JP4382898B2 - Peroxide derivatives with antimalarial activity - Google Patents

Description

［式中、Ｃは炭素数１〜６の直鎖状または分枝状の低級アルキル基を置換基として有しても良い炭素数３〜１２の単環の脂環式炭化水素環基またはアマダンチリデン基、ｎは０〜６の整数を示す。］
【０００５】
上記一般式（１）において、Ｃの置換基を有してもよい脂環式炭化水素環基としては、例えばシクロプロピリデン、シクロブチリデン、シクロペンチリデン、シクロヘキシリデン、シクロへプチリデン、シクロオクチリデン、シクロノニリデン、シクロデシリデン、シクロウンデシリデン、シクロドデシリデン基等の炭素数３〜１２の単環の脂環式炭化水素基；ビシクロブチリデン、ビシクロオクチリデン、ビシクロノニリデン、ノルボルニリデン、ノルボレニリデン、アダマンチリデン、ノルアダマンチリデン基等の架橋環又は多環の脂環式炭化水素基等が挙げられ、好ましくは炭素数６〜１２の単環の脂環式炭化水素基又はアダマンチリデン基であり、より好ましくはシクロヘキシリデン、シクロドデシリデン又はアダマンチリデン基である。
また、Ｃの脂環式炭化水素環基が有してもよい置換基としては、例えばメチル、エチル、ｎ−プロピル、ｉ−プロピル、ｎ−ブチル、ｉ−ブチル、ｓｅｃ−ブチル、ｔｅｒｔ−ブチルとして例示される直鎖又は各種分枝したペンチル基等の炭素数１〜６の直鎖状又は分枝状の低級アルキル基であり、より好ましくはｔｅｒｔ−ブチル基である。
本発明化合物のうち、好ましい化合物としては一般式（１）において、Ｃが置換基として炭素数１〜６の直鎖状又は分枝状の低級アルキル基を有しても良い脂環式炭化水素環基である化合物であり、より好ましくはＣが４−ｔｅｒｔ−ブチルシクロヘキシリデン、シクロドデシリデン又はアダマンチリデン基であり、ｎが１〜４である化合物である。
【０００６】
【発明の実施の形態】
以下、本発明の実施の形態について詳細に説明する。
上記一般式（１）で表される本発明化合物は下記の方法により製造できる。
【化３】

［式中、Ｃ及びｎは上記に同じ。Ｘはハロゲン原子を示す。］
上記反応工程式中、Ｘで表されるハロゲン原子としては、フッ素原子、塩素原子、臭素原子、ヨウ素原子であり、好ましくは臭素原子、ヨウ素原子である。
【０００７】
＜反応工程（ｉ）＞
本反応工程は、Ｊ．Ｏｒｇ．Ｃｈｅｍ．，６２，４９４９（１９９７）記載の方法に準じて行われる。すなわち、一般式（２）で表される公知化合物を過酸化水素存在下、適当な溶媒中でオゾンと反応させることにより一般式（３）で表されるビスヒドロペルオキシド化合物を得る。本工程で用いられる溶媒としては反応に関与しないものであれば特に制限はなく、エーテル、テトラヒドロフラン、アセトニトリル等を例示でき、好ましくはエーテルである。過酸化水素は３０〜１００％のものが使用できる。反応に際しては、化合物（２）に対して、過酸化水素を１〜１０倍モル量、好ましくは１〜３倍モル量使用し、オゾンを０．５〜５倍モル量、好ましくは１〜２倍モル量使用する。反応温度は−７０〜２０℃であり、反応時間は５〜３０分である。得られた化合物（３）は、通常の分離手段、例えばカラムクロマトグラフィー、再結晶等により反応混合物から容易に単離精製することが出来る。
上記反応工程（ｉ）で得られた化合物（３）は単離又は単離することなく反応工程（ｉｉ）に使用できる。
【０００８】
＜反応工程（ｉｉ）＞
上記反応工程（ｉ）で得られた化合物（３）と一般式（４）で表される化合物を、塩基存在下、適当な溶媒中で反応させることにより、一般式（１）で表される本発明化合物を得る。
本工程で使用される塩基としては、例えば水酸化カリウム、水酸化ナトリウム、水酸化セシウム等のアルカリ金属の水酸化物；ナトリウムメトキシド、ナトリウムエトキシド等のアルカリ金属アルコキシド、またトリエチルアミン、ジイソプロピルエチルアミン等の第三級アミンが用いられ、好ましくは水酸化セシウムである。溶媒としては、非水溶媒であれば特に制限はないが、特にジメチルホルムアミド、ジメチルスルホキシドの様な極性の高いものが好まれる。また１８−クラウン−６の様なクラウンエーテル類を反応促進剤として添加することも可能である。反応に際しては、化合物（３）に対して化合物（４）及び塩基をそれぞれ１〜３倍モル使用する。反応促進剤を添加する場合には、化合物（３）に対して１〜１０モル量を使用する。反応温度は０〜５０℃、好ましくは１０〜３０℃であり、反応時間は１〜４８時間である。得られた化合物（１）は、通常の分離手段、例えばカラムクロマトグラフィー、再結晶等により反応混合物から容易に単離精製することが出来る。
【０００９】
本発明化合物をマラリア原虫類による感染症の予防及び治療に使用する場合、投与経路としては、経口、皮下注射、静脈注射、局所投与等のいずれでもよい。また、製剤としては、通常、製薬的に許容される担体や賦形剤、その他添加剤を用いて製造した散剤、錠剤、細粒剤、丸剤、カプセル剤、顆粒剤等の経口剤、点眼剤、注射剤、坐剤等の非経口剤が挙げられる。製薬的に許容される担体や賦形剤、その他添加剤としては、グルコース、ラクトース、ゼラチン、マンニトール、でんぷんペースト、トリケイ酸マグネシウム、コーンスターチ、ケラチン、コロイド状シリカ等があり、さらには、安定剤、増量剤、着色剤及び芳香剤の様な補助剤を含有してもよい。これらの製剤は、各々当業者に公知慣用の製造方法により製造できる。
本発明化合物の製剤中の配合量としては、０．１〜１００重量％が好ましく、さらに好ましくは０．１〜８０重量％であり、０．１〜５０重量％が好適である。
また、１日当たりの投与量は、患者の症状、体重、年齢、性別等によって異なり一概に決定できないが、通常成人１日当り本発明化合物を０．１〜１０００ｍｇ、好ましくは１〜６００ｍｇを１回又は２〜４回程度に分けて投与するのが好ましい。
【００１０】
【実施例】
次に本発明を製造例、実施例、試験例により具体的に説明する。
＜製造例１＞（シクロドデシリデン）ビスヒドロペルオキシド（化合物ａ）の合成
斉藤等の方法（Saito，I.；Nagata，R.；Yuba，K.；Matuura，T．Tetrahedron Lett．1983，24，1737）で調整した過酸化水素の２．５ｍｏｌエーテル溶液２５ｍｌに公知化合物であるメトキシメチレンシクロドデカン６３０ｍｇ（３．００ｍｍｏｌ）を溶かし、−７０℃でオゾン化を行った（通常のオゾン化装置（Ｎｉｐｐｏｎ Ｏｚｏｎｅ Ｍｏｄｅｌ ＯＮ−１−２（日本オゾン株式会社製））を使い、１５分間５０ｌ／ｈｒの流速で酸素を吹き込むことにより、使用したメトキシメチレンシクロドデカンと等量のオゾンを発生させた)。反応終了後、７０ｍｌのエーテルを加え、有機層を重曹水、次いで飽和食塩水で洗った後、無水硫酸マグネシウムで乾燥させた。次いで、シリカゲルカラムクロマトグラフィー（留出液：エーテル−ヘキサン、２：８)により、標記化合物ａを２３２ｍｇ（収率３３％）得た。物性値を以下に示す。
融点：１４０−１４１℃
^１Ｈ ＮＭＲ（ＣＤＣｌ_３）δ： 1.2-1.8 (m, 22 H), 8.13 (br s, 2 H)
^１３Ｃ ＮＭＲ（ＣＤＣｌ_３）δ： 19.28, 21.86, 22.15, 6.02, 26.19, 26.29, 112.64.
