynurenine was used as an experimental group.
(i) L-15 vs 10 ^-6 M kynurenine L-15
(ii) L-15 vs 10 ^-4 M kynurenine L-15

<Sampling of scales>
In order to collect regenerated scales from goldfish, goldfish were anesthetized with an anesthetic solution and collected with tweezers. The collected scales were placed one by one in a predetermined position on a 96-well plate (manufactured by Falcon) in which 200 μL of medium without solute was placed in each well in advance. It was placed in an incubator at 15 ° C. and pre-cultured for 1 hour. Thereafter, the medium was discarded, and 100 μL of each medium was added and cultured at 15 ° C. for a fixed time. After culturing, all the medium is removed, the scale is washed with 100 μL of PBS (Wako Pure Chemical Industries, Ltd.), and an alkaline buffer (ALP buffer: 1 mM MgCl2) is used for the scale for measuring osteoblast activity. ₂ ; 0.1 mM ZnCl ₂ ; 100 mM Tris (both manufactured by Wako Pure Chemical Industries, Ltd.) (pH 9.5), acid scale tartrate resistant acid buffer (TRAP buffer: 20 mM Tartrate resistant acid) 100 mM Acetic Acid-Sodium Acetate Buffer Solution (each Wako Pure Chemical Industries, Ltd.) (pH 5.3) was added in an amount of 100 μL. The plate was sealed and stored frozen at −80 ° C. until measurement.

<Measurement of enzyme activity>
As a phosphatase substrate, disodium p-nitrophenyl phosphate (Disodium p-Nitrophenylphosphate Hexahydrate: pNPP, manufactured by Wako Pure Chemical Industries, Ltd.) was used. pNPP undergoes degradation by phosphatase to produce p-nitrophenol (pNP), which exhibits an absorption wavelength of 405 nm under alkaline conditions. The frozen sample was thawed at room temperature, and 100 μL each of ALP buffer or TRAP buffer supplemented with 20 mM pNPP was added. The mixture was placed on a shaker and allowed to react for 30 minutes while gently shaking. After the reaction, 50 μL of 3N NaOH (manufactured by Wako Pure Chemical Industries, Ltd.) was added to each well to stop the reaction. The reaction solution was stirred by pipetting and then transferred to a corresponding position on a new 96-well plate by 100 μL. The plate was measured with a plate reader having an absorption wavelength of 405 nm, and the concentration was calculated using a standard curve prepared in advance with a pNP solution having a known concentration.

<Measurement of scale area>
It is known that the enzyme activities of ALP and TRAP in the goldfish scale are proportional to the area of the scale. Therefore, using this, the enzyme activity of scales per unit time unit area was calculated and compared. After the absorbance measurement, the remaining reaction solution was removed, the scale was washed with 100 μL of PBS, and 100 μL of methylene blue was added and stained for 10 minutes or longer. The stained scale was taken in as image data with a scanner, and the area was measured with image analysis software Image J.

<Calculation of enzyme activity per unit time unit area and statistical processing>
The measured pNP concentration was divided by the area per scale and the reaction time, and the enzyme activity per unit time per unit area (μM / h / mm ² ) was calculated. A paired t-test was performed between the control group and the experimental group. For the resulting p-value, p <0.05 was the statistical significance level, and p <0.10 was the statistical significance trend.

<Result>
The scales of ALP and TRAP were cultured for 24 hours in L-15 medium supplemented with SSW or DOW for 24 hours, and the activity of ALP and TRAP was measured. The activity showed an upward trend, but the activity of TRAP was not changed (FIGS. 8 and 9). In addition, scales that had been regenerated for 10 days were cultured in L-15 medium supplemented with SSW or DOW for 6, 12 and 24 hours, and the ALP activity was measured. The activity of ALP in the scales tended to increase (FIG. 10). In addition, L-15 medium supplemented with 10 ^-4 M or 10 ^-6 M kynurenine, SSW or DOW, and scales regenerated for 10 days were cultured for 24 hours, and the ALP and TRAP activities were measured. Assuming that the medium without kynurenine was 1, the ALP activity of the scales added with kynurenine was calculated and compared. As a result, the ALP activity increased with the addition of kynurenine. Furthermore, when SSW was set to 1, the activity of ALP in scales cultured in a medium supplemented with DOW was calculated and compared. When DOW was added, ALP activity was significantly increased (FIG. 11).

  As with the results of the enzyme activity of the goldfish scales osteoblasts and osteoclasts (FIGS. 8-9), flounder bred in artificial seawater without addition of kynurenine to bred artificial seawater The blood cortisol concentration tended to be low (p = 0.09) (FIG. 7A). This suggested that the addition of kynurenine reduces the stress caused by aquarium breeding. This is the same result as the effect of DOW breeding, suggesting that the effect of DOW breeding is due to kynurenine.

