JP6910188B2 - TRPA1 activator - Google Patents

Description

The present invention relates to TRPA1 activators, preventive or ameliorating agents for intestinal obstruction, and preventive or ameliorating agents for constipation.

TRPA1 (Transient receptor potential cation channel subfamily A member 1) is a non-selective cation channel belonging to the superfamily of transient receptor potential (TRP) ion channels, and is a cryoreceptor (17 ° C) in nociceptive neurons. (Non-Patent Document 1). After that, TRPA1 is a chemical receptor that reacts with mustard oil and allyl isothiocyanate (AITC), cinnamon, garlic, methylsalicylate, eugenol, alcohols, etc. contained therein, and further, low temperature and mechanical stimulation, chemistry. It has been reported that it is a pain receptor that responds to stimuli (Non-Patent Documents 2, 3, 4 and 5).

Intestinal obstruction, also called ileus, refers to a condition in which the movement of intestinal contents to the anal side is impaired. When the intestinal tract is occluded, the oral side of the occluded area is dilated by gas or intestinal juice, and venous return is impaired. As a result, the intestinal wall edema, water and sodium leak into the intestinal cavity, the pressure in the intestinal tract rises, and arterial blood flow is impaired, causing necrosis and perforation of the intestinal tract. Symptoms include abdominal bloating, nausea, vomiting, abdominal pain, and disruption of defecation / exhaust gas due to loss of intestinal motility, that is, paralysis.
Patients with intestinal obstruction cannot take food or drugs by mouth, are hydrated intravenously, and are given gastric decompression using a nasal gastric suction tube. Due to the discomfort and inconvenience of such treatment methods, a less burdensome method is required.

TRPA1 is known to be abundantly expressed in enterochromaffin cells, but recently, Daikenchuto, which is said to be useful for postoperative ileus (POI), has been introduced. It has been reported to increase intestinal blood flow through stimulation of TRPA1 in epithelial cells of the small intestine. It was found that in POI model mice, AITC and the like, which are TRPA1 agonists, improve passage through the small intestine, and in TRPA1-deficient mice, periodic peristaltic movements in the small intestine are suppressed. It has been suggested that it is useful for improving the motor disorder of the mouse (Non-Patent Document 6).
Therefore, activation of TRPA1 is expected to prevent or ameliorate intestinal obstruction.

Constipation (constipation) is an autonomic dysfunction that occurs in the lower gastrointestinal tract, overabsorption of water in the intestinal tract, decreased intestinal mucus secretion, motor disorders, gastrointestinal psychosomatic disorders (eg, irritable bowel syndrome), and decreased defecation reflex function. Defecation is difficult or rare due to such factors as organic constipation caused by physical passage obstruction such as stenosis or mass, and functional constipation caused by decreased intestinal motility function or rectal and anal discharge function. being classified.
Oral administration of the TRPA1 agonist ASP7663 to the spastic constipation model mice to which loperamide was administered and the flaccid constipation model mice to which clonidine was administered improved colon motility. This phenomenon disappears with the first oral administration of the TRPA1 antagonist HC-030031, suggesting that TRPA1 stimulation is useful for spastic and flaccid constipation (non-TPA1). Patent Document 7).
Therefore, activation of TRPA1 is expected to prevent or ameliorate constipation.

Rosmarinic acid, which is a type of tannin contained in a large amount in Labiatae plants, has an effect of suppressing the onset and progression of amyotrophic lateral sclerosis (ALS) (Patent Document 1) and an insulin secretion promoting effect. It has been reported that there is (Patent Document 2) and that it has an effect of improving pollinosis (Non-Patent Document 8).
Further, it has been reported that dimethylrosmarinic acid excreted in human urine as a metabolite of rosmarinic acid has an induction inhibitory activity of cyclooxygenase 2 and has an anti-inflammatory effect (Patent Document 3).
However, it is not known at all that rosmarinic acid or dimethylrosmarinic acid activates TRPA1 and is useful for preventing or ameliorating intestinal obstruction and constipation.

