JP5476321B2 - Tricine amino acid derivatives and antiviral and anticancer agents containing the same - Google Patents

Description

  The present invention relates to a tricine amino acid derivative having higher cell permeability, water solubility and blood migration than tricine, and an antiviral agent and an antitumor agent containing the same.

Tricine (4 ′, 5,7-trihydroxy-3 ′, 5′-dimethoxyflavone) (tricin (4 ′, 5,7-trihydroxy-3 ′, 5′-dimethyflavone)), a kind of flavonoid, It has been confirmed that it is contained in grasses and has various physiological activities such as antioxidant, anticancer, and antiviral effects.
For example, Non-Patent Document 1 and the like report that tricine isolated from wheat leaves, grain maize seeds, etc. is an antioxidant substance.
Rice bran-derived tricine inhibits cyclooxygenase and suppresses carcinogenesis in the small intestine (Non-patent document 2), and exhibits anticancer activity in vitro against human-derived colon cancer and breast cancer cells (non-patent document) 3) It has been reported that an anticancer activity is exhibited in a mouse P388 leukemia model (Non-patent Document 4).
Furthermore, it has been reported that tricine contained in the extract of Kumazasa exhibits an antiviral effect (Patent Document 1).

  While many useful physiological activities have been confirmed in in vitro studies and their clinical application is expected, Tricine is very hydrophobic and needs to be dissolved in dimethyl sulfoxide (DMSO). Thus, it is anticipated that there will be limitations in its use in the preparation of drugs for administration to patients in clinical trials.

  Moreover, in vitro studies, tricine has low cell permeability, and therefore, it is expected that tissue transferability and bioavailability in vivo are low. Further, in order to effectively act as an antiviral agent, it is required that the drug penetrates into the host cell. Therefore, development of a prodrug having high cell permeability, water solubility and blood migration is expected.

In general, there are many unclear points about the pharmacokinetics when flavonoids are orally administered. Flavonoids exist in plants as glycosides to which various sugars are bound. The theory that this glycoside was hydrolyzed by intestinal bacteria in the digestive tract and that the aglycone from which the sugar was removed was absorbed in the large intestine was the mainstream. However, using Caco-2 cells, a human small intestine model, we investigated quercetin, a kind of flavonoid. As a result, it was shown that aglycone is more than 10 times more easily absorbed than glucose glycosides. It has been suggested that a highly flavonoid derivative may be more efficiently absorbed in the small intestine (Non-patent Document 5).
In addition, it has been reported that amino acid derivatives of quercetin show high water solubility and cell permeability in vitro (Non-patent Document 6).

  The present inventor believes that amino acids have a carboxyl group which is a hydrophilic group, amino acid derivatives are actively absorbed in the small intestine, and are hydrolyzed even if the amino acid is cleaved by esterase in the blood after absorption. Focusing on the fact that the produced amino acid itself is harmless, we developed a novel amino acid derivative of tricine as a prodrug having high cell penetrability, water solubility, and blood permeability.

WO / 2008/123102

J. Agric. Food Chem. 47: 4500-4505 (1999) Molecular Cancer Therapeutics, 4: 1287-1292 (2005). Cancer Epidemiol Biomarkers Prev, 9: 1163-70 (2000). J Nat Prod. 44: 530-5 (1981). Chemistry and Biology 39: 213 (2001). Bioorg. Med. Lett. 17: 1164-1171 (2009).

An object of the present invention is to provide a tricine amino acid derivative.
Another object of the present invention is to provide a tricine amino acid derivative having high cell permeability, water solubility and blood migration.
Another object of the present invention is to provide an antiviral agent and an anticancer agent comprising a tricine amino acid derivative.
Another object of the present invention is to provide an anticancer agent for treating cervical cancer comprising tricine or a tricine amino acid derivative.
Another object of the present invention is selected from tricine or a tricine amino acid derivative and gancyclovir, acyclovir, cidofovir, famciclovir, fomivirsen, foscalnet, idoxuridine, pencyclovir, trifluridine, valacyclovir, valganciclovir and vidarabine It is to provide an antiviral agent containing at least one kind.

で表される化合物（式中、Ａは任意のアミノ酸残基であり、Ｂは任意のアミノ酸残基であり、ＡとＢは同じ又は異なっても良く、ｎ＝０〜４である。）。
（２）Ａが任意のアミノ酸残基であり、ｎ＝０又は１である上記（１）記載の化合物。
（３）Ａがグリシン、アラニン、バリン、フェニルアラニン、アスパラギン酸及びグルタミン酸からなる群から選択されるアミノ酸の残基である上記（１）又は（２）記載の化合物。
（４）Ａがアラニン残基であり、Ｂが任意のアミノ酸残基である上記（１）記載の化合物。
（５）Ａがアラニン残基であり、Ｂがアスパラギン酸又はグルタミン酸残基である上記（１）記載の化合物。
（６）４’−Ｏ−ＣＯ−（Ｇｌｙ−ＯＨ）−トリシン、４’−Ｏ−ＣＯ−（Ａｌａ−ＯＨ）−トリシン、４’−Ｏ−ＣＯ−（Ｖａｌ−ＯＨ）−トリシン、４’−Ｏ−ＣＯ−（Ｐｈｅ−ＯＨ）−トリシン、４’−Ｏ−ＣＯ−（Ａｓｐ−ＯＨ）−トリシン、４’−Ｏ−ＣＯ−（Ｇｌｎ−ＯＨ）−トリシン、４’−Ｏ−ＣＯ−（Ａｌａ−Ａｓｐ−ＯＨ）−トリシン及び４’−Ｏ−ＣＯ−（Ａｌａ−Ｇｌｕ−ＯＨ）−トリシンからなる群から選択される上記（１）記載の化合物。
（７）上記（１）〜（６）のいずれか一に記載の化合物を含む抗ウイルス剤。
（８）対象のウイルスが、ヘルペスウイルス、サイトメガロウイルスあるいはインフルエンザウイルスである、上記７記載の抗ウイルス剤。
（９）対象のウイルスが、オセルタミビル耐性インフルエンザウイルス株、アマンタジン耐性インフルエンザウイルス株、ガンシクロビル耐性ヒトサイトメガロウイルスからなる群より選択されるウイルスである、上記（７）記載の抗ウイルス剤。
（１０）経口投与用である上記７〜９のいずれか一に記載の抗ウイルス剤。
（１１）上記（１）〜（６）のいずれか一に記載の化合物を含む抗癌剤。
（１２）対象の癌が大腸癌または子宮頸癌である、上記（１１）記載の抗癌剤。
（１３）経口投与用である、上記（１１）または（１２）記載の抗癌剤。
（１４）（Ａ）トリシン又はそのアミノ酸誘導体、及び（Ｂ）ガンシクロビル、アシクロビル、シドフォビル、ファムシクロビル、フォミビルセン、ホスカルネット、イドクスウリジン、ペンシクロビル、トリフルリジン、バラシクロビル、バルガンシクロビル及びビダラビンからなる群から選択される少なくとも一種の化合物、を有効成分として含むことを特徴とする、抗ウイルス剤。
（１５）前記（Ｂ）成分がガンシクロビルである、上記（１４）に記載の抗ウイルス剤。
（１６）前記ウイルスがヘルペスウイルスである、上記（１４）または（１５）に記載の抗ウイルス剤。
（１７）前記ウイルスがサイトメガロウイルスである、上記（１４）〜（１６）のいずれかに記載の抗ウイルス剤。
（１８）前記ウイルスが、ヒトサイトメガロウイルスである上記（１４）〜（１７）のいずれかに記載の抗ウイルス剤。
（１９）前記（Ａ）成分と（Ｂ）成分とのモル比が１００：１〜１０００：１（（Ａ）成分：（Ｂ）成分）の範囲内である、上記（１４）〜（１８）のいずれかに記載の抗ウイルス剤。 In order to achieve the above object, the present invention employs the following configuration.
(1)
Formula 1:

(Wherein A is an arbitrary amino acid residue, B is an arbitrary amino acid residue, A and B may be the same or different, and n = 0-4).
(2) The compound according to (1) above, wherein A is any amino acid residue and n = 0 or 1.
(3) The compound according to (1) or (2) above, wherein A is an amino acid residue selected from the group consisting of glycine, alanine, valine, phenylalanine, aspartic acid and glutamic acid.
(4) The compound according to (1) above, wherein A is an alanine residue and B is any amino acid residue.
(5) The compound according to (1) above, wherein A is an alanine residue and B is an aspartic acid or glutamic acid residue.
(6) 4'-O-CO- (Gly-OH) -tricine, 4'-O-CO- (Ala-OH) -tricine, 4'-O-CO- (Val-OH) -tricine, 4 ' -O-CO- (Phe-OH) -tricine, 4'-O-CO- (Asp-OH) -tricine, 4'-O-CO- (Gln-OH) -tricine, 4'-O-CO- The compound according to (1) above, which is selected from the group consisting of (Ala-Asp-OH) -tricine and 4'-O-CO- (Ala-Glu-OH) -tricine.
(7) An antiviral agent comprising the compound according to any one of (1) to (6) above.
(8) The antiviral agent according to 7 above, wherein the target virus is a herpes virus, cytomegalovirus or influenza virus.
(9) The antiviral agent according to (7) above, wherein the target virus is a virus selected from the group consisting of an oseltamivir resistant influenza virus strain, an amantadine resistant influenza virus strain, and a ganciclovir resistant human cytomegalovirus.
(10) The antiviral agent according to any one of the above 7 to 9, which is for oral administration.
(11) An anticancer agent comprising the compound according to any one of (1) to (6) above.
(12) The anticancer agent according to (11) above, wherein the target cancer is colon cancer or cervical cancer.
(13) The anticancer agent according to (11) or (12), which is for oral administration.
(14) the group consisting of (A) tricine or an amino acid derivative thereof, and (B) ganciclovir, acyclovir, cidofovir, famciclovir, fomivirsen, foscarnet, idoxuridine, pencyclovir, trifluridine, valacyclovir, valganciclovir and vidarabine An antiviral agent comprising at least one compound selected from the above as an active ingredient.
(15) The antiviral agent according to (14) above, wherein the component (B) is ganciclovir.
(16) The antiviral agent according to (14) or (15) above, wherein the virus is a herpes virus.
(17) The antiviral agent according to any one of (14) to (16), wherein the virus is a cytomegalovirus.
(18) The antiviral agent according to any one of (14) to (17), wherein the virus is a human cytomegalovirus.
(19) Said (14)-(18) whose molar ratio of said (A) component and (B) component exists in the range of 100: 1-1000: 1 ((A) component: (B) component). The antiviral agent in any one of.

Since the tricine amino acid derivative of the present invention has higher cell permeability, water solubility, and blood migration than tricine, it exhibits high tissue migration and bioavailability. In addition, the tricine amino acid derivative of the present invention exhibits high tricine transferability in the blood by oral administration, but no toxicity is observed even when administered at a considerably high concentration.
Based on the above, it can be used for the prevention and treatment of related diseases based on the anticancer action and antiviral action of tricine.
The tricine or tricine amino acid derivative of the present invention and at least selected from the group consisting of ganciclovir, acyclovir, cidofovir, famciclovir, fomivirsen, foscalnet, idoxuridine, pencyclovir, trifluridine, valacyclovir, valganciclovir and vidarabine One type of antiviral agent has a significantly higher antiviral activity than an antiviral agent containing only tricine as an active ingredient.

