JP4599526B2 - Novel chromone derivative derivative compound and pharmaceutical composition containing the same - Google Patents
Novel chromone derivative derivative compound and pharmaceutical composition containing the same Download PDFInfo
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本発明は、抗炎症剤、抗アレルギー剤として有用な新規なクロモン誘導体化合物及びそれを含む医薬に関する。 The present invention relates to a novel chromone derivative compound useful as an anti-inflammatory agent and an antiallergic agent, and a medicament containing the same.
阜康阿魏（学名 Ferula fukanensis K M shen）は、アジアの乾燥地帯に分布するセリ科の多年草であり、その根茎から採取した樹脂は、リウマチ、婦人病、痙攣の治療に使用され、ヒステリー症の鎮静剤として用いられている。
コジマ（Kojima）らは、Ferula ferulioidesの根から分離したプレニルベンゾイルフラノン型のセスキテルペン誘導体及びその生合成の経路を報告しているが、その有用性については述べていない（非特許文献１）。
バオ−ニン ス（Bao-Ning Su）らは、Ferula pallidaの根から抽出したセスキテルペンクマリン及びその誘導体並びにその生合成経路を報告しているが、その有用性につては述べていない（非特許文献２）。
本発明者らは、阜康阿魏の根茎から抽出した化合物のいくつかにＮＯ産生抑制活性があることを見出している（非特許文献３）が、それらはクマリン誘導体又はオキソフラン誘導体であり、本発明の化合物とは骨格を異にする。
Fukuyasu Asuna (scientific name: Ferula fukanensis KM shen) is a perennial plant belonging to the family Aceraceae distributed in dry areas of Asia. It is used as a sedative.
Kojima et al. Have reported prenylbenzoylfuranone-type sesquiterpene derivatives isolated from the roots of Ferula ferulioides and their biosynthetic pathways, but do not mention their usefulness (Non-patent Document 1).
Bao-Ning Su et al. Reported sesquiterpene coumarin and its derivatives extracted from the roots of Ferula pallida and their biosynthetic pathways, but did not mention their usefulness (non-patented). Reference 2).
The inventors of the present invention have found that some of the compounds extracted from the roots of Fuyasu-Aki have NO production inhibitory activity (Non-Patent Document 3), which are coumarin derivatives or oxofuran derivatives. The skeleton is different from the compound of the invention.
本発明の目的は、ＮＯ産生抑制作用を有する新規なクロモン誘導体化合物及びこれを含む医薬を提供することである。 An object of the present invention is to provide a novel chromone derivative compound having an NO production inhibitory action and a medicament containing the same.
本発明の新規なクロモン誘導体化合物は以下の一般式（Ｉ）を有する。
式中、
Ｒ1、Ｒ2及びＲ3はそれぞれ水素原子を表すか、又はＲ1とＲ2が一緒になって結合を表し、又はＲ2とＲ3が一緒になって結合を表す。
一般式（Ｉ）の化合物は、ＮＯ産生抑制活性並びにｉＮＯＳ、インターロイキン−１β（ＩＬ−１β）、インターロイキン−６（ＩＬ−６）及び腫瘍壊死因子−α（ＴＮＦ−α）のｍＲＮＡ発現を抑制する活性を有し、抗炎症剤、抗アレルギー剤として使用することができる。
The novel chromone derivative compound of the present invention has the following general formula (I).
Where
R 1 , R 2 and R 3 each represent a hydrogen atom, or R 1 and R 2 together represent a bond, or R 2 and R 3 together represent a bond.
The compound of general formula (I) exhibits NO production inhibitory activity and mRNA expression of iNOS, interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). It has an inhibitory activity and can be used as an anti-inflammatory agent or anti-allergic agent.