Japanese Unexamined Patent Publication No. 2009-256282 Japanese Unexamined Patent Publication No. 2005-139136 Japanese Unexamined Patent Publication No. 2005-272362

Story et al. 2003, Cell 112, 819-829 Kwan et al. 2006, Neuron 50, 277-289 Journal of Japanese Pharmacology, Vol. 124, pp. 219-227, 2004, published by the Japanese Pharmacological Society Fijita et al. 2010, IFSCC Congress 2010 Komatsu et al. 2012, Eur J Physio 463, 549-559 K. Tsuchiya et al. Neurogastroenterology & Motility (2016) DOI: 10.1111 / nmo.12877 Kojima et al. 2014, European Journal of Pharmacology 723, 288-293 Osakabeet al., Biofactors. 2004 21,127-31

The present invention relates to providing a TRPA1 activator, a preventive or ameliorating agent for intestinal obstruction, and a preventive or ameliorating agent for constipation, which activate TRPA1 and are useful for preventing or ameliorating intestinal obstruction or constipation.

As a result of diligent studies on a material that activates TRPA1, the present inventors have dimethylrosmarinic acid, which is a human metabolite of rosmarinic acid, having an action of activating TRPA1 to prevent or improve intestinal obstruction and constipation. Found to be useful for.

That is, the present invention relates to the following 1) to 6).
1) A TRPA1 activator containing at least one selected from rosmarinic acid, dimethylrosmarinic acid and salts or solvates thereof as an active ingredient.
2) A preventive or ameliorating agent for intestinal obstruction containing at least one selected from rosmarinic acid, dimethylrosmarinic acid and salts or solvates thereof as an active ingredient.
3) A constipation preventive or ameliorating agent containing at least one selected from rosmarinic acid, dimethylrosmarinic acid and salts or solvates thereof as an active ingredient.
4) A food for activating TRPA1 containing at least one selected from rosmarinic acid, dimethylrosmarinic acid and salts or solvates thereof as an active ingredient.
5) A food for preventing or improving intestinal obstruction containing at least one selected from rosmarinic acid, dimethylrosmarinic acid and salts or solvates thereof as an active ingredient.
6) A food for preventing or improving constipation containing at least one selected from rosmarinic acid, dimethylrosmarinic acid and salts or solvates thereof as an active ingredient.

The TRPA1 activator of the present invention can effectively activate TRPA1 and is useful for preventing or ameliorating diseases caused by decreased activity of TRPA1 channels, such as intestinal obstruction and constipation.

In the present invention, "TRPA1" means "Transient receptor potential cation channel subfamily A member 1". TRPA1 is a protein encoded by the TRPA1 gene in humans.

The "rosmarinic acid" of the present invention is a kind of tannin contained in a large amount in Labiatae plants, and is represented by the following formula (A). The "dimethylrosmarinic acid" of the present invention is a human metabolite of the rosmarinic acid (Patent Document 3), and means 3,3'-dimethylrosmarinic acid represented by the following formula (B).

In the present invention, rosmarinic acid may be a commercially available product or chemically synthesized according to a conventional method (SYNTHESIS, 755-762 (1996), Can. J. Chem., 75, 1783-1794 (1997). ), Or it can be obtained by extracting and purifying from a plant such as a rosmarinic acid-containing plant or cultured cells (Japanese Patent Laid-Open No. 62-32889).
Dimethylrosmarinic acid can be synthesized, for example, according to the following reaction scheme, as shown in the production example described later.

Optical isomers are present in the rosmarinic acid and dimethyl rosmarinic acid of the present invention. The rosmarinic acid and dimethylrosmarinic acid of the present invention may be any of these optical isomers or a mixture of the optical isomers. In the present invention, an R-form or a mixture of optical isomers is preferable.

The rosmarinic acid and dimethylrosmarinic acid of the present invention can be pharmaceutically acceptable salts or solvates thereof. Preferred examples of such salts include alkali metal and alkaline earth metal salts such as sodium salt, potassium salt and calcium salt, acid addition salts and the like. Preferred examples of the solvate include hydrates, alcohol solvates, acetone hydrates and the like.

