JP2022025029A - Novel compounds and pharmaceutical compositions - Google Patents
Novel compounds and pharmaceutical compositions Download PDFInfo
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- JP2022025029A JP2022025029A JP2021115706A JP2021115706A JP2022025029A JP 2022025029 A JP2022025029 A JP 2022025029A JP 2021115706 A JP2021115706 A JP 2021115706A JP 2021115706 A JP2021115706 A JP 2021115706A JP 2022025029 A JP2022025029 A JP 2022025029A
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Abstract
Description
本発明は、新規化合物、及び、その新規化合物を有効成分とする医薬組成物に関する。 The present invention relates to a novel compound and a pharmaceutical composition containing the novel compound as an active ingredient.
シクロオキシゲナーゼ(COX)の作用によりアラキドン酸から産生されるプロスタグランジンH2(PGH2)は、各種PG合成酵素の作用により、PGD2やPGE2等のプロスタノイドへと変換される。中でもPGD2は、血小板凝集や睡眠誘導等さまざまな生理作用を有するだけでなく、さらなる代謝を受けて、シクロペンテノン構造を有するJ2型PG類へと変換されることが知られている(非特許文献１)。 Prostaglandin H 2 (PGH 2 ) produced from arachidonic acid by the action of cyclooxygenase (COX) is converted to prostanoids such as PGD 2 and PGE 2 by the action of various PG synthases. Among them, PGD 2 is known not only to have various physiological actions such as platelet aggregation and sleep induction, but also to be converted into J 2 type PGs having a cyclopentenone structure by further metabolism (). Non-Patent Document 1).
プロスタグランジンD2(PGD2)の過剰な産生は、アレルギー疾患、睡眠障害、デュシェンヌ筋ジストロフィー等様々な疾患に関連しており、造血器型プロスタグランジンD合成酵素(Hematopoietic prostaglandin(PG)D synthase: H-PGDSと記載することがある。)はPGD2を産生する酵素の一つである(非特許文献２～５)。食物アレルギーの主役となるマスト細胞は、炎症を引き起こすヒスタミンやセロトニンといった生理活性物質とともに、H-PGDSを強く発現しており、大量のPGD2を産生している。また、デュシェンヌ型筋ジストロフィーの骨格筋では、H-PGDSによりPGD2が活発に産生され、DP1あるいはDP2受容体を介した二次的炎症により筋壊死が拡大し、集団的筋壊死が起こる。
Excessive production of prostaglandin D 2 (PGD 2 ) is associated with various diseases such as allergic diseases, sleep disorders, and Duchenne muscle dystrophy, and hematopoietic prostaglandin (PG) D synthase. : H-PGDS may be described) is one of the enzymes producing PGD 2 (
H-PGDSは上記疾患の創薬ターゲットとして注目されており、H-PGDSを標的とした阻害剤はアレルギー反応及び炎症反応の治療のために有用であることが報告されている(非特許文献６)。しかしながら、臨床試験では患者の病態により望む治療効果が得られない等の問題が報告されていることから(非特許文献７)、新たな作用機序による薬剤開発が求められている。 H-PGDS is attracting attention as a drug discovery target for the above diseases, and it has been reported that an inhibitor targeting H-PGDS is useful for the treatment of allergic and inflammatory reactions (Non-Patent Document 6). ). However, since clinical trials have reported problems such as the inability to obtain the desired therapeutic effect depending on the pathological condition of the patient (Non-Patent Document 7), drug development by a new mechanism of action is required.
近年、細胞内のユビキチン-プロテアソームシステム(UPS)を利用して標的タンパク質を分解誘導できる革新的な分子標的薬が国内外で開発されている。これらの薬物はPROTAC(Proteolysis targeting chimeras)(非特許文献８)及びSNIPER(specific and non-genetic inhibitor of apoptosis protein-dependent protein erasers)(非特許文献９)と呼ばれており、標的タンパク質リガンド(X)とユビキチンリガーゼリガンド(Y)をリンカー(-)で繋いだキメラ分子(X-Y)である。PROTACやSNIPERは、標的タンパク質とユビキチンリガーゼを物理的に近づけることで、標的タンパク質がポリユビキチン化されプロテアソームにより分解誘導を受ける。また、PROTACやSNIPERは従来の阻害剤と比較して、標的タンパク質により引き起こされる細胞応答を持続的に抑制できることも報告されている(非特許文献１０)。PROTACやSNIPERは創薬モダリティのひとつとして期待されており、特に、がんに関連するタンパク質を標的とした開発が盛んである。しかしながらH-PGDSを標的とした分解誘導剤は未だ報告されておらず、H-PGDSを標的としたRROTACやSNIPERの有効性も実証されていない。
In recent years, innovative molecular-targeted drugs that can induce degradation of target proteins using the intracellular ubiquitin-proteasome system (UPS) have been developed at home and abroad. These drugs are called PROTAC (Proteolysis targeting chimeras) (Non-Patent Document 8) and SNIPER (specific and non-genetic inhibitor of apoptosis protein-dependent protein erasers) (Non-Patent
本発明はかかる問題点に鑑みてなされたものであって、H-PGDSを標的とした分解誘導剤としての新規化合物及びその新規化合物を有効成分とする医薬組成物を提供することを目的とする。 The present invention has been made in view of the above problems, and an object of the present invention is to provide a novel compound as a decomposition inducer targeting H-PGDS and a pharmaceutical composition containing the novel compound as an active ingredient. ..
本発明にかかる新規化合物は、E3ユビキチンリガーゼのリガンドと、造血器型プロスタグランジンD合成酵素のリガンドとを、接続したことを特徴とする。 The novel compound according to the present invention is characterized in that a ligand of E3 ubiquitin ligase and a ligand of a hematopoietic prostaglandin D synthase are linked.
本発明にかかる医薬組成物は、本発明にかかる新規化合物を有効成分とし、アレルギー疾患を予防及び／又は治療するための医薬組成物である。 The pharmaceutical composition according to the present invention is a pharmaceutical composition for preventing and / or treating an allergic disease, using the novel compound according to the present invention as an active ingredient.
本発明にかかる医薬組成物は、本発明にかかる新規化合物を有効成分とし、炎症性疾患を予防及び／又は治療するための医薬組成物である。 The pharmaceutical composition according to the present invention is a pharmaceutical composition for preventing and / or treating an inflammatory disease by using the novel compound according to the present invention as an active ingredient.
本発明によれば、H-PGDSを標的とした分解誘導剤としての新規化合物が得られる。またその新規化合物を有効成分とする医薬組成物が得られる。 According to the present invention, a novel compound as a decomposition inducer targeting H-PGDS can be obtained. Further, a pharmaceutical composition containing the novel compound as an active ingredient can be obtained.
以下、添付の図面を参照して本発明の実施形態について具体的に説明するが、当該実施形態は本発明の原理の理解を容易にするためのものであり、本発明の範囲は、下記の実施形態に限られるものではなく、当業者が以下の実施形態の構成を適宜置換した他の実施形態も、本発明の範囲に含まれる。 Hereinafter, embodiments of the present invention will be specifically described with reference to the accompanying drawings, but the embodiments are for facilitating the understanding of the principles of the present invention, and the scope of the present invention is as follows. The present invention is not limited to the embodiments, and other embodiments in which those skilled in the art appropriately replace the configurations of the following embodiments are also included in the scope of the present invention.
本発明にかかる新規化合物は、図１(a)に示されるように、E3ユビキチンリガーゼのリガンドと、造血器型プロスタグランジンD合成酵素のリガンドとを、リンカーで接続したものである。 As shown in FIG. 1 (a), the novel compound according to the present invention is obtained by linking a ligand of E3 ubiquitin ligase and a ligand of a hematopoietic prostaglandin D synthase with a linker.
細胞内のUPSを利用して標的タンパク質を分解誘導できる薬物はPROTACやSNIPERと呼ばれており、標的タンパク質リガンド(X)とユビキチンリガーゼリガンド(Y)をリンカー(-)で繋いだキメラ分子(X-Y)である。PROTACは、標的タンパク質とユビキチンリガーゼを物理的に近づけることで、標的タンパク質がポリユビキチン化されプロテアソームにより分解誘導を受ける。本件発明者は、造血器型プロスタグランジンD合成酵素のリガンド(X)とE3ユビキチンリガーゼのリガンド(Y)とをリンカー(-)で接続した新規化合物(X-Y)を合成し、かかる新規化合物がH-PGDSタンパク質を効率的に分解することを新知見として見出し、かかる事実に基づいて本件発明を完成させた。 Drugs that can induce degradation of the target protein using intracellular UPS are called PROTAC and SNIPER, and are chimeric molecules (XY) in which the target protein ligand (X) and the ubiquitin ligase ligand (Y) are linked by a linker (-). ). PROTAC physically brings the target protein and ubiquitin ligase closer to each other, so that the target protein is polyubiquitinated and induced to be degraded by the proteasome. The present inventor synthesizes a novel compound (XY) in which a ligand (X) of a hematopoietic prostaglandin D synthase and a ligand (Y) of E3 ubiquitin ligase are linked by a linker (-), and the novel compound is obtained. We have found as a new finding that the H-PGDS protein is efficiently degraded, and completed the present invention based on this fact.
また本発明にかかる新規化合物は、図１(b)に示されるように、E3ユビキチンリガーゼのリガンドと、造血器型プロスタグランジンD合成酵素のリガンドとを、リンカー介さずに接続したものである。 Further, in the novel compound according to the present invention, as shown in FIG. 1 (b), a ligand of E3 ubiquitin ligase and a ligand of a hematopoietic prostaglandin D synthase are linked without a linker. ..
標的タンパク質リガンド(X)とユビキチンリガーゼリガンド(Y)をリンカーを介さずに繋いだキメラ分子(XY)である。本件発明者は、造血器型プロスタグランジンD合成酵素のリガンド(X)とE3ユビキチンリガーゼのリガンド(Y)とをリンカーを介さずに接続した新規化合物(XY)を合成し、かかる新規化合物がH-PGDSタンパク質を効率的に分解することを新知見として見出し、かかる事実に基づいて本件発明を完成させた。 It is a chimeric molecule (XY) in which a target protein ligand (X) and a ubiquitin ligase ligand (Y) are linked without a linker. The present inventor has synthesized a novel compound (XY) in which a ligand (X) of a hematopoietic prostaglandin D synthase and a ligand (Y) of E3 ubiquitin ligase are linked without a linker, and the novel compound is obtained. We have found as a new finding that the H-PGDS protein is efficiently degraded, and completed the present invention based on this fact.
H-PGDSは、ヒトでは胎盤、肺、胎児肝、及びリンパ節に多く発現し、脳、胸腺、心臓、脾臓、及び骨髄にも発現が認められる。H-PGDSにより合成されるPGD2がアレルギー反応の進展に密接に関与していることが示唆されている。またアレルギー反応以外については、デュシェンヌ型筋ジストロフィーや多発性筋炎患者の壊死筋、外傷性脳損傷により活性化されたミクログリア細胞においてH-PGDSの誘導が観察されている。 H-PGDS is abundantly expressed in the placenta, lungs, fetal liver, and lymph nodes in humans, and is also expressed in the brain, thymus, heart, spleen, and bone marrow. It has been suggested that PGD 2 synthesized by H-PGDS is closely involved in the development of allergic reactions. In addition to allergic reactions, induction of H-PGDS has been observed in Duchenne muscular dystrophy, necrotic muscles in patients with polymyositis, and microglial cells activated by traumatic brain injury.
造血器型プロスタグランジンD合成酵素のリガンドは、造血器型プロスタグランジンD合成酵素に対して特異的な結合作用を有する物質を用いることが好ましい。ここで、特異的とは、造血器型プロスタグランジンD合成酵素に対して結合作用を有し、リポカリン型プロスタグランジンD合成酵素に対しては実質的に結合作用を有していないことである。造血器型プロスタグランジンD合成酵素のリガンドは、H-PGDSに対して阻害作用を有する化合物候補をスクリーニング手法にて取得することができる。 As the ligand of the hematopoietic prostaglandin D synthase, it is preferable to use a substance having a specific binding action to the hematopoietic prostaglandin D synthase. Here, the specific is that it has a binding action to the hematopoietic prostaglandin D synthase and has substantially no binding action to the lipocalin type prostaglandin D synthase. be. For the ligand of the hematopoietic prostaglandin D synthase, compound candidates having an inhibitory effect on H-PGDS can be obtained by a screening method.