【００１１】
＜製造例２＞（４−ｔｅｒｔ−ブチルシクロヘキシリデン）ビスヒドロペルオキシド（化合物ｂ）の合成
製造例１と同様に調整した過酸化水素を含むエーテル溶液２５ｍｌに４−ｔｅｒｔ−ブチル−２−メトキシメチレンシクロヘキサン５４６ｍｇ（３．００ｍｍｏｌ）を溶かし、−７０℃でオゾン化を行った。反応終了後、上記製造例１と同様の方法で処理した後、シリカゲルカラムクロマトグラフィー（留出液：エーテル−ヘキサン、２：８）により、標記化合物ｂを２８５ｍｇ（収率４７％）得た。以下に物性値を示す。
融点：８３−８４℃(エーテル−ヘキサン)
^１Ｈ ＮＭＲ(ＣＤＣｌ_３)δ： 0.87 (s, 9 H), 1.1-1.8 (m, 9 H), 9.27 (s, 2 H)
^１３Ｃ ＮＭＲ(ＣＤＣｌ_３)δ： 23.32, 27.58, 29.70, 32.31, 47.39, 110.00.
元素分析：Anal.Calcd. for C₁₀H₂₀O₄: C, 58.80; H, 9.87. Found: C, 58.87; H, 9.80.
【００１２】
＜製造例３＞（２−アダマンチリデン）ビスヒドロペルオキシド（化合物ｃ）の合成
製造例１と同様に調整した過酸化水素を含むエーテル溶液２５ｍｌに２−メトキシメチレンアダマンタン７１２ｍｇ（４．００ｍｍｏｌ）を溶かし、−７０℃でオゾン化を行った。反応終了後、上記製造例１と同様の方法で処理した後、シリカゲルカラムクロマトグラフィー（留出液：エーテル−ヘキサン、２：８）により、標記化合物ｃを３３５ｍｇ（収率４２％）得た。以下に物性値を示す。
融点：１４４−１４５℃(エーテル−ヘキサン)
^１Ｈ ＮＭＲ (ＣＤＣｌ_３)δ：1.7-2.1 (m, 14 H), 8.82 (s, 2 H)
^１３Ｃ ＮＭＲ (ＣＤＣｌ_３)δ：26.94, 31.14, 33.68, 36.98, 112.88.
【００１３】
＜実施例１＞１，２，６，７−テトラオキサスピロ［７．１１］ノナデカン（化合物１）の合成
アルゴン雰囲気下で、水酸化セシウムの一水塩５０４ｍｇ（３．００ｍｍｏｌ）のジメチルホルムアミド溶液１０ｍｌを調整した。この溶液に、シリンジを用いて、製造例１で得られた化合物ａ３４８ｍｇ（１．５０ｍｍｏｌ）のジメチルホルムアミド溶液５ｍｌを、次いで１，３−ジヨードプロパン６６６ｍｇ（２．２５ｍｍｏｌ）のジメチルホルムアミド溶液５ｍｌを、０℃で、それぞれ１０分間かけて加えた。その後、室温で１６時間撹拌した。反応終了後、７０ｍｌのエーテルを加え、有機層を重曹水、次いで飽和食塩水で洗った後、無水硫酸マグネシウムで乾燥させた。溶媒を減圧で留去後、残渣のシリカゲルカラムクロマトグラフィー（留出液：エーテル−ヘキサン、１：２５）により、標記化合物１を１６４ｍｇ（収率４０％）得た。以下に物性値を示す。
融点：８３−８４℃(ヘキサン)
^１Ｈ ＮＭＲ(ＣＤＣｌ_３)δ：1.3-1.7 (m, 22 H), 2.1-2.2 (m, 2 H), 4.12 (dt, J = 12.5, 5.0 Hz, 2 H), 4.31 (dt, J = 12.5, 5.0 Hz, 2 H)
^１３Ｃ ＮＭＲ(ＣＤＣｌ_３)δ： 19.36, 21.90, 22.17, 25.91, 26.07, 26.25, 30.40, 73.94, 112.13.
元素分析：Anal. Calcd. for C₁₅H₂₆O₄: C, 66.14; H, 10.36. Found: C, 65.91; H, 10.42.
【００１４】
＜実施例２＞１，２，６，７−テトラオキサスピロ［８．１１］イコサン（化合物２）の合成
製造例１で得られた化合物ａ３４８ｍｇ（１．５０ｍｍｏｌ）と水酸化セシウムの一水塩 ５０４ｍｇ（３．００ｍｍｏｌ）をジメチルホルムアミド２５ｍｌに溶かし、これに１，３−ジヨードブタン６９８ｍｇ（２．２５ｍｍｏｌ）を１０分間かけて加えた。その後、反応を室温で１６時間行った。反応終了後、上記実施例１と同様の処理法で反応混合物を処理した後、シリカゲルカラムクロマトグラフィー（留出液：エーテル−ヘキサン、１：２５）により、標記化合物２を１２９ｍｇ（収率３０％）得た。以下に物性値を示す。
融点：９７−９８℃(ヘキサン)
^１Ｈ ＮＭＲ(ＣＤＣｌ_３)δ：1.3-1.7 (m, 24 H), 2.2-2.3 (m, 2 H), 3.66 (t, J = 12.2 Hz, 2 H), 4.26 (dd, J = 12.2, 3.9 Hz, 2 H)
^１３Ｃ ＮＭＲ(ＣＤＣｌ_３)δ：19.30, 21.89, 22.17, 25.95, 26.11, 26.43, 30.93, 32.51, 73.85, 111.82 .
元素分析： Anal. Calcd. for C₁₆H₃₀O₄: C, 67.10; H, 10.56. Found: C, 66.66; H, 10.49 .
