JP4176150B2 - Glutamate release inhibitors and novel compounds - Google Patents
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Description
技術分野
本発明は、医薬組成物、特に神経伝達物質の中で興奮性アミノ酸に分類されているグルタミン酸の放出阻害剤に関する。さらに詳しくは、細胞内のグルタミン酸が細胞外へ放出されることを阻害することにより、グルタミン酸の過剰放出による神経細胞障害を防ぐ作用を有する芳香族ケトン誘導体に関する。
背景技術
グルタミン酸は中枢神経系に高濃度に存在し、ニューロン活動に興奮作用を起こすアミノ酸で興奮性伝達物質として知られている。すなわち、シナプス前部より刺激によって放出されたグルタミン酸が、シナプス後部のＮ−メチル−D−アスパラギン酸（ＮＭＤＡ）受容体等に結合することにより、神経細胞の興奮を伝達している。
細胞外に放出されたグルタミン酸は、シナプス後細胞やグリア細胞の細胞膜に存在するグルタミン酸輸送体により細胞外から取り除かれ、細胞外のグルタミン酸濃度は低濃度に維持されている。
しかし、脳虚血時においては細胞外のカリウムイオン濃度が上昇し、ナトリウムイオン濃度が低下することから膜が脱分極し、グルタミン酸の逆向きの輸送が起こり、グルタミン酸が細胞内から細胞外へと放出される（グルタミン酸輸送体の逆転）。この逆向きの輸送は、細胞外のグルタミン酸濃度を神経細胞が興奮毒性を発揮するのに十分な濃度まで引き上げる。そのことが、脳虚血後の神経細胞障害の一つの要因と考えられている。
従来より、脳虚血時の神経細胞障害を防ぐために二種類のアプローチがされてきた。すなわち、過剰放出されたグルタミン酸に対し、ＮＭＤＡ受容体等のグルタミン酸受容体の拮抗薬等を用いて興奮の伝達を阻害する方法と、グルタミン酸の過剰放出自体を阻害する方法である。
グルタミン酸の放出を阻害する方法としては、現在、ナトリウムチャンネルブロッカー、カルシウムチャンネルブロッカー、抗酸化剤などが使用が挙げられる。しかし、これらの薬剤の使用は結果的にグルタミン酸の放出を阻害しているが、それぞれのチャンネルがブロックされることにより、一次的にグルタミン酸の放出を阻害するのではなく、二次的あるいは三次的に阻害するため、他の個所に悪影響を及ぼす可能性を有している。
このように、従来の方法では脳虚血による神経細胞障害を引き起こす一因であるグルタミン酸の過剰放出において、最も直接的に抑制すべきグルタミン酸放出のみを押さえることができなかった。
発明の開示
上記に鑑み、本発明者らは神経細胞壊死の原因の一つであるグルタミン酸の過剰放出を、より直接的に抑制するグルタミン酸放出阻害剤について研究を行ってきた。
本発明者らは鋭意研究の結果、従来から知られているナトリウムチャンネルブロッカーやカルシウムチャンネルブロッカーの作用を全く有さない、グルタミン酸放出阻害作用を有する芳香族ケトン誘導体化合物を見出した。これらの化合物は、グルタミン酸輸送体の逆転を阻害していると考えられる。
すなわち本発明は、一般式（Ｉ）：
からなる医薬に関する。
さらに詳しくは、
ａ）一般式（Ｉ）において、Ｒ1は水素原子、ヒドロキシ、低級アルコキシまたは式：
ｂ）一般式（ＩＩ）：
ｃ）前記の一般式で表わされる化合物を有効成分として含有するグルタミン酸放出阻害剤。
ｄ）一般式（ＩＩＩ）：
ｅ）一般式（ＩＩＩ）：
ｆ）一般式（ＩＩ）：
ｇ）一般式（ＩＩ）：
ｈ）請求項ｄ）〜ｇ）のいずれかに記載の化合物を有効成分として含有する医薬組成物。
ｉ）請求項ｄ）〜ｇ）のいずれかに記載の化合物を有効成分として含有するグルタミン酸放出阻害剤、に関する。
本明細書中、「ハロゲン」とはフッ素、塩素、臭素、およびヨウ素を意味する。
本明細書中、「低級アルキル」とは、直鎖状または分枝状のＣ1〜Ｃ6アルキルを意味する。例えば、メチル、エチル、ｎ−プロピル、イソプロピル、ｎ−ブチル、イソブチル、ｓｅｃ−ブチル、ｔｅｒｔ−ブチル等が挙げられる。
本明細書中、「低級アルコキシ」とは、アルキル部分が前記「低級アルキル」であるアルコキシを意味する。例えば、メトキシ、エトキシ、ｎ−プロポキシ、イソプロポキシ、ｎ−ブトキシ、イソブトキシ、ｓｅｃ−ブトキシ、ｔｅｒｔ−ブトキシ等が挙げられる。
本明細書中、「置換されていてもよい低級アルコキシ」とは、アルキル部分が低級アルコキシ、低級アルキルオキシカルボニル、カルボキシ、モノ低級アルキル置換アミノ、ジアルキル置換アミノ等で置換されていてもよい前記「低級アルキル」であるアルコキシを意味する。例えば、メトキシカルボニルメトキシ、メトキシカルボニルエトキシ、エトキシカルボニルメトキシ、エトキシカルボニルエトキシ、ジメチルアミノメトキシ、ジメチルアミノエトキシ等が挙げられる。
本明細書中、「アシル」とは、アルキル部分が前記「低級アルキル」であるアルカノイル、アリール部分がフェニルであり、さらに低級アルキル、ハロゲン等で置換されていてもよいアロイルを意味する。例えば、アセチル、プロピオニル、ベンゾイル、トルオイル等が挙げられる。
本明細書中、「ヘテロアリール」とは、５〜６員環でＮ、ＯまたはＳ原子を環内に１個以上含む単環のヘテロ芳香族を意味する。例えば、ピロール、ピロリル、ピリジル、チエニル、フリル等が挙げられる。
本明細書中、「置換されていてもよいヘテロアリール」における炭素上の置換基としては、ハロゲン、低級アルキル、低級アルコキシ、アルコキシカルボニル等が挙げられる。ただし、ヘテロ原子がＮである場合は、そのＮ原子が低級アルコキシ、低級アルコキシカルボニル、カルボキシ、モノアルキル置換アミノ、ジアルキル置換アミノ等で置換されていてもよい「低級アルキル」、または「アシル」等で置換されていてもよい。
【図面の簡単な説明】
第１図は、化合物（１８）、（１９）の培養小脳細胞に対するグルタミン酸放出容量依存性を示すグラフである。
第２図は、化合物（１８）、（１９）の海馬スライスに対するグルタミン酸放出容量依存性を示すグラフである。
第３図は、正常ラット、虚血ラット、および化合物（１８）を投与後虚血にしたラットについて、右前大脳動脈灌流域大脳皮質、右大脳動脈灌流域大脳皮質、および右線状体における脳虚血障害の指標としての水分含有量を示すグラフである。
発明を実施するための最良の形態
本発明化合物において、一般式（Ｉ）のＲ1およびＲ4が同一または異なってヒドロキシまたは置換されていてもよい低級アルコキシであり、Ａがピロール誘導体である化合物は、例えば、下式で示す２種類の方法で合成される。
上記式中、第１工程（（ＩＶ）→（Ｖ），（ＶＩ）および（ＸＩＶ）→（ＸＶ），（ＸＶＩ））はピロール等のヘテロアリール誘導体とハロゲン化ベンゾイル誘導体とのフリーデル−クラフト反応により、芳香族ケトン誘導体へと導く反応である。ハロゲン化ベンゾイル誘導体およびヘテロアリール誘導体を塩化メチレン、クロロホルム、四塩化炭素等の溶媒に溶解し、−３０〜８０℃、好ましくは氷冷下にて塩化アルミニウム等のルイス酸を加え、氷冷下〜８０℃、好ましくは室温で３０分〜５時間、好ましくは１〜３時間攪拌する。反応液に氷水を加え有機溶媒で抽出後、有機層を飽和食塩水等で洗浄し、有機層を硫酸マグネシウム等で乾燥する。減圧下溶媒を留去し、必要に応じて残査をシリカゲルカラムクロマトグラフィーで精製することにより目的物を得る。
上記式中、「ハロゲン化」（（Ｖ）→（ＶＩＩ）、（ＸＶ）→（ＸＩＸ）、および（ＸＶＩＩ），（ＸＶＩＩＩ）→（ＸＩＸ））は通常用いられる芳香族のハロゲン化により行うことができる。例えば、出発原料を塩化メチレン、クロロホルム等の溶媒に溶解し、−３０〜５０℃、好ましくは氷冷下にてＮ−クロロサクシンイミド、Ｎ−ブロモサクシンイミド等のハロゲン化剤を加え、氷冷下〜５０℃、好ましくは室温にて１〜６時間、好ましくは２〜４時間攪拌する。反応液に重亜硫酸ナトリウム水溶液を加え５〜３０分間攪拌し、有機溶媒で抽出後、有機層を飽和食塩水等で洗浄し、有機層を硫酸マグネシウム等で乾燥する。減圧下溶媒を留去し、必要に応じて残査をシリカゲルカラムクロマトグラフィーで精製することにより目的物を得る。