The result of the cell permeability test of a tricine and a tricine amino acid derivative is shown. 3 shows the transition of plasma tricine concentration after single oral administration of a tricine derivative in male rats. A representative chromatogram before administration of a tricine derivative is shown. A represents animal number 101, B: animal number 301. A representative chromatogram 30 minutes after administration of a tricine derivative is shown. A represents animal number 101, B: animal number 301 (use of 5-fold diluted sample). A representative chromatogram 2 hours after administration of a tricine derivative is shown. A represents animal number 101, B: animal number 301 (using a 2-fold diluted sample). A representative chromatogram 4 hours after administration of a tricine derivative is shown. A represents animal number 101 (using a 3-fold diluted sample), and B: animal number 301 (using a 3-fold diluted sample). A representative chromatogram 8 hours after administration of a tricine derivative is shown. A represents animal number 101, B: animal number 301 (3-fold diluted sample used). It represents the inhibitory effect of tricine on the proliferation of TIG-3 cells (fetal lung fibroblasts) (normal cells, control). It represents the inhibitory effect of tricine on the proliferation of CA cells. It represents the inhibitory effect of tricine on the growth of CaSki cells. It represents the inhibitory effect of tricine on the proliferation of HeLa cells. It represents the inhibitory effect of tricine on the growth of SiHa cells. The virus titer (%) of ganciclovir relative to the control is shown. The virus titer (%) relative to the control when tricine and ganciclovir are used and when tricine alone is used is shown.

<< Tricine amino acid derivative >>
The first aspect of the present invention is a tricine amino acid derivative characterized in that one or a plurality of amino acids are bonded to the 4′-position OH of tricine via a carboxyl group.
Examples of amino acids that give amino acid residues represented by A or B in the general formula of the present invention include alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys). , Glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro) , Natural amino acids such as serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Val).
More preferably, the amino acid is selected from the group consisting of glycine, alanine, valine, serine, phenylalanine, tyrosine, aspartic acid and glutamic acid.
More preferably, the amino acid is selected from the group consisting of glycine, alanine, valine, phenylalanine, aspartic acid and glutamic acid.
The amino acid may be either L-form or D-form, but is preferably L-form.

のように表されるアミノ酸から誘導される２価の基を意味する。
ここでＲはアミノ酸の側鎖であり、例えばアラニンの場合ＲはＣＨ₃であり、アスパラギン酸の場合ＲはＣＨ₂ＣＯＯＨとなる。 In the present invention, “amino acid residue” means

Is a divalent group derived from an amino acid represented by
Here, R is a side chain of an amino acid. For example, R is CH ₃ in the case of alanine, and R is CH ₂ COOH in the case of aspartic acid.

部分において、Ａとｎ個のＢはペプチド結合しており、これはより具体的には

と表される。Ｒ₁及びＲ₂はアミノ酸側鎖を表す。 Equation 1

In part, A and n B are peptide-bonded, more specifically

It is expressed. R ₁ and R ₂ represent amino acid side chains.

ｎ＝２の場合

と表される。 -A (B) nOH is, for example, when n = 1

When n = 2

It is expressed.

(Synthesis method)
The tricine amino acid derivative of the present invention can be produced by combining the above-described amino acids by various known methods using tricine as a raw material.
For example, the carboxyl group of the raw material amino acid may be protected in advance by esterification or the like, and reacted with the —OH group at the 4 ′ position of tricine using a reagent such as bis (4-nitrophenyl) carbonate in the presence of a base. Good. The amino acid protecting group may then be appropriately deprotected.
In addition, when preparing a derivative in which two or more amino acids are joined, the amino acids may be produced by binding to tricine one by one in order, or tricine is prepared after preparing a plurality of amino acids bonded in advance. May be combined.

(Cell permeability)
MDCK cells (cell lines derived from canine kidney tubular epithelial cells) form tight junctions when cultured in a monolayer, and actively transport salts and water. It has been recognized that the MDCK cell membrane permeability of drugs absorbed by passive diffusion correlates with the gastrointestinal absorption rate in humans. Therefore, the cell permeability of the tricine derivative in MDCK cells was examined.
When tested on various tricine amino acid derivatives, all showed higher cell permeability than tricine.

(Blood migration)
In order to study the in vivo kinetics of the tricine amino acid derivative, particularly the blood transferability when orally administered, the tricine concentration in plasma obtained by orally administering the tricine derivative and collecting blood after a predetermined time was measured.
When tricine was orally administered, the transfer of tricine into the blood was hardly observed, but in the test where the tricine amino acid derivative was orally administered, a maximum of 4448 ng / mL of tricine was detected, and the transfer into the blood was observed. It was found that the sex was much higher than tricine.
After the tricine amino acid derivative is taken into the body, it is considered that the amino acid part is hydrolyzed in the body to be converted into tricine and transferred into the blood.
From the results of cell permeability and blood migration, it is clear that the tricine amino acid derivative of the present invention exhibits higher tissue migration and higher bioavailability than tricine.

(Toxicity test)
The tricine amino acid derivative orally administered with tricine had a much higher transferability of tricine into the blood than that with tricine administered. Therefore, a toxicity test was conducted to confirm its safety. As a result, when the tricine derivative was orally administered to male mice once, no abnormal findings were found even in the high dose group of 2000 mg / kg. In addition, the no-effect amount when the tricine derivative was orally administered to male mice for 14 consecutive days was 300 mg / kg / day, suggesting that the tricine derivative is a safe drug similar to tricine.

(Antiviral agent)
Tricine exhibits an antiviral action against herpes virus, cytomegalovirus, influenza virus and the like, as described in WO / 2008/123102.
On the other hand, the tricine amino acid derivative of the present invention exhibits high cell permeability, water solubility, and blood migration, and exhibits a high blood concentration of tricine. Therefore, the tricine amino acid derivative of the present invention can be used as an antiviral agent.
The dose of the antiviral agent of the present invention can be appropriately selected depending on the usage, patient age, sex, disease severity, and other conditions. Usually, the amount of the tricine amino acid derivative in the range of about 1 to 2000 mg / kg / day, preferably about 5 to 1000 mg / kg / day, and more preferably about 10 to 600 mg / kg / day should be used as a guide. These antiviral agents of the present invention can be administered once a day or divided into about 2 to 4 times a day.

In the present invention, “virus” means animal viruses, insect viruses, plant viruses, bacterial viruses (bacteriophages), and particularly viruses that have an unfavorable effect on animals and plants including mammals including humans, birds and the like. Viruses for which the antiviral agent of the present invention is particularly effective include herpes virus and influenza virus, and in particular, cytomegalovirus, particularly cytomegalovirus such as human cytomegalovirus, sarcytomegalovirus, mouse cytomegalovirus. Human influenza viruses such as influenza virus A, influenza virus B, and influenza virus C, and avian influenza virus.

  The antiviral agent of the present invention is effective as an antiviral agent for mammals other than human beings, birds, shellfish, crustaceans, insects, amphibians, reptiles and the like. Therefore, the antiviral agent of the present invention can be used as an antiviral agent for these animals (for example, pet medicine, animal feed, pet food, etc.). Furthermore, the antiviral agent of the present invention is useful as an antiviral agent for various plants in addition to animals, and is also useful as a preservative.

The antiviral agent of the present invention is effective against various viruses, particularly herpes viruses, especially cytomegalovirus, particularly human cytomegalovirus; influenza virus, particularly human influenza virus A, influenza virus B, influenza virus C and avian influenza virus. And high antiviral activity.
The antiviral agent of the present invention has excellent antiviral activity against resistant viruses in addition to the excellent advantages of high cell permeability, high blood migration, and low cytotoxicity.

(Anticancer agent)
Tricine inhibits cyclooxygenase and suppresses carcinogenesis in the small intestine (Molecular Cancer Therapeutics, 4: 1287-1292 (2005)), and exhibits anticancer activity in vitro against human colon cancer and breast cancer cells ( Cancer Epidemiol Biomarkers Prev, 9: 1163-70 (2000)), an anticancer activity in a mouse P388 leukemia model (J Nat Prod. 44: 530-5 (1981)) and the like have been reported. Tricine has also been reported to be effective against azoxymethane and dextran sulfate-induced colon cancer (Cancer Prev Res., 2 (12), 1031-1038 (2009)).
As shown in the examples, tricine is an anticancer agent for treating cervical cancer because it suppresses the growth of cervical cancer cell lines.
The tricine amino acid derivative of the present invention can be used as an anticancer agent because it has high cell permeability, water solubility and blood migration, and is converted to tricine in the body.

  The dosage of the anticancer agent of the present invention can be appropriately selected depending on the usage, patient age, sex, disease severity, and other conditions. Usually, the amount of the tricine amino acid derivative in the range of about 1 to 2000 mg / kg / day, preferably about 5 to 1000 mg / kg / day, and more preferably about 10 to 600 mg / kg / day should be used as a guide. These anticancer agents of the present invention can be administered once a day or divided into about 2 to 4 times a day.

  There are no particular limitations on the cancer that can be treated or alleviated by administration of the anticancer agent of the present invention. Cancer, rectal cancer, head and neck cancer, lung cancer, breast cancer, cervical cancer, ovarian cancer, bladder cancer, prostate cancer, testicular tumor, bone / soft tissue sarcoma, skin cancer, malignant lymphoma, acute leukemia, brain tumor and the like.

  The antiviral agent and anticancer agent of the present invention are effective not only as a human but also as an anticancer agent for mammals other than humans, birds, fish, reptiles and the like. Therefore, the anticancer agent of the present invention can be used as an anticancer agent for these animals (for example, pet medicine, pet food, etc.).

  The dosage form of the antiviral agent or anticancer agent of the present invention can be appropriately selected according to the therapeutic purpose. Specific examples include tablets, pills, powders, solutions, suspensions, emulsions, granules, capsules, ointments and the like. Examples of the pharmaceutical carrier used here include various substances commonly used for ordinary drugs, such as diluents or excipients such as fillers, extenders, binders, disintegrants, surfactants, lubricants, and the like. It can be illustrated.

  Oral administration forms include non-food forms such as tablets, pills, powders, liquids, chewing gum, candy, chocolate, bread, cookies, rice crackers, biscuits, buckwheat, udon, various drinks, jelly, miso soup, soup, potage, etc. Examples of the oral administration form include injections, topical administration agents (creams, ointments etc.), suppositories and the like. Examples of the dosage form of the topical administration agent include those obtained by impregnating the anticancer agent of the present invention with a carrier such as gauze made of natural fiber or synthetic fiber, or those incorporated in cosmetics such as lipstick.

  When forming into tablets, for example, lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid and other excipients, water, ethanol, simple syrup, glucose Solution, starch solution, gelatin solution, carboxymethylcellulose, ceramic, methylcellulose, potassium phosphate, polyvinylpyrrolidone and other binders, dried starch, sodium alginate, agar powder, laminaran powder, sodium bicarbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid Absorption of disintegrants such as esters, sodium lauryl sulfate, stearic acid monoglyceride, starch, lactose, disintegration inhibitors such as sucrose, stearic acid, cocoa butter, hydrogenated oil, quaternary ammonium salts, sodium lauryl sulfate, etc. Accelerator, glycerol, humectants, such as starch, starch, lactose, kaolin, bentonite, adsorbent such as colloidal silicic acid, purified talc, a lubricant such as stearic acid salts may be used. Further, the tablets can be made into tablets with ordinary coatings as necessary, for example, sugar-coated tablets, gelatin-encapsulated tablets, enteric-coated tablets, film-coated tablets, double tablets, multilayer tablets and the like.