As shown in Examples below, dimethylrosmarinic acid, which is a metabolite of rosmarinic acid, activates TRPA1. That is, when TRPA1 gene transfer brings a transformed cell (TRPA1-expressing cell) in which TRPA1 is functionally expressed into contact with dimethylrosmarinic acid, TRPA1 increases the influx of intracellular cations. It has an activating effect.
Therefore, dimethyl rosmarinic acid, its precursor rosmarinic acid and salts or solvates thereof (hereinafter, also referred to as "compounds of the present invention") serve as TRPA1 activators for TRPA1 activation or TRPA1 activity. Can be used to make agents.

As described above, TRPA1 stimulation improves gastrointestinal motility disorders in postoperative ileus model mice because TRPA1 agonists improve small intestinal passage and in TRPA1-deficient mice, periodic peristaltic movements in the small intestine are suppressed. It is considered to be useful for improving the above (Non-Patent Document 6).
In addition, oral administration of the TRPA1 agonist ASP7663 to the spastic constipation model mouse to which loperamide was administered and the flaccid constipation model mouse to which clonidine was administered improved the motility of the colon. Since this phenomenon disappears by first oral administration of the TRPA1 antagonist HC-030031, TRPA1 stimulation is considered to be useful for spastic constipation and flaccid constipation (the non-existent constipation described above). Patent Document 7).
Therefore, it is considered that the TRPA1 activator can be used for the prevention or amelioration of intestinal obstruction or constipation as a preventive or ameliorating agent for intestinal obstruction and a preventive or ameliorating agent for constipation.

In the present invention, "activation of TRPA1" refers to activation of the receptor TRPA1. Specifically, the inherent action of TRPA1, such as the ability to regulate ion flux (for example, the ability to transport cations such as calcium ions and sodium ions from extracellular to intracellular), and the ability to regulate membrane potential. It means activating (for example, the ability to generate an electric current).

In the present invention, "ileus" refers to a condition in which the movement of intestinal contents toward the anus is impaired. Intestinal obstruction is mechanical intestinal obstruction in which the intestine is twisted or a tumor is formed inside the intestinal tract, and the peristaltic movement of the intestine is lost due to the effects of nerve abnormalities and inflammation, and the contents are in the intestine. It is classified as a functional intestinal obstruction that is stagnant, but in the present invention, any of them may be used.
In the present invention, "constipation" refers to a condition in which stool is difficult or rare to be discharged, and is organic constipation caused by physical passage obstruction such as stenosis or mass, and intestinal motility function or rectal-anal discharge function. It is classified as functional constipation due to deterioration or the like, but in the present invention, any of them may be used.

In the present invention, for TRPA1 activation, "use" for prevention or amelioration of intestinal obstruction or constipation may be administration or ingestion to animals including humans, and non-therapeutic use even for therapeutic use. May be. "Non-therapeutic" is a concept that does not include medical practice, that is, a concept that does not include a method of operating, treating or diagnosing a human, more specifically, a doctor or a person who has been instructed to perform surgery on a human. , A concept that does not include a method of performing treatment or diagnosis.

In addition, in this specification, "prevention" means preventing or delaying the onset of a disease or a symptom in an individual, or reducing the risk of developing a disease or a symptom in an individual. Further, "improvement" means improvement of a disease, symptom or condition, prevention or delay of deterioration of the disease, symptom or condition, or reversal, prevention or delay of progression of the disease, symptom or condition.

The TRPA1 activator, the preventive or ameliorating agent for intestinal obstruction, and the preventive or ameliorating agent for constipation of the present invention have a TRPA1 activating effect, a preventive or ameliorating effect on intestinal obstruction, and prevention of constipation when ingested or administered to animals including humans. Alternatively, it can be a drug, a non-medicinal product, a supplement or a food that exerts an improving effect, or can be a material or a preparation to be incorporated into these.
In addition to general foods and drinks, the foods of the present invention include foods labeled to that effect, functional foods, foods for the sick, foods for specified health uses, foods with functional claims, and supplements as necessary. ..