本発明の化合物はＮＯ産生抑制活性並びにｉＮＯＳ、インターロイキン−１β（ＩＬ−１β）、インターロイキン−６（ＩＬ−６）及び腫瘍壊死因子−α（ＴＮＦ−α）のｍＲＮＡ発現を抑制する活性を有する新規なクロモン誘導体化合物であり、抗炎症剤、抗アレルギー剤として有用である。 The compound of the present invention has an NO production inhibitory activity and an activity of suppressing mRNA expression of iNOS, interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). It is a novel chromone derivative compound that is useful as an anti-inflammatory agent and anti-allergic agent.
一般式（Ｉ）の化合物において、Ｒ1、Ｒ2及びＲ3はそれぞれ水素原子を表すか、又はＲ1とＲ2が一緒になって結合を表し、又はＲ2とＲ3が一緒になって結合を表す。
例えば一般式（Ｉ）の化合物において、Ｒ1とＲ2が一緒になって結合を表し、Ｒ3が水素原子である以下の化合物：
及び一般式（Ｉ）の化合物において、Ｒ2とＲ3が一緒になって結合を表し、Ｒ1が水素原子である以下の化合物を挙げることができる。
For example, in the compound of the general formula (I), R 1 and R 2 together represent a bond, and R 3 is a hydrogen atom:
In the compounds of formula (I), R 2 and R 3 together represent a bond, and R 1 is a hydrogen atom.
本発明の望ましい化合物として、以下の式（II）で表される化合物１、式（III）表される化合物２、式（IV）で表される化合物３及び式（V）で表される化合物４を挙げることができる。
本発明の化合物１ないし４は、阜康阿魏の根茎をそのまま又は乾燥し、好ましくは粉砕し、有機溶媒（例えばメタノール、クロロホルム、酢酸エチル）を用いて室温で抽出して得ることができ、得られた抽出物を自体公知の方法で処理し、精製することにより製造することができる。
これらの化合物を出発原料として本発明の一般式（Ｉ）で表される他の化合物を製造することができる。
Compounds 1 to 4 of the present invention can be obtained by directly or drying, and preferably pulverizing, the roots of Fuyasu-Aki and extracting them at room temperature using an organic solvent (eg, methanol, chloroform, ethyl acetate), The obtained extract can be produced by treating and purifying it by a method known per se.
Using these compounds as starting materials, other compounds represented by the general formula (I) of the present invention can be produced.
製造例１
阜康阿魏（学名 Ferula fukanensis K M shen）の根茎５．９kgを粉砕し、これを８０％メタノール（メタノール：水＝８：２容量）により３８リットルの溶液とし、室温で１２０時間温浸した。温浸を同一条件で繰り返し、得られた浸出液を合わせて減圧下に４０℃で濃縮して７１１gのエキスを得た。
このエキスの４５０gを水１．５リットルに溶解し、クロロホルム、酢酸エチルの各１．５リットルで、順次それぞれ３回ずつ抽出した。これらの抽出液をそれぞれ減圧下に４０℃で濃縮し、クロロホルム画分から２７０g、酢酸エチル画分から１４０gの濃縮エキスを得た。
クロロホルム画分から得られた濃縮エキス７０gをシリカゲルカラム（Wako gel C-200、和光純薬社製、６φ×１７cm）にのせ、ヘキサン：酢酸エチル系の展開溶媒を使用し、酢酸エチル濃度０％、２％、５％、１０％、２０％、４０％、６０％及び１００％の溶媒をそれぞれ３リットル順次使用して展開し、２００mlずつ分取して第１〜第１１の１１画分に分画した。
得られた第６画分（４０％の酢酸エチルを含むヘキサン溶媒によって溶出した画分）の減圧濃縮物を、シリカゲルカラム（ワコーゲル C-300、和光純薬株式会社社製、４×２３cm）にのせ、ヘキサン：酢酸エチル系の展開溶媒を使用し、酢酸エチル濃度を０％、２％、５％、１０％、２０％、４０％、６０％及び１００％とした溶媒をそれぞれ１．５リットル順次使用して展開し、１００mlずつ分取して第１’〜第１１’の１１画分に分画した。