In shaping into a pill form, as a carrier, for example, excipients such as glucose, lactose, starch, cacao butter, hydrogenated vegetable oil, kaolin, talc, binders such as gum arabic powder, tragacanth powder, gelatin, laminaran, Disintegrants such as agar can be used.
Capsules are prepared by mixing the tricine amino acid derivative, which is the active ingredient of the present invention, with the various carriers exemplified above and filling them into hard gelatin capsules, soft capsules and the like.

  The amount of the tricine amino acid derivative contained in the antiviral agent or anticancer agent of the present invention is not particularly limited and may be appropriately selected. For example, it is 0.5 to 50% by mass, preferably about 1 to 8% by mass. It is good.

  The antiviral agent and anticancer agent of the present invention may be administered either orally or parenterally, and the administration method is determined according to each preparation form, patient age, sex, degree of patient symptom, and other conditions. For example, tablets, pills, powders, solutions, suspensions, emulsions, granules and capsules are administered orally, solutions are administered parenterally as injections, and ointments are administered topically.

  In the production of various dosage forms of the present invention, a coloring agent, a preservative, a fragrance, a flavoring agent, a sweetening agent, and other pharmaceuticals may be blended as necessary. In addition, base components such as oily components used in normal pharmaceutical compositions, cosmetics, dermatological compositions, etc., moisturizers, preservatives, stabilizers, wound healing agents, surfactants, etc. may be used. it can. When water is used for the anticancer agent, tap water, natural water, purified water and the like are not particularly limited, but generally high-purity water such as ion-exchanged water is preferable.

  Examples of oil components include animal oils such as squalane, beef tallow, pork tallow, horse oil, lanolin, beeswax, eucalyptus oil, olive oil, grape seed oil, palm oil, jojoba oil, germ oil (eg, rice germ oil), etc. Examples include vegetable oils, liquid paraffin, higher fatty acid esters (for example, octyl palmitate, isopropyl palmitate, octyldodecyl myristate), synthetic oils such as silicone oil, and semi-synthetic oils. Eucalyptus oil is preferable because it has a stimulating effect.

  The oily component is used in combination as appropriate according to the required performance such as skin protection, emollient imparting effect (effect of covering the skin surface with a thin film to prevent drying and imparting flexibility and elasticity), refreshing feeling and the like. A combination of squalane, olive oil and octyldodecyl myristate is one preferred example.

  In order to adjust the hardness and fluidity of each dosage form, solid oils such as stearic acid, stearyl alcohol, behenic acid, cetanol and petrolatum are used, and preferably stearic acid and cetanol are used in combination.

<< Antiviral agent (combination drug) >>
The second aspect of the present invention includes (A) tricine or an amino acid derivative thereof, and (B) ganciclovir, acyclovir, cidofovir, famciclovir, fomivirsen, foscarnet, idoxuridine, pencyclovir, trifluridine, valacyclovir, valcivir An antiviral agent (hereinafter sometimes referred to as an antiviral agent (combination agent)) characterized by using at least one selected from ganciclovir and vidarabine in combination.

  In the present invention, “virus” means animal viruses, insect viruses, plant viruses, bacterial viruses (bacteriophages), and particularly viruses that have an unfavorable effect on animals and plants including mammals including humans, birds and the like. To do.

  The antiviral agent (combination agent) of the present invention is effective not only as a human but also as an antiviral agent for mammals other than humans, birds, shellfish, crustaceans, insects, amphibians, reptiles, and the like. Therefore, the antiviral agent of the present invention can be used as an antiviral agent for these animals (for example, pet medicine, animal feed, pet food, etc.). Furthermore, the antiviral agent of the present invention is useful as an antiviral agent for various plants in addition to animals, and is also useful as a preservative.

  Viruses for which the antiviral agent (concomitant drug) of the present invention is particularly effective include herpes viruses, and particularly cytomegaloviruses such as human cytomegalovirus (HCMV), sarcytomegalovirus, and mouse cytomegalovirus. . Among these, the antiviral agent of the present invention effectively acts particularly on human cytomegalovirus.

<(A) component>
The active ingredient in the antiviral agent (concomitant drug) of the present invention is 4 ′, 5,7-trihydroxy-3 ′, 5′-dimethoxyflavone (tricine) or an amino acid derivative thereof. Although tricine is extracted from straw, it goes without saying that it may be a synthetic product or one collected from another source. For example, plants other than grapes, in particular, many plants belonging to the family Gramineae, such as rice (rice), wheat (wheat), corn, barley, rye, sugar cane, bamboo, Japanese pampas grass, pampas grass, reed (also called reed, reed) ) And other leaves and stems contain a large amount of 4 ′, 5,7-trihydroxy-3 ′, 5′-dimethoxyflavone (tricine) and can be extracted. Specific plant names are as follows.
Rice, wheat, barley, oats, rye, millet, millet, millet, corn, millet, sorghum, bamboo, makomo, sugarcane, pearl barley, reed, suki, sasa, danchiku, shiroganeyoshi, shiba.

  The plant leaves, stems and the like can be extracted using an appropriate solvent, and tricine can be separated and purified using separation and purification means such as HPLC. For example, the water extract can be concentrated, the solid can be extracted with alcohol or water-containing alcohol (for example, methanol, ethanol, water-containing methanol, water-containing ethanol), the solid content can be dissolved in water, and purified by partitioning with ethyl acetate or the like.

  The amino acid derivative of tricine is the tricine amino acid derivative represented by the general formula 1 described in the first aspect.

<(B) component>
The antiviral agent (concomitant drug) of the present invention includes, as an active ingredient, together with the component (A), gancyclovir, acyclovir, cidofovir, famciclovir, fomivirsen, foscarnet, idoxuridine, penciclovir, trifluridine, valacyclovir, At least one selected from valganciclovir and vidarabine. Among these, ganciclovir is particularly preferable.

The antiviral agent (combination agent) of the present invention exhibits a significantly superior antiviral effect than conventional antiviral agents using tricine as an active ingredient by combining the (A) component and the (B) component. I can do it.
In addition, conventional antiviral agents mainly containing only ganciclovir use a large amount of ganciclovir, which leads to problems such as the emergence of resistant viruses and side effects (strong cytotoxicity). On the other hand, the antiviral agent (concomitant drug) of the present invention uses tricine together, so that the cytotoxicity is weak and the problem of resistant strains hardly occurs. Furthermore, it has an antiviral activity equivalent to or better than that of ganciclovir.

The content ratio (molar ratio) between the component (A) and the component (B) is not particularly limited as long as the effect of the present invention is exhibited, but is 1: 1 to 1000: 1 (component (A): (B). Component), more preferably 10: 1 to 1000: 1, more preferably 100: 1 to 1000: 1. When the content ratio is within the above range, the component (A) and the component (B) exert a synergistic effect, and exhibit an antiviral effect far superior to that expected from the high viral activity of each component. I can do it.
The dose of the antiviral agent of the present invention can be appropriately selected depending on the usage, patient age, sex, disease severity, and other conditions. For example, in the case of intravenous injection, it may be administered so that the dose of component (B) is about 0.1 to 10 mg per day, and in the case of oral administration, the amount of component (B) is 0. What is necessary is just to administer so that it may become about 1-50 mg. Moreover, it can be administered once a day or divided into about 2 to 6 times as needed. Moreover, you may administer (A) component and (B) component each simultaneously or continuously.
With conventional antiviral agents containing ganciclovir, for example, ganciclovir in an amount of about 10 to 1000 mg per day for intravenous injection and about 10 to 5000 mg per day for oral administration has been administered to adults. In the present invention, the amount of the component (B) can be suppressed to about 100 to 1/1000 of the conventional ganciclovir dosage by using in combination with tricine, and the problem of cytotoxicity hardly occurs and the safety is excellent.

The antiviral agent (combination agent) of the present invention comprising the component (A) and the component (B) as active ingredients can be used in various forms. For example, it is suitable as an antiviral agent for mucosal protective compositions, viral infection prevention and / or treatment compositions, preservatives, filtration devices and the like.
The dosage form of the antiviral agent (concomitant drug) of the present invention may be any of liquid, solid, gas, gel, gel, and aerosol. The antiviral agent of the present invention may be administered in any oral or parenteral dosage form.
Examples of oral dosage forms include capsules, dry syrups, tablets, pills, powders, liquids and the like.
Further, it may be in the form of food such as chewing gum, koji, various drinks, drinking water, seasonings and the like.
Examples of parenteral dosage forms include nasal solutions and gels, aerosols such as oral sprays, nasal preparations directly applied to the nasal cavity and pharynx, ointments, emulsions and creams for transdermal absorption. Formulations, injections, suppositories, vaginal administration forms (tampons, etc.) and the like can be mentioned. The antiviral agent of the present invention impregnated with a carrier such as natural fiber or synthetic fiber gauze, cosmetics such as lipstick and other forms of cosmetics (lotion, oil, soap, lotion, cosmetic cream, etc.) Semi-solid or liquid forms such as those contained in bath preparations, cosmetic liquids, shampoos, body soaps, and facial cleansing foams may also be used. It is good also as forms, such as eye drops and a mouthwash. Forms such as a spray for treating wound sites and a spray for treating pharyngeal inflammation are also included.

  For the production of antiviral agents (combination agents) of various dosage forms according to the present invention, in addition to predetermined amounts of the above-mentioned components (A) and (B), ordinary pharmaceutical compositions, cosmetics, dermatological compositions, etc. Substrate components such as oily components used, humectants, preservatives, and the like can be used.

The antiviral agent (combination agent) of the present invention may be used in combination with other antiviral agents other than the component (A) and the component (B).
The antiviral agent (combination agent) of the present invention may further contain a stabilizer, a moisturizer, a wound healing agent, a preservative, a surfactant and the like as necessary.

Hereinafter, the present invention will be specifically described with reference to examples.
<< Tricine amino acid derivative >>
[Example 1]
(Synthesis of Compound 2: 4′-O—CO— (Gly-OH) -Tricine)

Under a nitrogen atmosphere, glycine t-butyl ester hydrochloride (55.3 mg, 0.33 mmol) was dissolved in THF (5 ml), and bis (4-nitrophenyl) carbonate (100.4 mg, 0.33 mmol) was cooled in an ice bath. ), N, N-diisopropylethylamine (115.0 μl, 0.66 mmol) was added, and the mixture was stirred at room temperature for 12 hours. Thereafter, tricine (100.0 mg, 0.30 mmol) and N, N-diisopropylethylamine (115.0 μl, 0.66 mmol) were added to the reaction solution, followed by stirring at room temperature for 12 hours. After confirming the completion of the reaction by TLC (chloroform / methanol = 10/1), the solvent was distilled off. This was isolated and purified by silica gel column chromatography (chloroform → chloroform / methanol = 50/1), and 4′-O—CO— (Gly-OtBu) -tricine (Compound 1) (92 mg, 63%) was yellow. Obtained as a powder.
Under a nitrogen atmosphere, this 4'-O-CO- (Gly-OtBu) -tricine (Compound 1) (40.0 mg, 0.082 mmol) was dissolved in dichloromethane (5 ml), and trifluoroacetic acid ( 2 ml) was added and stirred at room temperature for 4 hours. After confirming the completion of the reaction by TLC (chloroform / methanol = 5/1), the solvent was distilled off. Dichloromethane (5 ml) was added thereto, and suction filtration was performed to obtain 4′-O—CO— (Gly-OH) -tricine (Compound 2) (33 mg, 94%) as a yellow powder.