The above-mentioned pharmaceutical products (including quasi-drugs) containing the compound of the present invention can be administered in any administration form, but oral administration is preferable. Upon administration, the active ingredient can be mixed with a solid or liquid pharmaceutical non-toxic carrier suitable for administration methods such as oral administration, rectal administration, and injection, and administered in the form of a conventional pharmaceutical preparation.

Examples of such a preparation include solid preparations such as tablets, granules, powders and capsules, solutions, suspensions, liquids such as emulsions, lyophilizers and the like. These preparations can be prepared by conventional means on the preparation. Further, if necessary, conventional additives such as stabilizers, wetting agents, emulsifiers, binders, isotonic agents, and excipients can be appropriately added.

In addition, the form of the above-mentioned food containing the compound of the present invention includes foods and drinks such as soft drinks, tea-based beverages, coffee beverages, fruit juice beverages, carbonated beverages, jellies, wafers, biscuits, breads, noodles, sausages, and nutritional foods. In addition to various foods such as, further, a nutritional supplement composition in the same form as the above-mentioned orally administered preparation (solid preparation such as tablets, capsules, troches) can be mentioned. Among them, tablets are preferable, and chewable tablets are more preferable.
To prepare various forms of food, the compounds of the invention alone or with other food materials, solvents, softeners, oils, emulsifiers, preservatives, fragrances, stabilizers, colorants, antioxidants , Moisturizer, thickener and the like can be used in combination as appropriate.

The content of the compound of the present invention in the above-mentioned pharmaceutical products (including quasi-drugs) and foods varies depending on the mode of use thereof, but is usually preferably 0.01% by mass or more, more preferably 0.1% by mass. As mentioned above, it is more preferably 1% by mass or more, preferably 95% by mass or less, more preferably 90% by mass or less, still more preferably 70% by mass or less. Further, it is preferably 0.01 to 95% by mass, more preferably 0.1 to 90% by mass, and further preferably 1 to 70% by mass.

When the compound of the present invention is used as a drug or food, or in combination with a drug or food, the dose to humans may vary depending on the condition, weight, sex, age or other factors of the subject, but orally. In the case of administration, the daily dose per adult is usually 0.01 mg or more, more preferably 1 mg or more, still more preferably 100 mg or more, and preferably 100 mg or more, as dimethylrosmarinic acid or rosmarinic acid. It is 80,000 mg or less, more preferably 15,000 mg or less, still more preferably 5000 mg or less. Further, it is preferably 0.01 to 80,000 mg, more preferably 1 to 15,000 mg, and further preferably 100 to 5,000 mg.
Further, although the above-mentioned preparation can be administered according to an arbitrary administration plan, it is preferable to administer it once to several times a day and continuously for several weeks to several months.

The subject to be administered or ingested is not particularly limited as long as it is an animal including humans who needs or desires activation of TRPA1, prevention or improvement of intestinal obstruction or constipation, but the abdomen is abdominal due to loss of intestinal motility, that is, paralysis. It is effective to administer or ingest to humans with symptoms such as bloating, nausea, vomiting, abdominal pain, and disruption of defecation / exhaust gas, and constipation.

Regarding the above-described embodiment, the following aspects are disclosed in the present invention.
<1> A TRPA1 activator containing at least one selected from rosmarinic acid, dimethylrosmarinic acid and salts or solvates thereof as an active ingredient.
<2> A preventive or ameliorating agent for intestinal obstruction containing one or more selected from rosmarinic acid, dimethylrosmarinic acid and salts or solvates thereof as active ingredients.
<3> A constipation preventive or ameliorating agent containing at least one selected from rosmarinic acid, dimethylrosmarinic acid and salts or solvates thereof as an active ingredient.
<4> A food for activating TRPA1 containing at least one selected from rosmarinic acid, dimethylrosmarinic acid and salts or solvates thereof as an active ingredient.
<5> A food for preventing or improving intestinal obstruction containing at least one selected from rosmarinic acid, dimethylrosmarinic acid and salts or solvates thereof as an active ingredient.
<6> A food for preventing or improving constipation containing at least one selected from rosmarinic acid, dimethylrosmarinic acid and salts or solvates thereof as an active ingredient.