造血器型プロスタグランジンD合成酵素のリガンドは、特に限定されるものではないが、例えば、下記に示すものが挙げられ、好ましくはTFC-007である。
・TFC-007・・・N-(4-(4-(モルホリン-4-カルボニル)ピペリジン-1-イル)フェニル)-2-フェノキシピリミジン-5-カルボキサミド
・TAS-204・・・(N-メトキシ-N-メチル)-4-(5-ベンゾイルベンゾイミダゾール-2-イル)-3,5-ジメチルピロール-2-カルボキサミド塩酸塩
・TAS-205・・・4-(1-メチル-1H-ピロール-2-カルボニル)-N-(4-(4-(モルホリン-4-カルボニル)ピペリジン-1-イル)フェニル)ピペラジン-1-カルボキサミド
・4-((1-メチルピロール-2-イル)-カルボニル)-N-(1-(4-(3-(1,2,3-トリアゾール-1-イル)-プロピル)-フェニル)-ピペリジン-4-イル)-1-ピペラジンカルボキサミド
・4-((1-メチルピロール-2-イル)-カルボニル)-N-(1-(4-(3-モルホリノ-3-オキソプロペン-1-イル)-フェニル)-ピペリジン-4-イル)-1-ピペラジンカルボキサミド
・4-((1-メチルピロール-2-イル)-カルボニル)-N-(1-(4-(3-モルホリノ-3-オキソプロ
ピル)-フェニル)-ピペリジン-4-イル)-1-ピペラジンカルボキサミド
・6-(4-(4-((1-メチルピロール-2-イル)-カルボニル)-1-ピペラジンカルバモイル)-ピペリジン-1-イル)-ニコチン酸
・4-((1-メチルピロール-2-イル)-カルボニル)-N-(1-(5-(4-モルホリニルカルボニル)ピリジン-2-イル)-ピペリジン-4-イル)-1-ピペラジンカルボキサミド
・4-((1-エチルピロール-2-イル)-カルボニル)-N-(1-(4-(2-モルホリノエチルカルバモイル)フェニル)-ピペリジン-4-イル)-1-ピペラジンカルボキサミド
・4-((1-エチルピロール-2-イル)-カルボニル)-N-(1-(4-(2-(1,2,3-トリアゾール-1-イル)-エチル)-フェニル)-ピペリジン-4-イル)-1-ピペラジンカルボキサミド
・4-(4-(4-((1-メチルピロール-2-イル)-カルボニル)-1-ピペラジンカルバモイル)ピペリジン-1-イル)-安息香酸
・4-((1-メチルピロール-2-イル)-カルボニル)-N-(1-(4-(ピリジン-3-イルメチルカルバモイル)フェニル)-ピペリジン-4-イル)-1-ピペラジンカルボキサミド
・4-((1-メチルピロール-2-イル)-カルボニル)-N-(1-(4-(2-モルホリノエチルカルバモイル)-フェニル)-ピペリジン-4-イル)-1-ピペラジンカルボキサミド
・4-((1-メチルピロール-2-イル)-カルボニル)-N-(1-(4-(4-モルホリニルカルボニル)フェニル)-ピペリジン-4-イル)-1-ピペラジンカルボキサミド
・4-((1-メチルピロール-2-イル)-カルボニル)-N-(1-(4-(1-ピペリジニルカルボニル)フェニル)-ピペリジン-4-イル)-1-ピペラジンカルボキサミド
・4-((1-メチルピロール-2-イル)-カルボニル)-N-(1-(4-(1-ピロリジニルカルボニル)フェニル)-ピペリジン-4-イル)-1-ピペラジンカルボキサミド
・4-((1-メチルピロール-2-イル)-カルボニル)-N-(1-(4-(2-(1,2,3-トリアゾール-1-イル)-エチル)-フェニル)-ピペリジン-4-イル)-1-ピペラジンカルボキサミド
・4-((1-メチルピロール-2-イル)-カルボニル)-N-(1-(4-(3-(1,2,4-トリアゾール-1-イル)-プロピル)-フェニル)-ピペリジン-4-イル)-1-ピペラジンカルボキサミド
・4-((1-メチルピロール-2-イル)-カルボニル)-N-(1-(4-(3-(3,5-ジメチル-1,2,4-トリアゾール-1-イル)-プロピル)-フェニル)-ピペリジン-4-イル)-1-ピペラジンカルボキサミド
ユビキチン化は、ユビキチン活性化酵素(E1)、ユビキチン結合酵素(E2)、ユビキチンリガーゼ(E3)の３種類の酵素の働きにより、基質のリシン残基にユビキチンのＣ末端のグリシンをイソペプチド結合する反応である。これらの酵素のうちで、どの基質にユビキチンを付加するのかを決めるのはE3ユビキチンリガーゼである。
The ligand of the hematopoietic prostaglandin D synthase is not particularly limited, but examples thereof include those shown below, preferably TFC-007.
・ TFC-007 ・ ・ ・ N- (4- (4- (morpholin-4-carbonyl) piperidine-1-yl) phenyl) -2-phenoxypyrimidine-5-carboxamide ・ TAS-204 ・ ・ ・ (N-methoxy) -N-methyl) -4- (5-benzoylbenzoimidazol-2-yl) -3,5-dimethylpyrol-2-carboxamide hydrochloride ・ TAS-205 ・ ・ ・ 4- (1-methyl-1H-pirol-) 2-carbonyl) -N- (4- (4- (morpholin-4-carbonyl) piperidine-1-yl) phenyl) piperazine-1-carboxamide 4-((1-methylpyrol-2-yl) -carbonyl) -N-(1- (4- (3- (1,2,3-triazole-1-yl) -propyl) -phenyl) -piperidine-4-yl) -1-piperazin carboxamide 4-((1-1- Methylpyrrole-2-yl) -carbonyl) -N- (1- (4- (3-morpholino-3-oxopropen-1-yl) -phenyl) -piperidine-4-yl) -1-piperazin carboxamide · 4 -((1-Methylpyrol-2-yl) -carbonyl) -N-(1- (4- (3-morpholino-3-oxopropyl) -phenyl) -piperidine-4-yl) -1-piperazincarboxamide. 6-(4-(4-((1-Methylpyrol-2-yl) -carbonyl) -1-piperazincarbamoyl) -piperidine-1-yl)-nicotinic acid ・ 4-((1-Methylpyrol-2-yl) Ill) -carbonyl) -N- (1- (5- (4-morpholinylcarbonyl) pyridine-2-yl) -piperidine-4-yl) -1-piperazin carboxamide 4-((1-ethylpyrrole-) 2-yl) -carbonyl) -N- (1- (4- (2-morpholinoethylcarbamoyl) phenyl) -piperidine-4-yl) -1-piperazin carboxamide 4-((1-ethylpyrrole-2-yl) )-Maldon) -N- (1- (4- (2- (1,2,3-triazole-1-yl) -ethyl) -phenyl) -piperidine-4-yl) -1-piperazin carboxamide 4- (4- (4-((1-Methylpyrol-2-yl) -carbonyl) -1-piperazincarbamoyl) piperidine-1-yl)-benzoic acid ・ 4-((1-methylpyrol-2-yl)- Carbonyl) -N- (1- (4- (pyridin-3-ylmethylcarbamoyl) phenyl) -piperidine-4-yl) -1-piperazin Carboxamide 4-((1-Methylpyrol-2-yl) -carbonyl)-N-(1- (4- (2-Morholinoethylcarbamoyl) -phenyl) -piperidin-4-yl) -1-piperazin carboxamide. 4-((1-Methylpyrol-2-yl) -carbonyl) -N-(1- (4- (4-morpholinylcarbonyl) phenyl) -piperidin-4-yl) -1-piperazin carboxamide · 4- ((1-Methylpyrol-2-yl) -carbonyl) -N- (1- (4- (1-piperidinylcarbonyl) phenyl) -piperidin-4-yl) -1-piperazin carboxamide 4-((( 1-Methylpyrrol-2-yl) -carbonyl) -N-(1- (4- (1-pyrrolidinylcarbonyl) phenyl) -piperidin-4-yl) -1-piperazin carboxamide 4-((1-(1-) Methylpyrol-2-yl) -carbonyl) -N-(1- (4- (2- (1,2,3-triazol-1-yl) -ethyl) -phenyl) -piperidine-4-yl) -1 -Piperazine carboxamide 4-((1-methylpyrol-2-yl) -carbonyl)-N-(1-(4- (3- (1,2,4-triazol-1-yl) -propyl) -phenyl) )-Piperidine-4-yl) -1-Piperazinecarboxamide ・ 4-((1-Methylpyrol-2-yl) -carbonyl) -N- (1- (4- (3- (3,5-dimethyl-1) -1) , 2,4-Triazol-1-yl) -propyl) -phenyl) -piperidin-4-yl) -1-piperazin Carboxamide Ubiquitination is a ubiquitin activating enzyme (E1), ubiquitin binding enzyme (E2), ubiquitin ligase. This is a reaction in which the C-terminal glycine of ubiquitin is isopeptide-bound to the lysine residue of the substrate by the action of the three types of enzymes (E3). Of these enzymes, it is the E3 ubiquitin ligase that determines which substrate to add ubiquitin to.
E3ユビキチンリガーゼは、特に限定されるものではないが、例えば、セレブロン(CRBN)、MDM2、APC、UBR5、SOCS、LNX1、BIRC2、BIRC3、BIRC4、CBX4、CBLL1、HACE1、HECTD1、HECTD2、HECTD3、HECTD4、HECW1、HECW2、HERC1、HERC2、HERC3、HERC4、HERC5、HERC6、HUWE1、ITCH、NEDD4、NEDD4L、PPIL2、PRPF19、PIAS1、PIAS2、PIAS3、PIAS4、RANBP2、RNF4、RBX1、SMURF1、SMURF2、STUB、TOPORS、TRIP12、UBE3A、UBE3B、UBE3C、UBE3D、UBE4A、UBE4B、UBOX5、UBR5、WWP1、WWP2、又は、Parkin等が挙げられ、好ましくはセレブロン(CRBN)である。 The E3 ubiquitin ligase is not particularly limited, but is, for example, Cereblon (CRBN), MDM2, APC, UBR5, SOCS, LNX1, BIRC2, BIRC3, BIRC4, CBX4, CBLL1, HACE1, HECTD1, HECTD2, HECTD3, HECTD4. , HECW1, HECW2, HERC1, HERC2, HERC3, HERC4, HERC5, HERC6, HUWE1, ITCH, NEDD4, NEDD4L, PPIL2, PRPF19, PIAS1, PIAS2, PIAS3, PIAS4, RANBP2, RNF4, RBX1, SMURF1, SMURF1 , TRIP12, UBE3A, UBE3B, UBE3C, UBE3D, UBE4A, UBE4B, UBOX5, UBR5, WWP1, WWP2, Parkin and the like, preferably cereblon (CRBN).
E3ユビキチンリガーゼのリガンドは、ポマリドミド、リナリドミド、サリドマイド、又は、インジスラム等が挙げられ、好ましくはポマリドミドである。 Examples of the ligand of E3 ubiquitin ligase include pomalidomide, linaridomide, thalidomide, indismlam and the like, and pomalidomide is preferable.
リンカーは、造血器型プロスタグランジンD合成酵素のリガンドとE3ユビキチンリガーゼのリガンドとを接続できるものであれば特に限定されるものではなく、例えばポリエチレングリコールリンカー又はアルキルリンカー等が挙げられ、好ましくはポリエチレングリコールリンカーである。リンカーの構成単位数は、特に限定されるものではなく、例えば１乃至10ある。 The linker is not particularly limited as long as it can connect the ligand of the hematopoietic prostaglandin D synthase and the ligand of E3 ubiquitin ligase, and examples thereof include polyethylene glycol linkers and alkyl linkers, which are preferable. It is a polyethylene glycol linker. The number of constituent units of the linker is not particularly limited, and is, for example, 1 to 10.