[Example 2]
(Compound 4: Synthesis of 4′-O—CO— (Ala—OH) -tricine)

Under a nitrogen atmosphere, alanine t-butyl ester hydrochloride (59.9 mg, 0.33 mmol) was dissolved in THF (5 ml), and bis (4-nitrophenyl) carbonate (100.4 mg, 0.33 mmol) was cooled in an ice bath. ), N, N-diisopropylethylamine (115.0 μl, 0.66 mmol) was added, and the mixture was stirred at room temperature for 12 hours. Thereafter, tricine (100.0 mg, 0.30 mmol) and N, N-diisopropylethylamine (115.0 μl, 0.66 mmol) were added to the reaction solution, followed by stirring at room temperature for 12 hours. After confirming the completion of the reaction by TLC (chloroform / methanol = 10/1), the solvent was distilled off. This was isolated and purified by silica gel column chromatography (chloroform → chloroform / methanol = 50/1), and 4′-O—CO— (Ala-OtBu) -tricine (compound 3) (84 mg, 55%) was yellow. Obtained as a powder.
Under a nitrogen atmosphere, this 4′-O—CO— (Ala-OtBu) -tricine (compound 3) (41.1 mg, 0.082 mmol) was dissolved in dichloromethane (5 ml) and cooled in an ice bath while trifluoroacetic acid ( 2 ml) was added and stirred at room temperature for 4 hours. After confirming the completion of the reaction by TLC (chloroform / methanol = 5/1), the solvent was distilled off. Dichloromethane (5 ml) was added thereto, and suction filtration was performed to obtain 4′-O—CO— (Ala—OH) -tricine (Compound 4) (30 mg, 81%) as a yellow powder.

[Example 3]
(Compound 6: Synthesis of 4′-O—CO— (Val—OH) -tricine)

Under a nitrogen atmosphere, valine t-butyl ester hydrochloride (69.2 mg, 0.33 mmol) was dissolved in THF (5 ml) and cooled in an ice bath while bis (4-nitrophenyl) carbonate (100.4 mg, 0.33 mmol). ), N, N-diisopropylethylamine (115.0 μl, 0.66 mmol) was added, and the mixture was stirred at room temperature for 12 hours. Thereafter, tricine (100.0 mg, 0.30 mmol) and N, N-diisopropylethylamine (115.0 μl, 0.66 mmol) were added to the reaction solution, followed by stirring at room temperature for 12 hours. After confirming the completion of the reaction by TLC (chloroform / methanol = 10/1), the solvent was distilled off. This was isolated and purified by silica gel column chromatography (chloroform → chloroform / methanol = 50/1), and 4′-O—CO— (Val-OtBu) -tricine (compound 5) (94 mg, 52%) was yellow. Obtained as a powder.
Under a nitrogen atmosphere, this 4'-O-CO- (Val-OtBu) -tricine (compound 5) (44.6 mg, 0.082 mmol) was dissolved in dichloromethane (5 ml), and trifluoroacetic acid ( 2 ml) was added and stirred at room temperature for 4 hours. After confirming the completion of the reaction by TLC (chloroform / methanol = 5/1), the solvent was distilled off. Dichloromethane (5 ml) was added thereto, and suction filtration was performed to obtain 4′-O—CO— (Val—OH) -tricine (Compound 6) (35 mg, 90%) as a yellow powder.

[Example 4]
(Compound 8: Synthesis of 4′-O—CO— (Phe—OH) -tricine)

Under a nitrogen atmosphere, phenylalanine t-butyl ester hydrochloride (85.1 mg, 0.33 mmol) was dissolved in THF (5 ml) and bis (4-nitrophenyl) carbonate (100.4 mg, 0.33 mmol) while cooling in an ice bath. ), N, N-diisopropylethylamine (115.0 μl, 0.66 mmol) was added, and the mixture was stirred at room temperature for 12 hours. Thereafter, tricine (100.0 mg, 0.30 mmol) and N, N-diisopropylethylamine (115.0 μl, 0.66 mmol) were added to the reaction solution, followed by stirring at room temperature for 12 hours. After confirming the completion of the reaction by TLC (chloroform / methanol = 10/1), the solvent was distilled off. This was isolated and purified by silica gel column chromatography (chloroform → chloroform / methanol = 50/1), and 4′-O—CO— (Phe-OtBu) -tricine (Compound 7) (116 mg, 61%) was yellow. Obtained as a powder.
Under a nitrogen atmosphere, this 4′-O—CO— (Phe-OtBu) -tricine (Compound 7) (47.4 mg, 0.082 mmol) was dissolved in dichloromethane (5 ml), and trifluoroacetic acid (cooled) was cooled in an ice bath. 2 ml) was added and stirred at room temperature for 4 hours. After confirming the completion of the reaction by TLC (chloroform / methanol = 5/1), the solvent was distilled off. Dichloromethane (5 ml) was added thereto, and suction filtration was performed to obtain 4′-O—CO— (Phe—OH) -tricine (Compound 8) (48 mg, 80%) as a yellow powder.

[Example 5]
(Compound 10: Synthesis of 4′-O—CO— (Asp—OH) -tricine)

Under a nitrogen atmosphere, aspartic acid t-butyl ester hydrochloride (93.0 mg, 0.33 mmol) was dissolved in THF (5 ml) and cooled in an ice bath while bis (4-nitrophenyl) carbonate (100.4 mg, 0. 0). 33 mmol) and N, N-diisopropylethylamine (115.0 μl, 0.66 mmol) were added, and the mixture was stirred at room temperature for 12 hours. Thereafter, tricine (100.0 mg, 0.30 mmol) and N, N-diisopropylethylamine (115.0 μl, 0.66 mmol) were added to the reaction solution, followed by stirring at room temperature for 12 hours. After confirming the completion of the reaction by TLC (chloroform / methanol = 10/1), the solvent was distilled off. This was isolated and purified by silica gel column chromatography (chloroform → chloroform / methanol = 50/1), and 4′-O—CO— (Asp-OtBu) -tricine (Compound 9) (106 mg, 53%) was yellow. Obtained as a powder.
Under a nitrogen atmosphere, this 4'-O-CO- (Asp-OtBu) -tricine (Compound 9) (49.3 mg, 0.082 mmol) was dissolved in dichloromethane (5 ml) and cooled in an ice bath while cooling with trifluoroacetic acid ( 2 ml) was added and stirred at room temperature for 4 hours. After confirming the completion of the reaction by TLC (chloroform / methanol = 5/1), the solvent was distilled off. Dichloromethane (5 ml) was added thereto, and suction filtration was performed to obtain 4′-O—CO— (Asp—OH) -tricine (Compound 10) (36 mg, 90%) as a yellow powder.

[Example 6]
(Compound 12: Synthesis of 4'-O-CO- (Glu-OH) -tricine)

Under a nitrogen atmosphere, glutamic acid t-butyl ester hydrochloride (97.6 mg, 0.33 mmol) was dissolved in THF (5 ml), and bis (4-nitrophenyl) carbonate (100.4 mg, 0.33 mmol) was cooled in an ice bath. ), N, N-diisopropylethylamine (115.0 μl, 0.66 mmol) was added, and the mixture was stirred at room temperature for 12 hours. Thereafter, tricine (100.0 mg, 0.30 mmol) and N, N-diisopropylethylamine (115.0 μl, 0.66 mmol) were added to the reaction solution, followed by stirring at room temperature for 12 hours. After confirming the completion of the reaction by TLC (chloroform / methanol = 10/1), the solvent was distilled off. This was isolated and purified by silica gel column chromatography (chloroform → chloroform / methanol = 50/1), and 4′-O—CO— (Glu-OtBu) -tricine (Compound 11) (124 mg, 61%) was yellow. Obtained as a powder.
Under a nitrogen atmosphere, this 4′-O—CO— (Glu-OtBu) -tricine (Compound 11) (50.5 mg, 0.082 mmol) was dissolved in dichloromethane (5 ml) and cooled in an ice bath while cooling with trifluoroacetic acid ( 2 ml) was added and stirred at room temperature for 4 hours. After confirming the completion of the reaction by TLC (chloroform / methanol = 5/1), the solvent was distilled off. Dichloromethane (5 ml) was added thereto, and suction filtration was performed to obtain 4′-O—CO— (Glu—OH) -tricine (Compound 12) (34 mg, 83%) as a yellow powder.

[Example 7]
(Compound 14: Synthesis of 4'-O-CO- (Ala-Asp-OH) -Tricine)

Aspartic acid t-butyl ester hydrochloride (87.4 mg, 0.31 mmol), 4′-O—CO— (Ala—OH) -tricine (compound 4) (140 mg, 0) while cooling to 0 ° C. under a nitrogen atmosphere. .31 mmol), dissolved in DMF (5 ml), 1-hydroxy-1H-benzotriazole (94.9 mg, 0.62 mmol), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (65.4 mg, 0 .34 mmol) was added and stirred for 3 days. After confirming the completion of the reaction by TLC (chloroform / methanol = 10/1), the solvent was distilled off. This was isolated and purified by silica gel column chromatography (chloroform) to obtain 4'-O-CO- (Ala-Asp-OtBu) -tricine (Compound 13) (68 mg, 32%) as a yellow powder.
Under a nitrogen atmosphere, this 4'-O-CO- (Ala-Asp-OtBu) -tricine (Compound 13) (55.0 mg, 0.095 mmol) was dissolved in dichloromethane (5 ml), and trifluoro while cooling in an ice bath. Acetic acid (2 ml) was added and stirred at room temperature for 4 hours. After confirming the completion of the reaction by TLC (chloroform / methanol = 5/1), the solvent was distilled off. Dichloromethane (5 ml) was added thereto, and suction filtration was performed to obtain 4′-O—CO— (Ala-Asp—OH) -tricine (Compound 14) (42 mg, 91%) as a yellow powder.

[Example 8]
(Compound 16: Synthesis of 4'-O-CO- (Ala-Glu-OH) -tricine)

Glutamic acid t-butyl ester hydrochloride (91.7 mg, 0.31 mmol), 4′-O—CO— (Ala—OH) -tricine (compound 4) (140 mg, 0. 1) while cooling to 0 ° C. under nitrogen atmosphere. 31 mmol), dissolved in DMF (5 ml), 1-hydroxy-1H-benzotriazole (94.9 mg, 0.62 mmol), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (65.4 mg, 0. 34 mmol) was added and stirred for 3 days. After confirming the completion of the reaction by TLC (chloroform / methanol = 10/1), the solvent was distilled off. This was isolated and purified by silica gel column chromatography (chloroform) to obtain 4′-O—CO— (Ala-Glu-OtBu) -tricine (Compound 15) (73 mg, 33%) as a yellow powder.
Under a nitrogen atmosphere, this 4'-O-CO- (Ala-Glu-OtBu) -tricine (Compound 15) (55.0 mg, 0.078 mmol) was dissolved in dichloromethane (5 ml), and trifluoro while cooling in an ice bath. Acetic acid (2 ml) was added and stirred at room temperature for 4 hours. After confirming the completion of the reaction by TLC (chloroform / methanol = 5/1), the solvent was distilled off. Dichloromethane (5 ml) was added thereto, and suction filtration was performed to obtain 4′-O—CO— (Ala-Glu—OH) -tricine (Compound 16) (44 mg, 96%) as a yellow powder.