上記式中、「脱アルキル化」（（Ｖ）→（ＶＩＩＩ），（ＩＸ）、（ＸＶ）→（ＸＶＩＩ），（ＸＶＩＩＩ）、および（ＸＩＸ）→（ＸＸＩ））はエーテル結合のＯ−Ｃ結合を開裂させるのに通常用いる反応により行う。試薬としては、三塩化ホウ素、三臭化ホウ素、三フッ化ホウ素、トリメチルシリルクロリド等が用いられる。例えば、出発原料を塩化メチレン、クロロホルム等の溶媒に溶解し、−８０℃〜室温、好ましくは−３０℃〜氷冷下にて三臭化ホウ素の塩化メチレン溶液を加え、−３０℃〜５０℃、好ましくは氷冷下〜室温にて１０〜４０時間、好ましくは、１５〜３０時間攪拌する。反応液に氷水を加え有機溶媒で抽出後、有機層を飽和食塩水等で洗浄し、有機層を硫酸マグネシウム等で乾燥する。減圧下溶媒を留去し、必要に応じて残査をシリカゲルカラムクロマトグラフィーで精製することにより目的物を得る。
上記式中、「Ｏ−アルキル化」（（ＶＩＩＩ），（ＩＸ）→（Ｘ），（ＸＩ））はヒドロキシル基に対して通常用いられるアルキル化反応を用いることにより行う。例えば、出発原料をテトラヒドロフラン、ジオキサン、ジメチルホルムアミド等の溶媒に溶解し、炭酸カリウム、炭酸ナトリウムなどの塩基を加えた後、氷冷下〜８０℃、好ましくは室温〜６０℃にて目的のアルキル化に従ってそのハロゲン化物を前記溶媒に溶解した溶液を加え、室温〜８０℃、好ましくは室温〜６０℃にて１〜１０時間、好ましくは３〜７時間攪拌する。反応液に氷水を加え、１規定塩酸で中和し、有機溶媒で抽出後、有機層を飽和食塩水等で洗浄し、有機層を硫酸マグネシウム等で乾燥する。減圧下溶媒を留去し、必要に応じて残査をシリカゲルカラムクロマトグラフィーで精製することにより目的物を得る。
上記式中、「Ｎ−アルキル化」（（Ｘ），（ＸＩ）→（ＸＩＩ），（ＸＩＩＩ）および（ＸＩＸ）→（ＸＸ））はイミノ基に対する通常用いられるアルキル化反応を用いることに行う。例えば、水素化ナトリウム、水素化リチウム等の塩基をジメチルホルムアミド、テトラヒドロフラン、ジオキサン等の溶媒に懸濁し、−３０℃〜６０℃、好ましくは氷冷下〜室温にて前記溶媒に溶解した出発原料の溶液を加え、さらに同温度で目的のアルキル化に従ってそのハロゲン化物を加え１〜５時間、好ましくは２〜３時間攪拌する。反応液に氷水を加え有機溶媒で抽出後、有機層を飽和食塩水等で洗浄し、有機層を硫酸マグネシウム等で乾燥する。減圧下溶媒を留去し、必要に応じて残査をシリカゲルカラムクロマトグラフィーで精製することにより目的物を得る。
上記式中、「Ｎ−アシル化」（（Ｘ），（ＸＩ）→（ＸＩＩ），（ＸＩＩＩ）および（ＸＩＸ）→（ＸＸ））はイミノ基に対して通常用いられるアシル化反応を用いることにより行う。例えば、「Ｎ−アルキル化」の場合と同様に塩基を調製した後、−３０℃〜６０℃、好ましくは氷冷下〜室温にて前記溶媒に溶解した出発原料の溶液を加え、さらに同温度で目的のアシル化に従ってその酸ハロゲン化物を加え１〜５時間、好ましくは２〜３時間攪拌する。反応液に氷水を加え有機溶媒で抽出後、有機層を飽和食塩水等で洗浄し、有機層を硫酸マグネシウム等で乾燥する。減圧下溶媒を留去し、必要に応じて残査をシリカゲルカラムクロマトグラフィーで精製することにより目的物を得る。
上記式中、「アルキル化」（（ＸＸＩ）→（ＸＸＩＩ））は前記の「Ｏ−アルキル化」と同様にして行う。
上記式中、「加水分解」（（ＸＸ）→（ＸＸＩＶ）および（ＸＸＩＩ）→（ＸＸＩＩＩ））は通常用いられるエステルのカルボン酸への加水分解反応により行う。例えば、出発原料をメタノール、エタノール、テトラヒドロフラン、ジオキサン等の溶媒に溶解し、氷冷下〜６０℃、好ましくは氷冷下〜室温にて１〜３規定の水酸化ナトリウム水溶液を加え、１〜５時間、好ましくは２〜３時間攪拌する。反応液に有機溶媒および水を加えた後、塩酸水を加えｐＨを約２に調製し、有機溶媒で抽出後、有機層を飽和食塩水等で洗浄し、有機層を硫酸マグネシウム等で乾燥する。減圧下溶媒を留去し、必要に応じて残査をシリカゲルカラムクロマトグラフィーで精製することにより目的物を得る。
一般式（Ｉ）で表わされる化合物の中で、上記の例に含まれない化合物についても上記と同様の方法を用いて合成することができる。
また、既知化合物については、J.Agric.Food.Chem.(1990),38,1260-1263、J.Org.Chem.(1974),39(24),3559-3564、J.Chem.Res.S(Synopses)(197),(7),186等に記載の方法により得ることもできる。
「本発明化合物」という場合には、薬理学的に許容される塩、またはその水和物も抱合される。例えば、アルカリ金属（リチウム、ナトリウム、カリウム等）、アルカリ土類金属（マグネシウム、カルシウム等）、アンモニウム、有機塩基およびアミノ酸との塩、または無機酸（塩酸、臭化水素酸、りん酸、硫酸等）、および有機酸（酢酸、クエン酸、マレイン酸、フマル酸、ベンゼンスルホン酸、ｐ−トルエンスルホン酸等）との塩が挙げられる。これらの塩は、通常行われる方法によって形成させることができる。
本発明化合物は、グルタミン酸の放出阻害作用を有しているが、従来から知られているナトリウムブロッカー、カルシウムブロッカー、抗酸化剤の作用は全く有していない。このことから、グルタミン酸輸送体の逆転を阻害している全く新規な化合物であると考えられる。
本発明化合物は、神経伝達物質の中で興奮性アミノ酸に分類されているグルタミン酸の放出を阻害する作用を有することから、痙攣、てんかん、鎮痛、片頭痛、脳機能障害等の治療薬として使用することができる。
本発明化合物を、上記の疾患の治療あるいは予防を目的としてヒトに投与する場合は、散剤、顆粒剤、錠剤、カプセル剤、丸剤、液剤等として経口的に、または注射剤、坐剤、経皮吸収剤、吸入剤等として非経口的に投与することができる。また、本化合物の有効量にその剤型に適した賦形剤、結合剤、湿潤剤、崩壊剤、滑沢剤等の医薬用添加剤を必要に応じて混合し、医薬製剤とすることができる。注射剤の場合には、適当な担体と共に滅菌処理を行って製剤とする。
投与量は疾患の状態、投与ルート、患者の年齢、または体重によっても異なるが、成人に経口で投与する場合、通常０．１〜１００mg／kg／日であり、好ましくは１〜２０mg／kg／日である。
以下に実施例および試験例を挙げて本発明をさらに詳しく説明するが、本発明はこれらにより限定されるものではない。
実施例中、以下の略号を使用する。
Ｍｅ：メチル
Ｅｔ：エチル
ＤＭＳＯ：ジメチルスルホキシド
実施例
実施例１
（３，５−ジクロロ−２，６−ジメトキシ−フェニル）−（１Ｈ−ピロール−２−イル）−メタノン（化合物（１））および（３，５−ジクロロ−２，６−ジメトキシ−フェニル）−（１Ｈ−ピロール−３−イル）−メタノン（化合物（２））の調製
３，５−ジクロロ−２，６−ジメトキシ安息香酸（1.02g，4.07mmol）を塩化チオニル（3ml）に溶解し、３０分間還流撹拌した後、減圧下に溶媒を留去し、氷冷下得られた３，５−ジクロロ−２，６−ジメトキシ安息香酸クロリドにピロール（0.42ml，6.05mmol）のジクロロエタン溶液（10ml）を加えた。次いで本反応液に氷冷下塩化アルミニウム（0.60g，4.49mmol）の塩化メチレン懸濁液（10ml）を加え、室温下1.5時間撹拌した後、反応液を氷中に注ぎ、塩化メチレンで抽出した。有機層は飽和食塩水で洗浄、硫酸マグネシウムで乾燥した後、減圧下溶媒を留去して得られた残査をシリカゲルカラムクロマトグラフィー（メルク社キーゼルゲル60，70-230メッシュ）に付し、酢酸エチル：ヘキサン＝４：１の混合溶媒で溶出し、溶出順に化合物（１）（664mg，54%）と化合物（２）（133mg，11%）を得た。
同様の手法で化合物（３）〜化合物（１１）を合成した。その結果を表１〜表２に示した。
（２，６−ジメトキシ−フェニル）−（１Ｈ−ピロール−２−イル）−メタノン（化合物（１２））、（２，６−ジメトキシ−フェニル）−（１Ｈ−ピロール−３−イル）−メタノン（化合物（１３））の調製