得られた第８’画分（５０％の酢酸エチルを含むヘキサン溶媒によって溶出した画分）と第９’画分（６０％の酢酸エチルを含むヘキサン溶媒によって溶出した画分）を合わせて減圧下に濃縮し、この濃縮物をシリカゲルカラム（ワコーゲル C-300、和光純薬株式会社社製、３φ×２０cm）にのせ、ヘキサン：酢酸エチル系の展開溶媒を使用し、酢酸エチル濃度を０％、２％、５％、１０％、２０％、４０％、６０％及び１００％とした溶媒をそれぞれ３リットル順次使用して展開し、２００mlずつ分取して第１”〜第１０”の１０画分に分画した。第６”画分を逆相ＨＰＬＣ（YMC-PAK Pro C18、株式会社ＹＭＣ社製）により分離した。移動相として５６％アセトニトリル（アセトニトリル：水＝５６：４４）を用い、流速９．０ml／min（室温）で溶出させ、溶出液をＵＶ（２１０nm）で監視しつつ、保持時間１１分５４秒、１３分４２秒、１４分５７秒、１５分２０秒及び１６分５４秒にピークを示す溶出液を分取した。このうち１３分４２秒にピークを示す画分を順相ＨＰＬＣ（YMC-PAK SIL-06、株式会社ＹＭＣ社製）により分離した。移動相として４８％ヘキサン（ヘキサン：酢酸エチル＝４８：５２）を用い、流速３．０ml／min（室温）で溶出させ、溶出液をＵＶ（２５４nm）で監視しつつ、保持時間１５分２４秒にピークを示す溶出液を分取した。この溶出液を減圧下に濃縮して化合物１を得た。
Production Example 1
5.9 kg of rhizome of Fukuyasu Aki (scientific name: Ferula fukanensis KM shen) was pulverized, and this was made into a 38 liter solution with 80% methanol (methanol: water = 8: 2 volume) and digested at room temperature for 120 hours. Digestion was repeated under the same conditions, and the obtained leachates were combined and concentrated under reduced pressure at 40 ° C. to obtain 711 g of extract.
450 g of this extract was dissolved in 1.5 liters of water, and extracted successively with 1.5 liters of chloroform and ethyl acetate three times each. These extracts were concentrated under reduced pressure at 40 ° C. to obtain 270 g of concentrated extract from the chloroform fraction and 140 g of concentrated extract from the ethyl acetate fraction.
70 g of the concentrated extract obtained from the chloroform fraction was placed on a silica gel column (Wako gel C-200, manufactured by Wako Pure Chemical Industries, 6φ × 17 cm), and a developing solvent of hexane: ethyl acetate was used, with an ethyl acetate concentration of 0%, Using 3% each of 2%, 5%, 10%, 20%, 40%, 60%, and 100% solvent, develop them in succession, aliquot 200 ml, and divide into 1st to 11th fractions. I drew it.
The vacuum concentrate of the obtained sixth fraction (fraction eluted with a hexane solvent containing 40% ethyl acetate) was applied to a silica gel column (Wakogel C-300, manufactured by Wako Pure Chemical Industries, Ltd., 4 × 23 cm). And using a developing solvent of hexane: ethyl acetate, and 1.5 liters each of the solvents in which the ethyl acetate concentration is 0%, 2%, 5%, 10%, 20%, 40%, 60% and 100% The sample was sequentially used and developed, and 100 ml was collected and fractionated into 11 fractions of 1 'to 11'.