The physicochemical properties of the starting compounds, intermediates and products in Examples 1-8 are shown below.
NMR data
(Tricine)
¹ H NMR (400 MHz; acetone-d ₆ ): δ 7.39 (s, 2H), 6.74 (s, 1H), 6.56 (d, J = 2.0 Hz, 1H), 6.26 (d, J = 2.0 Hz, 1H ), 3.97 (s, 6H).
¹³ C NMR (100 MHz; acetone-d ₆ ): δ 183.1, 165.1, 164.9, 163.3, 158.8, 149.1 (2C), 140.9, 122.3, 105.4 (2C), 105.2, 104.7, 99.7, 94.9, 56.9 (2C) .
MS (FAB): m / z = 331 [M + H] ⁺

(Compound 1: 4′-O—CO— (Gly-OtBu) -tricine)
¹ H NMR (400 MHz; acetone-d ₆ ): δ 7.37 (s, 2H), 7.00 (br t, J = 6.0 Hz, 1H), 6.82 (s, 1H), 6.54 (d, J = 1.8 Hz, 1H), 6.24 (d, J = 1.8 Hz, 1H), 3.97 (s, 6H), 3.84 (d, J = 6.0 Hz, 2H), 1.46 (s, 9H).
¹³ C NMR (100 MHz; acetone-d ₆ ): δ 182.3, 168.8, 164.2, 163.4, 162.5, 158.0, 153.9, 153.7 (2C), 132.4, 129.0, 105.5, 104.7, 103.6 (2C), 99.0, 94.2, 80.9, 56.1 (2C), 43.5, 28.8 (3C).
MS (FAB): m / z = 488 [M + H] ⁺

(Compound 2: 4′-O—CO— (Gly-OH) -tricine)
¹ H NMR (400 MHz; DMSO-d ₆ ): δ 10.86 (br s, 1H), 8.05 (t, J = 6.0 Hz, 1H), 7.34 (s, 2H), 7.09 (s, 1H), 6.56 ( d, J = 1.9 Hz, 1H), 6.20 (d, J = 1.9 Hz, 1H), 3.85 (s, 6H), 3.72 (d, J = 6.0 Hz, 2H).
¹³ C NMR (100 MHz; DMSO-d ₆ ): δ 182.5, 171.7, 164.9, 163.3, 161.9, 158.0, 154.1, 153.5 (2C), 132.1, 128.9, 106.1, 104.5, 104.2 (2C), 99.5, 94.9, 56.9 (2C), 42.9.
MS (FAB): m / z = 432 [M + H] ⁺

(Compound 3: 4'-O-CO- (Ala-OtBu) -tricine)
¹ H NMR (400 MHz; acetone-d ₆ ): δ 7.36 (s, 2H), 7.00 (br d, J = 9.2 Hz, 1H), 6.81 (s, 1H), 6.53 (d, J = 2.3 Hz, 1H), 6.23 (d, J = 2.3 Hz, 1H), 4.14 (quin, J = 7.3 Hz, 1H), 3.92 (s, 6H), 1.46 (s, 9H), 1.41 (d, J = 7.3 Hz, 3H).
¹³ C NMR (100 MHz; acetone-d ₆ ): δ 183.2, 172.5, 165.0, 164.3, 163.3, 158.8, 154.5 (2C), 153.9, 133.3, 129.8, 106.4, 105.5, 104.5 (2C), 99.8, 95.0, 81.6, 56.9 (2C), 51.6, 28.1 (3C), 18.0.
MS (FAB): m / z = 502 [M + H] ⁺

(Compound 4: 4′-O—CO— (Ala—OH) -tricine)
¹ H NMR (400 MHz; DMSO-d ₆ ): δ 10.86 (br s, 1H), 8.11 (d, J = 7.8 Hz, 1H), 7.33 (s, 2H), 7.08 (s, 1H), 6.56 ( d, J = 1.9 Hz, 1H), 6.20 (d, J = 1.9 Hz, 1H), 4.03 (quin, J = 7.3 Hz, 1H), 3.84 (s, 6H), 1.29 (d, J = 7.3 Hz, 3H).
¹³ C NMR (100 MHz; DMSO-d ₆ ): δ 182.5, 174.5, 164.9, 163.3, 161.9, 158.0, 153.5 (3C), 132.2, 128.8, 106.1, 104.5, 104.2 (2C), 99.5, 94.9, 57.0 ( 2C), 50.0, 17.8.
MS (FAB): m / z = 446 [M + H] ⁺

(Compound 5: 4′-O—CO— (Val-OtBu) -tricine)
¹ H NMR (400 MHz; acetone-d ₆ ): δ 7.36 (s, 2H), 6.87-6.83 (m, 1H), 6.80 (s, 1H), 6.53 (d, J = 2.0 Hz, 1H), 6.23 (d, J = 2.0 Hz, 1H), 4.10-4.04 (m, 1H), 3.92 (s, 6H), 2.26-2.14 (m, 1H), 1.48 (s, 9H), 1.04-1.00 (m, 6H ).
¹³ C NMR (100 MHz; acetone-d ₆ ): δ 182.3, 170.6, 164.2, 163.4, 162.5, 158.0, 153.6 (3C), 132.5, 128.9, 105.5, 104.7, 103.7 (2C), 99.0, 94.2, 81.0, 60.5, 56.0 (2C), 31.1, 27.4 (3C), 18.6, 17.3.

(Compound 6: 4'-O-CO- (Val-OH) -tricine)
¹ H NMR (400 MHz; DMSO-d ₆ ): δ 10.91 (br s, 1H), 8.06 (d, J = 9.2 Hz, 1H), 7.38 (s, 2H), 7.12 (s, 1H), 6.59 ( d, J = 1.9 Hz, 1H), 6.23 (d, J = 1.9 Hz, 1H), 3.95-3.88 (m, 1H), 3.88 (s, 6H), 2.15-2.08 (m, 1H), 0.96 (d , J = 3.8 Hz, 3H), 0.94 (d, J = 3.8 Hz, 3H).
¹³ C NMR (100 MHz; DMSO-d ₆ ): δ 182.0, 172.9, 164.4, 162.9, 161.4, 157.5, 153.7, 153.0 (2C), 131.8, 128.3, 105.6, 104.0, 103.7 (2C), 99.0, 94.4, 59.8, 56.4 (2C), 30.0, 19.1, 18.0.

(Compound 7: 4′-O—CO— (Phe-OtBu) -tricine)
¹ H NMR (400 MHz; acetone-d ₆ ): δ 7.36 (s, 2H), 7.36-7.30 (m, 4H), 7.28-7.23 (m, 1H), 6.92-6.88 (m, 1H), 6.81 ( s, 1H), 6.55 (d, J = 1.9 Hz, 1H), 6.24 (d, J = 1.9 Hz, 1H), 4.40-4.34 (m, 1H), 3.90 (s, 6H), 3.19 (dd, J = 13.7, 6.0 Hz, 1H), 3.08 (dd, J = 13.7, 6.0 Hz, 1H), 1.42 (s, 9H).
¹³ C NMR (100 MHz; acetone-d ₆ ): δ 183.2, 171.2, 165.8, 164.4, 163.4, 158.9, 154.5 (2C), 153.8, 138.1, 133.2, 130.4 (2C), 129.2 (2C), 127.5, 126.9 , 106.4, 104.5 (3C), 99.9, 95.0, 82.0, 57.2, 56.9 (2C), 38.3, 28.1 (3C).
MS (FAB): m / z = 578 [M + H] ⁺

(Compound 8: 4′-O—CO— (Phe—OH) -tricine)
¹ H NMR (400 MHz; DMSO-d ₆ ): δ 10.86 (br s, 1H), 8.15 (d, J = 8.2 Hz, 1H), 7.30 (s, 2H), 7.29-7.20 (m, 5H), 7.07 (s, 1H), 6.55 (d, J = 1.9 Hz, 1H), 6.19 (d, J = 1.9 Hz, 1H), 4.20-4.13 (m, 1H), 3.78 (s, 6H), 3.08 (dd , J = 13.7, 4.6 Hz, 1H), 2.87 (dd, J = 13.7, 4.6 Hz, 1H).
¹³ C NMR (100 MHz; DMSO-d ₆ ): δ 182.5, 173.5, 164.9, 163.3, 161.9, 158.0, 153.7, 153.4 (2C), 138.2, 132.2, 129.7 (2C), 128.8 (3C), 127.0, 106.1 , 104.5, 104.2 (2C), 99.5, 94.9, 56.9 (2C), 56.2, 37.0.

(Compound 9: 4′-O—CO— (Asp-OtBu) -tricine)
¹ H NMR (400 MHz; acetone-d ₆ ): δ 7.37 (s, 2H), 6.98-6.93 (m, 1H), 6.81 (s, 1H), 6.54 (d, J = 2.0 Hz, 1H), 6.24 (d, J = 2.0 Hz, 1H), 4.49-4.44 (m, 1H), 3.93 (s, 6H), 2.83 (dd, J = 16.0, 6.0 Hz, 1H), 2.75 (dd, J = 16.0, 6.0 Hz, 1H), 1.47 (s, 9H), 1.46 (s, 9H).
¹³ C NMR (100 MHz; acetone-d ₆ ): δ 182.3, 169.6, 169.5, 164.2, 163.5, 163.4, 162.5, 158.0, 153.7 (2C), 132.4, 129.1, 105.6, 104.7, 103.7 (2C), 99.0, 94.2, 81.4, 80.7, 56.1 (2C), 51.9, 37.5, 27.4 (3C), 27.3 (3C).

(Compound 10: 4'-O-CO- (Asp-OH) -tricine)
¹ H NMR (400 MHz; DMSO-d ₆ ): δ 10.90 (br s, 1H), 8.15 (d, J = 8.2 Hz, 1H), 7.37 (s, 2H), 7.12 (s, 1H), 6.59 ( d, J = 1.9 Hz, 1H), 6.23 (d, J = 1.9 Hz, 1H), 4.35 (q, J = 7.8 Hz, 1H), 3.88 (s, 6H), 2.77 (dd, J = 16.5, 6.7 Hz, 1H), 2.63 (dd, J = 16.5, 6.7 Hz, 1H).
¹³ C NMR (100 MHz; DMSO-d ₆ ): δ 182.0, 172.3, 171.6, 164.4, 162.8, 161.4, 157.5, 153.0, 152.9 (2C), 131.7, 128.3, 105.6, 104.0, 103.8 (2C), 99.0, 94.4, 56.4 (2C), 55.8, 29.6.

(Compound 11: 4′-O—CO— (Glu-OtBu) -tricine)
¹ H NMR (400 MHz; acetone-d ₆ ): δ 7.36 (s, 2H), 7.01-6.97 (m, 1H), 6.80 (s, 1H), 6.53 (d, J = 2.0 Hz, 1H), 6.23 (d, J = 2.0 Hz, 1H), 4.24-4.18 (m, 1H), 3.93 (s, 6H), 2.45-2.41 (m, 2H), 2.20-2.11 (m, 1H), 1.98-1.89 (m , 1H), 1.47 (s, 1H), 1.44 (s, 1H).
¹³ C NMR (100 MHz; acetone-d ₆ ): δ 182.3, 171.6, 170.8, 164.2, 163.5, 163.4, 162.5, 158.0, 153.6 (2C), 153.4, 132.5, 129.0, 105.5, 104.7, 103.7 (2C), 99.0, 94.2, 81.1, 79.8, 56.1 (2C), 54.4, 31.1, 27.5 (3C), 27.3 (3C).