【００１５】
＜実施例３＞１，２，６，７−テトラオキサスピロ［９．１１］ヘニコサン（化合物３）の合成
水酸化セシウムの一水塩１００８ｍｇ（６．００ｍｍｏｌ）をジメチルホルムアミド溶液２０ｍｌに溶かし、これに製造例１で得られた化合物ａ６９６ｍｇ（３．００ｍｍｏｌ）を溶かしたジメチルホルムアミド溶液２０ｍｌ及び１，３−ジヨードペンタン１４５８ｍｇ（４．５０ｍｍｏｌ）を溶かしたジメチルホルムアミド溶液２０ｍｌを、別々のシリンジを用いて、０℃で１時間かけて加えた。その後、反応を室温で１６時間行った。反応終了後、上記実施例１と同様の処理法で反応混合物を処理した後、シリカゲルカラムクロマトグラフィー（留出液：エーテル−ヘキサン、１：２５）により、標記化合物３を１５３ｍｇ（収率１７％）得た。以下に物性値を示す。
融点：５１−５２℃(ヘキサン)
^１Ｈ ＮＭＲ(ＣＤＣｌ_３)δ：1.3-1.8 (m, 24 H), 19-2.1 (m, 4 H), 4.1-4.2 (m, 4 H)
^１３Ｃ ＮＭＲ(ＣＤＣｌ_３)δ：19.26, 21.92, 22.25, 24.89, 25.97, 26.06, 26.65, 28.45, 76.64, 110.89.
【００１６】
＜実施例４＞１，２，６，７−テトラオキサスピロ［８．１１］ドコサン（化合物４）の合成
水酸化セシウムの一水塩１００８ｍｇ（６．００ｍｍｏｌ）をジメチルホルムアミド溶液２０ｍｌに溶かし、これに製造例１で得られた化合物ａ６９６ｍｇ（３．００ｍｍｏｌ）を溶かしたジメチルホルムアミド溶液２０ｍｌ及び１，３−ジヨードヘキサン１５２１ｍｇ（４．５０ｍｍｏｌ）を溶かしたジメチルホルムアミド溶液２０ｍｌを、別々のシリンジを用いて、０℃で１時間かけて加えた。その後、反応を室温で１６時間行った。反応終了後、上記処理法で反応混合物を処理した後、シリカゲルカラムクロマトグラフィー（留出液：エーテル−ヘキサン、１：２５）により、標記化合物４を１６８ｍｇ（収率１８％）得た。以下に物性値を示す。
融点：４８−４９℃（ヘキサン)
^１Ｈ ＮＭＲ(ＣＤＣｌ_３)δ：1.3-1.8 (m, 30 H), 4.02 (t, J = 4.8 Hz, 4 H)^１３Ｃ ＮＭＲ(ＣＤＣｌ_３)δ：19.35, 21.89, 22.23, 25.00, 26.02, 26.13, 26.43, 26.99, 74.63, 112.06.
【００１７】
＜実施例５＞７，８，１２，１３−テトラオキサスピロ［６．８］トリデカン（化合物５）の合成
製造例２で得られた化合物ｂ１２５ｍｇ（０．６１ｍｍｏｌ）と水酸化セシウムの一水塩 ２０５ｍｇ（１．２２ｍｍｏｌ）をジメチルホルムアミド１５ｍｌに溶かし、これに１，３−ジヨードプロパン２７２ｍｇ（０．９２ｍｍｏｌ）を１０分間かけて加えた。その後、反応を室温で１６時間続けた。反応終了後、上記実施例１と同様の処理法で反応混合物を処理した後、シリカゲルカラムクロマトグラフィー（留出液：エーテル−ヘキサン、１：２５）により、標記化合物５を３８ｍｇ（収率２６％）得た。以下に物性値を示す。
融点：５８−５９℃（ヘキサン)
^１Ｈ ＮＭＲ(ＣＤＣｌ_３)δ：0.86 (s, 9 H), 1.1-1.8 (m, 9 H), 2.1-2.3 (m, 2 H), 4.10-4.2 (m, 2 H), 4.3-4.5 (m, 2 H)
^１３Ｃ ＮＭＲ(ＣＤＣｌ_３)δ：23.34, 23.61, 27.64, 28.59, 30.35, 31.93, 32.37, 47.44, 73.76, 74.02, 107.92.
元素分析：Anal. Calcd. for C₁₃H₂₄O₄ : C, 63.91; H, 9.90. Found: C, 63.60; H, 9.97.
【００１８】
＜実施例６＞スピロ［トリシクロ［３．３．１．１^３，７］デカン］２，３’−［１，２，４，５］テトロキソカン（化合物６）の合成
製造例３で得られた化合物ｃ３１３ｍｇ（１．５７ｍｍｏｌ）と水酸化セシウムの一水塩５２８ｍｇ（３．１４ｍｍｏｌ）をジメチルホルムアミド１５ｍｌに溶かし、これに１，３−ジヨードプロパン６９５ｍｇ（２．３５ｍｍｏｌ）を１０分間かけて加えた。その後、反応を室温で１６時間行った。反応終了後、上記処理法で反応混合物を処理した後、シリカゲルカラムクロマトグラフィー（留出液：エーテル−ヘキサン、１：２５）により、標記化合物６を７３ｍｇ（収率１９％）得た。以下に物性値を示す。
融点：３４−３５℃（ヘキサン)
^１Ｈ ＮＭＲ(ＣＤＣｌ_３)δ：1.6-2.3 (m, 16 H), 4.12 (dt, J = 12.9, 5.6 Hz, 2 H), 4.35 (dt, J = 12.5, 4.8 Hz, 2 H)
^１３Ｃ ＮＭＲ(ＣＤＣｌ_３)δ：27.06, 30.30, 31.81, 33.67, 33.98, 37.25, 73.94, 109.95.