ピロール（５ｍｌ，和光純薬製特級）と２，６−ジメトキシベンゾイル クロリド（１０ｇ，アルドリッチ製）をクロロホルム（１００ｍｌ）に溶解し、氷冷下撹拌する。クロロホルム（１００ｍｌ）と無水塩化アルミニュウム（８．２９ｇ，ナカライ テスク製特級）を加え、氷冷下１５分間撹拌する。更に室温下、２時間撹拌する。水（１００ｍｌ）を氷冷下撹拌しながら加え、クロロホルム層を水洗、無水硫酸ナトリウムで乾燥し、減圧下溶媒留去して粗生成物（１２．０ｇ）を得た。粗生成物（１２．０ｇ）をシリカゲルカラムクロマトグラフィー（メルク社、Kieselgel 60，0.040〜0.063mm，110g，クロロホルム：メタノール＝２０：１）精製し、化合物（１２）（６．０ｇ）、化合物（１３）（１．７２ｇ）をそれぞれ得た。
化合物（１２）：
EIMS，m/z：231(M)+(base peak)
IR，λmax KBr cm-1：3275,3111,2937,2837,1615,1592,1546,1473,1431,1403,1334,1305,1282,1253,1129,1112,1032,891,875,855,786,758,745,721,641,602
1H NMR（CDCl3，300MHz）δ：3.74(6H,s),6.24(1H,m),6.55(1H,m),6.61(2H,d,J=8.4Hz),7.07(1H,m),7.32(1H,t,J=8.4Hz),9.46(1H,br.s)
化合物（１３）：
EIMS，m/z：231(M)+，94(base peak)
IR，λmax KBr cm-1：3206,2958,2840,1617,1592,1539,1505,1471,1430,1403,1339,1306,1282,1252,1184,1137,1110,1051,1027,981,979,783,754,743,719,683,605
1H NMR（d6-DMSO，300MHz）δ：3.65(6H,s),6.33(1H,m),6.79(1H,m),6.70(2H,d,J=8.4Hz),6.97(1H,br.s),7.32(1H,t,J=8.4Hz),11.33(1H,br.s)
13C NMR(d6-DMSO，50MHz）δ：55.50,104.14,107.74,119.64,119.75,125.43,126.30,129.76,156.56,187.70
実施例１１
（２−ヒドロキシ−６−メトキシ−フェニル）−（１Ｈ−ピロール−２−イル）−メタノン（化合物（１４））、（２，６−ジヒドロキシ−フェニル）−（１Ｈ−ピロール−３−イル）−メタノン（化合物（１５））の調製
化合物（１２）（１１６ｍｇ，０．５ｍＭ）をジクロロメタン（５ｍｌ）に溶解し、−７０℃冷却下撹拌する。１．０Ｍ三臭化ホウ素−ジクロロメタン溶液（１．１ｍｌ，アルドリッチ製）を加え、−７０℃冷却下３時間撹拌する。更に室温下、１７．５時間撹拌する。氷水中に空け、ジクロロメタン層を除き、更にジエチルエーテルで抽出し、有機層を合わせて水洗、無水硫酸ナトリウムで乾燥し、減圧下溶媒留去して粗生成物（９０ｍｇ）を得た。粗生成物を薄層シリカゲルプレート（メルク社、Pre-Coated TLC Plates,SILICA GEL F-254,0.5mm，トルエン：アセトン＝５：３）精製し、化合物（１４）（１２ｍｇ）、化合物（１５）（７５ｍｇ）をそれぞれ得た。
同様の手法で化合物（１６）、化合物（１７）を合成した。その結果を表３に示した。
（３−クロロ−２，６−ジメトキシ−フェニル）−（４−クロロ−１Ｈ−ピロール−２−イル）−メタノン（化合物（１８））、（３−クロロ−２，６−ジメトキシ−フェニル）−（４，５−ジクロロ−１Ｈ−ピロール−２−イル）−メタノン（化合物（１９））の調製
化合物（１２）（０．４６ｇ，１．９９ｍＭ）のクロロホルム（１０ｍｌ）溶液を氷冷下撹拌しながら、Ｎ−クロロサクシンイミド（０．６１６ｇ，４．９８ｍＭ）を加え、１時間氷冷下撹拌する。更に室温下３時間撹拌する。１０％重亜硫酸ナトリウム水溶液（６ｍｌ）を加え、１０分間撹拌後クロロホルム層を水洗、無水硫酸ナトリウムで乾燥後、減圧下溶媒留去して粗生成物（０．６８ｇ）を得た。粗生成物（０．６８ｇ）をシリカゲルカラムクロマトグラフィー（メルク社、ＬｉＣｈｒｏｐｒｅｐ Ｓｉ６０，ＳｉｚｅＢ，トルエン：酢酸エチル＝１４：１〜９：１）精製し、化合物（１８）（７３ｍｇ）、化合物（１９）（２００ｍｇ）それぞれ得た。
出発原料に化合物（１）、（３）を用いて、同様の手法で化合物（２０）、化合物（２１）を合成した。その結果を表４に示した。
（３−クロロ−２，６−ジヒドロキシ−フェニル）−（４−クロロ−１Ｈ−ピロール−２−イル）−メタノン（化合物（２２））の調製
化合物（１８）（５０ｍｇ）をジクロロメタン（８ｍｌ）に溶解し、氷冷下撹拌する。１．０Ｍ三臭化ホウ素−ジクロロメタン溶液（０．８ｍｌ，アルドリッチ製）を加え、氷冷下２時間撹拌する。更に室温下、１６時間撹拌する。氷水中に空け、ジクロロメタン層を除き、更に酢酸エチルで抽出し、有機層を合わせて水洗、無水硫酸ナトリウムで乾燥し、減圧下溶媒留去して残渣を酢酸エチル：ｎ−ヘキサンで再結晶し、淡黄色針状晶として化合物（２２）（４２ｍｇ）を得た。
出発原料に化合物（１９）、（３）を用いて、同様の手法で化合物（２３）、（２４）を合成した。その結果を表５に示した。
（３−クロロ−２−ヒドロキシ−６−エトキシカルボニルメトキシ−フェニル）−（４−クロロ−１Ｈ−ピロール−２−イル）−メタノン（化合物（２５））、（３−クロロ−２，６−ジエトキシカルボニルメトキシ−フェニル）−（４−クロロ−１Ｈ−ピロール−２−イル）−メタノン（化合物（２６））、（３−クロロ−２，６−ジエトキシカルボニルメトキシ−フェニル）−（Ｎ−エトキシカルボニルメチル−４−クロロ−１Ｈ−ピロール−２−イル）−メタノン（化合物（２７））の調製
化合物（２２）（５０ｍｇ）のＮ，Ｎ−ジメチルホルムアミド（１．７５ｍｌ）溶液に炭酸カリウム（３１．８ｍｇ）、ブロモ酢酸エチル（７６．９ｍｇ）を加え、窒素気流下油浴中８０℃に加熱し、７時間撹拌する。反応液に水（２ｍｌ）を加えた後、１Ｎ塩酸を加えて中和する。次いで、酢酸エチル（５ｍｌ）で２回抽出し、酢酸エチル層を水洗、無水硫酸ナトリウムで乾燥後、減圧下溶媒留去して粗生成物（８７ｍｇ）を得た。粗生成物を薄層シリカゲルプレート（メルク社、Pre-Coated TLC Plates，SILICA GEL F-254，0.5mm，クロロホルム：メタノール＝５０：１）精製し、淡黄色プリズムとして化合物（２５）（８ｍｇ）、無色油状物として化合物（２６）（４０ｍｇ）、化合物（２７）（２８ｍｇ）をそれぞれ得た。
出発原料に化合物（２３）、（１４）、（１５）を用いて、同様の手法で化合物（２８）〜（３３）を合成した。その結果を表６〜表７に示した。
（３−ブロモ−２，６−ジエトキシカルボニルメトキシ−フェニル）−（４，５−ジブロモ−１Ｈ−ピロール−２−イル）−メタノン（化合物（３４））の調製
化合物（３３）（78.0mg，0.208mmol）の酢酸溶液（5ml）に臭素酢酸溶液（0.64M，1ml）を加え、室温下４時間撹拌した。反応液にハイポ水を加え、クロロホルムで抽出を行い、有機層は飽和食塩水で洗浄、硫酸マグネシウムで乾燥した後、減圧下溶媒を留去して得られた残査をシリカゲルカラムクロマトグラフィー（メルク社キーゼルゲル60，70-230メッシュ）に付し、酢酸エチル：ヘキサン＝１：２の混合溶媒で溶出し化合物（３４）（75.0mg，59%）を得た。
融点：165-166℃
元素分析（C19H18Br3NO7・1/4H2O）として
計算値：C，37.01；H，3.02；Br，38.88；N，2.27(%）
実験値：C，36.89；H，3.06；Br，38.65；N，2.30(%）
1H-NMR(CDCl3）δ：1.27(6H,t,J=7,0Hz),4.17-4.30(4H,m),4.59(2H,s),4.62(2H,s),6.55(1H,d,J=9.0Hz),6.76(1H,d,J=2.6Hz),7.56(1H,d,J=9.0Hz),9.76(1H,br s).