  As is apparent from FIGS. 10 to 11 showing the results of the enzyme activity of the osteoblast on the goldfish scale, DOW and kynurenine have the effect of enhancing the alkaline phosphatase (ALLP) activity on the goldfish scale. Was suggested. ALP, an osteoblast marker enzyme, metabolizes diphosphate to monophosphate around osteoblasts. Diphosphate is known as an inhibitor of the formation of hydroxyapatite crystals, the main component of teeth and bones, and ALP metabolizes this diphosphate to convert it into a form that can be used for bone mineralization as a hydroxyapatite material. It is known that From the results of this Example, there was a tendency that ALP activity increased in the medium supplemented with DOW and kynurenine by culturing for 24 hours. This indicates that DOW enhanced osteoblast function.

(Example 10)
Next, in Example 10, the indoles represented by the chemical formula (I) (compounds [1] to [18] in Tables 2 and 3) are disclosed in Japanese Patent Application Laid-Open No. 2006-104152 and International Patent Publication No. WO20007 / 010723. Brochures and M. Somei, et al., Heterocycles, 36, p1859-1866 (1993), M. Somei, et al., Heterocycles, 53, p1725-1736 (2000) and M. Somei, et al., Advances in Heterocyclic Chemistry, Vol. 82, ed. By AR Katritzky, Elsevier Science (USA), 2002, p101-155 According to the method described in the literature or synthesized by a conventional method, the indole is alkaline phosphatase (ALP). Activity was measured. Compounds [1] to [5] are those described in Heterocycles, 53, p1725-1736 (2000). Compound [17] is described in Advances in Heterocyclic Chemistry, Vol. 82, 2002, p101-155. As typical examples of other compounds, synthesis examples of compounds [6] to [8] and [12] are specifically shown. In addition, other compounds were also synthesized according to the methods described in the literature and conventional methods.

30.2mg(0.064mmol)の2,4,6-トリブロモメラトニン(N-アセチル-2,4,6-トリブロモ-5-メトキシインドール-3-エタナミン)（M. Somei, et al., Heterocycles, 53, p1725-1736 (2000)）を2.0mLのDMFに溶解した溶液に、31.1mg(0.22mmol)の炭酸カリウムを加えて室温下5分間撹拌した。この溶液に、0.11mL(1.28mmol)のアリルブロミドを加えて室温下1.5時間撹拌した。反応液に水及び酢酸エチルエステル−メタノール(95:5,v/v)混合溶媒を加えて撹拌後、有機相を分離した。水相を更に酢酸エチルエステル−メタノール(95:5,v/v)混合溶媒で3回抽出した。有機相と抽出液を合し、飽和食塩水で洗浄し、硫酸ナトリウムで乾燥後、溶媒を減圧留去して黄色油状物を得た。シリカゲルを担体とし、酢酸エチルエステルを溶出溶媒とするカラムクロマトグラフィーで精製すると31.0mg(95%)の収率で目的物の2,4,6-トリフ゛ロモメラトニンから1-アリル-2,4,6-トリブロモメラトニン(N-アセチル-1-アリル-2,4,6-トリブロモ-5-メトキシインドール-3-エタナミン）（化合物[６]）が得られた。酢酸エチルエステル−ヘキサンから再結晶して、無色の針状晶を得た。
mp 142-143℃.
IR (KBr): 3284, 1633, 1562, 1456, 1412, 1298, 1018 cm^-1.
¹H-NMR (CDCl₃)δ: 1.93 (3H, s), 3.24 (2H, t, J=6.6 Hz), 3.58 (2H, q, J=6.6 Hz), 3.89 (3H, s), 4.76 (2H, dt, J=4.9, 1.7 Hz), 4.89 (1H, d, J=16.6 Hz), 5.20 (1H, d, J=10.3 Hz), 5.55 (1H, br t, 重水添加により消失), 5.87 (1H, ddt, J=16.6, 10.3, 4.9 Hz), 7.4 (1H, s).
Anal. Calcd for C₁₆H₁₇Br₃N₂O₂: C, 37.75; H, 3.37; N, 5.50. Found: C, 37.75; H, 3.37; N, 5.42. <Synthesis of Compound [6]>