The obtained 8 'fraction (fraction eluted with hexane solvent containing 50% ethyl acetate) and 9' fraction (fraction eluted with hexane solvent containing 60% ethyl acetate) were combined and reduced in pressure. Concentrate down and place this concentrate on a silica gel column (Wakogel C-300, manufactured by Wako Pure Chemical Industries, Ltd., 3φ × 20 cm), using a developing solvent of hexane: ethyl acetate, with an ethyl acetate concentration of 0% 2%, 5%, 10%, 20%, 40%, 60%, and 100% of each solvent was developed using 3 liters sequentially, and 200 ml was dispensed to obtain the first to 10th 10 Fractions were fractionated. The 6th "fraction was separated by reverse phase HPLC (YMC-PAK Pro C18, manufactured by YMC Corporation). 56% acetonitrile (acetonitrile: water = 56: 44) was used as the mobile phase, and the flow rate was 9.0 ml / min. Elution is performed at room temperature, and the eluate is monitored by UV (210 nm), and peaks are observed at retention times of 11 minutes 54 seconds, 13 minutes 42 seconds, 14 minutes 57 seconds, 15 minutes 20 seconds and 16 minutes 54 seconds. Among these, a fraction having a peak at 13 minutes 42 seconds was separated by normal phase HPLC (YMC-PAK SIL-06, manufactured by YMC Co., Ltd.) 48% hexane (hexane: acetic acid) as a mobile phase. The mixture was eluted with a flow rate of 3.0 ml / min (room temperature) using ethyl = 48: 52), and the eluate showing a peak at a retention time of 15 minutes and 24 seconds was collected while monitoring the eluate with UV (254 nm). The eluate was concentrated under reduced pressure to obtain Compound 1. It was.
製造例２
製造例１において、第６”画分を逆相ＨＰＬＣにより分離した画分のうち、１４分５７秒にピークを示す画分を順相ＨＰＬＣ（YMC-PAK SIL-06、株式会社ＹＭＣ社製）により分離した。移動相として４７％ヘキサン（ヘキサン：酢酸エチル＝４７：５３）を用い、流速３．０ml／min（室温）で溶出させ、溶出液をＵＶ（２５４nm）で監視しつつ、保持時間１５分２４秒にピークを示す溶出液を分取した。この溶出液を減圧下に濃縮して化合物３を得た。
Production Example 2
In Production Example 1, among the fractions obtained by separating the 6th ”fraction by reverse phase HPLC, the fraction showing a peak at 14 minutes 57 seconds was obtained by normal phase HPLC (YMC-PAK SIL-06, manufactured by YMC Corporation). Using 47% hexane (hexane: ethyl acetate = 47: 53) as a mobile phase, elution was performed at a flow rate of 3.0 ml / min (room temperature), and the eluate was monitored by UV (254 nm) while maintaining the retention time. The eluate having a peak at 15 minutes and 24 seconds was collected, and the eluate was concentrated under reduced pressure to obtain
製造例３
製造例１において、第６”画分を逆相ＨＰＬＣにより分離した画分のうち、１６分５４秒にピークを示す画分を順相ＨＰＬＣ（YMC-PAK SIL-06、株式会社ＹＭＣ社製）により分離した。移動相として４７％ヘキサン（ヘキサン：酢酸エチル＝４７：５３）を用い、流速３．０ml／min（室温）で溶出させ、溶出液をＵＶ（２５４nm）で監視しつつ、保持時間１６分５４秒にピークを示す溶出液を分取した。この溶出液を減圧下に濃縮して化合物４を得た。
Production Example 3
In Production Example 1, among the fractions obtained by separating the 6th ”fraction by reverse phase HPLC, the fraction showing a peak at 16 minutes and 54 seconds is a normal phase HPLC (YMC-PAK SIL-06, manufactured by YMC Corporation). Using 47% hexane (hexane: ethyl acetate = 47: 53) as a mobile phase, elution was performed at a flow rate of 3.0 ml / min (room temperature), and the eluate was monitored by UV (254 nm) while maintaining the retention time. The eluate having a peak at 16 minutes and 54 seconds was collected, and the eluate was concentrated under reduced pressure to obtain Compound 4.