<7> Use of one or more selected from rosmarinic acid, dimethylrosmarinic acid and salts or solvates thereof for producing a TRPA1 activator.
<8> Use of one or more selected from rosmarinic acid, dimethylrosmarinic acid and salts or solvates thereof for producing an agent for preventing or improving intestinal obstruction.
<9> Use of one or more selected from rosmarinic acid, dimethylrosmarinic acid and salts or solvates thereof for producing a preventive or ameliorating agent for constipation.
<10> Use of one or more selected from rosmarinic acid, dimethylrosmarinic acid and salts or solvates thereof for producing foods for TRPA1 activation.
<11> Use of one or more selected from rosmarinic acid, dimethylrosmarinic acid and salts or solvates thereof for producing foods for preventing or improving intestinal obstruction.
<12> Use of one or more selected from rosmarinic acid, dimethylrosmarinic acid and salts or solvates thereof for producing foods for preventing or improving constipation.

<13> One or more selected from rosmarinic acid, dimethylrosmarinic acid, and salts or solvates thereof for use in TRPA1 activation.
<14> One or more selected from rosmarinic acid, dimethylrosmarinic acid, and salts or solvates thereof for use in prevention or amelioration of intestinal obstruction.
<15> One or more selected from rosmarinic acid, dimethylrosmarinic acid, and salts or solvates thereof for use in the prevention or improvement of constipation.

<16> A TRPA1 activation method in which one or more selected from rosmarinic acid, dimethylrosmarinic acid and salts or solvates thereof are administered or ingested in an effective amount to a subject who needs them.
<17> A method for preventing or ameliorating intestinal obstruction in which one or more selected from rosmarinic acid, dimethylrosmarinic acid and salts or solvates thereof are administered or ingested in an effective amount to a subject who needs them.
<18> A method for preventing or ameliorating constipation in which one or more selected from rosmarinic acid, dimethylrosmarinic acid and salts or solvates thereof are administered or ingested in an effective amount to a subject who needs them.

<19> <1> and <7> TRPA1 activators, <2> and <8> preventive or ameliorating agents for intestinal obstruction, <3> and <9> preventive or ameliorating agents for constipation, <4> and The content of the active ingredient in the TRPA1 activation food of <10>, the food for preventing or improving intestinal obstruction of <5> and <11>, and the food for preventing or improving constipation of <6> and <12> is The total amount is preferably 0.01% by mass or more, more preferably 0.1% by mass or more, still more preferably 1% by mass or more, and preferably 95% by mass or less, more preferably 90% by mass or less, further. It is preferably 70% by mass or less. Further, it is preferably 0.01 to 95% by mass, more preferably 0.1 to 90% by mass, and further preferably 1 to 70% by mass.

In <20> <16> to <18>, the daily dose per adult is preferably 0.01 mg or more, more preferably 1 mg or more, still more preferably 100 mg or more as rosmarinic acid or dimethylrosmarinic acid. Also, it is preferably 80,000 mg or less, more preferably 15,000 mg or less, still more preferably 5000 mg or less. Further, it is preferably 0.01 to 80,000 mg, more preferably 1 to 15,000 mg, and further preferably 100 to 5,000 mg.

Hereinafter, the present invention will be described in more detail based on examples, but the present invention is not limited thereto.
Production example Synthesis of dimethylrosmarinic acid (1) Reagent 3- (3-methoxy-4-hydroxyphenyl) sodium lactate (1) (racemic mixture) from Tokyo Kasei, ferulic acid (3) from Wako Pure Chemical Industries, Ltd. Purchased and used.