実施例２７
（３，５−ジブロモ−２，６−ジメトキシ−フェニル）−（１−メチル−１Ｈ−ピロール−２−イル）−メタノン（化合物（３５））の調製
60%水素化ナトリウム（24.8mg，0.620mmol）に氷冷下化合物（３）（214mg，0.549mmol）のジメチルホルムアミド溶液（4ml）を加え１０分間撹拌した後、ヨウ化メチル（244mg，1.65mmol）のジメチルホルムアミド溶液（1ml）を加え、更に室温下３時間撹拌した。反応液は氷冷下水を加え、酢酸エチルで抽出し、有機層は飽和食塩水で洗浄、硫酸マグネシウムで乾燥した後、減圧下溶媒を留去して得られた残査をシリカゲルカラムクロマトグラフィー（メルク社キーゼルゲル60，70-230メッシュ）に付し、酢酸エチル：ヘキサン＝１：２の混合溶媒で溶出し化合物（３５）（218mg，99%）を得た。
出発原料に化合物（１８）を用いて、同様の手法で化合物（３６）を合成した。その結果を表８に示した。
（３−クロロ−２，６−ジヒドロキシカルボニルメトキシ−フェニル）−（４−クロロ−１Ｈ−ピロール−２−イル）−メタノン（化合物（３７））の調製
化合物（２６）（２０ｍｇ）のジオキサン（０．８ｍｌ）溶液に１Ｎ水酸化ナトリウム水溶液（０．２ｍｌ）を加え、窒素気流下室温で２．６時間撹拌する。酢酸エチル（１０ｍｌ）水（１０ｍｌ）を加え、１Ｎ塩酸水溶液で酸性（ｐＨ２．０）に調製する。酢酸エチル層を水洗、無水硫酸ナトリウムで乾燥後、減圧下溶媒留去し、残査に酢酸エチル、ｎ−ヘキサンを加え、無色粉末として化合物（３７）（２０ｍｇ）を得た。
出発原料に化合物（２７）、（２８）、（３６）を用いて、同様の手法で化合物（３８）、（３９）、（４０）を合成した。その結果を表９に示した。
（２，６−ジメトキシ−フェニル）−２−ピリジル−メタノン（化合物（４１））の調製
２−ブロモピリジン（８ｇ，和光純薬製特級）を−７０℃冷却下１．６６Ｍ ｎ−ブチルリチュウム−ｎ−ヘキサン溶液（３０ｍｌ，アルドリッチ製）を加え撹拌する。２０分後、２，６−ジメトキシベンゾニトリル（６．６５ｇ，アルドリッチ製）のジエチルエーテル溶液（８０ｍｌ）を加え、−７０℃冷却下２時間撹拌する。更に室温下３時間撹拌する。氷冷下水飽和ジエチルエーテルを加え、沈殿を瀘取し、粗生成物（１１．０ｇ）を得た。粗生成物（１１．０ｇ）を酢酸エチル（３００ｍｌ）と０．５Ｎ塩酸（３００ｍｌ）で分配する。水層は更に酢酸エチル（３００ｍｌ）で抽出し、酢酸エチルは合わせて減圧下溶媒留去して残渣（２．０ｇ）を得た。得られた残渣をシリカゲルカラムクロマトグラフィー（メルク社、Kieselgel 60，0.040〜0.063mm，150g，アセトン：ｎ−ヘキサン＝１：３）精製し、メタノールから再結晶して化合物（４１）（１．４ｇ）を得た。
物理恒数は表１０に示す。
実施例３４
（３−クロロ−２，６−ジメトキシ−フェニル）−２−ピリジル−メタノン（化合物（４２））、（３，５−ジクロロ−２，６−ジメトキシ−フェニル）−２−ピリジル−メタノン（化合物（４３））の調製
化合物（４１）（１２２ｍｇ）のクロロホルム（３ｍｌ）溶液を氷冷下撹拌しながら、Ｎ−クロロサクシイミド（２０３ｍｇ）を加え、４時間氷冷下撹拌する。更に室温下撹拌する。１２時間後Ｎ−クロロサクシイミド（１３５ｍｇ）、更に２４時間後Ｎ−クロロサクシイミド（１３５ｍｇ）を加える。室温下撹拌合計３６時間後１０％重亜硫酸ナトリウム水溶液（３ｍｌ）を加え、１０分間撹拌後クロロホルム層を水洗、無水硫酸ナトリウムで乾燥後、減圧下溶媒留去して粗生成物（２９５ｍｇ）を得た。粗生成物（０．６８ｇ）をシリカゲルカラムクロマトグラフィー（メルク社キーゼルゲル60，0.040〜0.063mm，90g，トルエン：酢酸エチル＝３：１）精製し、化合物（４２）（３２ｍｇ）、化合物（４３）（１０９ｍｇ）をそれぞれ得た。
物理恒数は表１０に示す。
（３，５−ジブロモ−２，６−ジメトキシ−フェニル）−２−ピリジル−メタノン（化合物（４４））の調製
化合物（４１）（１２２ｍｇ）のクロロホルム（５ｍｌ）溶液を室温下撹拌しながら、Ｎ−ブロモサクシイミド（４４５ｍｇ）を加え、室温下２４時間撹拌する。１０％重亜硫酸ナトリウム水溶液（３ｍｌ）を加え、１０分間撹拌後クロロホルム層を水洗、無水硫酸ナトリウムで乾燥後、減圧下溶媒留去して粗生成物（２３９ｍｇ）を得た。粗生成物（２３９ｍｇ）をシリカゲルカラムクロマトグラフィー（メルク社キーゼルゲル60，0.040〜0.063mm，90g，トルエン：酢酸エチル＝１９：１）精製し、酢酸エチル−ｎ−ヘキサンから結晶化し化合物（４４）（１４４ｍｇ）を得た。
物理恒数は表１１に示す。
実施例３６
（２，６−ジメトキシ−フェニル）−３−ピリジル−メタノン（化合物（４５））の調製
ニコチン酸クロリド塩酸塩（３．５６ｇ，アルドリッチ製）、Ｎ，Ｏ−ジメチルヒドロキシルアミン塩酸塩（１．９９ｇ，アルドリッチ製）、トリエチルアミン（６．０７ｇ，和光純薬工業製特級）のテトラヒドロフラン（２０ｍｌ）溶液を３時間加熱還流する。室温下クロロホルム（５０ｍｌ）を加え３時間撹拌する。反応液中にジクロロメタン：ジエチルエーテル（１：１）溶液（１５０ｍｌ）、次いで飽和食塩水（３０ｍｌ）を加え、分液し、有機層を無水硫酸ナトリウムで乾燥後、減圧下溶媒留去してニコチン酸アミド誘導体（３．１８ｇ）を得た。次に、メタージメトキシベンゼン（１３８ｍｇ，アルドリッチ製）のテトラヒドロフラン（２ｍｌ）溶液に、１．６６Ｍ ｎ−ブチルリチュウム−ｎ−ヘキサン溶液（１．２ｍｌ，アルドリッチ製）を加え室温下撹拌する。１時間後、反応液中に先に調製したニコチン酸アミド誘導体（２８４ｍｇ）のテトラヒドロフラン（２ｍｌ）溶液を滴下し、１．５時間撹拌する。反応液に水飽和ジエチルエーテル（２ｍｌ）次いで水（２ｍｌ）を加え、得られた有機層を無水硫酸ナトリウムで乾燥後、減圧下溶媒留去して残渣（３５０ｍｇ）を得た。残渣をシリカゲルカラムクロマトグラフィー（メルク社キーゼルゲル60，0.040〜0.063mm，50g，アセトン：ｎ−ヘキサン＝１：３）精製し、化合物（４５）（１６０ｍｇ）を得た。
物理恒数は表１１に示す。
実施例３７
（３，５−ジクロロ−２，６−ジメトキシ−フェニル）−３−ピリジル−メタノン（化合物（４６））の調製
化合物（４５）（９７ｍｇ）のクロロホルム（４ｍｌ）溶液を室温下撹拌しながら、Ｎ−クロロサクシンイミド（５３４ｍｇ）を加える。４日間撹拌後１０％重亜硫酸ナトリウム水溶液（３ｍｌ）を加え、１０分間撹拌後クロロホルム層を水洗、無水硫酸ナトリウムで乾燥後、減圧下溶媒留去して粗生成物（２１３ｍｇ）を得た。粗生成物をシリカゲルカラムクロマトグラフィー（メルク社キーゼルゲル，0.040〜0.063mm，30g，アセトン：ｎ−ヘキサン＝１：３）精製し、化合物（４６）（７７ｍｇ）を得た。
物理恒数は表１１に示す。
グルタミン酸放出抑制効果（ｉｎ ｖｉｔｒｏ）
８日令のSD（Sprague Dawley（日本SLC，静岡））系ラットの小脳を２０匹分摘出し、０．２５％トリプシン液および１mM EDTA・４Na液で10分間37℃で処理、ピペッティングにより細胞を分散させる。ナイロンメッシュ（Cell Strainer 70μm Nyron,FALCON）を通し、大きな組織塊を除去する。ポリ−Ｌ−リジン（poly-L-Lysine）を使用して表面をコーティングした２４穴培養皿２０枚に分散した細胞を分注する。培地はα-MEM培地に１０％ウシ血清、25mM KClを添加したものを用いる。細胞を24穴培養皿に分注後24-48時間に、終濃度10μMシトシンβ-D-アラビノフラノシド（Cytosine β-D-arabinofuranoside）を添加し、増殖性細胞を除去する。その後、３日置きに培地交換を行い、１・２週目の小脳顆粒細胞を実験に用いた（Manual of the Nervous System，pages 203-206，1989 Alan R.Liss,Inc.）。各種濃度の化合物をクレブス−リンゲル液に添加して37℃10分添加してプレインキュベーションする。さらにベラトリジンを添加して37℃10分処理することによって細胞外に放出されたグルタミン酸を測定した。測定方法としては、２４穴培養皿の細胞をクレブス−リンゲル液１mlで２回洗浄し、検体を含む500μlのクレブス−リンゲル液を添加し37℃10分間処理する。250μlを取り、既報（Life Sciences 43，pages 913-922，1988）に記載されているｏ−フタルアルデヒド（phthalaldehyde）を用いたHPLC法により、細胞から放出されたグルタミン酸の濃度を測定する方法を用いた。
試験例２
グルタミン酸放出抑制効果（ｓｌｉｃｅ）
４週令のSD系ラットの海馬を摘出し0.35mmの厚さにスライスし、酸素を負荷した24℃のグルコースを含むクレブス−リンゲル液中に1時間静置してから、実験に使用した。上記の海馬スライスを、窒素置換することにより酸素を除き、さらにグルコースを除いた37℃のクレブス−リンゲル液に入れ、一定時間インキュベーションした後、クレブス−リンゲル液中に放出されたグルタミン酸濃度を上記のHPLC法によって測定した。
試験例３
中大脳動脈結紮モデルによる脳虚血実験
SD系ラットの中大脳動脈を1時間結紮し、部分的な脳虚血モデルを作る。１時間後に結紮した血管を再開通させ、18時間後の脳を取り出し各梗塞領域に分け、標本の湿重量を測定する。更に標本の水分を完全に除去し乾燥重量を測定し、組織の水分含量を測定した。さらに詳しくは、ラットの脳内に検体化合物を5nmol/5μl(0.5%DMSO/生理食塩水）で直接投与し、その後中大脳動脈に栓子をいれて血流を止める。１時間後再開通（栓子を除去）し、24時間後ラットを断頭、脱血、血液灌流して脳を取り出す。その後脳内の各部位をとる（右前大脳動脈灌流域大脳皮質、右大脳動脈灌流域大脳皮質、右線状体）。各々の湿重量を測定後、乾燥機で完全に乾燥して重量を測定し、水分含量を計算した。
図１からわかるように、それぞれの検体は容量依存的にグルタミン酸の放出を抑制した。また、この結果からＩＣ50値を算出した。
図２からわかるように、いずれの検体化合物も実験的虚血条件下で海馬スライスから放出されるグルタミン酸の放出を抑制した。
図３からわかるように、化合物（１８）を投与した群では有意に水分含量の増加が抑制されている。虚血障害を受けた脳領域では、顕著にその組織の水分含量が増加する（浮腫）ことが知られているので、本化合物の投与により脳虚血障害が改善されていることがわかる。
製剤例
一般式（Ｉ）で表わされる化合物 １０ｍｇ
でんぷん ２４ｍｇ
乳糖 １２ｍｇ
ヒドロキシプロピルセルロース ０．８ｍｇ
ステアリン酸マグネシウム ０．４ｍｇ
産業上の利用可能性
細胞内のグルタミン酸が細胞外へ放出されることを阻害することにより、グルタミン酸の過剰放出による神経細胞障害を防ぐ作用を有する芳香族ケトン誘導体を提供する。Technical field
The present invention relates to a pharmaceutical composition, in particular, a glutamate release inhibitor classified as an excitatory amino acid among neurotransmitters. More specifically, the present invention relates to an aromatic ketone derivative having an action of preventing neuronal cell damage due to excessive release of glutamate by inhibiting intracellular release of glutamate.
Background art
Glutamic acid is an amino acid that is present in high concentrations in the central nervous system and excites neuronal activity and is known as an excitatory transmitter. That is, glutamic acid released by stimulation from the anterior part of the synapse is converted into N-methyl- D -It transmits the excitement of nerve cells by binding to aspartate (NMDA) receptors and the like.
The glutamic acid released to the outside of the cell is removed from the outside by a glutamate transporter present in the cell membrane of the post-synaptic cell or glial cell, and the extracellular glutamate concentration is maintained at a low concentration.
However, during cerebral ischemia, the extracellular potassium ion concentration increases and the sodium ion concentration decreases, causing the membrane to depolarize and reverse glutamate transport, leading to glutamate transport from the cell to the cell. Released (reversal of glutamate transporter). This reverse transport raises the extracellular glutamate concentration to a concentration sufficient for neuronal cells to exhibit excitotoxicity. This is considered to be one factor of neuronal damage after cerebral ischemia.