【００１９】
＜熱帯熱マラリア原虫の培養検定試験＞
本実験では、熱帯熱マラリア原虫としてP.falciparum FCR-3 strain（ATCC 30932)を用いた。また抗マラリア剤として市販されているクロロキンの耐性株に対する本発明化合物の効果を検証するためにP.falciparum K1 strainのクロロキン耐性マラリア原虫を用いた。実験に用いた培地は、濾過滅菌したRPMI1640培地で、ｐＨを７．４に合わせ、ヒト血清を１０％となるように添加した。マラリア原虫の培養はＯ_２濃度５％、ＣＯ_２濃度５％、Ｎ_２濃度９０％、温度は３６．５℃で行った。ヘマトクリット値（赤血球浮遊液中に占める赤血球の体積の割合）は５％にして用いた。培養開始時の熱帯熱マラリア原虫の初期感染率は０．１％とした。２４穴培養プレートを用いて培養し、培地は毎日交換し、感染率４％で植え継ぎを行った。感染率は薄層塗沫標本を作成し、ギムザ染色あるいはDiff-Qick染色を行った後、顕微鏡（油浸、１０００×）下で計測し、マラリア原虫感染率を下記式より算出した。
マラリア原虫感染率（％）＝｛（感染赤血球数）／（総赤血球数）｝×１００
【００２０】
＜試験例１＞マラリア原虫増殖阻害スクリーニング試験
培養したマラリア原虫感染赤血球を遠心で集め、血清を含む培地で洗浄を行った後、非感染赤血球を加え、初期感染率を０．３％とした。この時のヘマトクリット値は３％である。実験に用いるサンプルは滅菌水、ジメチルホルムアミド（ＤＭＦ）あるいはジメチルスルホキシド（ＤＭＳＯ）に溶解し、所定濃度のサンプルとした。
２４穴培養プレートにサンプルを５〜１０μｌずつ加えた。サンプルはduplicateあるいはtriplicateにとった。コントロールは滅菌水、ＤＭＦあるいはＤＭＳＯを１０μｌ／ウエル加えた。
次に、あらかじめ用意しておいた熱帯熱マラリア原虫培養液を９９０〜９９５μｌずつ加え、静かにピペッティングを行い培地に一様に懸濁させた。培養プレートはＣＯ_２−Ｏ_２−Ｎ_２（５％、５％、９０％）インキュベーター中で７２時間培養した後、それぞれのウエルについて薄層塗沫標本を作成し、染色した後、顕微鏡下で観察し、試薬を加えたものの感染率およびコントロールの感染率を算出した。
上記で求めたマラリア原虫感染率から次の式によって増殖率を算出することにより、マラリア原虫に対する５０％増殖阻害濃度（ＥＣ_５０）を求めた。結果を表１に示す。
増殖率（％）＝｛（［ｂ］−［ａ］）／（［ｃ］−［ａ］）｝×１００
ａ：初期感染率
ｂ：サンプル添加時の感染率
ｃ：サンプル非添加時（コントロール）の感染率
【００２１】
＜試験例２＞マウスＦＭ３Ａ細胞増殖阻害試験
マウス乳がん由来ＦＭ３Ａ細胞の野生株であるＦ２８−７株を用いた。培地はＥＳ培地に非働化した胎児牛血清を２％となるように添加し、ＣＯ_２濃度５％、３７℃で培養した。この条件下でのＦＭ３Ａ細胞の倍加時間は約１２時間であった。
前培養を行い、対数増殖期に入った細胞を５×１０^４ｃｅｌｌｓ／ｍｌになるように培地で希釈した。サンプルはマラリア原虫の抗マラリア活性測定時調製したものを用いた。２４穴培養プレートにサンプル溶液を５〜１０μｌずつ加えた（培地等を加えると最終濃度は１×１０^−４〜１×１０^−６となった）。化合物はduplicateあるいはtriplicateにとり、コントロールとして滅菌水、ＤＭＦあるいはＤＭＳＯを１０μｌ加えたウエルも同時に用意した。次に、用意しておいた培養細胞浮遊液を９９０〜９９５μｌずつ加え、静かにピペッティングを行い培地に一様に懸濁させた。４８時間培養した後、それぞれのウエルについて細胞数をセルコントローラー（ＣＣ−１０８、Ｔｏａ Ｍｅｄｉｃａｌ Ｅｌｅｃｔｒｉｃｓ社製）で計数し、下記式より増殖率を算出した。
増殖率（％）＝｛（［Ｃ］−［Ａ］）／（［Ｂ］−［Ａ］）｝×１００
Ａ:初期細胞数
Ｂ:４８時間後のコントロールの細胞数
Ｃ:サンプル添加した４８時間後の細胞数
細胞増殖阻害活性は、サンプルを添加したウエルの細胞数およびコントロールの細胞数から算出した。これにより、サンプルの細胞毒性を評価し、細胞増殖阻害濃度（ＥＣ_５０）で示した。ＥＣ_５０値とはマラリア原虫、あるいはＦＭ３Ａ細胞の培地にサンプルを添加していないコントロールの増殖率、あるいはマラリア原虫感染率を１００％とし、サンプル添加によってコントロールの増殖率を５０％阻害するサンプルの濃度のことである（モル濃度で表示する）。結果を表１に示す。
サンプルの抗マラリア作用は、ＦＭ３Ａ細胞に対するマラリア原虫のサンプルのＥＣ_５０値の比（化学療法係数、下記式参照）から評価し、薬効判定を行った。また、参考までに抗マラリア剤として利用されているクロロキンについても同様の試験を行った。結果を表１に示す。
化学療法係数＝（マウスＦＭ３Ａ細胞に対するサンプルのＥＣ_５０値）
÷（熱帯熱マラリア原虫に対するサンプルのＥＣ_５０値）
【００２２】
【表１】

【００２３】
以上の結果、本発明化合物は、細胞毒性が低く、マラリア原虫増殖阻害活性を有することが判明した。また、クロロキン耐性原虫に対しても効果が保持されていることが判明した。
【００２４】
＜試験例３＞ネズミマラリア原虫感染マウスを用いた原虫抑制試験
本実験はPeters，W．and Richrds，W．H．G．Antimalarial drugs Ｉ，in：W．Peters，W．H．G．Richards（Eds．），Springer-Verlag，Berlin，1984，pp．229-230に記載の4-day suppressive testに準じて行った。用いたネズミマラリア原虫（P.berghei NK65株）は強毒原虫株で、このネズミマラリア原虫を感染させるとマウスは感染してから１０日以内に全部死亡する。継代しているネズミマラリア原虫感染マウスの血中感染率が１０％となった時点で実験を開始した。エーテル麻酔を行ったマウスの心臓採血を行い、リン酸緩衝生理食塩水１ｍｌあたり５×１０^６原虫/ｍｌとなるように調製した原虫浮遊液を感染していないマウスに２００μｌ腹腔内感染（i.p.）した。原虫感染２時間後、オリーブ油に懸濁した化合物を経口投与又は腹腔内投与（化合物５は腹腔内投与のみ）した。投与期間は、１日１回を連続４日間行い、実験開始４日目にマウスの尾より採血する。採血した血液で薄層塗抹標本を作製し、ギムザ染色により顕微鏡下で赤血球感染率を求めた（上記の熱帯熱マラリア原虫の培養検定試験と同様の方法で行った）。溶媒のみを投与したコントロール群に対する薬剤投与群の感染率の割合を調べることで５０％原虫抑制濃度（ＥＤ_５０値）と９０％原虫抑制濃度（ＥＤ_９０値）を求めた。結果を表２に示す。
【００２５】
【表２】

【００２６】
以上の結果、本発明化合物は、優れたマラリア原虫増殖抑制活性を有することが判明した。
【００２７】
【発明の効果】
本発明化合物は優れた抗マラリア作用を有し、マラリア等の原虫類による感染症の予防及び治療薬として有用である。またクロロキン耐性マラリアに対しても有効である。
【図面の簡単な説明】
【図１】各製造例、実施例における化合物の化学構造式の対照表を示す表図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to novel compounds useful for the prevention and treatment of infectious diseases caused by malaria parasites.