本発明にかかる新規化合物は、造血器型プロスタグランジンD合成酵素のリガンド(X)がTFC-007で、E3ユビキチンリガーゼのリガンド(Y)がポマリドミドで、リンカー(-)の構成単位がポリエチレングリコールリンカーの場合、下記にて示される。ここでポリエチレングリコールリンカーの構成単位数は、特に限定されるものではなく、例えばnは1～10であり、好ましくは1～5であり、より好ましくは1又は2であり、最も好ましくは1である。 In the novel compound according to the present invention, the ligand (X) of the hematopoietic prostaglandin D synthase is TFC-007, the ligand (Y) of E3 ubiquitin ligase is pomalidemid, and the constituent unit of the linker (-) is polyethylene glycol. In the case of a linker, it is shown below. Here, the number of constituent units of the polyethylene glycol linker is not particularly limited, and for example, n is 1 to 10, preferably 1 to 5, more preferably 1 or 2, and most preferably 1. be.
n=1の場合、PROTAC(H-PGDS)-6と記載することがあり、下記にて示される。 When n = 1, it may be described as PROTAC (H-PGDS) -6, which is shown below.
n=2の場合、PROTAC(H-PGDS)-5と記載することがある。 When n = 2, it may be described as PROTAC (H-PGDS) -5.
n=3の場合、PROTAC(H-PGDS)-4と記載することがある。 When n = 3, it may be described as PROTAC (H-PGDS) -4.
n=4の場合、PROTAC(H-PGDS)-3と記載することがある。 When n = 4, it may be described as PROTAC (H-PGDS) -3.
n=5,N-methyl pomalidomideの場合、PROTAC(H-PGDS)-2と記載することがある。 In the case of n = 5, N-methyl pomalidomide, it may be described as PROTAC (H-PGDS) -2.
n=5の場合、PROTAC(H-PGDS)-1と記載することがあり、下記にて示される。 When n = 5, it may be described as PROTAC (H-PGDS) -1, which is shown below.
本発明にかかる新規化合物は、造血器型プロスタグランジンD合成酵素のリガンド(X)がTFC-007で、E3ユビキチンリガーゼのリガンド(Y)がポマリドミドで、リンカーを介さずに接続する場合、下記にて示される(本明細書において、下記化合物をPROTAC(H-PGDS)-7と記載することがある。)。 The novel compound according to the present invention is described below when the ligand (X) of the hematopoietic prostaglandin D synthase is TFC-007 and the ligand (Y) of E3 ubiquitin ligase is pomalidemid, which is connected without a linker. (In the present specification, the following compounds may be referred to as PROTAC (H-PGDS) -7).
本発明にかかる新規化合物は、造血器型プロスタグランジンD合成酵素のリガンド(X)がTAS-205で、E3ユビキチンリガーゼのリガンド(Y)がポマリドミドで、リンカーを介さずに接続する場合、下記にて示される(本明細書において、下記化合物をPROTAC(H-PGDS)-10と記載することがある。)。 The novel compound according to the present invention is described below when the ligand (X) of the hematopoietic prostaglandin D synthase is TAS-205 and the ligand (Y) of E3 ubiquitin ligase is pomalidemid, which is connected without a linker. (In the present specification, the following compounds may be referred to as PROTAC (H-PGDS) -10).
本発明においては、標的タンパク質であるH-PGDSとユビキチンリガーゼを物理的に近づけることで、H-PGDSがポリユビキチン化されプロテアソームにより分解誘導を受ける。具体的には、H-PGDSを発現している細胞に、PROTAC(H-PGDS)-1、PROTAC(H-PGDS)-7又はPROTAC(H-PGDS)-10を添加すると、細胞内でPROTAC(H-PGDS)-1又はPROTAC(H-PGDS)-7構造中のTFC-007部位がH-PGDSタンパク質へ結合し、又は、PROTAC(H-PGDS)-10構造中のTAS-205部位がH-PGDSタンパク質へ結合する。そして、ポマリドミド部位が細胞内に存在している酵素であるユビキチンリガーゼ(セレブロン)へ結合する。これによりH-PGDSとユビキチンリガーゼ(セレブロン)とが近接し、H-PGDSがポリユビキチン化され、プロテアソームによって分解される。即ち、本発明にかかる新規化合物は、H-PGDSを標的とした分解誘導剤として機能する。 In the present invention, by physically bringing H-PGDS, which is a target protein, and ubiquitin ligase close to each other, H-PGDS is polyubiquitinated and induced to be degraded by the proteasome. Specifically, when PROTAC (H-PGDS) -1, PROTAC (H-PGDS) -7 or PROTAC (H-PGDS) -10 is added to cells expressing H-PGDS, PROTAC is intracellularly added. The TFC-007 site in the (H-PGDS) -1 or PROTAC (H-PGDS) -7 structure binds to the H-PGDS protein, or the TAS-205 site in the PROTAC (H-PGDS) -10 structure It binds to the H-PGDS protein. Then, the pomalidomide site binds to ubiquitin ligase (cereblon), which is an enzyme present in cells. As a result, H-PGDS and ubiquitin ligase (cereblon) are in close proximity, and H-PGDS is polyubiquitinated and degraded by the proteasome. That is, the novel compound according to the present invention functions as a decomposition inducer targeting H-PGDS.
本発明にかかる医薬組成物は、本発明にかかる新規化合物を有効成分とする、アレルギー疾患を予防及び／又は治療するための医薬組成物である。アレルギー疾患は、特に限定されるものではなく、例えばアトピー性皮膚炎、気管支喘息、花粉症、アレルギー性鼻炎、副鼻腔炎、中耳炎、又は、アレルギー性結膜炎等が挙げられる。 The pharmaceutical composition according to the present invention is a pharmaceutical composition for preventing and / or treating an allergic disease, which comprises a novel compound according to the present invention as an active ingredient. The allergic disease is not particularly limited, and examples thereof include atopic dermatitis, bronchial asthma, hay fever, allergic rhinitis, sinusitis, middle ear inflammation, and allergic conjunctivitis.
本発明にかかる医薬組成物は、本発明にかかる新規化合物を有効成分とする、炎症性疾患を予防及び／又は治療するための医薬組成物である。炎症性疾患は、特に限定されるものではなく、例えば筋ジストロフィー、筋肉炎、慢性閉塞性動脈性疾患、関節リウマチ、変形性関節症、多発性硬化症、筋萎縮性側索硬化症、炎症性腸疾患、慢性閉塞性肺疾患、間質性肺炎、過敏性肺炎、又は、好酸球性肺炎等が挙げられる。筋ジストロフィーには、例えば顔面肩甲上腕型筋ジストロフィー、デュシェンヌ型筋ジストロフィー、又は、ベッカー型筋ジストロフィーが含まれ、好ましくはデュシェンヌ型筋ジストロフィーである。 The pharmaceutical composition according to the present invention is a pharmaceutical composition for preventing and / or treating an inflammatory disease, which comprises the novel compound according to the present invention as an active ingredient. The inflammatory disease is not particularly limited, and is not particularly limited, for example, muscular dystrophy, myitis, chronic obstructive arterial disease, rheumatoid arthritis, osteoarthritis, multiple sclerosis, muscular atrophic lateral sclerosis, and inflammatory bowel. Diseases, chronic obstructive pulmonary disease, interstitial pneumonia, hypersensitivity pneumonitis, eosinophilic pneumonitis and the like can be mentioned. The muscular dystrophy includes, for example, facial scapulohumeral dystrophy, Duchenne muscular dystrophy, or Becker muscular dystrophy, preferably Duchenne muscular dystrophy.
本明細書において「予防」には疾患の発症を抑えること及び遅延させることが含まれ、疾患になる前の予防だけでなく、治療後の疾患の再発に対する予防も含まれる。一方、「治療」には、症状を治癒すること、症状を改善すること及び症状の進行を抑えることが含まれる。 As used herein, "prevention" includes suppressing and delaying the onset of the disease, including not only prevention before the disease develops, but also prevention against recurrence of the disease after treatment. On the other hand, "treatment" includes healing the symptoms, improving the symptoms and suppressing the progression of the symptoms.
本発明にかかる医薬組成物は、公知の方法に従って製剤化し、投与する薬剤とすることができる。例えば、そのまま液剤として又は適当な剤型の薬剤として、ヒト又は哺乳類に対して経口的又は非経口的に投与することができる。ヒトに対する投与量は、特に限定されるものではないが、例えば0.01 mg/kg～50 mg/kgとすることが可能である。 The pharmaceutical composition according to the present invention can be a drug to be formulated and administered according to a known method. For example, it can be administered orally or parenterally to humans or mammals as it is as a liquid or as a suitable dosage form. The dose to humans is not particularly limited, but can be, for example, 0.01 mg / kg to 50 mg / kg.
薬剤は、微生物の増殖を抑制する防腐剤又はｐＨを許容範囲に保つのに役立つ緩衝剤を含んでもよい。防腐剤は、アジ化ナトリウム、オクタデシルジメチルベンジルアンモニウムクロライド、塩化ヘキサメトニウム、塩化ベンザルコニウム、塩化ベンゼトニウム、フェノール、ブチル若しくはベンジルアルコール、メチル若しくはプロピルパラベン等のアルキルパラベン、カテコール、レゾルシノール、シクロヘキサノール、３-ペンタノール、及びｍ-クレゾール等である。緩衝剤は、リン酸、クエン酸、及びその他の有機酸である。 The agent may contain a preservative that suppresses the growth of microorganisms or a buffer that helps keep the pH within acceptable limits. Preservatives include sodium azide, octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkylparabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, 3-Pentanol, m-cresol and the like. Buffering agents are phosphoric acid, citric acid, and other organic acids.
また、薬剤は、例えば、賦形剤、安定剤、ＥＤＴＡ等のキレート化剤、塩、又は抗菌剤を含んでもよい。他にも、アスコルビン酸及びメチオニン等の酸化防止剤、ポリペプチド、血清アルブミン、ゼラチン、若しくは非特異的免疫グロブリン等のタンパク質、ポリビニルピロリドン等の親水性ポリマー、グリシン、グルタミン、アスパラギン、ヒスチジン、アルギニン、若しくはリジン等のアミノ酸、グルコース、マンノース、若しくはデキストリン等の単糖類、二糖類、及びその他の炭水化物、スクロース、マンニトール、トレハロース、若しくはソルビトール等の糖類を含むことが可能である。 The agent may also contain, for example, excipients, stabilizers, chelating agents such as EDTA, salts, or antibacterial agents. In addition, antioxidants such as ascorbic acid and methionine, proteins such as polypeptides, serum albumin, gelatin, or non-specific immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, glycine, glutamine, asparagine, histidine, arginine, Alternatively, it can contain amino acids such as lysine, monosaccharides such as glucose, mannose, or dextrin, disaccharides, and other carbohydrates, saccharides such as sucrose, mannitol, trehalose, or sorbitol.
1.PROTAC(H-PGDS)-1及びPROTAC(H-PGDS)-2の合成
下記合成手順に従い、ポリエチレングリコールリンカーを介して、H-PGDSのリガンドであるTFC-007とユビキチンリガーゼリガンド(ポマリドミド)を結合させたPROTAC(H-PGDS)-1を合成した。
1. Synthesis of PROTAC (H-PGDS) -1 and PROTAC (H-PGDS) -2 Following the following synthetic procedure, TFC-007, which is a ligand for H-PGDS, and a ubiquitin ligase ligand (pomalidomide) are used via a polyethylene glycol linker. PROTAC (H-PGDS) -1 was synthesized.