30.2 mg (0.064 mmol) 2,4,6-tribromomelatonin (N-acetyl-2,4,6-tribromo-5-methoxyindole-3-ethanamine) (M. Somei, et al., Heterocycles, 53 , p1725-1736 (2000)) was dissolved in 2.0 mL of DMF, and 31.1 mg (0.22 mmol) of potassium carbonate was added and stirred at room temperature for 5 minutes. To this solution, 0.11 mL (1.28 mmol) of allyl bromide was added and stirred at room temperature for 1.5 hours. Water and a mixed solvent of ethyl acetate-methanol (95: 5, v / v) were added to the reaction solution and stirred, and then the organic phase was separated. The aqueous phase was further extracted three times with a mixed solvent of ethyl acetate-methanol (95: 5, v / v). The organic phase and the extract were combined, washed with saturated brine, dried over sodium sulfate, and the solvent was evaporated under reduced pressure to give a yellow oil. Purification by column chromatography using silica gel as the carrier and ethyl acetate as the eluting solvent yields 31.0 mg (95%) of the desired 2,4,6-trifluoromelatonin to 1-allyl-2,4, 6-Tribromomelatonin (N-acetyl-1-allyl-2,4,6-tribromo-5-methoxyindole-3-ethanamine) (compound [6]) was obtained. Recrystallization from ethyl acetate-hexane gave colorless needle crystals.
mp 142-143 ° C.
IR (KBr): 3284, 1633, 1562, 1456, 1412, 1298, 1018 cm ^-1 .
¹ H-NMR (CDCl ₃ ) δ: 1.93 (3H, s), 3.24 (2H, t, J = 6.6 Hz), 3.58 (2H, q, J = 6.6 Hz), 3.89 (3H, s), 4.76 ( 2H, dt, J = 4.9, 1.7 Hz), 4.89 (1H, d, J = 16.6 Hz), 5.20 (1H, d, J = 10.3 Hz), 5.55 (1H, br t, disappeared by adding heavy water), 5.87 (1H, ddt, J = 16.6, 10.3, 4.9 Hz), 7.4 (1H, s).
Anal. Calcd for C ₁₆ H ₁₇ Br ₃ N ₂ O ₂ : C, 37.75; H, 3.37; N, 5.50. Found: C, 37.75; H, 3.37; N, 5.42.

30.1mg(0.064mmol)の2,4,6-トリブロモメラトニン(N-アセチル-2,4,6-トリブロモ-5-メトキシインドール-3-エタナミン）を2.0mLのDMFに溶解した溶液に、31.9mg(0.22mmol)の炭酸カリウムを加えて室温下5分間撹拌した。この溶液に、0.09mL(1.28mmol)のプロパルギルクロリドを加えて室温下4時間撹拌した。反応液に水及び酢酸エチルエステル−メタノール(95:5,v/v)混合溶媒を加えて撹拌後、有機相を分離した。水相を更に酢酸エチルエステル−メタノール(95:5,v/v)混合溶媒で3回抽出した。有機相と抽出液を合し、飽和食塩水で洗浄し、硫酸ナトリウムで乾燥後、溶媒を減圧留去して黄色油状物を得た。シリカゲルを担体とし、酢酸エチルエステルを溶出溶媒とするカラムクロマトグラフィーで精製すると31.6mg(97%)の収率で目的物2,4,6-トリブロモ-1-プロパルギルメラトニン(N-アセチル-2,4,6-トリブロモ-5-メトキシ-1-プロパルギルインドール-3-エタナミン）（化合物[７]）が得られた。酢酸エチルエステル−ヘキサンから再結晶して、無色の針状晶を得た。
mp 199-200℃.
IR (KBr): 3286, 1628, 1558, 1456, 1435, 1410, 1294, 1018 cm^-1.
¹H-NMR (CDCl₃) δ: 1.93 (3H, s), 2.34 (1H, t, J = 2.4 Hz), 3.23 (2H, t, J = 6.6 Hz), 3.58 (2H, dt, J = 2.9, 6.6 Hz, 重水添加によりt, J = 6.6 Hz に変化), 3.89 (3H, s), 4.91 (2H, d, J = 2.4 Hz), 5.54 (1H, br t, J = 6.6 Hz, 重水添加により消失), 7.58 (1H, s).
Anal. Calcd for C₁₆H₁₅Br₃N₂O₂: C, 37.90; H, 2.98; N, 5.53. Found: C, 37.78; H, 3.00; N, 5.44. <Synthesis of Compound [7]>