(Compound 12: 4'-O-CO- (Glu-OH) -tricine)
¹ H NMR (400 MHz; DMSO-d ₆ ): δ 10.93 (br s, 1H), 8.15 (d, J = 8.2 Hz, 1H), 7.38 (s, 2H), 7.12 (s, 1H), 6.60 ( d, J = 2.0 Hz, 1H), 6.24 (d, J = 2.0 Hz, 1H), 4.09-4.02 (m, 1H), 3.89 (s, 6H), 2.40-2.35 (m, 1H), 2.09-2.00 (m, 1H), 1.88-1.78 (m, 1H).
¹³ C NMR (100 MHz; DMSO-d ₆ ): δ 182.5, 174.3, 173.6, 164.9, 163.3, 161.9, 158.0, 153.9, 153.4 (2C), 132.3, 128.8, 106.1, 104.5, 104.2 (2C), 94.9 ( 2C), 56.9 (2C), 53.7, 30.4, 26.9.

(Compound 13: 4′-O—CO— (Ala-Asp-OtBu) -Tricine)
¹ H NMR (400 MHz; acetone-d ₆ ): δ 7.51 (br d, J = 8.2 Hz, 1H), 7.34 (s, 2H), 7.00 (br d, J = 7.3 Hz, 1H), 6.80 (s , 1H), 6.53 (d, J = 1.9 Hz, 1H), 6.23 (d, J = 1.9 Hz, 1H), 4.68-4.63 (m, 1H), 4.30 (quin, J = 6.8 Hz, 1H), 3.91 (s, 6H), 2.74 (d, J = 5.5 Hz, 2H), 1.44 (s, 9H), 1.43 (d, J = 6.8 Hz, 3H), 1.40 (s, 9H).
¹³ C NMR (100 MHz; acetone-d ₆ ): δ 182.3, 171.9, 169.6 (3C), 164.3, 163.4, 162.5, 158.0, 153.6 (2C), 131.9, 129.0, 105.5, 104.7, 103.5 (2C), 99.0 , 94.3, 81.3, 80.6, 56.0 (2C), 50.8, 49.5, 37.3, 27.4 (3C), 27.3 (3C), 18.1.

(Compound 14: 4'-O-CO- (Ala-Asp-OH) -tricine)
¹ H NMR (400 MHz; DMSO-d ₆ ): δ 10.91 (br s, 1H), 8.15 (d, J = 8.2 Hz, 1H), 7.99 (d, J = 7.8 Hz, 1H), 7.37 (s, 2H), 7.12 (s, 1H), 6.59 (d, J = 2.0 Hz, 1H), 6.23 (d, J = 2.0 Hz, 1H), 4.56 (q, J = 7.8 Hz, 1H), 4.13 (quin, J = 7.8 Hz, 1H), 3.88 (s, 6H), 2.69 (dd, J = 16.5, 6.0 Hz, 1H), 2.62 (dd, J = 16.5, 6.0 Hz, 1H), 1.29 (d, J = 6.8 Hz, 3H).
¹³ C NMR (100 MHz; DMSO-d ₆ ): δ 182.0, 172.3, 172.0, 171.6, 164.4, 162.8, 161.4, 157.5, 153.0 (2C), 152.7, 131.7, 128.3, 105.6, 104.0, 103.7 (2C), 99.0, 94.4, 56.4 (2C), 50.1, 48.5, 36.0, 18.4.

(Compound 15: 4′-O—CO— (Ala-Glu-OtBu) -Tricine)
¹ H NMR (400 MHz; acetone-d ₆ ): δ 7.46 (br d, J = 7.8 Hz, 1H), 7.34 (s, 2H), 6.97 (br d, J = 7.8 Hz, 1H), 6.80 (s , 1H), 6.53 (d, J = 1.9 Hz, 1H), 6.24 (d, J = 1.9 Hz, 1H), 4.42-4.36 (m, 1H), 4.29 (quin, J = 7.3 Hz, 1H), 3.90 (s, 6H), 2.34-2.30 (m, 2H), 2.14-2.05 (m, 1H), 1.92-1.82 (m, 1H), 1.45 (s, 9H), 1.43 (d, J = 7.3 Hz, 3H ), 1.38 (s, 9H).
¹³ C NMR (100 MHz; acetone-d ₆ ): δ 182.3, 172.1, 171.5, 170.7, 164.3, 163.4, 162.5, 158.0, 153.6 (2C), 153.1, 132.0, 128.9, 105.4, 104.7, 103.5 (2C), 99.0, 94.3, 81.2, 79.7, 56.0 (2C), 52.3, 50.7, 31.2, 27.4 (3C), 27.3 (3C), 27.2, 18.0.

(Compound 16: 4′-O—CO— (Ala-Glu-OH) -tricine)
¹ H NMR (400 MHz; DMSO-d ₆ ): δ 10.90 (br s, 1H), 8.09 (d, J = 7.8 Hz, 1H), 7.98 (d, J = 7.8 Hz, 1H), 7.37 (s, 2H), 7.12 (s, 1H), 6.59 (d, J = 2.0 Hz, 1H), 4.28-4.22 (m, 1H), 4.12 (quin, J = 7.3 Hz, 1H), 3.89 (s, 6H), 2.31-2.23 (m, 2H), 2.03-1.94 (m, 1H), 1.83-1.74 (m, 1H), 1.27 (d, J = 7.3 Hz, 1H).
¹³ C NMR (100 MHz; DMSO-d ₆ ): δ 182.0, 173.7, 173.1, 172.2, 164.4, 162.8, 161.4, 157.5, 153.0 (2C), 152.7, 131.7, 128.3, 105.5, 104.0, 103.7 (2C), 99.0, 94.4, 56.4 (3C), 51.1, 29.9, 26.4, 18.2.

[Example 9]
(Cell permeability test)
The MDCK cell culture was mixed with 89% E-MEM, 10% inactivated calf serum, and 1% penist (antibiotic).
MDCK cells were seeded at 2.7 × 10 ⁵ / cm ² (300 μl) in the upper layer of the 12-well cell penetration plate chamber, and 500 μl of the culture solution was added to the lower layer. The culture was performed at 37 ° C. and CO ₂ 5% a day, and then the upper culture solution was removed and a new culture solution was added. Again, after one day of culture, the upper and lower culture broths were removed. 500 μl of the culture solution was added to the lower layer, and 300 μl of the test substance 30 μM (solvent: PBS) was added to the upper layer. After culturing for 1 hour, 150 μl of the upper and lower layers were collected, and UV (355 nm) absorption was measured. The cell penetration rate of the test substance was calculated using a calibration curve. As a result of the cell permeability test, the relative cell penetration rate when the cell penetration rate of tricine is 1 is shown in FIG.
The state of the cell layer was confirmed by the absence of lucifer yellow permeation.
As a result, all amino acid derivatives of tricine showed higher cell permeability than tricine, suggesting the possibility of higher tissue migration and higher bioavailability.

[Reference Example 1]
(Tricine concentration in plasma after 14 days of continuous oral administration of tricine in rats)
Methods Three male and female Crl: CD (SD) rats (Nippon Charles River Co., Ltd.) were prepared for each group, and tricine 100, 300, and 1000 mg / kg / day were orally administered to each group for 14 days. The tricine concentration in plasma obtained by blood sampling was measured before administration on the 14th day of administration, 0.5, 2 and 4 hours after administration.
Analytical method Tricine concentration in rat plasma was measured by high performance liquid chromatography (HPLC) -UV method. Extraction of tricine from plasma and HPLC measurement conditions were as described by Hong Cai et al. (Hong Cai et al. Biomed. Chromatogr. 17, 435-439 (2003), Hong Cai, et al. Biomed. Chromatogr. 19, 518- 522 (2005)), and the tricine concentration in plasma was measured by partially modifying the extraction method.

Blood collection Heparinized blood was collected from the vaginal vein before administration on the 14th day of administration, 30 minutes after administration, and 2 and 4 hours after administration, and centrifuged at 4 ° C. and 6000 rpm for 10 minutes. The obtained plasma was stored as rat TK plasma in a freezer set at −25 ° C. from the day of blood collection to the day of use.
Extraction method Add 10 μL of internal standard solution (quercetin dihydrate DMSO solution, plasma concentration 800 ng / mL) to 50 μL of rat TK plasma, and after stirring, extract solvent (0.1 mol / L acetic acid / acetone solution) 40 μL Was added and stirred. After centrifuging at 15000 rpm for 10 minutes in a micro high speed cooling centrifuge, 25 μL of distilled water was added and stirred, followed by centrifugation in a micro high speed cooling centrifuge, and the resulting supernatant was injected into HPLC.
Also, 10 μL of standard solution for calibration curve (plasma tricine concentration: 50, 100, 200, 500, 1000, 2000 ng / mL) was added to 50 μL of rat pool plasma (derived from untreated male and female Crl: CD (SD) system) After the addition, a standard curve obtained by performing the same operation as rat TK plasma was used.
-HPLC condition YMC Pack ODS-AM (6.0 mm x 150 mm, YMC Co., Ltd.) column was injected with 40 μL of the supernatant after extraction, column temperature 40 ° C, wavelength 355 nm, flow rate 1.4 mL / min, mobile phase Was measured under the conditions of ammonium acetate / acetic acid (0.1 mol / L, pH 5.1): methanol = 2: 3, and an analysis time of 10 minutes.

Results The results of measuring the tricine concentration in rat TK plasma are shown in Tables 1 and 2.
All were values below the limit of quantification (500 ng / mL), but tricine was detected in plasma at 0.5, 2 and 4 hours after administration in males. The plasma tricine concentration correlated with the dose tended to be high at 2 and 4 hours after administration, but the plasma tricine concentration at 2 and 4 hours after administration in the 300 or 1000 mg / kg administration group was There was no tendency to increase or decrease over time. In contrast, no tricine was detected in plasma until 4 hours after administration in the female 100 and 300 mg / kg groups. In the female 1000 mg / kg administration group, tricine was detected in plasma at 0.5, 2 and 4 hours after administration, but the plasma tricine concentration tended to decrease with a peak at 0.5 hours after administration.

^*：定量限界（５００ｎｇ／ｍＬ）以下であることから参考値である。 Table 1: Plasma Tricine concentrations in male rats in a 14-day repeated oral dose toxicity study

^* : Reference value because it is below the limit of quantification (500 ng / mL).

^*：定量限界（５００ｎｇ／ｍＬ）以下であることから参考値である。 Table 2: Tricine plasma concentrations in female rats in a 14-day repeated oral dose toxicity study

^* : Reference value because it is below the limit of quantification (500 ng / mL).