上記合成手順において、化合物1及び化合物3は既出の手法にて合成した。
In the above synthesis procedure,
化合物2の合成では、化合物1(1.51 g, 5.99 mmol)のN,N-ジメチルホルムアミド溶液に、N-Bocピペラジン(1.69 g, 8.99 mmol)、EDCI(1.40 g, 7.19 mmol)、HOBt・H2O(1.02 g, 6.59 mmol)を加えて、室温で6時間撹拌した。その後、反応液を水に注ぎ、得られた固体を濾取し、乾燥して黄色固体を得た。得られた固体は精製せずにエタノール(60 ml)に溶解させ、パラジウム炭素(91.2 mg)を加え、水素雰囲気下、一晩撹拌した。反応液をセライトろ過し、赤褐色固体2(2.14 g, 92%)を得た。
In the synthesis of
化合物4の合成では、化合物2(2.10 g, 5.40 mmol)、化合物3(1.17 g, 6.49 mmol)、EDCI(1.25 g, 6.49 mmol)、HOBt・H2O(1.02 g, 6.59 mmolをN,N-ジメチルホルムアミド(20 mL)に溶解させた後、室温で6時間撹拌した。その後、反応液を水に注ぎ、得られた固体を濾取し、真空乾燥させ、灰色固体4(1.14 g, 36%)を得た。
In the synthesis of
化合物5の合成では、化合物4(20.7 mg, 0.0352 mmol)のCH2Cl2 溶液(1 mL)に、トリフルオロ酢酸(TFA, 40.2 mg, 0.352 mmol)を加えて室温で2時間撹拌した。反応液を減圧留去し、得られた固体をNHシリカゲルカラムクロマトグラフィー(CH2Cl2/MeOH=10/1)により精製し、無色透明のオイル状化合物5(19.3 mg, quant.)を得た。
In the synthesis of
PROTAC(HPGDS)-1の合成では、化合物5(21.2 mg, 0.0436 mmol)のN,N-ジメチルホルムアミド溶液(2.5 mL)に、市販の化合物6(21.9 mg, 0.0371 mmol), EDCI(12.0 mg, 0.0653 mmol)、DIPEA(11.0 mg, 0.0653 mmol)、HOBt (11.0 mg, 0.0872 mmol) を加えて室温で2日間撹拌し、反応液をHPLC(0.1% TFA MeCN/H2O = 15 : 85から50 : 50、40 min)により精製し, 白色固体PROTAC(HPGDS)-1 (2.88 mg, 2.78 μMmol, 7.5%) を得た。 For the synthesis of PROTAC (HPGDS) -1, commercially available compound 6 (21.9 mg, 0.0371 mmol), EDCI (12.0 mg, 12.0 mg,) was added to compound 5 (21.2 mg, 0.0436 mmol) in N, N-dimethylformamide (2.5 mL). 0.0653 mmol), DIPEA (11.0 mg, 0.0653 mmol), HOBt (11.0 mg, 0.0872 mmol) were added and stirred at room temperature for 2 days, and the reaction solution was HPLC (0.1% TFA MeCN / H 2 O = 15: 85 to 50). : 50, 40 min) to obtain a white solid PROTAC (HPGDS) -1 (2.88 mg, 2.78 μMmol, 7.5%).
また、下記合成手順に従い、ネガティブコントロールとして、ユビキチンリガーゼに結合できないN-Methylated pomalidomideを導入したPROTAC(H-PGDS)-2を合成した。 In addition, PROTAC (H-PGDS) -2 introduced with N-Methylated pomalidomide, which cannot bind to ubiquitin ligase, was synthesized as a negative control according to the following synthesis procedure.
上記合成手順において、化合物8の合成では、化合物5(56.7 mg, 0.119 mmol)のN,N-ジメチルホルムアミド溶液(2.0 ml)に、市販の化合物7(36.2 mg, 0.108 mmol)、EDCI(64.2 mg, 0.324 mmol)、DIPEA(49.2 mg, 0.324 mmol)を加えて、室温で1.5時間撹拌した。その後、反応液を減圧留去した後、飽和重曹水を加えて酢酸エチルで抽出し、有機層を飽和食塩水で洗浄、硫酸ナトリウムで乾燥させ、ろ過した後、減圧留去して得られた固体を、シリカゲルカラムクロマトグラフィー(CH2Cl2/MeOH = 10/1)により精製し、白色固体8 (23.7 mg, 25%)を得た。
In the above synthesis procedure, in the synthesis of
化合物9の合成では、化合物8(23.7 mg, 0.0295 mmol)をメタノール(4 ml)に溶解させた後、パラジウム炭素(6.9 mg)を加え、水素雰囲気下で24時間撹拌した。反応液をセライトろ過し、濾液を減圧留去し、白色固体9 (20.3 mg, 66%)を得た。
In the synthesis of
PROTAC(HPGDS)-2の合成では、化合物9(28.9 mg, 0.0276 mmol)のジメチルスルホキシド溶液(1 mL)に、化合物10(9.7 mg, 0.0308 mmol)、DIPEA(15.6 mg, 0.0772 mmol)を加えて90 °Cで30分間撹拌し、反応溶液を冷却し、室温で6時間撹拌した。得られた固体をHPLC(0.1% TFA MeCN/H2O = 10 : 90から90 : 10、40 min)により精製し、白色固体PROTAC(HPGDS)-2 (1.2 mg, 4.2%)を得た。 For the synthesis of PROTAC (HPGDS) -2, compound 10 (9.7 mg, 0.0308 mmol) and DIPEA (15.6 mg, 0.0772 mmol) were added to a dimethyl sulfoxide solution (1 mL) of compound 9 (28.9 mg, 0.0276 mmol). The reaction solution was stirred at 90 ° C for 30 minutes, the reaction solution was cooled, and the mixture was stirred at room temperature for 6 hours. The obtained solid was purified by HPLC (0.1% TFA MeCN / H 2 O = 10: 90 to 90: 10, 40 min) to obtain a white solid PROTAC (HPGDS) -2 (1.2 mg, 4.2%).
2.上記化合物のH-PGDS分解活性評価とメカニズム解析
H-PGDS発現細胞であるヒトKU812細胞にPROTAC(H-PGDS)-1(1～1000 nM)を添加し、37℃で3～24時間インキュベートした後、H-PGDSタンパク質量をウェスタンブロットにより評価した。その結果、PROTAC(H-PGDS)-1は濃度依存的且つ時間依存的にH-PGDSタンパク質を減少させることが明らかとなった。
2. Evaluation of H-PGDS decomposition activity and mechanism analysis of the above compounds
PROTAC (H-PGDS) -1 (1 to 1000 nM) was added to human KU812 cells expressing H-PGDS, incubated at 37 ° C for 3 to 24 hours, and then the amount of H-PGDS protein was evaluated by Western blotting. did. As a result, it was clarified that PROTAC (H-PGDS) -1 reduces the H-PGDS protein in a concentration-dependent and time-dependent manner.
結果を図２に示す。図２は、PROTAC(H-PGDS)-1を用いた分解誘導の検討結果であり、KU812細胞をPROTAC(H-PGDS)-1(0、1、10、100、1000 nM)で処理し、3/6/24時間培養したものである。H-PGDSのタンパク質量を内部標準であるβ-Actinのタンパク質量で補正し、DMSOコントロールを100とした相対値で評価した。実験は３回行い、棒グラフは平均±標準偏差を表す。 The results are shown in FIG. FIG. 2 shows the results of a study on induction of degradation using PROTAC (H-PGDS) -1, in which KU812 cells were treated with PROTAC (H-PGDS) -1 (0, 1, 10, 100, 1000 nM). It was cultured for 3/6/24 hours. The protein amount of H-PGDS was corrected by the protein amount of β-Actin, which is an internal standard, and evaluated by a relative value with DMSO control as 100. The experiment was performed 3 times and the bar graph shows the mean ± standard deviation.
次いで、PROTAC(H-PGDS)-1で処理したH-PGDSの減少のメカニズムを検討することとした。まず、PROTAC(H-PGDS)-1を処理した後のH-PGDSのターンオーバーを検討した。KU812細胞をタンパク質合成阻害剤であるCHXで処理すると、コントロールの細胞では6時間までH-PGDSタンパク質の量が保持される一方で、PROTAC(H-PGDS)-1を添加した細胞では6時間以内にH-PGDSタンパク質量が劇的に減少した。結果を図３に示す。図３は、CHXを用いたH-PGDSタンパク質のターンオーバーの検討結果であり、KU812細胞をCHX(10 μg/ml)存在下、PROTAC(H-PGDS)-1(0、100 nM)で処理し、0/1/2/4/6時間培養したものである。H-PGDSとcyclin-B1のタンパク質量を内部標準であるβ-Actinのタンパク質量で補正し、処理時間0での値を100とした相対値で評価した。実験は３回行い、棒グラフは平均±標準偏差を表す。
Next, we decided to investigate the mechanism of reduction of H-PGDS treated with PROTAC (H-PGDS) -1. First, the turnover of H-PGDS after processing PROTAC (H-PGDS) -1 was examined. Treatment of KU812 cells with the protein synthesis inhibitor CHX retains the amount of H-PGDS protein for up to 6 hours in control cells, whereas within 6 hours in cells supplemented with PROTAC (H-PGDS) -1. The amount of H-PGDS protein decreased dramatically. The results are shown in FIG. FIG. 3 shows the results of examining the turnover of H-PGDS protein using CHX. KU812 cells were treated with PROTAC (H-PGDS) -1 (0, 100 nM) in the presence of CHX (10 μg / ml). It was cultured for 0/1/2/4/6 hours. The protein amounts of H-PGDS and cyclin-B1 were corrected by the protein amount of β-Actin, which is an internal standard, and evaluated as a relative value with the value at
さらに、KU812細胞におけるH-PGDSのmRNAレベルを検討したところPROTAC(H-PGDS)-1の添加による影響は観測されなかったことから、PROTAC(H-PGDS)-1はH-PGDSタンパク質の分解剤であることが明らかとなった。結果を図４に示す。図４は、PROTAC(HPGDS)-1を添加した際のKU812細胞中のH-PGDS mRNAの発現量を示すものであり、KU812細胞をPROTAC(H-PGDS)-1(0、1、10、100、1000 nM)で処理し、6時間培養したものである。H-PGDSのタンパク質量を内部標準であるβ-Actinのタンパク質量で補正し、DMSOコントロールを100とした相対値で評価した。実験は３回行い、棒グラフは平均±標準偏差を表す。スチューデントのt-検定(両側検定)にて有意差を評価し、*はコントロールと比べP<0.01のものを表す。 Furthermore, when the mRNA level of H-PGDS in KU812 cells was examined, no effect was observed by the addition of PROTAC (H-PGDS) -1, so PROTAC (H-PGDS) -1 degraded the H-PGDS protein. It became clear that it was an agent. The results are shown in FIG. FIG. 4 shows the expression level of H-PGDS mRNA in KU812 cells when PROTAC (HPGDS) -1 was added, and KU812 cells were added to PROTAC (H-PGDS) -1 (0, 1, 10, 10). It was treated with 100, 1000 nM) and cultured for 6 hours. The protein amount of H-PGDS was corrected by the protein amount of β-Actin, which is an internal standard, and evaluated by a relative value with DMSO control as 100. The experiment was performed 3 times and the bar graph shows the mean ± standard deviation. Significant differences are evaluated by Student's t-test (two-sided test), and * indicates that P <0.01 compared to the control.
続いて、H-PGDSタンパク質に対するTFC-007とポマリドミドの効果を検討するために、TFC-007とポマリドミドの混合物(1 μM)を添加したところ、H-PGDSタンパク質の減少は観測されなかった。結果を図５(a)に示す。図５(a)は、KU812細胞をPROTAC(H-PGDS)-1(1 μM)又はTFC-007/ポマリドミド混合物(Ligand mixと記載。各1 μMずつ)で処理し、6時間培養したものである。これにより、2つのリガンド(TFC-007とポマリドミド)をコンジュゲートした本件新規化合物がH-PGDSタンパク質の分解にとって重要であることが明らかとなった。 Subsequently, in order to examine the effect of TFC-007 and pomalidomide on the H-PGDS protein, a mixture of TFC-007 and pomalidomide (1 μM) was added, and no decrease in H-PGDS protein was observed. The results are shown in FIG. 5 (a). FIG. 5 (a) shows KU812 cells treated with PROTAC (H-PGDS) -1 (1 μM) or TFC-007 / pomalidomide mixture (described as Ligand mix, 1 μM each) and cultured for 6 hours. be. This revealed that the novel compound conjugated with two ligands (TFC-007 and pomalidomide) is important for the degradation of H-PGDS protein.
また、H-PGDSタンパク質の分解に関わるセレブロンをリクルートする必要性を検討するために、PROTAC(H-PGDS)-1(100 nM)に過剰量のポマリドミド(10 μM)を共添加することにより競合阻害アッセイを行った。結果を図５(b)に示す。図５(b)は過剰量のポマリドミド(10 μM)を共添加した際のPROTAC(H-PGDS)-1によるH-PGDSタンパク質減少への影響を示すものであり、薬剤処理後にKU812細胞を6時間培養したものである。過剰量のポマリドミド存在下においては、PROTAC(H-PGDS)-1によるH-PGDSタンパク質の減少は観測されなかったことから、H-PGDSのタンパク質分解にはPROTAC(H-PGDS)-1のポマリドミド部位のセレブロンへの結合が重要であることが示唆された。 It also competes by co-adding an excess of pomalidomide (10 μM) to PROTAC (H-PGDS) -1 (100 nM) to investigate the need to recruit cereblon involved in the degradation of the H-PGDS protein. An inhibition assay was performed. The results are shown in FIG. 5 (b). FIG. 5 (b) shows the effect of PROTAC (H-PGDS) -1 on the reduction of H-PGDS protein when an excessive amount of pomalidomide (10 μM) was co-added. It was cultured for hours. Since no decrease in H-PGDS protein was observed by PROTAC (H-PGDS) -1 in the presence of an excessive amount of pomalidomide, PROTAC (H-PGDS) -1 pomalidomide was used for proteolysis of H-PGDS. It was suggested that the binding of the site to cereblon is important.