[0002]
[Prior art]
Malaria is a contagious disease caused by infection with a protozoa belonging to the genus Plasmodium, which is transmitted through an anemone mosquito, and exhibits symptoms such as intermittent heat stroke, anemia, and splenomegaly. In recent years, malaria has begun to intensify with changes in nature and the environment. Its estimated number of infected patients is 300 to 500 million per year and the number of deaths per year is 1.5 to 3 million. It is a disease. Malaria parasites that infect humans include Plasmodium falciparum (P. falciparum) distributed throughout the tropical regions of Africa, Asia and Latin America, and Plasmodium falciparum distributed in parts of the tropics and temperate regions around the world ( P. vivax), protozoan malaria parasite (P. malariae) distributed in various parts of the world, and egg-shaped malaria parasite (P. ovale) mainly distributed in tropical West Africa, among which P. falciparum malaria is Shows the most severe symptoms, and develops severe malaria easily with encephalopathy, nephropathy, hemolytic anemia, pulmonary edema, heart failure, severe enterocolitis, etc. within 1 to 2 weeks after onset. Often shows failure and causes the host to die.
Representative drugs currently in use include chloroquine, primaquine, artemisinin, mefloquine, and pyrimesamine, but these drugs are often highly toxic and the emergence of protozoa resistant to many drugs. The spread of drug-resistant malaria has become a recent problem of chemotherapy. Quinine exists as the only effective drug for drug-resistant malaria, but it is extremely likely to cause renal failure and is a high-risk therapeutic agent in view of current medical standards. Under such circumstances, development of a new drug with high antimalarial activity and high safety is desired.
As compounds similar to the present invention, for example, organic peroxide compounds described in JP-B-59-46266, JP-A-8-67704 and the like are known, and these are used as initiators in the production of polymers. Only.
[0003]
[Problems to be solved by the invention]
An object of the present invention is to provide a novel compound having low toxicity and extremely high antimalarial activity.
[0004]
[Means for Solving the Problems]
As a result of intensive studies to solve such problems, the present inventors have found that a peroxide derivative represented by the following general formula (1) has extremely high antimalarial activity, and completed the present invention. It came to do.
That is, the present invention relates to a peroxide derivative represented by the general formula (1).
[Chemical formula 2]

[ Wherein C is a monocyclic alicyclic hydrocarbon ring group having 3 to 12 carbon atoms which may have a linear or branched lower alkyl group having 1 to 6 carbon atoms as a substituent, or amadantylidene. Group and n show the integer of 0-6. ]
[0005]
In the general formula (1), examples of the alicyclic hydrocarbon ring group which may have a C substituent include, for example, cyclopropylidene, cyclobutylidene, cyclopentylidene, cyclohexylidene, cycloheptylidene, cyclo Monocyclic alicyclic hydrocarbon groups having 3 to 12 carbon atoms such as octylidene, cyclononylidene, cyclodecylidene, cycloundecylidene, cyclododecylidene; bicyclobutylidene, bicyclooctylidene, bicyclononylidene, norbornylidene , Norborenylidene, adamantylidene, a bridged ring such as a noradamantylidene group or a polycyclic alicyclic hydrocarbon group, preferably a monocyclic alicyclic hydrocarbon group having 6 to 12 carbon atoms or an adamant A tylidene group, more preferably a cyclohexylidene, cyclododecylidene or adamantylidene group. That.
Examples of the substituent that the C alicyclic hydrocarbon ring group may have include, for example, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, sec-butyl, tert-butyl. And a straight chain or branched lower alkyl group having 1 to 6 carbon atoms such as a straight chain or various branched pentyl groups, more preferably a tert-butyl group.
Among the compounds of the present invention, a preferred compound is an alicyclic hydrocarbon in which, in general formula (1), C may have a linear or branched lower alkyl group having 1 to 6 carbon atoms as a substituent. A compound that is a cyclic group, more preferably a compound in which C is 4-tert-butylcyclohexylidene, cyclododecylidene, or adamantylidene group, and n is 1 to 4.
[0006]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, embodiments of the present invention will be described in detail.
The compound of the present invention represented by the general formula (1) can be produced by the following method.
[Chemical 3]

[Wherein, C and n are the same as above. X represents a halogen atom. ]
In the above reaction process formula, the halogen atom represented by X is a fluorine atom, a chlorine atom, a bromine atom or an iodine atom, preferably a bromine atom or an iodine atom.
[0007]
<Reaction step (i)>
This reaction step is described in J. Org. Org. Chem. 62 , 4949 (1997). That is, the bishydroperoxide compound represented by the general formula (3) is obtained by reacting the known compound represented by the general formula (2) with ozone in the presence of hydrogen peroxide in an appropriate solvent. The solvent used in this step is not particularly limited as long as it does not participate in the reaction, and examples thereof include ether, tetrahydrofuran, acetonitrile and the like, and ether is preferable. Hydrogen peroxide of 30 to 100% can be used. In the reaction, hydrogen peroxide is used in an amount of 1 to 10 times mol, preferably 1 to 3 times mol, and ozone is used in an amount of 0.5 to 5 times mol, preferably 1 to 2 with respect to compound (2). Use double molar amount. The reaction temperature is -70 to 20 ° C, and the reaction time is 5 to 30 minutes. The obtained compound (3) can be easily isolated and purified from the reaction mixture by ordinary separation means such as column chromatography, recrystallization and the like.
The compound (3) obtained in the above reaction step (i) can be used in the reaction step (ii) without isolation or isolation.
[0008]
<Reaction step (ii)>
The compound represented by the general formula (1) is obtained by reacting the compound (3) obtained in the reaction step (i) with the compound represented by the general formula (4) in an appropriate solvent in the presence of a base. The compound of the present invention is obtained.
Examples of the base used in this step include alkali metal hydroxides such as potassium hydroxide, sodium hydroxide and cesium hydroxide; alkali metal alkoxides such as sodium methoxide and sodium ethoxide; and triethylamine and diisopropylethylamine. And tertiary amines are used, preferably cesium hydroxide. The solvent is not particularly limited as long as it is a non-aqueous solvent, but a highly polar one such as dimethylformamide and dimethyl sulfoxide is particularly preferred. It is also possible to add a crown ether such as 18-crown-6 as a reaction accelerator. In the reaction, the compound (4) and the base are used in an amount of 1 to 3 moles per mole of the compound (3). When adding a reaction accelerator, 1-10 mol amount is used with respect to a compound (3). The reaction temperature is 0 to 50 ° C., preferably 10 to 30 ° C., and the reaction time is 1 to 48 hours. The obtained compound (1) can be easily isolated and purified from the reaction mixture by ordinary separation means such as column chromatography, recrystallization and the like.