(2) Synthesis of α-hydroxy-3-methoxy-4- (2-propyrenyloxy) benzenepropanoic acid 2-propyrenyl ester (2)

7. Take 3- (3-methoxy-4-hydroxyphenyl) sodium lactate (1) (500 mg, 2.14 mmol) and potassium carbonate (885 mg, 6.41 mmol) in a 50 mL eggplant flask, replace with argon, and then super-dehydrated acetone. 1 mL was added and dispersed. Then, allyl bromide (2.77 mL, 32.0 mmol) was added, and _{after N 2} substitution, the mixture was stirred under reflux conditions for 20 hours.
After completion of the reaction, the filtration and the filtration residue were washed with methylene chloride (25 mL), and the filtrate and the washing liquid were combined and concentrated under reduced pressure. To this methylene chloride 20mL and _H 2 O 20mL was added, after layer separation, to recover the methylene chloride layer. The aqueous layer was further extracted 3 times with 5 mL of methylene chloride and recovered. The recovered methylene chloride layer was combined, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure (988.2 mg).
This was purified by silica gel column chromatography to obtain 513 mg (colorless, oily, yield 82%) of the purified product of the title compound (2). The conditions for silica gel column chromatography are as follows.

<Conditions for silica gel column chromatography>
Column: Universal Premium L (30 μm, 60 Å, 3.0x16.5 cm, 40 g) [YAMAZEN]
Inject Column: INJECT COLUMN S [YAMAZEN]
Eluent: Hexane / Ethyl acetate = 95/5 (4min) 95/5 (11min) 74/26 (14min) 74/26
Flow velocity: 20 mL / min, preparative time: 29 min, preparative capacity: 12 mL / piece, 48 preparative preparatives After confirming each Fr. With TLC, the target Fr. (Fr.43-53) is concentrated under reduced pressure. Dry.

(3) Synthesis of 3-methoxy-4- (2-propyleneyloxy) phenyl-2-propylene acid (4)

To ferulic acid (3) (500 mg, 2.58 mmol), 17 mL of super-dehydrated ethanol was added and dissolved. Then, allyl bromide (267μL, 3.09mmol) and potassium carbonate (427 mg, 3.09 mmol), and the mixture was stirred 5.5 hours reflux conditions after _{N 2} substitution. While confirming the reaction progress by TLC, the mixture was allowed to cool to room temperature, potassium hydroxide (434 mg, 7.74 mmol) was added, and the mixture was further stirred for 16 hours.
After completion of the reaction, concentrated under reduced pressure, dissolved again _H 2 O 25 mL, was added to 2N hydrochloric acid and concentrated hydrochloric acid and the pH = 0. This was extracted 5 times with ethyl acetate (25 mL), dried over anhydrous magnesium sulfate, and concentrated under reduced pressure (687.5 mg).
This was purified by silica gel column chromatography to obtain 221.4 mg (colorless, oily, 37% yield) of the purified product of the title compound (4). The conditions for silica gel column chromatography are as follows.

<Conditions for silica gel column chromatography>
Column: Universal Premium L (30 μm, 60 Å, 3.0x16.5 cm, 40 g) [YAMAZEN]
Inject Column: INJECT COLUMN M [YAMAZEN]
Eluent:
1% Acetic Acid in Hexane / 1% Acetic Acid in Ethyl Acetate = 100/0 (4min) 100/0 (11min) 84/16 (24min) 84/16 (0min) 80/20 (7min) 80/20
Flow velocity: 20 mL / min, preparative time: 46 min, preparative capacity: 12 mL / piece, 77 preparative preparatives After confirming each Fr. With TLC, the target Fr. (Fr.48-75) is concentrated under reduced pressure. Dry.

(4) α-[[3- [3-Methoxy-4- (2-propyrenyloxy) phenyl] -1-oxo-2-propyrenyl] oxy] -3-methoxy-4- (2-propyrenyloxy) benzenepropanoic acid 2 -Synthesis of propyrenyl ester (5)

To compound (2) (100 mg, 0.34 mmol), 1.7 mL of methylene chloride was added and dissolved. Then, compound (4) (104 mg, 0.44 mmol) and dimethylaminopyridine (6.3 mg, 0.051 mmol) were added in that order, cooled to −20 ° C., and then N, N'-dicyclohexylcarbodiimide (88.2 mg, 0.43 mmol) was added. After N ₂ substitution, the mixture was stirred at room temperature for 24 hours, the precipitated urea was removed by filtration, washed with ethyl acetate, and concentrated under reduced pressure (288.5 mg).
This was purified by silica gel column chromatography to obtain 151.8 mg (white, waxy) of the purified product of the title compound (5). The conditions for silica gel column chromatography are as follows.