To a solution of 30.1 mg (0.064 mmol) 2,4,6-tribromomelatonin (N-acetyl-2,4,6-tribromo-5-methoxyindole-3-ethanamine) in 2.0 mL DMF, mg (0.22 mmol) of potassium carbonate was added and stirred at room temperature for 5 minutes. To this solution, 0.09 mL (1.28 mmol) of propargyl chloride was added and stirred at room temperature for 4 hours. Water and a mixed solvent of ethyl acetate-methanol (95: 5, v / v) were added to the reaction solution and stirred, and then the organic phase was separated. The aqueous phase was further extracted three times with a mixed solvent of ethyl acetate-methanol (95: 5, v / v). The organic phase and the extract were combined, washed with saturated brine, dried over sodium sulfate, and the solvent was evaporated under reduced pressure to give a yellow oil. When purified by column chromatography using silica gel as a carrier and acetic acid ethyl ester as an elution solvent, the desired product 2,4,6-tribromo-1-propargylmelatonin (N-acetyl-2, 4,6-tribromo-5-methoxy-1-propargylindole-3-ethanamine) (compound [7]) was obtained. Recrystallization from ethyl acetate-hexane gave colorless needle crystals.
mp 199-200 ° C.
IR (KBr): 3286, 1628, 1558, 1456, 1435, 1410, 1294, 1018 cm ^-1 .
¹ H-NMR (CDCl ₃ ) δ: 1.93 (3H, s), 2.34 (1H, t, J = 2.4 Hz), 3.23 (2H, t, J = 6.6 Hz), 3.58 (2H, dt, J = 2.9 , 6.6 Hz, changed to t, J = 6.6 Hz by adding heavy water), 3.89 (3H, s), 4.91 (2H, d, J = 2.4 Hz), 5.54 (1H, br t, J = 6.6 Hz, heavy water added ), 7.58 (1H, s).
Anal. Calcd for C ₁₆ H ₁₅ Br ₃ N ₂ O ₂ : C, 37.90; H, 2.98; N, 5.53. Found: C, 37.78; H, 3.00; N, 5.44.

40.1mg(0.086mmol)の2,4,6-トリブロモメラトニン(N-アセチル-2,4,6-トリブロモ-5-メトキシインドール-3-エタナミン)を2.0mLのDMFに溶解した溶液に、41.4mg(0.30mmol)の炭酸カリウムを加えて室温下5分間撹拌した。この溶液に、0.20mL(1.72mmol)のベンジルブロミドを加えて室温下1時間撹拌した。反応液に水及び酢酸エチルエステル−メタノール(95:5,v/v)混合溶媒を加えて撹拌後、有機相を分離した。水相を更に酢酸エチルエステル−メタノール(95:5,v/v)混合溶媒で3回抽出した。有機相と抽出液を合し、飽和食塩水で洗浄し、硫酸ナトリウムで乾燥後、溶媒を減圧留去して黄色油状物を得た。シリカゲルを担体とし、酢酸エチルエステルを溶出溶媒とするカラムクロマトグラフィーで精製すると40.3mg(83%)の収率で目的物1-ベンジル-2,4,6-トリブロモメラトニン(N-アセチル-1-ベンジル-2,4,6-トリブロモ-5-メトキシインドール-3-エタナミン）（化合物[８]）が得られた。酢酸エチルエステル−ヘキサンから再結晶して、無色の針状晶を得た。
mp 218-219℃.
IR (KBr): 3280, 1630, 1547, 1454, 1414, 1360, 1298, 1014 cm^-1.
¹H-NMR (CDCl₃)δ: 1.91 (3H, s), 3.26 (2H, t, J=6.6 Hz), 3.61 (2H, td, J=12.7, 6.6 Hz), 3.88 (3H, s), 5.36 (2H, s), 5.54 (1H, br t, J=6.6 Hz, 重水添加により消失), 7.01 (2H, d, J=6.6 Hz), 7.27-7.33 (3H, m), 7.39 (1H, s).
Anal. Calcd for C₂₀H₁₉Br₃N₂O₂: C, 42.97; H, 3.43; N, 5.01. Found: C, 42.76; H, 3.40; N, 4.86. <Synthesis of Compound [8]>

To a solution of 40.1 mg (0.086 mmol) 2,4,6-tribromomelatonin (N-acetyl-2,4,6-tribromo-5-methoxyindole-3-ethanamine) in 2.0 mL DMF, mg (0.30 mmol) of potassium carbonate was added and stirred at room temperature for 5 minutes. To this solution, 0.20 mL (1.72 mmol) of benzyl bromide was added and stirred at room temperature for 1 hour. Water and a mixed solvent of ethyl acetate-methanol (95: 5, v / v) were added to the reaction solution and stirred, and then the organic phase was separated. The aqueous phase was further extracted three times with a mixed solvent of ethyl acetate-methanol (95: 5, v / v). The organic phase and the extract were combined, washed with saturated brine, dried over sodium sulfate, and the solvent was evaporated under reduced pressure to give a yellow oil. Purification by column chromatography using silica gel as a carrier and acetic acid ethyl ester as an elution solvent yielded the desired product 1-benzyl-2,4,6-tribromomelatonin (N-acetyl-1) in a yield of 40.3 mg (83%). -Benzyl-2,4,6-tribromo-5-methoxyindole-3-ethanamine) (compound [8]) was obtained. Recrystallization from ethyl acetate-hexane gave colorless needle crystals.
mp 218-219 ° C.
IR (KBr): 3280, 1630, 1547, 1454, 1414, 1360, 1298, 1014 cm ^-1 .
¹ H-NMR (CDCl ₃ ) δ: 1.91 (3H, s), 3.26 (2H, t, J = 6.6 Hz), 3.61 (2H, td, J = 12.7, 6.6 Hz), 3.88 (3H, s), 5.36 (2H, s), 5.54 (1H, br t, J = 6.6 Hz, disappeared by adding heavy water), 7.01 (2H, d, J = 6.6 Hz), 7.27-7.33 (3H, m), 7.39 (1H, s).
Anal. Calcd for C ₂₀ H ₁₉ Br ₃ N ₂ O ₂ : C, 42.97; H, 3.43; N, 5.01. Found: C, 42.76; H, 3.40; N, 4.86.