[0009]
When the compound of the present invention is used for the prevention and treatment of infectious diseases caused by malaria parasites, the administration route may be any of oral, subcutaneous injection, intravenous injection, local administration, and the like. In addition, as preparations, oral preparations such as powders, tablets, fine granules, pills, capsules, granules, etc., usually produced using pharmaceutically acceptable carriers and excipients and other additives, eye drops And parenteral agents such as suppositories, injections, and suppositories. Examples of pharmaceutically acceptable carriers and excipients, and other additives include glucose, lactose, gelatin, mannitol, starch paste, magnesium trisilicate, corn starch, keratin, colloidal silica, and more. Adjuvants such as extenders, colorants and fragrances may be included. Each of these preparations can be produced by a conventional production method known to those skilled in the art.
As a compounding quantity in the formulation of this invention compound, 0.1 to 100 weight% is preferable, More preferably, it is 0.1 to 80 weight%, and 0.1 to 50 weight% is suitable.
The daily dose varies depending on the patient's symptoms, weight, age, sex, etc., and cannot be determined unconditionally, but usually 0.1 to 1000 mg, preferably 1 to 600 mg of the compound of the present invention per day for an adult or It is preferable to administer in 2 to 4 divided doses.
[0010]
【Example】
Next, the present invention will be specifically described with reference to production examples, examples and test examples.
<Production Example 1> Synthesis of (cyclododecylidene) bishydroperoxide (Compound a) Method by Saito et al. (Saito, I .; Nagata, R .; Yuba, K .; Matuura, T. Tetrahedron Lett. 1983, 24 , 1737), 630 mg (3.00 mmol) of methoxymethylenecyclododecane, a known compound, was dissolved in 25 ml of a 2.5 mol ethereal solution of hydrogen peroxide prepared in 1737) and ozonized at −70 ° C. (normal ozonizer ( Nippon Ozone Model ON-1-2 (manufactured by Nippon Ozone Co., Ltd.)) was blown with oxygen at a flow rate of 50 l / hr for 15 minutes to generate ozone equivalent to the methoxymethylenecyclododecane used). After completion of the reaction, 70 ml of ether was added, and the organic layer was washed with an aqueous sodium bicarbonate solution and then with a saturated saline solution, and then dried over anhydrous magnesium sulfate. Then, 232 mg (yield 33%) of the title compound a was obtained by silica gel column chromatography (distillate: ether-hexane, 2: 8). Physical property values are shown below.
Melting point: 140-141 ° C
¹ H NMR (CDCl ₃ ) δ: 1.2-1.8 (m, 22 H), 8.13 (br s, 2 H)
¹³ C NMR (CDCl ₃ ) δ: 19.28, 21.86, 22.15, 6.02, 26.19, 26.29, 112.64.
[0011]
<Production Example 2> Synthesis of (4-tert-butylcyclohexylidene) bishydroperoxide (compound b) 4-tert-butyl-2-methoxy was added to 25 ml of an ether solution containing hydrogen peroxide prepared in the same manner as in Production Example 1. Methylenecyclohexane 546 mg (3.00 mmol) was dissolved and ozonized at -70 ° C. After completion of the reaction, the reaction mixture was treated in the same manner as in Production Example 1, and then 285 mg (yield 47%) of the title compound b was obtained by silica gel column chromatography (distillate: ether-hexane, 2: 8). The physical property values are shown below.
Melting point: 83-84 ° C. (ether-hexane)
¹ H NMR (CDCl ₃ ) δ: 0.87 (s, 9 H), 1.1-1.8 (m, 9 H), 9.27 (s, 2 H)
¹³ C NMR (CDCl ₃ ) δ: 23.32, 27.58, 29.70, 32.31, 47.39, 110.00.
Elemental analysis: Anal.Calcd. For C ₁₀ H ₂₀ O ₄ : C, 58.80; H, 9.87. Found: C, 58.87; H, 9.80.
[0012]
<Production Example 3> Synthesis of (2-adamantylidene) bishydroperoxide (Compound c) To 25 ml of an ether solution containing hydrogen peroxide prepared in the same manner as in Production Example 1, 712 mg (4.00 mmol) of 2-methoxymethyleneadamantane was added. Dissolved and ozonized at -70 ° C. After completion of the reaction, the reaction mixture was treated in the same manner as in Production Example 1, and then 335 mg (yield 42%) of the title compound c was obtained by silica gel column chromatography (distillate: ether-hexane, 2: 8). The physical property values are shown below.
Melting point: 144-145 ° C. (ether-hexane)
¹ H NMR (CDCl ₃ ) δ: 1.7-2.1 (m, 14 H), 8.82 (s, 2 H)
¹³ C NMR (CDCl ₃ ) δ: 26.94, 31.14, 33.68, 36.98, 112.88.
[0013]
Example 1 Synthesis of 1,2,6,7-tetraoxaspiro [7.11] nonadecane (Compound 1) A dimethylformamide solution of 504 mg (3.00 mmol) of cesium hydroxide monohydrate under an argon atmosphere 10 ml was adjusted. To this solution, using a syringe, 5 ml of a dimethylformamide solution of 348 mg (1.50 mmol) of compound a obtained in Preparation Example 1, and then 5 ml of a dimethylformamide solution of 666 mg (2.25 mmol) of 1,3-diiodopropane were added. At 0 ° C. for 10 minutes each. Then, it stirred at room temperature for 16 hours. After completion of the reaction, 70 ml of ether was added, and the organic layer was washed with an aqueous sodium bicarbonate solution and then with a saturated saline solution, and then dried over anhydrous magnesium sulfate. After the solvent was distilled off under reduced pressure, 164 mg (yield 40%) of the title compound 1 was obtained by silica gel column chromatography of the residue (distillate: ether-hexane, 1:25). The physical property values are shown below.
Melting point: 83-84 ° C. (hexane)
¹ H NMR (CDCl ₃ ) δ: 1.3-1.7 (m, 22 H), 2.1-2.2 (m, 2 H), 4.12 (dt, J = 12.5, 5.0 Hz, 2 H), 4.31 (dt, J = (12.5, 5.0 Hz, 2 H)
¹³ C NMR (CDCl ₃ ) δ: 19.36, 21.90, 22.17, 25.91, 26.07, 26.25, 30.40, 73.94, 112.13.
Elemental analysis: Anal. Calcd. For C ₁₅ H ₂₆ O ₄ : C, 66.14; H, 10.36. Found: C, 65.91; H, 10.42.
[0014]
Example 2 Synthesis of 1,2,6,7-tetraoxaspiro [8.11] icosane (Compound 2) 348 mg (1.50 mmol) of Compound a obtained in Production Example 1 and monohydrate of cesium hydroxide 504 mg (3.00 mmol) was dissolved in 25 ml of dimethylformamide, and 698 mg (2.25 mmol) of 1,3-diiodobutane was added to this over 10 minutes. The reaction was then carried out for 16 hours at room temperature. After completion of the reaction, the reaction mixture was treated in the same manner as in Example 1 above, and then 129 mg of the title compound 2 was obtained by silica gel column chromatography (distillate: ether-hexane, 1:25) (yield 30%). )Obtained. The physical property values are shown below.