[Example 10]
(Toxicity test by single oral administration of tricine derivatives in male rats and changes in plasma concentration of tricine)
Methods Three male Crl: CD (SD) rats (Nippon Charles River Co., Ltd.) were prepared for each group, and each group had a tricine concentration of 0 mg / kg (control group), 100, 300 and 1000 mg / kg. A single tricine derivative equivalent to kg, compound 16 (4′-O—CO— (Ala-Glu-OH) -tricine) was administered once, before administration, 30 minutes, 2 hours, 4 hours and 8 hours after administration. The transition of plasma tricine concentration obtained by collecting blood was examined.

Observation, measurement and inspection items a. Observation of general condition In all cases, the life, death, appearance, behavior, etc. of the animals were observed at the time of blood collection before administration, immediately after administration, 30 minutes, 2 hours, 4 hours and 8 hours after administration.
b. Body weight measurement For all cases, the body weight of the animals was measured using an electronic top balance before administration on the day of administration and recorded in units of 1 g.

Analytical method Tricine concentration in rat plasma was measured by high performance liquid chromatography (HPLC) -UV method. Extraction of tricine from plasma and HPLC measurement conditions were as described by Hong Cai et al. (Hong Cai et al. Biomed. Chromatogr. 17, 435-439 (2003), Hong Cai, et al. Biomed. Chromatogr. 19, 518- 522 (2005)), plasma tricine concentration was measured by partially modifying the extraction method.

Blood collection Heparinized blood was collected from the vaginal vein at 30 minutes, 2 hours, 4 hours, and 8 hours after administration on the day of administration and centrifuged at 4 ° C. and 6000 rpm for 10 minutes. The obtained plasma was stored as rat TK plasma in a freezer set at −25 ° C. from the day of blood collection to the day of use.
・ Extraction method Add 10 μl of internal standard solution (1.25 μg / l quercetin dihydrate DMSO solution) to 50 μl of rat TK plasma, mix, and then add 40 μl of extraction solvent (0.1 mol / L acetic acid / acetone solution). Stir. Centrifugation was performed at 15000 rpm at 10 ° C. for 10 minutes. After adding 50 μl of distilled water, the mixture was stirred and centrifuged at 15000 rpm at 10 ° C. for 10 minutes. The supernatant after centrifugation was collected, and the resulting supernatant was injected into HPLC.
HPLC conditions: 400 μL of the supernatant after extraction was injected into a YMC Pack ODS-AM (4.6 mm × 150 mm, YMC Co.) column, column temperature 40 ° C., wavelength 355 nm, flow rate 1.4 mL / min, mobile phase. Was measured under the conditions of ammonium acetate solution: methanol = 2: 3 and analysis time of 10 minutes.

Results and general condition Table 3 shows the results of the general condition.
In each administration group, there was no abnormality in the general state before administration on the day of administration and at each time of blood collection at 30 minutes, 2 hours, 4 hours and 8 hours after administration.
-Body weight Table 4 shows the body weight on the day before and on the day of administration.
The body weight on the administration day increased in all administration groups compared to the day before administration, and the body weight increase until the administration day was steady.
Therefore, like tricine, tricine derivatives are presumed to be highly safe drugs.

-Plasma tricine concentration FIG. 2 and Table 5 show changes in plasma tricine concentration. Representative chromatograms are shown in FIGS.
In the plasma tricine concentration transition of the 100 mg / kg administration group, a bimodal transition was observed that increased 30 minutes after administration, decreased 2 hours after administration, and increased again 4 hours after administration. Compared to 30 minutes after administration, the tricine concentration at 4 hours after administration was about 3.6 times higher. The plasma concentration decreased 8 hours after administration, but tricine was detected in the same degree as 30 minutes after administration.

  Also in the plasma concentration transition of the 300 mg / kg administration group, a bimodal transition peaking at 30 minutes and 4 hours after administration was observed, but unlike the 100 mg / kg administration group, 30 minutes and 4 minutes after administration. Plasma concentrations over time were about the same. Compared with the 100 mg / kg administration group, the plasma tricine concentration at 30 minutes after administration was about 4.2 times, and about 2.5 times higher at 2 hours after administration, indicating an increase according to the dose. However, the concentration at 4 hours after administration was about 1.1 times, and at about 8 hours after administration, it was about 1.4 times, and the plasma concentration after 4 hours after administration was significantly different from the 100 mg / kg administration group. There wasn't.

Even in the plasma concentration transition of the 1000 mg / kg administration group, a bimodal transition was observed that increased 30 minutes after administration, decreased 2 hours after administration, and increased again 4 hours after administration. The value for 30 minutes after administration is about 1.6 times higher than 4 hours after administration, unlike the four polysaccharide groups, about 6.4 times that of the 100 mg / kg administration group, and about 1.5 times that of the 300 mg / kg administration group. It was high. Further, the concentration at 8 hours after administration was similar to that at 4 hours after administration in the same group or 4 hours after administration in the 300 mg / kg administration group.
In the pharmacokinetic parameters, the Cmax of the 1000 mg / kg administration group was high, but the values of the 100 and 300 mg / kg administration groups were similar, and Tmax tended to become shorter as the dosage increased. It was seen. AUC _0-8 was about 1.5 and 2.1 times higher in the 300 and 1000 mg / kg administration groups than in the 100 mg / kg administration group, respectively.

In the 100, 300, and 1000 mg / kg dose groups of tricine derivatives, a dose-related increase was observed in plasma concentrations 30 minutes after administration and AUC _0-8 , and 30 minutes after administration and 4 minutes after administration in all administration groups. A bimodal transition with a peak in time suggests the possibility of reabsorption by intestinal circulation.
In addition, the plasma concentrations of the tricine derivatives in the 100, 300, and 1000 mg / kg administration groups for 4 hours after administration are all similar, and in the 1000 mg / kg administration group, the plasma for 4 hours after administration and the plasma for 8 hours after administration. There was no difference in the medium concentration, and all were high. This suggested the possibility of delayed absorption from the intestinal tract associated with large doses.
For comparison, tricine was orally administered to rats for 14 consecutive days, and the plasma concentration transition was measured in the same manner, but only 61 ng / mL of tricine was detected at the maximum. In this study in which a tricine derivative was orally administered, a maximum of 4448 ng / mL of tricine was detected, and it was verified that the transferability into blood was much higher than that of tricine.

Ｎ：異常所見無し Table 3: General status in a single oral dose study of a tricine derivative in male rats

N: No abnormal findings

Table 4: Transition of body weight in a single oral administration test of a tricine derivative to male rats

Table 5: Plasma tricine concentrations in a single oral dose study of tricine derivatives in male rats

[Example 11]
(Toxicity test by single oral administration of tricine derivative in mice)
Method Five male and female Crlj: CD1 (ICR) mice (Nippon Charles River Co., Ltd.) were prepared per group, and the tricine concentrations in each group were 0 mg / kg (control group) and 500 mg / kg (control group), respectively. Low dose group), tricine derivative corresponding to 1000 mg / kg (medium dose group) and 2000 mg / kg (high dose group), compound 16 (4′-O—CO— (Ala-Glu-OH) -tricine) Orally administered twice.

Observation, measurement and inspection items a. General condition observation In all cases, the life and death, appearance, behavior, etc. of animals were counted from the administration start date as the administration day 0. From the administration day 1 to the administration day 13, at least twice a day in the morning and afternoon, and after the administration day 14 Observed once in the morning.
b. Body weight measurement For all cases, the body weight of the animals was measured on the first, third, fifth, seventh, tenth and 14th day of administration using an electronic precision balance and recorded in units of 0.1 g.
c. Necropsy findings All cases were euthanized on the 14th day of administration, and organs and tissues of the whole body were visually observed, and necessary organs and tissues were fixed and preserved.

Results and general condition No abnormality was observed during the administration period in the control group and any administration group.
-Body weight transition Table 6 shows the body weight transition. There was no significant difference between the administration groups and the control group during the administration period.
-Autopsy findings No abnormal findings were observed in either the control group or any of the treatment groups.
Based on the above, in a single oral dose toxicity test for mice with a tricine derivative, changes related to test substance administration were not observed in general conditions, body weight changes, and autopsy findings.

Table 6-1: Transition of body weight in single oral dose toxicity test of tricine derivative to male mice

Table 6-2: Transition of body weight in single oral toxicity test of tricine derivative to female mice

[Example 12]
(Toxicity test by continuous oral administration of tricine derivative in male mice for 14 days)
Method Five male Crlj: CD1 (ICR) mice (Charles River Japan Co., Ltd.) were prepared for each group, and each group had tricine concentrations of 0 mg / kg (control group), 300 and 1000 mg / kg / The tricine derivative corresponding to day, compound 16 (4'-O-CO- (Ala-Glu-OH) -tricine) was orally administered repeatedly for 14 days.

Observation, measurement and inspection items a. General condition observation Animal life and death, appearance, behavior, etc. are counted twice a day in the morning and afternoon from the first day of administration to the day after the 14th day of administration, starting from the first day of administration. did.
b. Body weight measurement For all cases, the body weight of the animals was measured using an electronic precision pan balance before administration and on the day of necropsy on days 1, 2, 7, 10 and 14 and recorded in units of 0.1 g.
c. Food intake For all cases, food consumption was measured using an electronic pan balance prior to dosing 1, 2, 7, 10 and 14 days and recorded in units of 0.1 g. The appropriate amount is measured on the day before the start of administration and then fed for each cage. The amount of food intake is calculated by the following formula.
Feeding amount (g / mouse / day) = (feed amount (g / mouse) −remaining amount (g / mouse)) / number of days in measurement day (day)

d. Hematological examination Blood was collected from all cases at necropsy, and the red blood cell count (RBC), hematocrit value (HCT), hemoglobin concentration (HGB), average red blood cell volume (MCV), average red blood cell hemoglobin content (MCH), average red blood cell hemoglobin concentration ( MCHC), platelet count (Platelet), and white blood cell count (WBC) were measured using an automated blood cell analyzer XT-2000 iV (Sysmex).
e. Blood chemistry test Blood samples were collected at the time of necropsy for all cases, and automated analyzer 7080 (AST, ALT, alkaline phosphatase (ALP), glucose (Glucose), urea nitrogen (UN), creatinine (Crea), total protein (TP) ( Hitachi High-Technologies Corporation).
f. Necropsy findings All patients were euthanized on the 14th day after administration, and whole body organs and tissues were observed macroscopically, and necessary organs and tissues were fixed and preserved.
g. Organ weights In all cases, the weights of brain, heart, heart, liver, kidney (left and right), spleen, adrenal (left and right), and testis (left and right) were measured and recorded at the time of necropsy. The relative weight was calculated from the following formula.
Relative weight (%) = (absolute weight (g) / body weight on autopsy day (g)) × 100

Results / General Conditions Table 7 shows the results of the general conditions. In the control group and the 300 mg / kg administration group, no abnormality was observed during the administration period.
In the 1000 mg / kg administration group, only one stool was observed only on the 8th day of administration in one case, and it was judged that there was no relevance to the administration of the test substance.
-Body weight transition Table 8 shows the body weight transition. There was no significant difference between the administration groups and the control group during the administration period.

-Food intake Table 9 shows food intake. In the 300 mg / kg administration group, a significantly high value was observed on the 7th day compared with the control group, but since there was no dose dependency, it was considered that there was no toxicological significance.
There was no significant change in the 1000 mg / kg administration group compared to the control group.
-Hematological examination No significant difference was observed in each administration group compared to the control group during the administration period.
-Blood chemistry test No significant difference was observed in each administration group compared to the control group during the administration period, but high values of AST and ALT were observed in one case of the 1000 mg / kg administration group.