Conventionally, two approaches have been taken to prevent nerve cell damage during cerebral ischemia. That is, there are a method of inhibiting excitatory transmission using glutamate receptor antagonists such as NMDA receptor and the like, and a method of inhibiting the excessive release of glutamate itself against the excessively released glutamate.
As a method for inhibiting the release of glutamic acid, use of a sodium channel blocker, a calcium channel blocker, an antioxidant and the like is currently used. However, the use of these drugs results in inhibition of glutamate release, but blocking each channel does not inhibit glutamate release primarily, but secondary or tertiary It has the potential to adversely affect other parts.
Thus, in the conventional method, in the excessive release of glutamate, which is a cause of causing neuronal damage due to cerebral ischemia, it was not possible to suppress only the release of glutamate that should be most directly suppressed.
Disclosure of the invention
In view of the above, the present inventors have studied a glutamate release inhibitor that more directly suppresses excessive release of glutamate, which is one of the causes of neuronal necrosis.
As a result of diligent research, the present inventors have found an aromatic ketone derivative compound having a glutamate release inhibitory action and having no conventionally known sodium channel blocker or calcium channel blocker action. These compounds are thought to inhibit the reversal of the glutamate transporter.
That is, the present invention relates to the general formula (I):
It relates to a medicine consisting of
For more details,
a) In the general formula (I), R 1 Is a hydrogen atom, hydroxy, lower alkoxy or the formula:
b) General formula (II):
c) A glutamate release inhibitor containing the compound represented by the above general formula as an active ingredient.
d) General formula (III):
e) General formula (III):
f) General formula (II):
g) General formula (II):
h) A pharmaceutical composition comprising the compound according to any one of claims d) to g) as an active ingredient.
i) A glutamic acid release inhibitor comprising the compound according to any one of claims d) to g) as an active ingredient.
In the present specification, “halogen” means fluorine, chlorine, bromine, and iodine.
In the present specification, “lower alkyl” means linear or branched C 1 ~ C 6 Means alkyl. For example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl and the like can be mentioned.
In the present specification, “lower alkoxy” means alkoxy in which the alkyl moiety is the “lower alkyl”. For example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy and the like can be mentioned.
In the present specification, “optionally substituted lower alkoxy” means that the alkyl moiety may be substituted with lower alkoxy, lower alkyloxycarbonyl, carboxy, mono-lower alkyl-substituted amino, dialkyl-substituted amino, or the like. It means alkoxy which is “lower alkyl”. Examples include methoxycarbonylmethoxy, methoxycarbonylethoxy, ethoxycarbonylmethoxy, ethoxycarbonylethoxy, dimethylaminomethoxy, dimethylaminoethoxy and the like.
In the present specification, “acyl” means alkanoyl in which the alkyl part is the above “lower alkyl”, and aroyl in which the aryl part is phenyl and may be further substituted with lower alkyl, halogen or the like. Examples include acetyl, propionyl, benzoyl, toluoyl and the like.
In the present specification, the “heteroaryl” means a monocyclic heteroaromatic which is a 5- to 6-membered ring and contains one or more N, O or S atoms in the ring. Examples include pyrrole, pyrrolyl, pyridyl, thienyl, furyl and the like.
In the present specification, examples of the substituent on carbon in “optionally substituted heteroaryl” include halogen, lower alkyl, lower alkoxy, alkoxycarbonyl and the like. However, when the heteroatom is N, the “lower alkyl”, “acyl” or the like which may be substituted with lower alkoxy, lower alkoxycarbonyl, carboxy, monoalkyl-substituted amino, dialkyl-substituted amino, etc. May be substituted.
[Brief description of the drawings]
FIG. 1 is a graph showing the glutamate release capacity dependence of compounds (18) and (19) on cultured cerebellar cells.
FIG. 2 is a graph showing the glutamate release capacity dependence of compounds (18) and (19) on hippocampal slices.
FIG. 3 shows the brain in the right anterior cerebral artery perfusion zone, the right cerebral artery perfusion zone, and the right striatum for normal rats, ischemic rats, and rats that are ischemic after administration of compound (18). It is a graph which shows the water content as a parameter | index of ischemic injury.
BEST MODE FOR CARRYING OUT THE INVENTION
In the compounds of the present invention, R of the general formula (I) 1 And R Four Are the same or different and are hydroxy or optionally substituted lower alkoxy, and the compound in which A is a pyrrole derivative is synthesized by, for example, two methods shown in the following formulae.
In the above formula, the first step ((IV) → (V), (VI) and (XIV) → (XV), (XVI)) is a Friedel-Craft of a heteroaryl derivative such as pyrrole and a halogenated benzoyl derivative. This reaction leads to an aromatic ketone derivative. A halogenated benzoyl derivative and a heteroaryl derivative are dissolved in a solvent such as methylene chloride, chloroform, carbon tetrachloride, and a Lewis acid such as aluminum chloride is added under ice cooling, preferably under ice cooling. Stir at 80 ° C., preferably at room temperature for 30 minutes to 5 hours, preferably 1 to 3 hours. Ice water is added to the reaction mixture, and the mixture is extracted with an organic solvent. The organic layer is washed with saturated brine, and the organic layer is dried over magnesium sulfate. The target product is obtained by evaporating the solvent under reduced pressure and purifying the residue by silica gel column chromatography as necessary.