製造例４
製造例１において、第２回目のシリカゲルカラムによる分離から得られた第１”〜１０”画分のうちの第７”画分を、順相ＨＰＬＣ（YMC-PAK SIL-06、株式会社ＹＭＣ社製）により分離した。移動相として４５％ヘキサン（ヘキサン：酢酸エチル＝４５：５５）を用い、流速８．０ml／min（室温）で溶出させ、溶出液をＵＶ（２５４nm）で監視しつつ、保持時間１９分００秒にピークを示す溶出液を分取した。この溶出液の減圧濃縮物をさらに逆相ＨＰＬＣ（YMC-PAK Pro C18、株式会社ＹＭＣ社製）により分離した。移動相として５５％アセトニトリル（アセトニトリル：水＝５５：４５）を用い、流速３．０ml／min（室温）で溶出させ、溶出液をＵＶ（２１０nm）で監視しつつ、保持時間１１分２３秒にピークを示す溶出液を分取した。この溶出液を減圧下に濃縮して化合物２を得た。
Production Example 4
In Production Example 1, the seventh “fraction” of the first “10” fraction obtained from the second silica gel column separation was subjected to normal phase HPLC (YMC-PAK SIL-06, YMC Corporation). Using 45% hexane (hexane: ethyl acetate = 45: 55) as a mobile phase, eluting at a flow rate of 8.0 ml / min (room temperature), and monitoring the eluate with UV (254 nm), An eluate having a peak at a retention time of 19:00 was collected, and the vacuum concentrate of the eluate was further separated by reverse-phase HPLC (YMC-PAK Pro C18, manufactured by YMC Co., Ltd.) As a mobile phase, 55 Elution with% acetonitrile (acetonitrile: water = 55: 45) and elution at a flow rate of 3.0 ml / min (room temperature), and monitoring the eluate with UV (210 nm), showing a peak at a retention time of 11 minutes 23 seconds The eluate was reduced. Concentration under pressure gave compound 2.
以下に、得られた化合物の1Ｈ−及び13Ｃ−ＮＭＲのデータを示す。なお、溶媒はすべてCDCl3である。
表１ 化合物１及び２のＮＭＲスペクトル
The 1 H- and 13 C-NMR data of the obtained compound are shown below. All solvents are CDCl 3 .
Table 1 NMR spectra of compounds 1 and 2
表２ 化合物３のNMRスペクトル
Table 2 NMR spectrum of
表３ 化合物４のNMRスペクトル
これらのデータから化合物１ないし４はそれぞれ下記の式（II）及び（V）の構造を有することが確認された。
本発明の化合物の活性を、以下の各種試験により評価した。
〔一酸化窒素産生抑制試験〕
生体内の一酸化窒素（ＮＯ）はＬ−アルギニンを前駆体とする。アルギニンのグアニジノ基に分子酸素が添加し、Ｌ−シトルリンへ変換され、同時にＮＯが産生される。この反応を触媒するのがＮＯ合成酵素（ＮＯＳ）であり、神経型ＮＯＳ、内皮型ＮＯＳとｉＮＯＳの３種類のアイソフォームが存在する。
ｉＮＯＳは通常発現しておらず、マクロファージ、白血球、血管平滑筋、内皮細胞、腎メンサギウム細胞、心筋細胞など多くの細胞で、インターフェロンγ（ＩＦＮ−γ）、ＩＬ−１、ＴＮＦ−αなどのサイトカインやリポポリサッカライド（ＬＰＳ）の刺激により誘導される。
炎症巣において、好中球やマクロファージはＮＯと同時に活性酸素の一種であるＯ2 -を産生する。ＮＯは速やかにＯ2 -と反応し、ＮＯより反応性の高いＯＮＯＯ-を生成する。この結果、慢性的な炎症やアレルギー、糖尿病、動脈硬化などが引き起こされる。
The activity of the compounds of the present invention was evaluated by the following various tests.
[Nitric oxide production suppression test]
In vivo nitric oxide (NO) uses L-arginine as a precursor. Molecular oxygen is added to the guanidino group of arginine and converted to L-citrulline, and NO is produced at the same time. NO synthase (NOS) catalyzes this reaction, and there are three types of isoforms: neuronal NOS, endothelial NOS and iNOS.
iNOS is not normally expressed and is a cytokine such as interferon γ (IFN-γ), IL-1, and TNF-α in many cells such as macrophages, leukocytes, vascular smooth muscle, endothelial cells, renal mensagium cells, and cardiomyocytes. It is induced by stimulation with lipopolysaccharide (LPS).