-Autopsy findings Table 10 shows the autopsy findings. There were no abnormal findings in the control group.
In the 300 mg / kg administration group, one male showed a reduction in the size of the left and right testis, but this was a dose-independent change.
No abnormal findings were observed in the 1000 mg / kg administration group.
-Organ weight The results of organ weight are shown in Table 11.
There was no significant change in the 300 mg / kg administration group compared to the control group.

In the 1000 mg / kg administration group, significant high values were observed in the absolute and relative weights of the liver. The liver function test items AST and ALT showed high values in only one case, and there was no statistically significant difference. Therefore, the possibility of weight increase due to induction of metabolic enzymes and the like was considered.
In addition, a significant lower value was observed in the absolute heart weight, but it was not accompanied by a significant difference in relative weight, and it was assumed that there was no toxicological significance.
Increased liver weight was observed in the 1000 mg / kg group, but changes related to the administration of the test substance were observed in the general condition, body weight change, food consumption, hematology, blood chemistry, and autopsy findings. There wasn't.
From the above, the no-effect amount of the tricine derivative under the present test conditions was estimated to be 300 mg / kg / day at present.

数値は動物数を示す。 Table 7: General status in a 14-day continuous oral toxicity study in male mice

Numbers indicate the number of animals.

Table 8: Transition of body weight in a 14-day continuous oral toxicity study of tricine derivatives in male mice

Table 9: Food intake in a 14-day continuous oral dose toxicity study of tricine derivatives in male mice

数値は動物数を示す。 Table 10: Autopsy findings in a 14-day continuous oral dose toxicity study of a tricine derivative in male mice

Numbers indicate the number of animals.

^※:対照群に対しp≦0.05 (Dunnett’s test)
^※※:対照群に対しp≦0.01 (Dunnett’s test) Table 11: Organ weight in a 14-day continuous oral dose toxicity study of a tricine derivative in male mice

^* : P ≦ 0.05 compared to the control group (Dunnett's test)
^** : p ≤ 0.01 (Dunnett's test) relative to the control group

[Example 13]
(Hydrolysis test)
The hydrolyzability of the ester bond of the tricine amino acid derivative was tested using an intracellular hydrolase.
Lysates of MDCK cells (1.33 × 10 ⁶ cells) were manually prepared (Harlow, E .; Lane, D. Antibodies: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1988, p 449.) Created according to
Tricine amino acid derivatives were treated with cell lysate to determine the time required to release tricine (t _1/2 : half-life) (Cai, H .; Verschoyle, RD; Steward, WP; Gescher, AJ Determination of the flavone tricin in human plasma by high-performance liquid chromatography. Biomed. Chromatogr. 2003, 17, 435-439.).
The half-life of the tricine-Ala-Glu derivative was 165 minutes. It is apparent that the tricine amino acid derivative is converted to tricine in the cell.
The half-life (165 minutes) of the tricine-Ala-Glu derivative was considerably longer than the half-life (110 minutes) of the quercetin-Ala-Glu derivative having a similar structure. It is considered that the tricine amino acid derivative is sterically hindered by the methoxy group at the 3 ′ position and the 5 ′ position, has a low affinity for the enzyme active site, and has a long hydrolysis half-life.

[Example 14]
(Cervical cancer cell line proliferation test)
In order to investigate the effect of tricine on the growth of cervical cancer cell line of tricine, the following experiment was conducted.
The following cell lines were used.
(i) TIG-3 strain Fetal lung fibroblasts (normal cells, as control)
(ii) CA strain HPV-p53- (cervical cancer cell line)
(See “Establishment of a HPV and p53-mutation-nagative human cell line (CA) derived from a squamous carcinoma of the uterine cervix”, Keiichi Isaka et al., Gynecologic Oncology 92, 15-21 (2004))
(iii) CaSki strain HPV16 type high copy p53 + (cervical cancer cell line)
(iv) HeLa strain HPV18 p53 + (cervical cancer cell line)
(v) SiHa strain HPV16 low copy p53 + (cervical cancer cell line)

Each cell line was seeded at 4,000 cells / well in a 96-well plate 24 hours before the addition of tricine, and precultured under normal culture conditions (37 ° C., 5% CO ₂ ).
Tricine was dissolved in DMSO and added to the culture to a final concentration of 0, 1, 10, 50 μM.

After the addition of tricine, assay and measurement were carried out every 24 hours until day 7 using a protocol according to CellTiter96 (registered trademark) Non-Radioactive Cell Proliferation Assay.
More specifically, 15 μl of staining solution was added to each well and incubated at 37 ° C. in a humidified CO ₂ incubator. 100 μl of lysis / stop solution was then added to each well. The assay was performed without exchanging the culture solution until completion.
The absorbance at 570 nm of the colored formazan product was measured with a multi-well spectrophotometer (SpectraMax 190 spectrophotometer (Molecular Device)). A wavelength of 630 to 750 nm was used as a reference wavelength. As the analysis software, Softmax (registered trademark) Pro5 (manufactured by Molecular Devices) was used.
The relationship between culture time and absorbance is shown in FIGS.
As is clear from FIGS. 8 to 12, cell growth was suppressed depending on the tricine concentration (0 μM to 50 μM) in all four cervical cancer cell lines and fetal lung fibroblast cell lines.
From the above results, it can be seen that tricine exhibits an excellent anticancer action against cervical cancer.
It is clear that the tricine amino acid derivative which shows high cell permeability and also changes to tricine in the body to give a high blood concentration of tricine also exhibits an excellent anticancer action against cervical cancer.

<< Antiviral action of antiviral agent (combination drug) against herpes virus >>
[Example 15]
The antiviral action against the herpes virus of the antiviral agent (combination agent) of the present invention was examined by the following method.
<Method>
Human fetal fibroblasts (HEL cells) cultured in a monolayer on a 24-well plate were adsorbed and infected with human cytomegalovirus (Town strain) (1 Moi) for 1 hour, and then ganciclovir 0.0001 μM and tricine (0, 0.01, 0.03 or 0.1 μM) was added, and the culture was started in a 5% carbon dioxide incubator.
The supernatant on the 5th day after virus infection was taken out and a plaque assay experiment was conducted.
The presence or status of cytopathic effect (CPE) was observed with a microscope, and the virus titer relative to the control of each sample (%) when the virus titer of control (both tricine and ganciclovir) was set to 100 Asked. The results are shown in Table 12 and FIG.

[Comparative Example 1]
Instead of a culture solution containing tricine (0, 0.01, 0.03, 0.1 μM) and ganciclovir (0.0001 μM), only tricine (0, 0.01, 0.03, or 0.1 μM) is contained. The experiment was performed in the same manner as in Example 15 except that the culture solution was used. The results are shown in Table 12 and FIG.

Table 12: Antiviral effect when tricine and GCV are used in combination and when tricine alone is used

[Reference Example 2]
As a reference, the antiviral action against herpes virus when ganciclovir alone was used as an active ingredient was examined by the following method. The experiment was replaced with a culture solution containing 4 ′, 5,7-trihydroxy-3 ′, 5′-dimethoxyflavone (0, 0.01, 0.03, 0.1 μM) and ganciclovir (0.0001 μM). This was carried out in the same manner as in Example 15 except that a culture solution containing ganciclovir (0, 0.00001, 0.0001, 0.001, 0.01, 0.1, or 1 μM) was used. The results are shown in Table 13 and FIG.

Table 13: Antiviral effects when only ganciclovir (GCV) is used

From the results in Table 12, it can be seen that the antiviral activity of the present invention using a combination of tricine and ganciclovir significantly improves the antiviral activity compared to the case of using tricine alone.
Moreover, it can be seen that the cytopathic effect of the antiviral agent (combination agent) of the present invention is far superior to the results that can be expected from the cytopathic effect of ganciclovir 0.0001 μM and the cytopathic effect of tricine. . For example, the relative virus titer when only tricine 0.01 μM is used is about 75%, and the relative virus titer when only ganciclovir 0.0001 μM is used is about 62%. The effect of the combined preparation of tricine and ganciclovir (0.0001 μM) that can be expected from these values is around 47% (calculated by 0.75 × 0.62). However, the actual relative virus titer with the antiviral agent (1) of the present invention was about 31%, and a relative virus titer much lower than expected could be obtained. From this result, it can be seen that tricine and ganciclovir exert a synergistic effect and, when combined, a significantly superior antiviral effect can be obtained than would normally be expected.
Furthermore, the antiviral agent of the present invention has an excellent antiviral action equivalent to or higher than that when ganciclovir alone is used at the same concentration, and also improves the problems (cytotoxicity and tolerance) of ganciclovir. It can be used as a possible new antiviral agent.

Claims (19)

Formula 1:

(Wherein A is an arbitrary amino acid residue, B is an arbitrary amino acid residue, A and B may be the same or different, and n = 0-4).   The compound according to claim 1, wherein A is any amino acid residue, and n = 0 or 1.   The compound according to claim 1 or 2, wherein A is a residue of an amino acid selected from the group consisting of glycine, alanine, valine, phenylalanine, aspartic acid and glutamic acid.   The compound according to claim 1, wherein A is an alanine residue and B is any amino acid residue.   The compound according to claim 1, wherein A is an alanine residue and B is an aspartic acid or glutamic acid residue. 4'-O-CO- (Gly-OH) -tricine,
4'-O-CO- (Ala-OH) -tricine,
4'-O-CO- (Val-OH) -tricine,
4'-O-CO- (Phe-OH) -tricine,
4'-O-CO- (Asp-OH) -tricine,
4'-O-CO- (Gln-OH) -tricine,
The compound of claim 1 selected from the group consisting of 4'-O-CO- (Ala-Asp-OH) -tricine or 4'-O-CO- (Ala-Glu-OH) -tricine.   The antiviral agent containing the compound as described in any one of Claims 1-6.   The antiviral agent according to claim 7, wherein the target virus is a herpes virus, cytomegalovirus or influenza virus.   The antiviral agent according to claim 7, wherein the target virus is a virus selected from the group consisting of an oseltamivir resistant influenza virus strain, an amantadine resistant influenza virus strain, and a ganciclovir resistant human cytomegalovirus. The antiviral agent according to any one of claims 7 to 9, which is for oral administration.   An anticancer agent comprising tricine or the compound according to any one of claims 1 to 6.   The anticancer agent according to claim 11, wherein the target cancer is colon cancer or cervical cancer.   The anticancer agent according to claim 11 or 12, which is for oral administration. (A) Tricine or the compound according to any one of claims 1 to 6, and (B) Ganciclovir, Acyclovir, Cidofovir, Famciclovir, Fomivirsen, Foscarnet, Idoxuridine, Penciclovir, Trifluridine, Valacyclovir At least one compound selected from the group consisting of valganciclovir and vidarabine,
An antiviral agent comprising: as an active ingredient.   The antiviral agent according to claim 14, wherein the component (B) is ganciclovir.   The antiviral agent according to claim 14 or 15, wherein the virus is a herpes virus.   The antiviral agent according to any one of claims 14 to 16, wherein the virus is a cytomegalovirus.   The antiviral agent according to any one of claims 14 to 17, wherein the virus is a human cytomegalovirus.   The molar ratio of said (A) component and (B) component exists in the range of 100: 1-1000: 1 ((A) component: (B) component), It is any one of Claims 14-18 The antiviral agent described.

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