Melting point: 97-98 ° C (hexane)
¹ H NMR (CDCl ₃ ) δ: 1.3-1.7 (m, 24 H), 2.2-2.3 (m, 2 H), 3.66 (t, J = 12.2 Hz, 2 H), 4.26 (dd, J = 12.2, (3.9 Hz, 2 H)
¹³ C NMR (CDCl ₃ ) δ: 19.30, 21.89, 22.17, 25.95, 26.11, 26.43, 30.93, 32.51, 73.85, 111.82.
Elemental Analysis: Anal. Calcd. For C ₁₆ H ₃₀ O ₄ : C, 67.10; H, 10.56. Found: C, 66.66; H, 10.49.
[0015]
Example 3 Synthesis of 1,2,6,7-tetraoxaspiro [9.11] henicosane (Compound 3) 1008 mg (6.00 mmol) of cesium hydroxide monohydrate was dissolved in 20 ml of a dimethylformamide solution. Separately, 20 ml of a dimethylformamide solution in which 696 mg (3.00 mmol) of the compound a obtained in Production Example 1 was dissolved and 20 ml of a dimethylformamide solution in which 1458 mg (4.50 mmol) of 1,3-diiodopentane was dissolved were used with separate syringes. And added at 0 ° C. over 1 hour. The reaction was then carried out for 16 hours at room temperature. After completion of the reaction, the reaction mixture was treated in the same manner as in Example 1 above, and 153 mg (17% yield) of the title compound 3 was obtained by silica gel column chromatography (distillate: ether-hexane, 1:25). )Obtained. The physical property values are shown below.
Melting point: 51-52 ° C. (hexane)
¹ H NMR (CDCl ₃ ) δ: 1.3-1.8 (m, 24 H), 19-2.1 (m, 4 H), 4.1-4.2 (m, 4 H)
¹³ C NMR (CDCl ₃ ) δ: 19.26, 21.92, 22.25, 24.89, 25.97, 26.06, 26.65, 28.45, 76.64, 110.89.
[0016]
Example 4 Synthesis of 1,2,6,7-tetraoxaspiro [8.11] docosane (Compound 4) 1008 mg (6.00 mmol) of cesium hydroxide monohydrate was dissolved in 20 ml of dimethylformamide solution. Using a separate syringe, 20 ml of a dimethylformamide solution in which 696 mg (3.00 mmol) of the compound a obtained in Production Example 1 was dissolved and 20 ml of a dimethylformamide solution in which 1521 mg (4.50 mmol) of 1,3-diiodohexane were dissolved. And added at 0 ° C. over 1 hour. The reaction was then carried out for 16 hours at room temperature. After completion of the reaction, the reaction mixture was treated by the above-mentioned treatment method, and then 168 mg (yield 18%) of the title compound 4 was obtained by silica gel column chromatography (distillate: ether-hexane, 1:25). The physical property values are shown below.
Melting point: 48-49 ° C. (hexane)
¹ H NMR (CDCl ₃ ) δ: 1.3-1.8 (m, 30 H), 4.02 (t, J = 4.8 Hz, 4 H) ¹³ C NMR (CDCl ₃ ) δ: 19.35, 21.89, 22.23, 25.00, 26.02, 26.13, 26.43, 26.99, 74.63, 112.06.
[0017]
<Example 5> Synthesis of 7,8,12,13-tetraoxaspiro [6.8] tridecane (Compound 5) 125 mg (0.61 mmol) of Compound b obtained in Production Example 2 and monohydrate of cesium hydroxide 205 mg (1.22 mmol) was dissolved in 15 ml of dimethylformamide, and 272 mg (0.92 mmol) of 1,3-diiodopropane was added thereto over 10 minutes. The reaction was then continued for 16 hours at room temperature. After completion of the reaction, the reaction mixture was treated in the same manner as in Example 1, and then 38 mg (yield 26%) of the title compound 5 was obtained by silica gel column chromatography (distillate: ether-hexane, 1:25). )Obtained. The physical property values are shown below.
Melting point: 58-59 ° C (hexane)
¹ H NMR (CDCl ₃ ) δ: 0.86 (s, 9 H), 1.1-1.8 (m, 9 H), 2.1-2.3 (m, 2 H), 4.10-4.2 (m, 2 H), 4.3-4.5 (m, 2 H)
¹³ C NMR (CDCl ₃ ) δ: 23.34, 23.61, 27.64, 28.59, 30.35, 31.93, 32.37, 47.44, 73.76, 74.02, 107.92.
Elemental analysis: Anal. Calcd. For C ₁₃ H ₂₄ O ₄ : C, 63.91; H, 9.90. Found: C, 63.60; H, 9.97.
[0018]
Example 6 Synthesis of Spiro [Tricyclo [3.3.1.1 ^3,7 ] decane] 2,3 ′-[1,2,4,5] tetroxocan (Compound 6) Obtained in Production Example 3 313 mg (1.57 mmol) of compound c and 528 mg (3.14 mmol) of cesium hydroxide monohydrate were dissolved in 15 ml of dimethylformamide, and 695 mg (2.35 mmol) of 1,3-diiodopropane was added thereto over 10 minutes. . The reaction was then carried out for 16 hours at room temperature. After completion of the reaction, the reaction mixture was treated by the above treatment method, and then 73 mg (yield 19%) of the title compound 6 was obtained by silica gel column chromatography (distillate: ether-hexane, 1:25). The physical property values are shown below.
Melting point: 34-35 ° C. (hexane)
¹ H NMR (CDCl ₃ ) δ: 1.6-2.3 (m, 16 H), 4.12 (dt, J = 12.9, 5.6 Hz, 2 H), 4.35 (dt, J = 12.5, 4.8 Hz, 2 H)
¹³ C NMR (CDCl ₃ ) δ: 27.06, 30.30, 31.81, 33.67, 33.98, 37.25, 73.94, 109.95.
[0019]
<Culture test of P. falciparum>
In this experiment, P. falciparum FCR-3 strain (ATCC 30932) was used as a Plasmodium falciparum. In addition, a chloroquine resistant malaria parasite of P. falciparum K1 strain was used to verify the effect of the compound of the present invention on a resistant strain of chloroquine marketed as an antimalarial agent. The medium used for the experiment was RPMI1640 medium sterilized by filtration, and the pH was adjusted to 7.4, and human serum was added to 10%. The malaria parasite was cultured at an O ₂ concentration of 5%, a CO ₂ concentration of 5%, an N ₂ concentration of 90%, and a temperature of 36.5 ° C. The hematocrit value (ratio of the volume of erythrocytes in the erythrocyte suspension) was 5%. The initial infection rate of P. falciparum at the start of culture was 0.1%. The culture was performed using a 24-well culture plate, the medium was changed every day, and transplantation was performed at an infection rate of 4%. The infection rate was measured under a microscope (oil immersion, 1000 ×) after preparing a thin-layer smear sample and stained with Giemsa or Diff-Qick, and the malaria parasite infection rate was calculated from the following formula.