In the inflamed foci, neutrophils and macrophages produce O 2 − , a kind of active oxygen, simultaneously with NO. NO is rapidly reacts with the O 2 - and, ONOO more reactive NO - generating a. This results in chronic inflammation, allergies, diabetes, arteriosclerosis and the like.
マクロファージとしてアベルソン ロイケミア（Abelson leukemia）ウィルスで形質転換したマウス腹腔より得られたマクロファージ様株化細胞RAW264.7細胞（ATCCより購入）を使用した。この細胞を、ウシ胎児血清を１０％含むHam's培地中に１〜５×１０5個／mlの濃度に調製し、９６穴プレート（住友ベークライト製、8096R）に２００μlずつ分注し、１時間、ＣＯ2インキュベーターで細胞を接着させた。各種濃度の試験化合物と共にマウスＩＦＮ−γ（ジェンザイム（Genzyme）製）及びＬＰＳ（シグマ（Sigma）製、055:B5）を加えた。最終濃度がＩＦＮ−γは０．３３ng／ml、ＬＰＳは１００ng／mlとなるように調整した。試験化合物はＤＭＳＯに溶解し、培地に対する含量が０．２％となるように調整した。
３７℃、５％ＣＯ2インキュベーターで１６時間培養後、培養上清を採取し、グリース法（L. J. Ignarro, et. al., Proc. Natl. Acad. Sci. USA, 84, 1987, pp. 9265-9269）により培地中のＮＯ2 -を定量した。得られたＮＯ2 -量から、抑制率を次式により算出した。
抑制率（％）＝｛１−（Ｘ−Ｙ）／（Ｚ−Ｙ）｝×１００
Ｘ：試験化合物の存在下でＩＦＮ−γとＬＰＳにより誘導されるＮＯ2 -の量
Ｙ：試験化合物、ＩＦＮ−γ及びＬＰＳがない状態で誘導されるＮＯ2 -の量
Ｚ：ＩＦＮ−γとＬＰＳにより誘導されるＮＯ2 -の量
試験化合物のＩＣ50、すなわち抑制率が５０％となる式（II）で表される化合物の濃度を表４に示す。
表４ 一酸化窒素産生抑制作用
After culturing at 37 ° C. in a 5% CO 2 incubator for 16 hours, the culture supernatant was collected, and the grease method (LJ Ignarro, et. Al., Proc. Natl. Acad. Sci. USA, 84, 1987, pp. 9265- 9269), NO 2 − in the medium was quantified. The resulting NO 2 - from the amount, the inhibition rate was calculated by the following equation.
Inhibition rate (%) = {1- (X−Y) / (Z−Y)} × 100
X: NO 2 induced by IFN-gamma and LPS in the presence of test compound - amount of Y: test compound, IFN-gamma and NO 2 LPS-induced in the absence - of the amount Z: and IFN-gamma The amount of NO 2 − induced by LPS Table 4 shows the IC 50 of the test compound, that is, the concentration of the compound represented by the formula (II) at which the inhibition rate is 50%.