フェニルイソシアネート(0.9 mL, 8.28 mmol) を無水テトラヒドロフラン (1 mL) に溶かして、1,8-ジメチル-1,2,3,3a,8,8a-ヘキサヒドロピロロ [2,3-b]インドール-5-オール・ボランコンプレックス (94.1 mg, 0.43 mmol) を無水テトラヒドロフラン (9 mL) に溶かした溶液に、氷冷撹拌下加えた後、全体を65°Cで1.5時間撹拌した。減圧下に溶媒を留去して得られた固形物を、クロロホルムを溶出溶液として、シリカゲルカラムクロマトグラフィを行い、溶出順にN-フェニル-N’-メチル-N’-[5-(N-フェニルカルバモイル)オキシ-1-メチルインドール-3-イル]-エチルウレア（化合物[１２]）(76.1 mg, 40%) およびN-フェニル-N’-[5-ヒドロキシ-1-メチルインドール-3-イル]-エチル-N’-メチルウレア (70.5 mg, 51%) を得た。
なおフェニルイソシアネート(0.07 mL, 0.64 mmol) を無水テトラヒドロフラン(1 mL) に溶かして、N-フェニル-N’-[5-ヒドロキシ-1-メチルインドール-3-イル]-エチル-N’-メチルウレア(10.0 mg, 0.03 mmol) を無水テトラヒドロフラン(1 mL) に溶かした溶液に加え、5時間還流撹拌した。減圧下に溶媒を留去し、得られた固形物を、酢酸エチルエステル-ヘキサン(1:1, v/v)混合溶媒を溶出溶液として、シリカゲルカラムクロマトグラフィを行い、N-フェニル-N’-メチル-N’-[5-(N-フェニルカルバモイル)オキシ-1-メチルインドール-3-イル]-エチルウレア（化合物[１２]）(12.6 mg, 92%)を得た。クロロホルム-メタノールから再結晶して無色プリズム晶を得た。
mp 182-184℃.
IR (KBr): 3375, 1710, 1645, 1526, 1484, 1443, 1227, 746 cm^-1.
¹H-NMR (DMSO-d₆)δ: 2.89 (2H, t, J=7.6 Hz), 2.98 (3H, s), 3.55 (2H, t, J=7.6 Hz), 3.74 (3H, s), 6.90 (1H, t, J=7.3 Hz), 6.98 (1H, dd, J=8.5, 2.2 Hz), 7.03 (1H, t, J=7.3 Hz), 7.19 (2H, t, J=7.5 Hz), 7.22 (1H, s), 7.31 (2H, t, J=7.5Hz), 7.38-7.44 (4H, m), 7.52 (2H, d, J=8.3 Hz), 8.16 (1H, s), 10.12 (1H, s, 重水添加により消失). High-resolution MS (FAB⁺) m/z: Calcd for C₂₆H₂₇N₄O₃: 443.2083. Found: 443.2044.
Anal. Calcd for C₂₆H₂₆N₄O_3・1/4H₂O: C, 69.86; H, 5.98; N, 12.53. Found: C, 69.83; H, 5.92; N, 12.43. <Synthesis of Compound [12]>