さらに、PROTAC(H-PGDS)-1によるH-PGDSタンパク質の分解に対するUPSの関与を検討するために、プロテアソーム阻害剤(MG132)又はユビキチン活性化酵素阻害剤(MLN7243)を共添加した際のH-PGDSタンパク質の量を測定した。結果を図５(c)に示す。図５(c)は、KU812細胞にてプロテアソーム阻害剤(MG132)又はユビキチン活性化酵素阻害剤(MLN7243)共添加におけるPROTAC(H-PGDS)-1によるH-PGDSタンパク質減少への影響を示すものであり、薬剤処理後にKU812細胞を6時間培養したものである。H-PGDSのタンパク質量を内部標準であるβ-Actinのタンパク質量で補正し、コントロールを100とした相対値で評価した。実験は３回行い、棒グラフは平均±標準偏差を表す。スチューデントのt-検定(両側検定)にて有意差を評価し、*はコントロールと比べP<0.01のものを表す。各種阻害剤の添加により、PROTAC(H-PGDS)-1によって誘導されるH-PGDSタンパク質の分解が抑制されたことから、H-PGDSタンパク質の分解にはUPSが関与していることが示唆された。 Furthermore, in order to investigate the involvement of UPS in the degradation of H-PGDS protein by PROTAC (H-PGDS) -1, H when a proteasome inhibitor (MG132) or a ubiquitin activating enzyme inhibitor (MLN7243) was co-added. -The amount of PGDS protein was measured. The results are shown in FIG. 5 (c). FIG. 5 (c) shows the effect of PROTAC (H-PGDS) -1 on the reduction of H-PGDS protein in KU812 cells when co-added with a proteasome inhibitor (MG132) or a ubiquitin activating enzyme inhibitor (MLN7243). KU812 cells were cultured for 6 hours after drug treatment. The protein amount of H-PGDS was corrected by the protein amount of β-Actin, which is an internal standard, and evaluated by a relative value with the control as 100. The experiment was performed 3 times and the bar graph shows the mean ± standard deviation. Significant differences are evaluated by Student's t-test (two-sided test), and * indicates that P <0.01 compared to the control. The addition of various inhibitors suppressed the degradation of the H-PGDS protein induced by PROTAC (H-PGDS) -1, suggesting that UPS is involved in the degradation of the H-PGDS protein. rice field.
3.PROTAC(H-PGDS)-1、PROTAC(H-PGDS)-2のH-PGDSへの結合能評価
上述したように、PROTAC(H-PGDS)-1はUPSを介した強力なH-PGDSタンパク質分解誘導剤であることを明らかとしたが、PROTAC(H-PGDS)-1はH-PGDSの酵素活性を阻害するTFC-007構造を含むため、H-PGDSの酵素活性も阻害する可能性がある。今後PROTAC(H-PGDS)-1によるPGD2産生抑制効果を検討する上で分解誘導活性の寄与を評価するために有用な、TFC-007とN-Methylated pomalidomide(セレブロンへの結合活性のないポマリドミド誘導体)から構成されるPROTAC(H-PGDS)-2を使用して結合能を評価した。
3. Evaluation of binding ability of PROTAC (H-PGDS) -1 and PROTAC (H-PGDS) -2 to H-PGDS As mentioned above, PROTAC (H-PGDS) -1 is a powerful H- via UPS. Although it was revealed that it is a PGDS proteolysis inducer, PROTAC (H-PGDS) -1 contains a TFC-007 structure that inhibits the enzymatic activity of H-PGDS, so that it can also inhibit the enzymatic activity of H-PGDS. There is sex. TFC-007 and N-Methylated pomalidomide (pomalidomide without binding activity to cereblon), which is useful for evaluating the contribution of degradation-inducing activity in investigating the effect of PROTAC (H-PGDS) -1 on suppressing PGD 2 production in the future. The binding ability was evaluated using PROTAC (H-PGDS) -2 composed of the derivative).
まずはPROTAC(H-PGDS)-1とPROTAC(H-PGDS)-2のH-PGDSへの結合活性、H-PGDSタンパク質の分解活性を評価した。結果を図６(a)に示す。図６(a)はProstaglandin D Synthase FP-Based Inhibitor Screening Assay Kit-Green (Cayman)を使用した蛍光偏光アッセイによるH-PGDSへの結合評価を示すものである。各化合物のH-PGDSへの結合親和性を蛍光偏光アッセイにより評価した結果、TFC-007、PROTAC(H-PGDS)-1とPROTAC(H-PGDS)-2は、H-PGDSに対して同等の親和性(0.32 μM、0.32 μM、0.30 μM)を示した(それぞれR2=0.9942, R2=0.9841, R2=0.9683, なおH-PGDSに対する特異的阻害剤であるHQL-79ではR2=0.9569)。 First, the binding activity of PROTAC (H-PGDS) -1 and PROTAC (H-PGDS) -2 to H-PGDS and the degrading activity of H-PGDS protein were evaluated. The results are shown in FIG. 6 (a). FIG. 6 (a) shows the evaluation of binding to H-PGDS by a fluorescence polarization assay using the Prostaglandin D Synthase FP-Based Inhibitor Screening Assay Kit-Green (Cayman). As a result of evaluating the binding affinity of each compound for H-PGDS by a fluorescence polarization assay, TFC-007, PROTAC (H-PGDS) -1 and PROTAC (H-PGDS) -2 are equivalent to H-PGDS. Affinities (0.32 μM, 0.32 μM, 0.30 μM) were shown (R 2 = 0.9942, R 2 = 0.9841, R 2 = 0.9683, respectively, and R 2 in HQL-79, which is a specific inhibitor for H-PGDS. = 0.9569).
また、H-PGDSタンパク質の分解を検討した。結果を図６(b)に示す。図６(b)はPROTAC(H-PGDS)-2によるH-PGDS分解活性評価を示すものであり、KU812細胞をPROTAC(H-PGDS)-1(100 nM)又はPROTAC(H-PGDS)-2(100 nM)で処理し、6時間培養したものである。H-PGDSのタンパク質量を内部標準であるβ-Actinのタンパク質量で補正し、DMSOコントロールを100とした相対値で評価した。実験は３回行い、棒グラフは平均±標準偏差を表す。スチューデントのt-検定(両側検定)にて有意差を評価し、*はコントロールと比べP<0.01のものを表す。図６(b)に示されるようにPROTAC(H-PGDS)-2はH-PGDSタンパク質の量を減少させなかった。 We also investigated the degradation of H-PGDS protein. The results are shown in FIG. 6 (b). FIG. 6 (b) shows the evaluation of H-PGDS degradation activity by PROTAC (H-PGDS) -2, and KU812 cells were subjected to PROTAC (H-PGDS) -1 (100 nM) or PROTAC (H-PGDS)-. It was treated with 2 (100 nM) and cultured for 6 hours. The protein amount of H-PGDS was corrected by the protein amount of β-Actin, which is an internal standard, and evaluated by a relative value with DMSO control as 100. The experiment was performed 3 times and the bar graph shows the mean ± standard deviation. Significant differences are evaluated by Student's t-test (two-sided test), and * indicates that P <0.01 compared to the control. PROTAC (H-PGDS) -2 did not reduce the amount of H-PGDS protein as shown in FIG. 6 (b).
以上よりPROTAC(H-PGDS)-2はH-PGDSの酵素阻害活性を持つが分解誘導活性のない化合物であることが示唆され、PROTAC(H-PGDS)-1によるPGD2産生抑制効果を検討する際のネガティブコントロール化合物になると考えられた。 From the above, it is suggested that PROTAC (H-PGDS) -2 is a compound that has the enzyme inhibitory activity of H-PGDS but not the degradation-inducing activity, and the effect of PROTAC (H-PGDS) -1 on suppressing PGD 2 production was investigated. It was thought that it would be a negative control compound when doing so.
4. PROTAC(H-PGDS)-3～PROTAC(H-PGDS)-10の合成
PROTAC(H-PGDS)-1をリードとしてPEG数の異なるPROTAC(H-PGDS)-3～PROTAC(H-PGDS)-7を設計、合成した。またリンカーを持たない化合物PROTAC(H-PGDS)-7、対応するネガティブコントロールPROTAC(H-PGDS)-8を同様のルートにより合成した。またリンカーを持たない化合物PROTAC(H-PGDS)-10を合成した。更にPomalidomideとTFC-007類縁体を連結したPROTAC(H-PGDS)-9を合成した。
4. Synthesis of PROTAC (H-PGDS) -3 to PROTAC (H-PGDS) -10
PROTAC (H-PGDS) -3 to PROTAC (H-PGDS) -7 with different PEG numbers were designed and synthesized with PROTAC (H-PGDS) -1 as the read. In addition, the compound PROTAC (H-PGDS) -7 having no linker and the corresponding negative control PROTAC (H-PGDS) -8 were synthesized by the same route. In addition, the compound PROTAC (H-PGDS) -10 having no linker was synthesized. Furthermore, PROTAC (H-PGDS) -9 in which Pomalidomide and TFC-007 analog were linked was synthesized.
合成手順を下記に示す。 The synthesis procedure is shown below.
NMRはECZ600(JEOL社)を使用した。化学シフト値(δ，ppm)は残留溶媒シグナル(DMSO-d6: 2.49 for 1H NMR, 39.5 for 13C NMR)を用いて補正した。 ECZ600 (JEOL) was used for NMR. The chemical shift values (δ, ppm) were corrected using a residual solvent signal (DMSO-d 6 : 2.49 for 1 H NMR, 39.5 for 13 C NMR).
分析HPLCはInertsil(登録商標) WP300 C18, 5 μm, 4.6 mm × 250 mm, solvent A: 0.1% TFA/water, solvent B: 0.1% TFA/MeCN, gradient: 10-90% gradient of solvent B over 30 min, flow rate: 2 mL/min, 40°C
General Procedure A
アミン体をジメチルホルムアミドに溶解させ、化合物5 (1.5 eq)とジイソプロピルエチルアミン(1.5 eq)を加えた。マイクロ波照射下80度で40分間反応させ、50度にて42時間撹拌後、反応液を冷却し、ろ過した。HPLC (gradient 30-60% MeCN-H2O containing 0.1% TFA in 40 min)にて精製した。
Analytical HPLC is Inertsil® WP300 C18, 5 μm, 4.6 mm × 250 mm, solvent A: 0.1% TFA / water, solvent B: 0.1% TFA / MeCN, gradient: 10-90% gradient of solvent B over 30 min, flow rate: 2 mL / min, 40 ° C
General Procedure A
The amine compound was dissolved in dimethylformamide and compound 5 (1.5 eq) and diisopropylethylamine (1.5 eq) were added. The reaction was carried out at 80 ° C. for 40 minutes under microwave irradiation, and after stirring at 50 ° C. for 42 hours, the reaction solution was cooled and filtered. Purified by HPLC (gradient 30-60% MeCN-H 2 O containing 0.1% TFA in 40 min).
General Procedure B
アミン体をジメチルホルムアミドに溶解させ、化合物5または7 (3 eq)とジイソプロピルエチルアミン(3 eq)を加えた。マイクロ波照射下80度で2時間反応させた。反応液を冷却し、ろ過した。HPLC (gradient 30-60% MeCN-H2O containing 0.1% TFA in 40 min)にて精製した。
General Procedure B
The amine compound was dissolved in dimethylformamide and
PROTAC(H-PGDS)-6の合成
化合物4 (32.2 mg, 53.5 μmol)をジメチルホルムアミド(0.5 mL)に溶解させ、General Procedure Bの方法に従い、PROTAC(H-PGDS)-6を黄色固体として得た(14.5 mg, 32%)。
1H NMR (600 MHz, DMSO-d6) : δ 11.1 (s, 1H), 10.5 (br, 1H), 9.10 (s, 2H), 7.78-7.71 (br, 3H), 7.58 (t, J = 7.2 Hz, 1H), 7.47 (t, J = 8.4 Hz, 2H), 7.38 (br, 1H), 7.29 (t, J = 7.2 Hz, 1H), 7.25 (d, J = 8.4 Hz, 2H), 7.13 (d, J = 9.0 Hz, 1H), 7.40 (d, J = 7.8 Hz, 1H), 6.57 (br, 1H), 5.05 (dd, J = 7.2, 5.4 Hz, 1H), 3.69-3.60 (m, 6H), 3.53-3.39 (m, 10H), 3.26 (br, 1H), 2.99-2.84 (m, 2H), 2.61 (br, 3H), 2.18-2.12 (m, 1H), 2.11-2.06 (m, 1H), 2.06-1.99 (m, 1H), 1.86 (br, 4H).