Table 4 Nitric oxide production inhibitory action
〔ｉＮＯＳ及びＩＬ−６等のサイトカインのｍＲＮＡ発現抑制試験〕
RAW264.7細胞(1.2×106細胞/mL)を試験化合物、ＩＮＦ−γ (10 U/mL)及びＬＰＳ（100 ng/mL）共存下、6時間培養した。RNA isolationキット（QIAGEN社製）を用いて、total RNAを抽出した。RNA量を260 nmの吸収により定量し、250 ngのRNAをオリゴ(dT)12-18プライマーを用いて、逆転写させcDNAを作製した。このcDNAを鋳型としてPCR反応を行った。反応は、50 mM KCl、5 mM MgCl2、0.2 mM dNTP、0.6 unit Ampli Taq gold（Applied Biosstems）、0.4 mMmolセンスプライマー及びアンチセンスプライマーを含む30 μlの反応溶液中で行った。ｉＮＯＳのプライマーに5’-ACCTACTTCCTGGACATTACGACCC-3’ [s]、5’-AAGGGAGCAATGCCCGTACCAGGCC-3' [a]を、またＧＡＰＤＨのプライマーに5'-ACCACAGTCCATGCCATCAC-3’ [s]、 5’-TCCACCACCCTGTTGCTGTA-3' [a]、ＩＬ−１βのプライマーに、5'-TGAAGGGCTGCTTCCAAACCTTTGACC-3’ [s]、5’-TGTCCATTGAGGAGAGCTTTCAGC-3’ [a]、ＩＬ−６のプライマーに、5'-TGCAGTCACAGAAGGAGTGGCTAAG3' [s]、5'-TCTGACCACAGTGAGGAATGTCCAC-3’ [a]、ＴＮＦ−αのプライマーに、5’-AGGCGCCACCACGCTCTTCT-3’ [s]、5’-GGCAGCCTTGGCCCTTGAA-5’ [a]を用いた。PCR反応は94℃、1分間 (denaturation)、60℃、1分間（annealing）、72℃、1.5分間を1サイクルとし25サイクル行った。PCR産物を2 %アガロースにて電気泳動し、エチジウムブロイマイドで染色し検出した。化合物４についての結果を図１に示す。図１から明らかなように、化合物４はｉＮＯＳ、インターロイキン−１β（ＩＬ−１β）、インターロイキン−６（ＩＬ−６）及び腫瘍壊死因子−α（ＴＮＦ−α）のｍＲＮＡ発現を化合物の濃度に依存して抑制する。
[Test for suppressing mRNA expression of cytokines such as iNOS and IL-6]
RAW264.7 cells (1.2 × 10 6 cells / mL) were cultured for 6 hours in the presence of a test compound, INF-γ (10 U / mL) and LPS (100 ng / mL). Total RNA was extracted using an RNA isolation kit (QIAGEN). The amount of RNA was quantified by absorption at 260 nm, and 250 ng of RNA was reverse transcribed using oligo (dT) 12-18 primer to prepare cDNA. A PCR reaction was performed using this cDNA as a template. The reaction was performed in 30 μl of a reaction solution containing 50 mM KCl, 5 mM MgCl 2 , 0.2 mM dNTP, 0.6 unit Ampli Taq gold (Applied Biosstems), 0.4 mM mol sense primer and antisense primer. 5'-ACCTACTTCCTGGACATTACGACCC-3 '[s], 5'-AAGGGAGCAATGCCCGTACCAGGCC-3' [a] as iNOS primer, and 5'-ACCACAGTCCATGCCATCAC-3 '[s], 5'-TCCACCACCCTGTTGCTGTA-3' as GAPDH primer [a], 5′-TGAAGGGCTGCTTCCAAACCTTTGACC-3 ′ [s], 5′-TGTCCATTGAGGAGAGCTTTCAGC-3 ′ [a] as a primer for IL-1β, 5′-TGCAGTCACAGAAGGAGTGGCTAAG3 ′ [s], 5 as a primer for IL-6 '-TCTGACCACAGTGAGGAATGTCCAC-3' [a] and 5'-AGGCGCCACCACGCTCTTCT-3 '[s] and 5'-GGCAGCCTTGGCCCTTGAA-5' [a] were used as primers for TNF-α. The PCR reaction was carried out for 25 cycles with 94 ° C for 1 minute (denaturation), 60 ° C for 1 minute (annealing), 72 ° C for 1.5 minutes. PCR products were electrophoresed with 2% agarose and stained with ethidium bromide for detection. The results for compound 4 are shown in FIG. As is apparent from FIG. 1, compound 4 shows mRNA expression of iNOS, interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). Suppress depending on.