<Conditions for silica gel column chromatography>
Column: Universal Premium L (30 μm, 60 Å, 3.0x16.5 cm, 40 g) [YAMAZEN]
Inject Column: INJECT COLUMN S [YAMAZEN]
Eluent:
Hexane / Ethyl Acetate = 87/13 (4min) 87/13 (11min) 66/34 (14min) 66/34
Flow velocity: 20 mL / min, preparative time: 29 min, preparative capacity: 12 mL / piece, 48 preparative preparatives After confirming each Fr. With TLC, the target Fr. (Fr.34-36) is concentrated under reduced pressure. Dry.

(5) Synthesis of 3,3'-dimethylrosmarinic acid (B)

Compound (5) (87.3 mg, 0.172 mmol) taken to 50mL round-bottomed flask in a glove box under _{N 2} atmosphere, ultra-dry tetrahydrofuran 8.6mL and tetrakis (triphenylphosphine) palladium (0) complex (Pd ( PPh ₃ ) ₄ ) (19.8 mg, 17.2 μmol) and morpholine (450 μL, 5.15 mmol) were added in that order. The reaction mixture, N ₂ atmosphere and stirred at room temperature for 18 hours.
After completion of the reaction, the mixture was concentrated under reduced pressure, acidified with 20 mL of 1N hydrochloric acid, and extracted 3 times with ethyl acetate (20 mL). The ethyl acetate layer was also dried over anhydrous magnesium sulfate and concentrated under reduced pressure (117.0 mg). This was purified by preparative TLC to obtain 61.4 mg (white, powdery, yield 92%) of the purified product of the title compound (B). The conditions for preparative TLC are as follows.
In the following examples, the synthesized dimethylrosmarinic acid was appropriately diluted with dimethyl sulfoxide (DMSO) and used for evaluation.
<Conditions for preparative TLC>
TLC: PLC Silica gel 60 F ₂₅₄ (1mm, 20x20cm, # 1.13895.0001) [Merck]
Developing solvent: Chloroform / Acetone / 88% Formic acid = 75 / 16.5 / 8.5 200mL
Elimination solution: Chloroform / Acetone / 88% Formic acid = 75 / 16.5 / 8.5 15mL

Example 1 Evaluation of TRPA1 activating action (1) Preparation of vector for human TRPA1 gene expression cDNA obtained by reverse transcription of totalRNA extracted from human duodenal-derived cell line (Hutu-80 cells, purchased from American Type Culture Collection) A polymerase chain reaction (PCR) was carried out under the following conditions using a primer set consisting of oligonucleotides represented by the nucleotide sequences shown below, which was synthesized with reference to the published human TRPA1 gene sequence using the above as a template. went.

<Primer set>
Forward primer; 5'-CACCATGAAGCGCAGCCTGAGGAAGATGTG-3'(SEQ ID NO: 1)
Reverse primer; 5'-GAGGAGCTAAGGCTCAAGATGGTGTG -3'(SEQ ID NO: 2)
<PCR conditions>
a) PCR solution composition
cDNA (Template) 15 μL
5x PrimeStar GXL Buffer 10 μL
dNTPs mixture (2.5mM) 4 μL
PrimeStar GXL DNA Polymerase (Takara Bio) 1 μL
Forward Primer (10 μM) 1 μL
Reverse Primer (10 μM) 1 μL
Water 18 μL
b) Temperature and cycle conditions
95 ℃ 2min
↓
98 ℃ 10sec 33 cycles
70 ℃ 2min

The obtained PCR product was purified using the High Pure PCR Product Purification Kit (trade name, manufactured by Roche). A vector for human TRPA1 gene expression was prepared using the purified PCR product and pcDNA3.1 Directional TOPO Expression Kit (trade name, manufactured by Invitrogen).