In the above formula, “halogenation” ((V) → (VII), (XV) → (XIX), and (XVII), (XVIII) → (XIX)) is performed by a commonly used aromatic halogenation. Can do. For example, a starting material is dissolved in a solvent such as methylene chloride and chloroform, and a halogenating agent such as N-chlorosuccinimide and N-bromosuccinimide is added at −30 to 50 ° C., preferably under ice cooling. Lower to 50 ° C., preferably at room temperature for 1 to 6 hours, preferably 2 to 4 hours. A sodium bisulfite aqueous solution is added to the reaction solution, and the mixture is stirred for 5 to 30 minutes. After extraction with an organic solvent, the organic layer is washed with a saturated saline solution and the organic layer is dried with magnesium sulfate and the like. The target product is obtained by evaporating the solvent under reduced pressure and purifying the residue by silica gel column chromatography as necessary.
In the above formula, “dealkylation” ((V) → (VIII), (IX), (XV) → (XVII), (XVIII), and (XIX) → (XXI)) is an O—C of an ether bond. It is carried out by a reaction usually used for cleaving the bond. As the reagent, boron trichloride, boron tribromide, boron trifluoride, trimethylsilyl chloride and the like are used. For example, a starting material is dissolved in a solvent such as methylene chloride or chloroform, and a solution of boron tribromide in methylene chloride is added at −80 ° C. to room temperature, preferably −30 ° C. to ice cooling, and −30 ° C. to 50 ° C. The mixture is preferably stirred under ice-cooling to room temperature for 10 to 40 hours, preferably 15 to 30 hours. Ice water is added to the reaction mixture, and the mixture is extracted with an organic solvent. The organic layer is washed with saturated brine, and the organic layer is dried over magnesium sulfate. The target product is obtained by evaporating the solvent under reduced pressure and purifying the residue by silica gel column chromatography as necessary.
In the above formula, “O-alkylation” ((VIII), (IX) → (X), (XI)) is carried out by using an alkylation reaction usually used for hydroxyl groups. For example, the starting material is dissolved in a solvent such as tetrahydrofuran, dioxane, dimethylformamide, a base such as potassium carbonate or sodium carbonate is added, and then the desired alkylation is performed at -80 ° C, preferably at room temperature to 60 ° C under ice-cooling. Then, a solution in which the halide is dissolved in the solvent is added, and the mixture is stirred at room temperature to 80 ° C, preferably at room temperature to 60 ° C for 1 to 10 hours, preferably 3 to 7 hours. Ice water is added to the reaction solution, neutralized with 1N hydrochloric acid, extracted with an organic solvent, the organic layer is washed with saturated brine, and the organic layer is dried over magnesium sulfate. The target product is obtained by evaporating the solvent under reduced pressure and purifying the residue by silica gel column chromatography as necessary.
In the above formula, “N-alkylation” ((X), (XI) → (XII), (XIII) and (XIX) → (XX)) is performed by using a commonly used alkylation reaction for an imino group. . For example, a starting material prepared by suspending a base such as sodium hydride or lithium hydride in a solvent such as dimethylformamide, tetrahydrofuran or dioxane and dissolving in the solvent at −30 ° C. to 60 ° C., preferably under ice cooling to room temperature. The solution is added and the halide is further added at the same temperature according to the desired alkylation and stirred for 1 to 5 hours, preferably 2-3 hours. Ice water is added to the reaction mixture, and the mixture is extracted with an organic solvent. The organic layer is washed with saturated brine, and the organic layer is dried over magnesium sulfate. The target product is obtained by evaporating the solvent under reduced pressure and purifying the residue by silica gel column chromatography as necessary.
In the above formula, “N-acylation” ((X), (XI) → (XII), (XIII) and (XIX) → (XX)) uses an acylation reaction usually used for imino groups. To do. For example, after preparing the base in the same manner as in the case of “N-alkylation”, a solution of the starting material dissolved in the solvent at −30 ° C. to 60 ° C., preferably under ice cooling to room temperature, is added, and the same temperature is further added. In accordance with the desired acylation, the acid halide is added and stirred for 1 to 5 hours, preferably 2 to 3 hours. Ice water is added to the reaction mixture, and the mixture is extracted with an organic solvent. The organic layer is washed with saturated brine, and the organic layer is dried over magnesium sulfate. The target product is obtained by evaporating the solvent under reduced pressure and purifying the residue by silica gel column chromatography as necessary.
In the above formula, “alkylation” ((XXI) → (XXII)) is carried out in the same manner as the above “O-alkylation”.
In the above formula, “hydrolysis” ((XX) → (XXIV) and (XXII) → (XXIII)) is performed by a hydrolysis reaction of a commonly used ester to a carboxylic acid. For example, the starting material is dissolved in a solvent such as methanol, ethanol, tetrahydrofuran, dioxane and the like, and 1 to 3 N aqueous sodium hydroxide solution is added under ice cooling to 60 ° C., preferably under ice cooling to room temperature. Stir for hours, preferably 2-3 hours. After adding an organic solvent and water to the reaction solution, aqueous hydrochloric acid is added to adjust the pH to about 2. After extraction with the organic solvent, the organic layer is washed with saturated saline and the like, and the organic layer is dried with magnesium sulfate and the like. . The target product is obtained by evaporating the solvent under reduced pressure and purifying the residue by silica gel column chromatography as necessary.
Among the compounds represented by the general formula (I), compounds not included in the above examples can be synthesized using the same method as described above.
As for known compounds, J. Agric. Food. Chem. (1990), 38, 1260-1263, J. Org. Chem. (1974), 39 (24), 3559-3564, J. Chem. Res. It can also be obtained by the method described in S (Synopses) (197), (7), 186, etc.
In the case of “the compound of the present invention”, a pharmacologically acceptable salt or a hydrate thereof is also conjugated. For example, alkali metals (lithium, sodium, potassium, etc.), alkaline earth metals (magnesium, calcium, etc.), ammonium, salts with organic bases and amino acids, or inorganic acids (hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, etc.) ) And salts with organic acids (acetic acid, citric acid, maleic acid, fumaric acid, benzenesulfonic acid, p-toluenesulfonic acid, etc.). These salts can be formed by a conventional method.
The compound of the present invention has a glutamate release inhibitory effect, but has no known sodium blocker, calcium blocker or antioxidant activity. From this, it is considered that this is a completely new compound that inhibits the reversal of the glutamate transporter.
Since the compound of the present invention has an action of inhibiting the release of glutamic acid classified as an excitatory amino acid among neurotransmitters, it is used as a therapeutic agent for convulsions, epilepsy, analgesia, migraine, brain dysfunction and the like. be able to.
When the compound of the present invention is administered to humans for the purpose of treating or preventing the above-mentioned diseases, it can be administered orally as a powder, granule, tablet, capsule, pill, liquid, etc., or as an injection, suppository, It can be administered parenterally as a skin absorbent, inhalant and the like. In addition, excipients, binders, wetting agents, disintegrants, lubricants and the like suitable for the dosage form may be mixed with an effective amount of this compound as necessary to obtain a pharmaceutical preparation. it can. In the case of an injection, it is sterilized with an appropriate carrier to obtain a preparation.
The dose varies depending on the disease state, administration route, patient age, or body weight, but is usually 0.1 to 100 mg / kg / day, preferably 1 to 20 mg / kg / day when orally administered to an adult. Day.
Hereinafter, the present invention will be described in more detail with reference to Examples and Test Examples, but the present invention is not limited thereto.
In the examples, the following abbreviations are used.
Me: methyl
Et: ethyl
DMSO: Dimethyl sulfoxide
Example
Example 1
(3,5-dichloro-2,6-dimethoxy-phenyl)-(1H-pyrrol-2-yl) -methanone (compound (1)) and (3,5-dichloro-2,6-dimethoxy-phenyl)- Preparation of (1H-pyrrol-3-yl) -methanone (compound (2))
3,5-Dichloro-2,6-dimethoxybenzoic acid (1.02 g, 4.07 mmol) was dissolved in thionyl chloride (3 ml) and stirred at reflux for 30 minutes, and then the solvent was distilled off under reduced pressure to obtain under ice cooling. A dichloroethane solution (10 ml) of pyrrole (0.42 ml, 6.05 mmol) was added to the obtained 3,5-dichloro-2,6-dimethoxybenzoic acid chloride. Next, a methylene chloride suspension (10 ml) of aluminum chloride (0.60 g, 4.49 mmol) was added to the reaction mixture under ice cooling, and the mixture was stirred at room temperature for 1.5 hours. The reaction mixture was poured into ice and extracted with methylene chloride. . The organic layer was washed with saturated brine, dried over magnesium sulfate, and the residue obtained by evaporating the solvent under reduced pressure was subjected to silica gel column chromatography (
Compound (3) to compound (11) were synthesized in the same manner. The results are shown in Tables 1 and 2.