Phenyl isocyanate (0.9 mL, 8.28 mmol) is dissolved in anhydrous tetrahydrofuran (1 mL) and 1,8-dimethyl-1,2,3,3a, 8,8a-hexahydropyrrolo [2,3-b] indole- To a solution of 5-ol borane complex (94.1 mg, 0.43 mmol) in anhydrous tetrahydrofuran (9 mL) was added with stirring under ice cooling, the whole was stirred at 65 ° C. for 1.5 hours. The solid obtained by distilling off the solvent under reduced pressure was subjected to silica gel column chromatography using chloroform as an elution solution, and N-phenyl-N'-methyl-N '-[5- (N-phenylcarbamoyl) in the order of elution. ) Oxy-1-methylindol-3-yl] -ethylurea (compound [12]) (76.1 mg, 40%) and N-phenyl-N ′-[5-hydroxy-1-methylindol-3-yl]- Ethyl-N′-methylurea (70.5 mg, 51%) was obtained.
Phenyl isocyanate (0.07 mL, 0.64 mmol) was dissolved in anhydrous tetrahydrofuran (1 mL), and N-phenyl-N '-[5-hydroxy-1-methylindol-3-yl] -ethyl-N'-methylurea ( 10.0 mg, 0.03 mmol) was added to a solution of anhydrous tetrahydrofuran (1 mL), and the mixture was stirred at reflux for 5 hours. The solvent was distilled off under reduced pressure, and the resulting solid was subjected to silica gel column chromatography using a mixed solvent of ethyl acetate-hexane (1: 1, v / v) as an elution solution, and N-phenyl-N'- Methyl-N ′-[5- (N-phenylcarbamoyl) oxy-1-methylindol-3-yl] -ethylurea (compound [12]) (12.6 mg, 92%) was obtained. Recrystallization from chloroform-methanol gave colorless prism crystals.
mp 182-184 ° C.
IR (KBr): 3375, 1710, 1645, 1526, 1484, 1443, 1227, 746 cm ^-1 .
¹ H-NMR (DMSO-d ₆ ) δ: 2.89 (2H, t, J = 7.6 Hz), 2.98 (3H, s), 3.55 (2H, t, J = 7.6 Hz), 3.74 (3H, s), 6.90 (1H, t, J = 7.3 Hz), 6.98 (1H, dd, J = 8.5, 2.2 Hz), 7.03 (1H, t, J = 7.3 Hz), 7.19 (2H, t, J = 7.5 Hz), 7.22 (1H, s), 7.31 (2H, t, J = 7.5Hz), 7.38-7.44 (4H, m), 7.52 (2H, d, J = 8.3 Hz), 8.16 (1H, s), 10.12 (1H , s, disappeared by adding heavy water) .High-resolution MS (FAB ⁺ ) m / z: Calcd for C ₂₆ H ₂₇ N ₄ O ₃ : 443.2083. Found: 443.2044.
Anal. Calcd for C ₂₆ H ₂₆ N ₄ O _{3 ・} 1 / 4H ₂ O: C, 69.86; H, 5.98; N, 12.53. Found: C, 69.83; H, 5.92; N, 12.43.

<Effects of osteoblasts: measurement of alkaline phosphatase (ALP) activity>
Goldfish females (weighing around 30 g) were anesthetized with MS222 (ethyl 3-aminobenzoate, methane sulfonic acid salt) (Aldrich), and the required number of scales were peeled off. The scales were washed twice with Eagles minimal medium (Dainippon Pharmaceutical Co., Ltd.) containing 1% antibiotic (penicillin-streptomycin mixture). Place 1 ml of the same medium in a 24-well plate, add multiple scales (usually 8), and add 10 ^-8 , 10 ^-6 , and 10 ^-4 M indoles to each hole. did. They were then cultured at 15 ° C. for 6 hours. In addition, an indole-free group (control) was also prepared, and the action on bone cells was compared. A total of 8 holes of control, 10 ⁻⁸ , 10 ⁻⁶ , and 10 ⁻⁴ M indoles (2 holes each) were prepared. Therefore, we can examine three types of indoles on a 24-well plate.

  After the culture, the medium was removed, and 0.05 M cacodylate buffer solution (pH 7.4) containing 10% formalin was added and fixed. This scale was stored at 4 ° C. in 0.05 M cacodylate buffer until measurement of enzyme activity.

  The scale subjected to the fixing treatment was taken out, and the weight of the scale was measured. After the measurement, place the scales in a 96-well microplate, and add 100 mM Tris-HCl buffer (pH 9.5) containing 10 mM paranitrophenol phosphate (substrate), 1 mM magnesium chloride, and 0.1 mM zinc chloride to each hole. 200 μl was added and reacted at 25 ° C. for 1 hour, and 2N aqueous sodium hydroxide solution (50 μl) was added to stop the reaction. Thereafter, 150 μl of the reaction completed solution was transferred to another microplate, and the amount of pNP generated by ALP was measured with a spectrophotometer (405 nm) to determine the activity.

  For the p-value by t-test (student's t-test), p <0.05 was assigned a statistical significance level with **, p <0.10 was a statistical significance trend, and * was attached.

  The results are shown in Tables 2-3 below.

  As shown in Tables 2 to 3, the compounds [1] to [18] represented by the chemical formula (I) had ALP activity, like kynurenine and indoleacetic acid.