(2) Preparation of human TRPA1-expressing cells Using DMEM / F12 medium (manufactured by Invitrogen) containing 10% fetal bovine serum, culture of human cervical cancer-derived cell lines (HeLa cells, purchased from American Type Culture Collection) was performed. went. HeLa cells were seeded in T-75 cell culture flasks at 5 × 10 ⁵ cells / Flask. After 3 days of culturing, the human TRPA1 gene expression vector (8 μg) prepared in (1) above was transfected into cells using TransIT-HeLaMONSTER Transfection Kit (manufactured by Mirus) and cultured for 1 day.
The cells were peeled off with Detachin (manufactured by Genlantis), and seeded in DMEM / F12 medium containing 10% fetal bovine serum in 96-well Optical bottom plate (manufactured by Nunc) at ^{a cell density of 1.5 × 10 4} cells / 90 μL / well. It was cultured for another day.

(3) Measurement of intracellular calcium ion influx activity The intracellular calcium ion influx activity was measured using Calcium Kit II-fluo 4 (trade name, DOJINDO). A loading buffer containing Fluo4-AM was added to the human TRPA1-expressing cells prepared in (2) above at 90 μL / well, and incubated at 37 ° C. for 1 hour. Then, the fluorescence intensity (excitation wavelength: 488 nm, fluorescence wavelength: 524 nm) was measured every 2 seconds using a fluorescent plate reader FDSS3000 (trade name, manufactured by Hamamatsu Photonics Co., Ltd.) at 37 ° C. Thirty seconds after the start of measurement, the dimethylrosmarinic acid solution prepared in the above production example as a sample was diluted with the dilution buffer of the measurement kit, 20 μL / well was added to the diluted solution, and the change in fluorescence intensity was measured every 2 seconds for up to 300 seconds. .. In addition, as a positive control for TRPA1 activation, allyl isothiocyanate (AITC), which is an agonist of TRPA1, was added at a final concentration of 1 μM.
The TRPA1 activity of the sample was calculated as the ratio of the fluorescence intensity (F0) before the addition of the sample solution to the maximum value (Fmax) of the fluorescence intensity after the addition of the sample.

The calcium ion influx rate when the sample was added was compared with that when the solvent was added and tested using Dunnett's test. Table 2 shows the results when dimethylrosmarinic acid (MeRA) was added.

From Table 2, dimethylrosmarinic acid significantly increased calcium ion influx, similar to the positive control AITC (TRPA1 agonist).
From the above results, TRPA1 activity is significantly activated by applying dimethylrosmarinic acid. This indicates that dimethylrosmarinic acid is effective in activating TRPA1 activity. Furthermore, dimethylrosmarinic acid, which has an action of activating TRPA1 activity, has been shown to be effective in preventing or ameliorating intestinal obstruction or constipation.
As described above, since dimethylrosmarinic acid is a metabolite of rosmarinic acid in the body, it is considered that the precursor rosmarinic acid is also effective in activating TRPA1 activity and preventing or ameliorating intestinal obstruction or constipation.

Claims (6)

3,3'-dimethyl Roz Marine acid and TRPA1 activator as an active ingredient one or more selected from salt or solvate thereof. 3,3'-dimethyl Roz Marine acid and prophylactic or improving agent of the obstruction as an active ingredient one or more selected from salt or solvate thereof. 3,3'-dimethyl Roz Marine acid and prophylactic or improving agent of constipation as an active ingredient one or more selected from salt or solvate thereof. 3,3'-dimethyl Roz Marine acid and TRPA1 activation food for at least one selected from salt or solvate thereof as an active ingredient. 3,3'-dimethyl Roz Marine acid and intestinal obstruction preventing or ameliorating a food to at least one selected from salt or solvate thereof as an active ingredient. 3,3'-dimethyl Roz Marine acid and preventing or ameliorating food constipation for at least one selected from salt or solvate thereof as an active ingredient.