このように、本発明の化合物はＮＯの産生を抑制し、ｉＮＯＳ、インターロイキン−１β（ＩＬ−１β）、インターロイキン−６（ＩＬ−６）及び腫瘍壊死因子−α（ＴＮＦ−α）のｍＲＮＡ発現を抑制するので、抗アレルギー剤、抗炎症剤として有用である。
本発明の化合物を経口、非経口又は経皮投与することができる。その投与量は患者の年齢、疾病の状況等によって変化するが、成人１日１人当たり１mg／kg〜１００mg／kgである。
本発明の化合物を種々の剤形で投与することができ、例えば錠剤、散剤、顆粒剤、カプセル剤、注射剤、座剤、軟膏剤、パップ剤等の形態で投与することができる。本発明の化合物をこれらの剤形に形成する場合、これらの製剤化に通常使用する担体や添加物、例えば溶剤、基剤、希釈剤、充填剤などの賦形剤、溶解補助剤、乳化剤、分散剤、崩壊剤、可溶化剤、増粘剤、滑沢剤等の補助剤、抗酸化剤、保存剤、芳香剤、甘味剤等の添加剤を常法に従って使用し、製剤化することができる。
Thus, the compound of the present invention suppresses NO production, and mRNA of iNOS, interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). Since its expression is suppressed, it is useful as an antiallergic agent and anti-inflammatory agent.
The compounds of the present invention can be administered orally, parenterally or transdermally. The dose varies depending on the age of the patient, the condition of the disease, etc., but is 1 mg / kg to 100 mg / kg per adult per day.
The compound of the present invention can be administered in various dosage forms such as tablets, powders, granules, capsules, injections, suppositories, ointments, poultices and the like. When the compound of the present invention is formed into these dosage forms, carriers and additives that are usually used in these formulations, such as solvents, bases, diluents, fillers and other excipients, solubilizers, emulsifiers, Additives such as dispersants, disintegrants, solubilizers, thickeners, lubricants, antioxidants, preservatives, fragrances, sweeteners, etc. can be formulated according to conventional methods. it can.
製剤例
５５０mgの化合物１、１０５５０mgの乳糖、４５００mgのデンプンを均一に混合し、これにヒドロキシプロピルセルロース液０．１５mlを添加し、練合して軟塊を製造し、顆粒機を通して顆粒を製造し、乾燥する。乾燥後の顆粒に３００mgのステアリン酸マグネシウムを加え、均一に混合した後、打錠機によって錠剤とする。
Formulation Example 550 mg of Compound 1, 10550 mg of lactose and 4500 mg of starch are uniformly mixed, 0.15 ml of hydroxypropylcellulose solution is added thereto, kneaded to produce a soft mass, and granules are produced through a granulator. ,dry. 300 mg of magnesium stearate is added to the dried granules and mixed uniformly, and then tableted by a tableting machine.
本発明の化合物はＮＯの産生を抑制し、ｉＮＯＳ、インターロイキン−１β（ＩＬ−１β）、インターロイキン−６（ＩＬ−６）及び腫瘍壊死因子−α（ＴＮＦ−α）のｍＲＮＡ発現を抑制する作用を有する新規なクロマン誘導体化合物であり、抗アレルギー剤、抗炎症剤として有用である。 The compounds of the present invention suppress NO production and suppress mRNA expression of iNOS, interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α). It is a novel chroman derivative compound having an action, and is useful as an antiallergic agent and antiinflammatory agent.
Claims (10)
式中、
Ｒ1、Ｒ2及びＲ3はそれぞれ水素原子を表すか、又はＲ1とＲ2が一緒になって結合を表し、又はＲ2とＲ3が一緒になって結合を表す。 The chromone derivative compound represented by the following general formula (I):
Where
R 1 , R 2 and R 3 each represent a hydrogen atom, or R 1 and R 2 together represent a bond, or R 2 and R 3 together represent a bond.
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