  As is apparent from Examples 1 to 10 and Tables 2 and 3, as stress-reducing agents to which the present invention is applied, deep sea water and artificial seawater containing kynurenine and indole-3-acetic acid, represented by the above chemical formula (I) Indoles, pharmaceutically acceptable salts thereof, and / or metabolites thereof can lower cortisol, raise calcitonin, lower calcium, and activate osteoblasts. Although it is an example in seafood, the mechanism of action is similar, so stress is reduced when raising various animals such as amphibians, birds, domestic animals such as non-human mammals, food animals and pets, Can survive for a long time in health.

  The effects of the stress-reducing agent of the present invention, for example, deep sea water containing kynurenine, on fish physiology are estimated as follows. The kynurenine absorbed in the intestine is consumed as a starting material of the kynurenine pathway instead of tryptophan, thereby indirectly increasing the substrate for serotonin synthesis, and from the osteoblasts on fish scales to the kynurenine present in deep ocean water. Calcitonin is released to reduce chronic stress. For animals other than fish, it is considered that stress is reduced by the same mechanism of action.

  Therefore, according to this stress-reducing agent, live fish collected from fishing grounds are bred with breeding water containing stress-reducing agent in the aquarium, and since they are not stressed, they live long and stay fresh and remain high quality. , Can be delivered to retailers, restaurants and consumers. Moreover, since no stress is applied when raising various animals, they can be kept healthy. In addition, the taste and quality of edible livestock can be improved, and livestock can be bred safely.

  This stress-reducing drug can be used for people who are socially stressed to reduce stress and maintain a healthy state, thereby contributing to medical cost reduction.

  In addition, this stress-reducing drug is safe without any particular appearance, physiological or pathological abnormality.

  The stress-reducing drug of the present invention is useful for preventing or treating human stress, and also for various animals such as seafood, amphibians, birds, domestic animals such as non-human mammals, food animals, and pets. It is useful for breeding and transporting live fish while preventing and treating stress.

Claims (5)

The following chemical formula (I)