Malaria parasite infection rate (%) = {(number of infected red blood cells) / (total number of red blood cells)} × 100
[0020]
<Test Example 1> Plasmodium growth inhibition screening test The cultured malaria parasite-infected erythrocytes were collected by centrifugation, washed with a medium containing serum, and then uninfected erythrocytes were added to make the initial infection rate 0.3%. The hematocrit value at this time is 3%. The sample used for the experiment was dissolved in sterilized water, dimethylformamide (DMF) or dimethyl sulfoxide (DMSO) to obtain a sample having a predetermined concentration.
Samples were added to a 24-well culture plate in an amount of 5 to 10 μl. Samples were duplicated or triplicate. As a control, sterile water, DMF or DMSO was added at 10 μl / well.
Next, 980 to 995 μl of the P. falciparum culture solution prepared in advance was added and gently pipetted to suspend uniformly in the medium. Culture plates _{CO 2 -O 2 -N 2 (5} %, 5%, 90%) after incubation for 72 hours in an incubator, for each well to create a thin layer smear after staining, under a microscope Observing and calculating the infection rate and the control infection rate of the reagent added.
By calculating the growth rate from the malaria parasite infection rate obtained above by the following formula, the 50% growth inhibitory concentration (EC ₅₀ ) for the malaria parasite was obtained. The results are shown in Table 1.
Growth rate (%) = {([b] − [a]) / ([c] − [a])} × 100
a: initial infection rate b: infection rate when the sample is added c: infection rate when the sample is not added (control)
Test Example 2 Mouse FM3A Cell Growth Inhibition Test F28-7 strain, which is a wild strain of mouse breast cancer-derived FM3A cells, was used. As the medium, fetal calf serum deactivated in ES medium was added to ₂ %, and cultured at a CO ₂ concentration of 5% at 37 ° C. The doubling time for FM3A cells under these conditions was about 12 hours.
Pre-culture was performed, and cells that entered the logarithmic growth phase were diluted with a medium so as to be 5 × 10 ⁴ cells / ml. A sample prepared at the time of measuring the antimalarial activity of the malaria parasite was used. The sample solution was added to each 24-well culture plate in an amount of 5 to 10 μl (the final concentration became 1 × 10 ⁻⁴ to 1 × 10 ⁻⁶ when medium or the like was added). The compound was duplicated or triplicate, and a well containing 10 μl of sterilized water, DMF or DMSO was also prepared at the same time as a control. Next, 990 to 995 μl of the prepared cultured cell suspension was added and gently pipetted to suspend uniformly in the medium. After culturing for 48 hours, the number of cells in each well was counted with a cell controller (CC-108, manufactured by Toa Medical Electrics), and the growth rate was calculated from the following formula.
Growth rate (%) = {([C] − [A]) / ([B] − [A])} × 100
A: Initial number of cells B: Number of control cells 48 hours later C: Number of cells 48 hours after addition of sample The cell growth inhibitory activity was calculated from the number of cells in the well to which the sample was added and the number of control cells. Thereby, the cytotoxicity of the sample was evaluated and expressed as a cell growth inhibitory concentration (EC ₅₀ ). EC ₅₀ value is the concentration of a sample that inhibits the growth rate of the control by adding 50% to the growth rate of the malaria parasite or control without adding the sample to the culture medium of FM3A cells, or the malaria parasite infection rate to 100%. (Expressed in molar concentration). The results are shown in Table 1.
The anti-malarial action of the sample was evaluated from the ratio of EC ₅₀ values of the malaria parasite sample to FM3A cells (chemotherapeutic coefficient, see formula below), and drug efficacy was determined. For reference, a similar test was conducted for chloroquine, which is used as an antimalarial agent. The results are shown in Table 1.
Chemotherapeutic index = (EC ₅₀ value of the sample for mouse FM3A cells)
÷ (Sample EC ₅₀ value against Plasmodium falciparum)
[0022]
[Table 1]

[0023]
As a result, it was found that the compound of the present invention has low cytotoxicity and has malaria parasite growth inhibitory activity. It was also found that the effect was retained against chloroquine resistant protozoa.
[0024]
<Test Example 3> Protozoa suppression test using mice infected with murine malaria parasite This experiment was conducted by Peters, W. et al. and Richrds, W. H. G. Antimalarial drugs I, in: W. Peters, W. H. G. Richards (Eds.), Springer-Verlag, Berlin, 1984, pp. The test was performed according to the 4-day suppressive test described in 229-230. The murine malaria parasite used (P. berghei NK65 strain) is a virulent protozoan strain. When this murine malaria parasite is infected, all mice die within 10 days after infection. The experiment was started when the blood infection rate of the subcultured murine malaria parasite-infected mice reached 10%. Blood was collected from mice subjected to ether anesthesia and 200 μl of intraperitoneal infection (ip) was administered to mice not infected with the protozoa suspension prepared to 5 × 10 ⁶ protozoa / ml per 1 ml of phosphate buffered saline. did. Two hours after protozoan infection, the compound suspended in olive oil was orally or intraperitoneally administered (compound 5 was only intraperitoneally administered). The administration period is once a day for 4 consecutive days, and blood is collected from the tail of the mouse on the fourth day of the experiment. A thin-layer smear was prepared from the collected blood, and the erythrocyte infection rate was determined under a microscope by Giemsa staining (performed in the same manner as the culture assay test for P. falciparum above). The 50% protozoa inhibitory concentration (ED ₅₀ value) and 90% protozoa inhibitory concentration (ED ₉₀ value) were determined by examining the ratio of the infection rate of the drug administration group to the control group to which only the solvent was administered. The results are shown in Table 2.
[0025]
[Table 2]

[0026]
As a result, the compound of the present invention was found to have excellent malaria parasite growth inhibitory activity.
[0027]
【The invention's effect】
The compound of the present invention has an excellent antimalarial activity and is useful as a preventive and therapeutic agent for infectious diseases caused by protozoa such as malaria. It is also effective against chloroquine resistant malaria.
[Brief description of the drawings]
FIG. 1 is a table showing a comparison table of chemical structural formulas of compounds in production examples and examples.

Claims (3)

A peroxide derivative represented by the general formula (1).

[ Wherein C is a monocyclic alicyclic hydrocarbon ring group having 3 to 12 carbon atoms which may have a linear or branched lower alkyl group having 1 to 6 carbon atoms as a substituent, or amadantylidene. Group and n show the integer of 0-6. ] In the general formula (1), C is 4-tert-butyl-cyclohexylidene, cycloalkyl dodecylcarbamoyl isopropylidene or adamantylidene group, peroxide derivative according to claim 1 Symbol placement n is 1-4.   The antimalarial agent containing the compound represented by General formula (1).

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