(2,6-dimethoxy-phenyl)-(1H-pyrrol-2-yl) -methanone (compound (12)), (2,6-dimethoxy-phenyl)-(1H-pyrrol-3-yl) -methanone ( Preparation of compound (13))
Pyrrole (5 ml, Wako Pure Chemical) and 2,6-dimethoxybenzoyl chloride (10 g, Aldrich) are dissolved in chloroform (100 ml) and stirred under ice cooling. Chloroform (100 ml) and anhydrous aluminum chloride (8.29 g, Nacalai Tesque special grade) are added and stirred for 15 minutes under ice cooling. The mixture is further stirred at room temperature for 2 hours. Water (100 ml) was added with stirring under ice-cooling, and the chloroform layer was washed with water, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure to obtain a crude product (12.0 g). The crude product (12.0 g) was purified by silica gel column chromatography (Merck,
Compound (12):
EIMS, m / z: 231 (M) + (base peak)
IR, λmax KBr cm -1 : 3275,3111,2937,2837,1615,1592,1546,1473,1431,1403,1334,1305,1282,1253,1129,1112,1032,891,875,855,786,758,745,721,641,602
1 H NMR (CDCl Three , 300MHz) δ: 3.74 (6H, s), 6.24 (1H, m), 6.55 (1H, m), 6.61 (2H, d, J = 8.4Hz), 7.07 (1H, m), 7.32 (1H, t , J = 8.4Hz), 9.46 (1H, br.s)
Compound (13):
EIMS, m / z: 231 (M) + , 94 (base peak)
IR, λmax KBr cm -1 : 3206,2958,2840,1617,1592,1539,1505,1471,1430,1403,1339,1306,1282,1252,1184,1137,1110,1051,1027,981,979,783,754,743,719,683,605
1 H NMR (d 6 -DMSO, 300 MHz) δ: 3.65 (6H, s), 6.33 (1H, m), 6.79 (1H, m), 6.70 (2H, d, J = 8.4Hz), 6.97 (1H, br.s), 7.32 (1H, t, J = 8.4Hz), 11.33 (1H, br.s)
13 C NMR (d 6 -DMSO, 50 MHz) δ: 55.50,104.14,107.74,119.64,119.75,125.43,126.30,129.76,156.56,187.70
Example 11
(2-hydroxy-6-methoxy-phenyl)-(1H-pyrrol-2-yl) -methanone (compound (14)), (2,6-dihydroxy-phenyl)-(1H-pyrrol-3-yl)- Preparation of methanone (compound (15))
Compound (12) (116 mg, 0.5 mM) is dissolved in dichloromethane (5 ml) and stirred while cooling at −70 ° C. Add 1.0 M boron tribromide-dichloromethane solution (1.1 ml, manufactured by Aldrich) and stir for 3 hours while cooling at -70 ° C. The mixture is further stirred at room temperature for 17.5 hours. The mixture was poured into ice water, the dichloromethane layer was removed, and the mixture was further extracted with diethyl ether. The organic layers were combined, washed with water, dried over anhydrous sodium sulfate, and evaporated under reduced pressure to obtain a crude product (90 mg). The crude product was purified by thin layer silica gel plates (Merck, Pre-Coated TLC Plates, SILICA GEL F-254, 0.5 mm, toluene: acetone = 5: 3), and compound (14) (12 mg), compound (15) (75 mg) was obtained respectively.
Compound (16) and compound (17) were synthesized in the same manner. The results are shown in Table 3.
(3-chloro-2,6-dimethoxy-phenyl)-(4-chloro-1H-pyrrol-2-yl) -methanone (compound (18)), (3-chloro-2,6-dimethoxy-phenyl)- Preparation of (4,5-dichloro-1H-pyrrol-2-yl) -methanone (compound (19))
While stirring a solution of compound (12) (0.46 g, 1.99 mM) in chloroform (10 ml) under ice-cooling, N-chlorosuccinimide (0.616 g, 4.98 mM) was added and stirred for 1 hour under ice-cooling. To do. The mixture is further stirred at room temperature for 3 hours. A 10% aqueous sodium bisulfite solution (6 ml) was added, and the mixture was stirred for 10 minutes. The chloroform layer was washed with water and dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure to obtain a crude product (0.68 g). The crude product (0.68 g) was purified by silica gel column chromatography (Merck, LiChroprep Si60, Size B, toluene: ethyl acetate = 14: 1 to 9: 1), and compound (18) (73 mg), compound (19) (200 mg) was obtained respectively.
Using compounds (1) and (3) as starting materials, compounds (20) and (21) were synthesized in the same manner. The results are shown in Table 4.
Preparation of (3-Chloro-2,6-dihydroxy-phenyl)-(4-chloro-1H-pyrrol-2-yl) -methanone (compound (22))
Compound (18) (50 mg) is dissolved in dichloromethane (8 ml) and stirred under ice cooling. Add 1.0 M boron tribromide-dichloromethane solution (0.8 ml, manufactured by Aldrich) and stir for 2 hours under ice cooling. The mixture is further stirred at room temperature for 16 hours. Empty in ice water, remove the dichloromethane layer, extract with ethyl acetate, combine the organic layers, wash with water, dry over anhydrous sodium sulfate, evaporate the solvent under reduced pressure, and recrystallize the residue with ethyl acetate: n-hexane. Compound (22) (42 mg) was obtained as pale yellow needles.
Compounds (23) and (24) were synthesized in the same manner using compounds (19) and (3) as starting materials. The results are shown in Table 5.
(3-chloro-2-hydroxy-6-ethoxycarbonylmethoxy-phenyl)-(4-chloro-1H-pyrrol-2-yl) -methanone (compound (25)), (3-chloro-2,6-di-) Ethoxycarbonylmethoxy-phenyl)-(4-chloro-1H-pyrrol-2-yl) -methanone (compound (26)), (3-chloro-2,6-diethoxycarbonylmethoxy-phenyl)-(N-ethoxy Preparation of carbonylmethyl-4-chloro-1H-pyrrol-2-yl) -methanone (compound (27))
To a solution of compound (22) (50 mg) in N, N-dimethylformamide (1.75 ml) was added potassium carbonate (31.8 mg) and ethyl bromoacetate (76.9 mg), and the mixture was heated to 80 ° C. in an oil bath under a nitrogen stream. And stir for 7 hours. Water (2 ml) is added to the reaction solution, and then neutralized with 1N hydrochloric acid. Next, the mixture was extracted twice with ethyl acetate (5 ml). The ethyl acetate layer was washed with water and dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure to obtain a crude product (87 mg). The crude product was purified on a thin layer silica gel plate (Merck, Pre-Coated TLC Plates, SILICA GEL F-254, 0.5 mm, chloroform: methanol = 50: 1), and compound (25) (8 mg) as a pale yellow prism, Compound (26) (40 mg) and compound (27) (28 mg) were obtained as colorless oils, respectively.
Compounds (28) to (33) were synthesized in the same manner using compounds (23), (14) and (15) as starting materials. The results are shown in Tables 6-7.
Preparation of (3-bromo-2,6-diethoxycarbonylmethoxy-phenyl)-(4,5-dibromo-1H-pyrrol-2-yl) -methanone (compound (34))
To a solution of compound (33) (78.0 mg, 0.208 mmol) in acetic acid (5 ml) was added bromoacetic acid solution (0.64 M, 1 ml), and the mixture was stirred at room temperature for 4 hours. Hypowater was added to the reaction solution, and the mixture was extracted with chloroform. The organic layer was washed with saturated brine and dried over magnesium sulfate, and the residue obtained by distilling off the solvent under reduced pressure was subjected to silica gel column chromatography (Merck). And eluted with a mixed solvent of ethyl acetate: hexane = 1: 2 to obtain Compound (34) (75.0 mg, 59%).
Melting point: 165-166 ° C
Elemental analysis (C 19 H 18 Br Three NO 7 ・ 1 / 4H 2 As O)
Calculated values: C, 37.01; H, 3.02; Br, 38.88; N, 2.27 (%)
Experimental value: C, 36.89; H, 3.06; Br, 38.65; N, 2.30 (%)
1 H-NMR (CDCl Three ) Δ: 1.27 (6H, t, J = 7,0Hz), 4.17-4.30 (4H, m), 4.59 (2H, s), 4.62 (2H, s), 6.55 (1H, d, J = 9.0Hz) , 6.76 (1H, d, J = 2.6Hz), 7.56 (1H, d, J = 9.0Hz), 9.76 (1H, br s).
Example 27
Preparation of (3,5-dibromo-2,6-dimethoxy-phenyl)-(1-methyl-1H-pyrrol-2-yl) -methanone (compound (35))
To 60% sodium hydride (24.8 mg, 0.620 mmol) was added a solution of compound (3) (214 mg, 0.549 mmol) in dimethylformamide (4 ml) under ice cooling, followed by stirring for 10 minutes, and then methyl iodide (244 mg, 1.65 mmol). Of dimethylformamide (1 ml) was added, and the mixture was further stirred at room temperature for 3 hours. Water was added to the reaction solution under ice-cooling, and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated brine and dried over magnesium sulfate, and the solvent was distilled off under reduced pressure.
Compound (36) was synthesized in the same manner using Compound (18) as a starting material. The results are shown in Table 8.