13C NMR (151 MHz, DMSO-d6) : δ 172.8, 172.0, 170.1, 169.0 (2C), 167.3, 165.8, 161.7, 159.8 (2C), 152.5, 146.4 (2C), 136.3 (2C), 132.1, 132.1, 129.8 (2C), 125.6, 123.9 (2C), 121.6 (2C), 121.4 (2C), 117.4 (2C), 114.6, 68.6 (2C), 66.8, 66.5, 55.0, 48.6 (6C), 41.6, 31.0 (2C), 26.9, 22.1.
HRMS (ESI) m/z calculated for C45H48N9O9[M + H]+ 858.3570 found 858.3563.
HPLC purity: >98% (tR = 13.0 min).
Synthesis of PROTAC (H-PGDS) -6 Compound 4 (32.2 mg, 53.5 μmol) was dissolved in dimethylformamide (0.5 mL) to obtain PROTAC (H-PGDS) -6 as a yellow solid according to the method of General Procedure B. (14.5 mg, 32%).
1 H NMR (600 MHz, DMSO-d 6) : δ 11.1 (s, 1H), 10.5 (br, 1H), 9.10 (s, 2H), 7.78-7.71 (br, 3H), 7.58 (t, J = 7.2 Hz, 1H), 7.47 (t, J = 8.4 Hz, 2H), 7.38 (br, 1H), 7.29 (t, J = 7.2 Hz, 1H), 7.25 (d, J = 8.4 Hz, 2H), 7.13 (d, J = 9.0 Hz, 1H), 7.40 (d, J = 7.8 Hz, 1H), 6.57 (br, 1H), 5.05 (dd, J = 7.2, 5.4 Hz, 1H), 3.69-3.60 (m, 6H), 3.53-3.39 (m, 10H), 3.26 (br, 1H), 2.99-2.84 (m, 2H), 2.61 (br, 3H), 2.18-2.12 (m, 1H), 2.11-2.06 (m, 1H), 2.06-1.99 (m, 1H), 1.86 (br, 4H).
13 C NMR (151 MHz, DMSO-d 6 ): δ 172.8, 172.0, 170.1, 169.0 (2C), 167.3, 165.8, 161.7, 159.8 (2C), 152.5, 146.4 (2C), 136.3 (2C), 132.1, 132.1, 129.8 (2C), 125.6, 123.9 (2C), 121.6 (2C), 121.4 (2C), 117.4 (2C), 114.6, 68.6 (2C), 66.8, 66.5, 55.0, 48.6 (6C), 41.6, 31.0 (2C), 26.9, 22.1.
HRMS (ESI) m / z calculated for C 45 H 48 N 9 O 9 [M + H] + 858.3570 found 858.3563.
HPLC purity:> 98% (t R = 13.0 min).
PROTAC(H-PGDS)-5の合成
化合物3 (9.2 mg, 14.2 μmol)をジメチルホルムアミド(0.5 mL)に溶解させ、General Procedure Aの方法に従い、PROTAC(H-PGDS)-5を黄色固体として得た(1.2 mg, 9%)。
HRMS (ESI) m/z calculated for C47H52N9O10[M + H]+ 902.3832 found 902.3838.
HPLC purity: >93% (tR = 13.1 min).
Synthesis of PROTAC (H-PGDS) -5 Compound 3 (9.2 mg, 14.2 μmol) was dissolved in dimethylformamide (0.5 mL) to obtain PROTAC (H-PGDS) -5 as a yellow solid according to the method of General Procedure A. (1.2 mg, 9%).
HRMS (ESI) m / z calculated for C 47 H 52 N 9 O 10 [M + H] + 902.3832 found 902.3838.
HPLC purity:> 93% (t R = 13.1 min).
PROTAC(H-PGDS)-4の合成
化合物2 (18.2 mg, 26.4 μmol)をジメチルホルムアミド(0.5 mL)に溶解させ、General Procedure Bの方法に従い、PROTAC(H-PGDS)-4を黄色固体として得た(3.0 mg, 12%)。
1H NMR (600 MHz, DMSO-d6) : δ 11.1 (s, 1H), 10.5 (br, 1H), 9.10 (s, 2H), 7.74 (br, 3H), 7.57 (t, J = 18.0 Hz, 1H), 7.47 (t, J= 7.8 Hz, 2H), 7.32 (br, 1H), 7.29 (t, J = 7.2 Hz, 1H), 7.25 (d, J= 7.2 Hz, 2H), 7.14 (d, J = 8.4 Hz, 1H), 7.03 (d, J = 7.2 Hz, 1H), 6.59 (br, 1H), 5.05 (dd, J = 7.8, 5.4 Hz, 1H), 3.68-3.63 (m, 2H), 3.62-3.34 (m, 24H), 2.90-2.84 (m, 1H), 2.61-2.55 (m, 5H), 2.04-1.99 (m, 1H), 1.84 (br, 4H).
13C NMR (151 MHz, DMSO-d6) : δ 172.8, 172.1, 170.1, 168.9 (2C), 167.3, 165.7, 161.6, 159.8 (2C), 152.5, 146.4 (2C), 136.2 (2C), 132.1, 129.8 (2C), 129.3, 125.6 , 123.9 (2C), 121.6 (2C), 121.4 (2C), 117.5 (2C), 115.2, 69.8-69.7 (5C), 68.9, 67.0, 66.7, 54.9, 48.5 (6C), 41.7, 31.0 (2C), 27.1, 22.1.
HRMS (ESI) m/z calculated for C49H56N9O11946.4094 [M + H]+ found 946.4098.
HPLC purity: >85% (tR = 13.3 min).
Synthesis of PROTAC (H-PGDS) -4 Compound 2 (18.2 mg, 26.4 μmol) was dissolved in dimethylformamide (0.5 mL) to obtain PROTAC (H-PGDS) -4 as a yellow solid according to the method of General Procedure B. (3.0 mg, 12%).
1 H NMR (600 MHz, DMSO-d 6 ): δ 11.1 (s, 1H), 10.5 (br, 1H), 9.10 (s, 2H), 7.74 (br, 3H), 7.57 (t, J = 18.0 Hz) , 1H), 7.47 (t, J = 7.8 Hz, 2H), 7.32 (br, 1H), 7.29 (t, J = 7.2 Hz, 1H), 7.25 (d, J = 7.2 Hz, 2H), 7.14 (d) , J = 8.4 Hz, 1H), 7.03 (d, J = 7.2 Hz, 1H), 6.59 (br, 1H), 5.05 (dd, J = 7.8, 5.4 Hz, 1H), 3.68-3.63 (m, 2H) , 3.62-3.34 (m, 24H), 2.90-2.84 (m, 1H), 2.61-2.55 (m, 5H), 2.04-1.99 (m, 1H), 1.84 (br, 4H).
13 C NMR (151 MHz, DMSO-d 6 ): δ 172.8, 172.1, 170.1, 168.9 (2C), 167.3, 165.7, 161.6, 159.8 (2C), 152.5, 146.4 (2C), 136.2 (2C), 132.1, 129.8 (2C), 129.3, 125.6, 123.9 (2C), 121.6 (2C), 121.4 (2C), 117.5 (2C), 115.2, 69.8-69.7 (5C), 68.9, 67.0, 66.7, 54.9, 48.5 (6C) , 41.7, 31.0 (2C), 27.1, 22.1.
HRMS (ESI) m / z calculated for C 49 H 56 N 9 O 11 946.4094 [M + H] + found 946.4098.
HPLC purity:> 85% (t R = 13.3 min).
PROTAC(H-PGDS)-3の合成
化合物1トリフルオロ酢酸塩 (30.5 mg, 36.0 μmol)をジメチルホルムアミド(0.5 mL)に溶解させ、General Procedure Aの方法に従い、PROTAC(H-PGDS)-3を黄色固体として得た(1.0 mg, 3%)。
HRMS (ESI) m/z calculated for C51H60N9O12[M + H]+ 990.4356 found 990.4332.
HPLC purity: >97% (tR = 13.4 min).
Synthesis of PROTAC (H-PGDS) -3
HRMS (ESI) m / z calculated for C 51 H 60 N 9 O 12 [M + H] + 990.4356 found 990.4332.
HPLC purity:> 97% (t R = 13.4 min).
PROTAC(H-PGDS)-7の合成
化合物6 (20 mg, 41.1 μmol)と化合物5を用いてGeneral Procedure Bの方法に従い、PROTAC(H-PGDS)-7を黄色固体として得た(10.7 mg, 35%)。
1H NMR (600 MHz, DMSO-d6) : δ 11.1 (s, 1H), 10.5 (br, 1H), 9.09 (s, 2H), 7.72 (m, 4H), 7.47 (t, J = 8.4 Hz, 2H), 7.40 (d, J = 6.6 Hz, 1H), 7.35 (d, J = 8.4 Hz, 1H), 7.29 (t, J= 7.8 Hz, 1H), 7.25-2.22 (m, 3H), 5.10 (dd, J = 7.2, 5.4 Hz, 1H), 3.75 (br, 2H), 3.67 (br, 4H), 3.34 (br, 2H), 3.26 (br, 2H), 3.15 (br, 1H), 2.98 (br, 1H), 3.15 (br, 1H), 2.90-2.84 (m, 1H), 2.60-2.51 (m, 2H), 2.02 (br, 1H), 1.86 (br, 4H).
13C NMR (151 MHz, DMSO-d6) : δ 172.7, 169.9, 166.9, 166.3, 165.7 (2C), 161.5, 159.7 (2C), 152.5, 149.3 (2C), 135.9 (2C), 133.5, 129.7 (2C), 125.5, 123.9, 123.8 (2C), 121.5 (2C), 121.3 (2C), 116.9 (2C), 115.2, 51.1 (2C), 48.9 (2C), 48.7 (2C), 44.8, 41.0, 30.9 (2C), 27.1, 21.7.
HRMS (ESI) m/z calculated for C40H39N8O7[M + H]+ 743.2936 found 743.2939
HPLC purity: >94% (tR= 12.6 min).
Synthesis of PROTAC (H-PGDS) -7 Using compound 6 (20 mg, 41.1 μmol) and
1 H NMR (600 MHz, DMSO-d 6 ): δ 11.1 (s, 1H), 10.5 (br, 1H), 9.09 (s, 2H), 7.72 (m, 4H), 7.47 (t, J = 8.4 Hz) , 2H), 7.40 (d, J = 6.6 Hz, 1H), 7.35 (d, J = 8.4 Hz, 1H), 7.29 (t, J = 7.8 Hz, 1H), 7.25-2.22 (m, 3H), 5.10 (dd, J = 7.2, 5.4 Hz, 1H), 3.75 (br, 2H), 3.67 (br, 4H), 3.34 (br, 2H), 3.26 (br, 2H), 3.15 (br, 1H), 2.98 ( br, 1H), 3.15 (br, 1H), 2.90-2.84 (m, 1H), 2.60-2.51 (m, 2H), 2.02 (br, 1H), 1.86 (br, 4H).
13 C NMR (151 MHz, DMSO-d 6 ): δ 172.7, 169.9, 166.9, 166.3, 165.7 (2C), 161.5, 159.7 (2C), 152.5, 149.3 (2C), 135.9 (2C), 133.5, 129.7 ( 2C), 125.5, 123.9, 123.8 (2C), 121.5 (2C), 121.3 (2C), 116.9 (2C), 115.2, 51.1 (2C), 48.9 (2C), 48.7 (2C), 44.8, 41.0, 30.9 ( 2C), 27.1, 21.7.
HRMS (ESI) m / z calculated for C 40 H 39 N 8 O 7 [M + H] + 743.2936 found 743.2939
HPLC purity:> 94% (t R = 12.6 min).