(In the formula (I),
R ¹ may be a hydrogen atom, a hydroxyl group, or a substituent, and may be an alkyl group, an alkyloxy group, an alkylthioether group, an alkenyl group, an alkenyloxy group, an alkenylthioether group, an alkynyl group, or an alkynyloxy group. , Alkynyl thioether group, hydrocarbon aromatic group, hydrocarbon aromatic group-containing oxy group, hydrocarbon aromatic group-containing thioether group, saturated heterocyclic group, saturated heterocyclic group-containing oxy group, saturated heterocyclic group-containing thioether group, Aromatic heterocyclic group, aromatic heterocyclic group-containing oxy group, aromatic heterocyclic group-containing thioether group, aralkyl group, aralkyloxy group, aralkyl thioether group, acyl group, acyloxy group, thioester group, thioamide group, alkylsulfonyl group , Alkylsulfonyloxy group, alkenylsulfonyl group Alkenylsulfonyloxy group, alkynylsulfonyl group, alkynylsulfonyloxy group, arylsulfonyl group, arylsulfonyloxy group, aralkylsulfonyl group, aralkylsulfonyloxy group, or amidino group;
R ² represents a hydrogen atom, a halogen atom, or an alkyl group which may be substituted with a substituent;
n1 is the number 0 or 1;
R ³ is a hydrogen atom, a halogen atom, —COOH, —COOR ^a , —CONH ₂ , or —CONHR ^b (where R ^a and R ^b are optionally substituted with an alkyl group, Alkenyl group, alkynyl group, hydrocarbon aromatic group, saturated heterocyclic group, aromatic heterocyclic group, aralkyl group, acyl group, alkylsulfonyl group, alkenylsulfonyl group, alkynylsulfonyl group, arylsulfonyl group, or aralkylsulfonyl group) ;
R ⁴ and R ⁵ are the same or different and may be substituted with a hydrogen atom or a substituent, and may be an alkyl group, an alkenyl group, an alkynyl group, a hydrocarbon aromatic group, a saturated heterocyclic group, an aromatic heterocyclic group. A cyclic group, an aralkyl group, an acyl group, an alkylsulfonyl group, an alkenylsulfonyl group, an alkynylsulfonyl group, an arylsulfonyl group, an aralkylsulfonyl group, an alkyloxycarbonyl group, an aryloxycarbonyl group, an alkylaminocarbonyl group, or an arylaminocarbonyl group;
n2 is a number of 0 or 1, n3 is a number of 0 or 1, and n2 + n3>0;
R ^{6 to} R ⁹ are the same or different and may be substituted with a hydrogen atom, a halogen atom, a hydroxyl group, a mercapto group, or a substituent, and an alkyl group, an alkyloxy group, an alkylthioether group, an alkenyl group, an alkenyl group Oxy group, alkenyl thioether group, alkynyl group, alkynyloxy group, alkynyl thioether group, hydrocarbon aromatic group, hydrocarbon aromatic group-containing oxy group, hydrocarbon aromatic group-containing thioether group, saturated heterocyclic group, saturated heterocyclic ring Group-containing oxy group, saturated heterocyclic group-containing thioether group, aromatic heterocyclic group, aromatic heterocyclic group-containing oxy group, aromatic heterocyclic group-containing thioether group, aralkyl group, aralkyloxy group, aralkyl thioether group, acyl group An acyloxy group, an alkylsulfonyl group, an alkylsulfonyloxy group, Lucenylsulfonyl group, thioester group, thioamide group, alkenylsulfonyloxy group, alkynylsulfonyloxy group, alkynylsulfonyloxy group, arylsulfonyl group, arylsulfonyloxy group, aralkylsulfonyloxy group, aralkylsulfonyloxy group, amidino group, -COOH,- COOR ^c , —CONH ₂ , or —CONHR ^d (where R ^c , R ^d may be substituted with a substituent, and an alkyl group, an alkenyl group, an alkynyl group, a hydrocarbon aromatic group, a saturated A cyclic group, an aromatic heterocyclic group, an aralkyl group, an acyl group, an alkylsulfonyl group, an alkenylsulfonyl group, an alkynylsulfonyl group, an arylsulfonyl group, or an aralkylsulfonyl group), —NH ₂ , —NHR ^e (where R ^e is alkyl) Group, alkenyl group, An alkynyl group, an aryl group, an aralkyl group, or an acyl group), —NR ^f R ^{f ′} (wherein R ^f and R ^{f ′} are the same or different, an alkyl group, an alkenyl group, an alkynyl group, an aryl group, an aralkyl group, or an acyl group) ), An alkylaminocarbonylamide group, or an arylaminocarbonylamide group;
Dashed / solid parallel lines are single bonds or double bonds,
The substituents in R ^{1 to} R ⁹ are each singular or plural, and are a hydroxyl group, mercapto group, halogen atom, alkyl group, alkyloxy group, alkylthioether group, alkenyl group, alkenyloxy group, alkenylthioether group. , Alkynyl group, alkynyloxy group, alkynyl thioether group, hydrocarbon aromatic group, hydrocarbon aromatic group-containing oxy group, hydrocarbon aromatic group-containing thioether group, saturated heterocyclic group, saturated heterocyclic group-containing oxy group, saturated Heterocyclic group-containing thioether group, aromatic heterocyclic group, aromatic heterocyclic group-containing oxy group, aromatic heterocyclic group-containing thioether group, aralkyl group, aralkyloxy group, aralkylthioether group, acyl group, thioester group, thioamide group , Acyloxy group, alkylsulfonyl group, alkylsulfonyloxy , Alkenylsulfonyl group, alkenylsulfonyl group, alkynyl-sulfonyl group, alkynylsulfonyl group, an arylsulfonyl group, an arylsulfonyl group, an aralkyl sulfonyl group, aralkyl sulfonyloxy groups, ^-COOH, -COOR g, -CONH _2, or - CONHR ^h (where R ^g and R ^h are an alkyl group, an alkenyl group, an alkynyl group, a hydrocarbon aromatic group, a saturated heterocyclic group, an aromatic heterocyclic group, an aralkyl group, an acyl group, an alkylsulfonyl group, an alkenylsulfonyl group) Group, alkynylsulfonyl group, arylsulfonyl group, or aralkylsulfonyl group), —NH ₂ , —NHR ⁱ (where R ⁱ is an alkyl group, alkenyl group, alkynyl group, aryl group, aralkyl group, or acyl group), —NR ^j R ^{j '(but j} and R ^{j 'are} the same or different alkyl group, an alkenyl group, an alkynyl group, an aryl group, an aralkyl group, or an acyl group), alkylaminocarbonyl amido group, an arylamino carbonyl amide group, or an amidino group. )
A stress-reducing drug comprising the indole represented by the formula (I), a pharmaceutically acceptable salt thereof, and / or a metabolite thereof as a stress-reducing component. The indole has a tryptophan skeleton, and its metabolite has a kynurenine skeleton,
The stress-reducing drug according to claim 1, wherein the indole has an indole acetic acid skeleton.   The stress-reducing drug according to claim 1, wherein the drug is used for human treatment and / or prevention, or for breeding non-human animals.   4. In blood and / or urine, cortisol is decreased, calcitonin is increased, calcium is decreased, and / or osteoblasts are activated. Stress-reducing drug.   The stress-reducing drug according to any one of claims 1 to 4, comprising deep ocean water and / or a component thereof.

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