Preparation of (3-chloro-2,6-dihydroxycarbonylmethoxy-phenyl)-(4-chloro-1H-pyrrol-2-yl) -methanone (compound (37))
To a solution of compound (26) (20 mg) in dioxane (0.8 ml) is added 1N aqueous sodium hydroxide solution (0.2 ml), and the mixture is stirred at room temperature for 2.6 hours under a nitrogen stream. Ethyl acetate (10 ml) and water (10 ml) are added, and the mixture is adjusted to acidic (pH 2.0) with 1N aqueous hydrochloric acid. The ethyl acetate layer was washed with water and dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. Ethyl acetate and n-hexane were added to the residue to obtain Compound (37) (20 mg) as a colorless powder.
Using compounds (27), (28) and (36) as starting materials, compounds (38), (39) and (40) were synthesized in the same manner. The results are shown in Table 9.
Preparation of (2,6-dimethoxy-phenyl) -2-pyridyl-methanone (compound (41))
2-Bromopyridine (8 g, Wako Pure Chemicals special grade) is added to a 1.66 M n-butyllithium-n-hexane solution (30 ml, manufactured by Aldrich) with stirring at -70 ° C. and stirred. After 20 minutes, a diethyl ether solution (80 ml) of 2,6-dimethoxybenzonitrile (6.65 g, manufactured by Aldrich) is added, and the mixture is stirred for 2 hours while cooling at -70 ° C. The mixture is further stirred at room temperature for 3 hours. Water-saturated diethyl ether was added under ice cooling, and the precipitate was collected by filtration to obtain a crude product (11.0 g). The crude product (11.0 g) is partitioned between ethyl acetate (300 ml) and 0.5N hydrochloric acid (300 ml). The aqueous layer was further extracted with ethyl acetate (300 ml), and the ethyl acetate was combined and the solvent was distilled off under reduced pressure to obtain a residue (2.0 g). The obtained residue was purified by silica gel column chromatography (Merck,
Physical constants are shown in Table 10.
Example 34
(3-chloro-2,6-dimethoxy-phenyl) -2-pyridyl-methanone (compound (42)), (3,5-dichloro-2,6-dimethoxy-phenyl) -2-pyridyl-methanone (compound ( 43))
While stirring a solution of compound (41) (122 mg) in chloroform (3 ml) under ice-cooling, N-chlorosuccinimide (203 mg) is added and stirred for 4 hours under ice-cooling. Stir at room temperature. After 12 hours N-chlorosuccinimide (135 mg) is added and after another 24 hours N-chlorosuccinimide (135 mg) is added. After a total of 36 hours of stirring at room temperature, 10% aqueous sodium bisulfite solution (3 ml) was added, and the mixture was stirred for 10 minutes. The chloroform layer was washed with water and dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure to obtain a crude product (295 mg). It was. The crude product (0.68 g) was purified by silica gel column chromatography (
Physical constants are shown in Table 10.
Preparation of (3,5-dibromo-2,6-dimethoxy-phenyl) -2-pyridyl-methanone (compound (44))
While stirring a solution of compound (41) (122 mg) in chloroform (5 ml) at room temperature, N-bromosuccinimide (445 mg) is added and stirred at room temperature for 24 hours. A 10% aqueous sodium bisulfite solution (3 ml) was added, and the mixture was stirred for 10 minutes. The chloroform layer was washed with water and dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure to obtain a crude product (239 mg). The crude product (239 mg) was purified by silica gel column chromatography (
Table 11 shows the physical constants.
Example 36
Preparation of (2,6-dimethoxy-phenyl) -3-pyridyl-methanone (compound (45))
Nicotinic acid chloride hydrochloride (3.56 g, manufactured by Aldrich), N, O-dimethylhydroxylamine hydrochloride (1.99 g, manufactured by Aldrich), triethylamine (6.07 g, special grade manufactured by Wako Pure Chemical Industries), tetrahydrofuran (20 ml) The solution is heated to reflux for 3 hours. Add chloroform (50 ml) at room temperature and stir for 3 hours. Dichloromethane: diethyl ether (1: 1) solution (150 ml) and then saturated brine (30 ml) were added to the reaction solution, and the mixture was separated. The organic layer was dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure to remove nicotine. An acid amide derivative (3.18 g) was obtained. Next, 1.66M n-butyllithium-n-hexane solution (1.2 ml, manufactured by Aldrich) is added to a tetrahydrofuran (2 ml) solution of meta-dimethoxybenzene (138 mg, manufactured by Aldrich), and the mixture is stirred at room temperature. After 1 hour, a solution of the previously prepared nicotinamide derivative (284 mg) in tetrahydrofuran (2 ml) is added dropwise to the reaction solution, and the mixture is stirred for 1.5 hours. Water saturated diethyl ether (2 ml) and then water (2 ml) were added to the reaction solution, and the obtained organic layer was dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure to obtain a residue (350 mg). The residue was purified by silica gel column chromatography (
Table 11 shows the physical constants.
Example 37
Preparation of (3,5-dichloro-2,6-dimethoxy-phenyl) -3-pyridyl-methanone (compound (46))
While stirring a solution of compound (45) (97 mg) in chloroform (4 ml) at room temperature, N-chlorosuccinimide (534 mg) is added. After stirring for 4 days, 10% aqueous sodium bisulfite solution (3 ml) was added, and after stirring for 10 minutes, the chloroform layer was washed with water, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure to obtain a crude product (213 mg). The crude product was purified by silica gel column chromatography (Merck Kieselgel, 0.040 to 0.063 mm, 30 g, acetone: n-hexane = 1: 3) to obtain Compound (46) (77 mg).
Table 11 shows the physical constants.
Inhibition of glutamate release (in vitro)
Eight days of SD (Sprague Dawley (Japan SLC, Shizuoka)) rat cerebellum was isolated from 20 rats, treated with 0.25% trypsin solution and 1 mM EDTA · 4Na solution at 37 ° C for 10 minutes, and the cells were pipetted. To disperse. Pass a nylon mesh (Cell Strainer 70 μm Nyron, FALCON) to remove large tissue mass. Disperse the cells dispersed in 20 24-well culture dishes whose surfaces are coated using poly-L-Lysine. The medium is an α-MEM medium supplemented with 10% bovine serum and 25 mM KCl. 24-48 hours after dispensing the cells into a 24-well culture dish, a final concentration of 10 μM cytosine β-D-arabinofuranoside is added to remove proliferating cells. Thereafter, the medium was changed every 3 days, and cerebellar granule cells at 1 and 2 weeks were used for the experiment (Manual of the Nervous System, pages 203-206, 1989 Alan R. Liss, Inc.). Various concentrations of compounds are added to the Krebs-Ringer solution and added at 37 ° C. for 10 minutes and preincubated. Further, glutamic acid released to the outside of the cell was measured by adding veratridine and treating at 37 ° C. for 10 minutes. As a measurement method, cells in a 24-well culture dish are washed twice with 1 ml of Krebs-Ringer solution, 500 μl of Krebs-Ringer solution containing the sample is added, and the cells are treated at 37 ° C. for 10 minutes. Take 250 μl and use the method of measuring the concentration of glutamic acid released from cells by HPLC using o-phthalaldehyde described in Life Sciences 43, pages 913-922, 1988. It was.
Test example 2
Glutamate release inhibitory effect (slice)
The hippocampus of 4-week-old SD rats was extracted, sliced to a thickness of 0.35 mm, and left in Krebs-Ringer solution containing glucose at 24 ° C. loaded with oxygen for 1 hour before use in the experiment. The above hippocampal slice was purged with nitrogen, oxygen was removed, glucose was removed, and the mixture was placed in a 37 ° C. Krebs-Ringer solution. After incubation for a certain period of time, the glutamic acid concentration released in the Krebs-Ringer solution was determined by the HPLC method described above. Measured by.
Test example 3
Cerebral ischemia experiment using middle cerebral artery ligation model
SD rat is ligated for 1 hour to create a partial cerebral ischemia model. One hour later, the ligated blood vessel is restarted, and the brain after 18 hours is removed and divided into infarcted areas, and the wet weight of the specimen is measured. Furthermore, the moisture of the specimen was completely removed, the dry weight was measured, and the moisture content of the tissue was measured. More specifically, the sample compound is directly administered into the brain of the rat in 5 nmol / 5 μl (0.5% DMSO / saline), and then the middle cerebral artery is plugged to stop blood flow. After 1 hour, reopen (remove the obturator). After 24 hours, remove the brain by decapitation, blood removal, and blood perfusion. Then, each part in the brain is taken (right anterior cerebral artery perfusion area cerebral cortex, right cerebral artery perfusion area cerebral cortex, right striatum). After each wet weight was measured, it was completely dried with a dryer, the weight was measured, and the water content was calculated.
As can be seen from FIG. 1, each specimen suppressed the release of glutamic acid in a dose-dependent manner. From this result, IC 50 The value was calculated.
As can be seen from FIG. 2, all the analyte compounds suppressed the release of glutamate released from hippocampal slices under experimental ischemic conditions.
As can be seen from FIG. 3, the increase in water content was significantly suppressed in the group administered with the compound (18). Since it is known that the water content of the tissue is markedly increased (edema) in the brain region affected by ischemic injury, it can be seen that the administration of this compound improves the cerebral ischemic injury.
Formulation example
10 mg of compound represented by general formula (I)
Starch 24mg
Lactose 12mg
Hydroxypropylcellulose 0.8mg
Magnesium stearate 0.4mg
Industrial applicability
Disclosed is an aromatic ketone derivative having an action of preventing nerve cell damage due to excessive release of glutamic acid by inhibiting the release of intracellular glutamic acid to the outside of the cell.
Claims (6)
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