PROTAC(H-PGDS)-8の合成
化合物6 (30 mg, 61.7 μmol)と化合物7をジメチルホルムアミド(0.50 mL)に溶解させ、General Procedure Bの方法に従い、PROTAC(H-PGDS)-8を黄色固体として得た(10.7 mg, 22%)。
1H NMR (600 MHz, DMSO-d6) : δ 10.4 (br, 1H), 9.09 (s, 2H), 7.73 (t, J = 7.2 Hz, 1H), 7.68 (br, 2H), 7.47 (t, J = 7.8 Hz, 2H), 7.40 (d, J = 7.8 Hz, 1H), 7.36 (d, J = 9.0 Hz, 1H), 7.29 (t, J = 6.6 Hz, 1H), 7.25 (d, J = 8.4 Hz, 2H), 7.16 (br, 1H), 5.18 (dd, J = 8.4, 4.8 Hz, 1H), 3.75-3.67 (m, 8H), 3.34 (s, 2H), 3.27 (s, 2H), 3.01 (s, 3H), 2.97-2.91 (m, 2H), 2.76 (d, J = 16.2 Hz, 1H), 2.60-2.53 (m, 2H), 2.04 (br, 1H), 1.82 (br, 4H).
13C NMR (151 MHz, DMSO-d6) : δ 172.3, 170.2, 167.5, 166.9, 166.3 (2C), 161.8, 160.3 (2C), 153.1, 149.9 (2C), 136.5 (2C), 134.4, 130.3 (2C), 126.2, 124.6, 124.5 (2C), 122.2 (2C), 122.0 (2C), 117.5 (2C), 115.8, 51.7 (2C), 49.9 (2C), 45.39 (2C), 43.68, 41.6, 31.7 (2C), 27.9, 27.2, 21.7.
HRMS (ESI) m/z calculated for C41H41N3O5757.3087 [M + H]+ found 757.3090.
HPLC purity: >98% (tR = 13.9 min).
Synthesis of PROTAC (H-PGDS) -8 Compound 6 (30 mg, 61.7 μmol) and
1 H NMR (600 MHz, DMSO-d 6 ): δ 10.4 (br, 1H), 9.09 (s, 2H), 7.73 (t, J = 7.2 Hz, 1H), 7.68 (br, 2H), 7.47 (t) , J = 7.8 Hz, 2H), 7.40 (d, J = 7.8 Hz, 1H), 7.36 (d, J = 9.0 Hz, 1H), 7.29 (t, J = 6.6 Hz, 1H), 7.25 (d, J) = 8.4 Hz, 2H), 7.16 (br, 1H), 5.18 (dd, J = 8.4, 4.8 Hz, 1H), 3.75-3.67 (m, 8H), 3.34 (s, 2H), 3.27 (s, 2H) , 3.01 (s, 3H), 2.97-2.91 (m, 2H), 2.76 (d, J = 16.2 Hz, 1H), 2.60-2.53 (m, 2H), 2.04 (br, 1H), 1.82 (br, 4H) ).
13 C NMR (151 MHz, DMSO-d 6 ): δ 172.3, 170.2, 167.5, 166.9, 166.3 (2C), 161.8, 160.3 (2C), 153.1, 149.9 (2C), 136.5 (2C), 134.4, 130.3 ( 2C), 126.2, 124.6, 124.5 (2C), 122.2 (2C), 122.0 (2C), 117.5 (2C), 115.8, 51.7 (2C), 49.9 (2C), 45.39 (2C), 43.68, 41.6, 31.7 ( 2C), 27.9, 27.2, 21.7.
HRMS (ESI) m / z calculated for C 41 H 41 N 3 O 5 757.3087 [M + H] + found 757.3090.
HPLC purity:> 98% (t R = 13.9 min).
化合物10の合成
化合物8(238 mg, 0.858 mmol)の無水DMF(4 mL)溶液に化合物9(204 mg, 0.944 mmol)、EDCI塩酸塩(181 mg, 0.944mmol)、HOBt(145 mg, 0.944 mmol)を加えた。室温にて6時間撹拌し、水を加えた。反応液をろ過し、固体を水で洗浄した。得られた固体を真空下で乾燥させて化合物10(288 mg, 71%)を灰色固体として得た。
1H NMR (600 MHz, DMSO-d6) : δ 10.3 (s, 1H), 9.08 (s, 2H), 7.57 (d, J = 9.0 Hz, 2H), 7.46 (t, J = 7.8 Hz, 2H), 7.29 (t, J = 7.8 Hz, 1H), 7.24 (d, J = 7.8 Hz, 2H), 6.96 (d, J = 9.0 Hz, 2H), 3.45 (br, 4H), 3.06 (br, 4H), 1.41 (s, 9H).
13C NMR (151 MHz, DMSO-d6) : δ 165.6, 161.1, 159.6 (2C), 153.8, 152.6, 147.6, 130.8, 129.8 (2C), 125.6, 124.1, 121.6 (2C), 121.4 (2C), 116.1 (2C), 79.0, 48.6 (4C), 28.0 (3C).
HRMS (ESI): m/z calcd. for C26H30N5O4 [M+H]+476.2292 found 476.2294.
Synthesis of
1 H NMR (600 MHz, DMSO-d 6 ): δ 10.3 (s, 1H), 9.08 (s, 2H), 7.57 (d, J = 9.0 Hz, 2H), 7.46 (t, J = 7.8 Hz, 2H) ), 7.29 (t, J = 7.8 Hz, 1H), 7.24 (d, J = 7.8 Hz, 2H), 6.96 (d, J = 9.0 Hz, 2H), 3.45 (br, 4H), 3.06 (br, 4H) ), 1.41 (s, 9H).
13 C NMR (151 MHz, DMSO-d 6 ): δ 165.6, 161.1, 159.6 (2C), 153.8, 152.6, 147.6, 130.8, 129.8 (2C), 125.6, 124.1, 121.6 (2C), 121.4 (2C), 116.1 (2C), 79.0, 48.6 (4C), 28.0 (3C).
HRMS (ESI): m / z calcd. For C 26 H 30 N 5 O 4 [M + H] + 476.2292 found 476.2294.
化合物11の合成
化合物10(50 mg, 0.105 mmol)のジクロロメタン(1.5 mL)溶液にトリフルオロ酢酸(0.5 mL)加えて室温にて撹拌した。4時間後、溶媒を減圧留去した。得られた残渣はアミノシリカゲルクロマトグラフィー(ジクロロメタン:メタノール=1:0 to 9:1)にて精製し、化合物11(38.4 mg, 97%)を淡茶色固体として得た。
1H NMR (600 MHz, DMSO-d6) : δ 10.2 (s, 1H), 9.08 (s, 2H), 7.56 (d, J = 9.0 Hz, 2H), 7.46 (t, J = 7.8 Hz, 2H), 7.29 (t, J = 7.8 Hz, 1H), 7.24 (d, J = 7.8 Hz, 2H), 6.91 (d, J = 9.0 Hz, 2H), 3.00 (br, 4H), 2.82 (br, 4H).
13C NMR (151 MHz, DMSO-d6) : δ 165.6, 161.0, 159.6 (2C), 152.6, 148.5, 130.2, 129.8 (2C), 125.6, 124.2, 121.6 (2C), 121.4 (2C), 115.4 (2C), 49.6 (2C), 45.6 (2C).
HRMS (ESI): m/z calcd. for C21H22N5O2 [M+H]+376.1768 found 376.1767.
Synthesis of Compound 11 Trifluoroacetic acid (0.5 mL) was added to a solution of Compound 10 (50 mg, 0.105 mmol) in dichloromethane (1.5 mL) and stirred at room temperature. After 4 hours, the solvent was distilled off under reduced pressure. The obtained residue was purified by amino silica gel chromatography (dichloromethane: methanol = 1: 0 to 9: 1) to obtain Compound 11 (38.4 mg, 97%) as a light brown solid.
1 H NMR (600 MHz, DMSO-d 6 ): δ 10.2 (s, 1H), 9.08 (s, 2H), 7.56 (d, J = 9.0 Hz, 2H), 7.46 (t, J = 7.8 Hz, 2H) ), 7.29 (t, J = 7.8 Hz, 1H), 7.24 (d, J = 7.8 Hz, 2H), 6.91 (d, J = 9.0 Hz, 2H), 3.00 (br, 4H), 2.82 (br, 4H) ).
13 C NMR (151 MHz, DMSO-d 6 ): δ 165.6, 161.0, 159.6 (2C), 152.6, 148.5, 130.2, 129.8 (2C), 125.6, 124.2, 121.6 (2C), 121.4 (2C), 115.4 ( 2C), 49.6 (2C), 45.6 (2C).
HRMS (ESI): m / z calcd. For C 21 H 22 N 5 O 2 [M + H] + 376.1768 found 376.1767.
PROTAC(H-PGDS)-9の合成
化合物11(38.4 mg, 0.103 mmol)の無水DMF(1 mL)溶液に化合物5(85.1 mg, 0.307 mmol)とジイソプロピルエチルアミン(53.5μL, 0.307 mmol)を加え、マイクロ波照射しながら80度で2時間反応させ、溶媒を減圧留去した。得られた残渣をHPLC(30-60% in 40min)で精製し、PROTAC(H-PGDS)-9(12.1 mg, 17%)を黄色固体として得た。
1H NMR (600 MHz, DMSO-d6) : δ 11.1(s, 1H), 10.3 (s, 1H), 9.09 (s, 2H), 7.73 (t, J = 7.2 Hz, 1H), 7.60 (d, J = 9.0 Hz, 2H), 7.47 (t, J = 7.2 Hz, 2H), 7.40 (t, J = 7.2 Hz, 2H), 7.29 (t, J = 7.26 Hz, 1H), 7.25 (d, J = 8.4 Hz, 2H), 7.03 (d, J = 9.0 Hz, 2H), 5.11 (dd, J = 7.8 Hz, 5.4Hz, 1H), 3.39 (br, 12H).
13C NMR (151 MHz, DMSO-d6) : δ 172.8,170.0,167.0,166.4,165.6, 161.1, 159.6 (2C), 152.6, 149.5, 147.6, 136.0, 133.7, 130.7, 129.8 (2C), 125.6, 124.2, 123.8, 121.6 (2C), 121.4, 116.8, 115.8, 115.1, 54.9, 50.4, 48.8 (2C), 48.6 (2C), 30.9, 29.0, 22.1.
HRMS (ESI): m/z calcd. for C34H30N7O6[M+H]+ 632.2252 found 632.2245.
Synthesis of PROTAC (H-PGDS) -9 Compound 5 (85.1 mg, 0.307 mmol) and diisopropylethylamine (53.5 μL, 0.307 mmol) were added to an anhydrous DMF (1 mL) solution of compound 11 (38.4 mg, 0.103 mmol). The reaction was carried out at 80 ° C. for 2 hours while irradiating with microwaves, and the solvent was distilled off under reduced pressure. The obtained residue was purified by HPLC (30-60% in 40 min) to obtain PROTAC (H-PGDS) -9 (12.1 mg, 17%) as a yellow solid.
1 H NMR (600 MHz, DMSO-d 6 ): δ 11.1 (s, 1H), 10.3 (s, 1H), 9.09 (s, 2H), 7.73 (t, J = 7.2 Hz, 1H), 7.60 (d) , J = 9.0 Hz, 2H), 7.47 (t, J = 7.2 Hz, 2H), 7.40 (t, J = 7.2 Hz, 2H), 7.29 (t, J = 7.26 Hz, 1H), 7.25 (d, J) = 8.4 Hz, 2H), 7.03 (d, J = 9.0 Hz, 2H), 5.11 (dd, J = 7.8 Hz, 5.4 Hz, 1H), 3.39 (br, 12H).
13 C NMR (151 MHz, DMSO-d 6 ): δ 172.8, 170.0, 167.0, 166.4, 165.6, 161.1, 159.6 (2C), 152.6, 149.5, 147.6, 136.0, 133.7, 130.7, 129.8 (2C), 125.6, 124.2, 123.8, 121.6 (2C), 121.4, 116.8, 115.8, 115.1, 54.9, 50.4, 48.8 (2C), 48.6 (2C), 30.9, 29.0, 22.1.
HRMS (ESI): m / z calcd. For C 34 H 30 N 7 O 6 [M + H] + 632.2252 found 632.2245.
PROTAC(H-PGDS)-10の合成
下記合成手順に従いGeneral Procedure Bの方法にて、H-PGDSのリガンドであるTAS-205とユビキチンリガーゼリガンド(ポマリドミド)を結合させたPROTAC(H-PGDS)-10を合成した。
Synthesis of PROTAC (H-PGDS) -10 PROTAC (H-PGDS)-by binding TAS-205, which is a ligand for H-PGDS, and a ubiquitin ligase ligand (pomalidomide) by the method of General Procedure B according to the following synthesis procedure. 10 was synthesized.
1H NMR (600 MHz, CD3OD) : δ 7.68 (t, J = 7.2 Hz, 2H), 7.60 (d, J = 9.0 Hz, 2H), 7.47 (t, J = 7.2 Hz, 2H), 7.42 (d, J = 7.2 Hz, 1H), 7.33 (d, J = 8.4 Hz, 1H), 7.21 (d, J = 8.4 Hz, 1H), 6.83 (t, J = 1.8 Hz, 1H), 6.41 (dd J = 1.8, 3.6 Hz, 1H), 6.08 (dd, J = 3.0, 3.6 Hz, 1H), 5.08-5.10 (m, 1H), 3.17-3.80 m, 23H), 2.81-2.86 (m, 1H), 2.68-2.76 (m, 2H), 2.07-2.19 (m, 6H). 1 H NMR (600 MHz, CD 3 OD): δ 7.68 (t, J = 7.2 Hz, 2H), 7.60 (d, J = 9.0 Hz, 2H), 7.47 (t, J = 7.2 Hz, 2H), 7.42 (d, J = 7.2 Hz, 1H), 7.33 (d, J = 8.4 Hz, 1H), 7.21 (d, J = 8.4 Hz, 1H), 6.83 (t, J = 1.8 Hz, 1H), 6.41 (dd) J = 1.8, 3.6 Hz, 1H), 6.08 (dd, J = 3.0, 3.6 Hz, 1H), 5.08-5.10 (m, 1H), 3.17-3.80 m, 23H), 2.81-2.86 (m, 1H), 2.68-2.76 (m, 2H), 2.07-2.19 (m, 6H).
分析HPLCはInertsil(登録商標) WP300 C18, 5 μm, 4.6 mm × 250 mm, solvent A: 0.1% TFA/water, solvent B: 0.1% TFA/MeCN, gradient: 10-90% gradient of solvent B over 30 min, flow rate: 2 mL/min, 40°C
HRMS (ESI) m/z calculated for C49H56N9O11 763.3442 [M + H]+ found 763.3462.
HPLC purity: >98% (tR = 10.8 min).
Analytical HPLC is Inertsil® WP300 C18, 5 μm, 4.6 mm × 250 mm, solvent A: 0.1% TFA / water, solvent B: 0.1% TFA / MeCN, gradient: 10-90% gradient of solvent B over 30 min, flow rate: 2 mL / min, 40 ° C
HRMS (ESI) m / z calculated for C49H56N9O11 763.3442 [M + H] + found 763.3462.
HPLC purity:> 98% (tR = 10.8 min).
5.上記化合物のH-PGDS分解活性評価とメカニズム解析
H-PGDS発現細胞(ヒトKU812細胞)に合成した化合物PROTAC(H-PGDS)-１，PROTAC(H-PGDS)-3～PROTAC(H-PGDS)-10を添加し、37℃で6時間インキュベートした後、H-PGDSタンパク質の量をウェスタンブロットにより評価し、化合物によるH-PGDSの分解に対するUPSの関与の検討は、プロテアソーム阻害剤(MG132)、ユビキチン活性化酵素阻害剤(MLN7243)を共添加することで評価した。さらにProstaglandin D Synthase FP-Based Inhibitor Screening Assay Kit-Green (Cayman)を使用した蛍光偏光アッセイにより、PROTAC(H-PGDS)-7~PROTAC(H-PGDS)-9H-PGDSへの結合能を評価した。
5. Evaluation of H-PGDS decomposition activity and mechanism analysis of the above compounds
Add the compound PROTAC (H-PGDS) -1, PROTAC (H-PGDS) -3 to PROTAC (H-PGDS) -10 synthesized to H-PGDS expressing cells (human KU812 cells), and incubate at 37 ° C for 6 hours. After that, the amount of H-PGDS protein was evaluated by Western blot, and the involvement of UPS in the degradation of H-PGDS by the compound was investigated by co-adding a proteasome inhibitor (MG132) and a ubiquitin activating enzyme inhibitor (MLN7243). Evaluated by doing. Furthermore, the ability to bind to PROTAC (H-PGDS) -7 to PROTAC (H-PGDS) -9H-PGDS was evaluated by a fluorescence polarization assay using the Prostaglandin D Synthase FP-Based Inhibitor Screening Assay Kit-Green (Cayman). ..
プロスタグランジン (PG) 類(D2,E2,F2α)産生に及ぼす作用は、ヒトKU812細胞にPROTAC(H-PGDS)-7，PROTAC(H-PGDS)-8を添加し、37℃で6時間インキュベートした後、培養液中からPROTAC(H-PGDS)-7，PROTAC(H-PGDS)-8を取り除くために、洗浄操作(遠心分離後に上清を除き、ここにPROTAC(H-PGDS)-7，PROTAC(H-PGDS)-8を含まない培養液を加える)を3回繰り返した後、カルシウムイオノフォア(A23187)を添加して、PG類の産生を惹起し、10分間に培養液中に遊離されたPG類をLC-MS/MS法によって定量分析して評価した。 The effect on prostaglandin (PG) type (D2, E2, F2α) production is that PROTAC (H-PGDS) -7 and PROTAC (H-PGDS) -8 are added to human KU812 cells at 37 ° C for 6 hours. After incubation, a washing operation (remove the supernatant after centrifugation, where PROTAC (H-PGDS)- 7. Add the culture solution containing PROTAC (H-PGDS) -8) 3 times, then add calcium ionophore (A23187) to induce the production of PGs, and put it in the culture solution for 10 minutes. The released PGs were quantitatively analyzed and evaluated by the LC-MS / MS method.
In vivo評価のプロトコルは下記であった。筋ジストロフィー性拡張型心筋症の抑制効果を調べるためデュシェンヌ型筋ジストロフィー(DMD)モデルマウス(mdx マウス, C57BL/6背景，9～10週齢，雄)を用いた。Andersonらの方法[Anderson JE, Liu L, Kardami E, The effects of hyperthyroidism on muscular dystrophy in the mdx mouse : Greater dystrophy in cardiac and soleus muscle. Muscle Nerve 17(1) : 64-73.1994]に従って、mdx マウスに3,4,3'-トリヨード-L-チロニン (T3，ナカライテスク株式会社)を2 mg/kg/day の用量で2週間皮下投与して、心肥大や心尖部の線維化を伴う拡張型心筋症を発症させた。 The protocol for in vivo evaluation was as follows. Duchenne muscular dystrophy (DMD) model mice (mdx mice, C57BL / 6 background, 9-10 weeks old, male) were used to investigate the inhibitory effect on muscular dystrophy dilated cardiomyopathy. According to Anderson et al.'S method [Anderson JE, Liu L, Kardami E, The effects of hyperthyroidism on muscular dystrophy in the mdx mouse: Greater dystrophy in cardiac and soleus muscle. Dilated cardiomyopathy with cardiac hypertrophy and apical fibrosis after subcutaneous administration of 3,4,3'-triiodo-L-tyronin (T3, Nakaraitesku Co., Ltd.) at a dose of 2 mg / kg / day for 2 weeks. I developed a disease.
PROTAC(HPGDS)-7或いは、HPGDS阻害薬 (TFC-007)をT3投与開始と同時に、1日1回、2週間皮下投与した。mdx マウスへのT3投与による拡張型心筋症に及ぼす効果は、心臓の臓器重量(体重比)、心筋炎症の生化学マーカーである血漿中の心筋トロポニンI (Cardiac Troponin I, cTnI)濃度測定、定量PCRによる心臓組織中の炎症性サイトカイン (TNF-α，TGF-β)、マクロファージの浸潤マーカーのCD11βの mRNA 発現量をウェスタンブロットにより評価した。 PROTAC (HPGDS) -7 or HPGDS inhibitor (TFC-007) was subcutaneously administered once daily for 2 weeks at the same time as the start of T3 administration. The effects of T3 administration on mdx mice on diastolic myocardial disease include cardiac organ weight (weight ratio), measurement and quantification of myocardial troponin I (Cardiac Troponin I, cTnI) concentration in plasma, which is a biochemical marker of myocardial inflammation. The mRNA expression levels of inflammatory cytokines (TNF-α, TGF-β) in cardiac tissue and CD11β, which is a marker for infiltration of macrophages, were evaluated by Western blot.
ヒトKU812細胞にPROTAC(H-PGDS)-1，PROTAC(H-PGDS)-3～PROTAC(H-PGDS)-7(10 pM ～ 10 nM)を添加し、37℃で6時間インキュベートした後、H-PGDSタンパク質量をウェスタンブロットにより評価した結果、PROTAC(H-PGDS)-1，PROTAC(H-PGDS)-3～PROTAC(H-PGDS)-7は濃度依存的にH-PGDSタンパク質を減少させることが明らかとなった(図７)。図７は化合物のリンカー長とH-PGDS分解活性の関係を示す図である。H-PGDSのタンパク質量を内部標準であるβ-Actinのタンパク質量で補正し、DMSOコントロールを100とした相対値で評価した。実験は３回行い、棒グラフは平均±標準偏差を表す。図７に示されるように、特にリンカー構造を持たないPROTAC(H-PGDS)-7は100 pMにおいても高いH-PGDS分解活性を有していた。 PROTAC (H-PGDS) -1, PROTAC (H-PGDS) -3 to PROTAC (H-PGDS) -7 (10 pM to 10 nM) were added to human KU812 cells, and the cells were incubated at 37 ° C for 6 hours. As a result of evaluating the amount of H-PGDS protein by Western blot, PROTAC (H-PGDS) -1, PROTAC (H-PGDS) -3 to PROTAC (H-PGDS) -7 decreased H-PGDS protein in a concentration-dependent manner. It became clear that this was done (Fig. 7). FIG. 7 is a diagram showing the relationship between the linker length of a compound and the H-PGDS degrading activity. The protein amount of H-PGDS was corrected by the protein amount of β-Actin, which is an internal standard, and evaluated by a relative value with DMSO control as 100. The experiment was performed 3 times and the bar graph shows the mean ± standard deviation. As shown in FIG. 7, PROTAC (H-PGDS) -7, which does not have a linker structure in particular, had high H-PGDS degradation activity even at 100 pM.
図８は、PROTAC(H-PGDS)-10によるH-PGDS分解活性評価を示す図である。KU812細胞をPROTAC(H-PGDS)-10 (10 pM-10000 pM)で処理し、6時間培養した後、H-PGDSタンパク質量への影響を評価した。H-PGDSのタンパク質量を内部標準であるβ-Actinのタンパク質量で補正し、DMSOコントロールを100とした相対値で評価した。実験は３回行い、棒グラフは平均±標準偏差を表す。PROTAC(H-PGDS)-10は濃度依存的にH-PGDSタンパク質を減少させることが明らかとなった(図８)。従ってH-PGDSのリガンドとしてTFC-007ばかりでなく、TAS-205をポマリドミドに接続した化合物もH-PGDS分解誘導活性を示すことが分かった。 FIG. 8 is a diagram showing the evaluation of H-PGDS degradation activity by PROTAC (H-PGDS) -10. KU812 cells were treated with PROTAC (H-PGDS) -10 (10 pM-10000 pM), cultured for 6 hours, and then the effect on the amount of H-PGDS protein was evaluated. The protein amount of H-PGDS was corrected by the protein amount of β-Actin, which is an internal standard, and evaluated by a relative value with DMSO control as 100. The experiment was performed 3 times and the bar graph shows the mean ± standard deviation. It was revealed that PROTAC (H-PGDS) -10 reduces the H-PGDS protein in a concentration-dependent manner (Fig. 8). Therefore, it was found that not only TFC-007 as a ligand for H-PGDS but also a compound in which TAS-205 was linked to pomalidomide showed H-PGDS degradation-inducing activity.
続いてH-PGDSタンパク質に対するTFC-007とPomalidomideの効果を検討した。KU812細胞をPROTAC(H-PGDS)-7(100 pM)またはTFC-007/Pomalidomide混合物(各100 pMずつ)で処理し、6時間培養した後、H-PGDSタンパク質量への影響を評価した。図９はPROTAC(H-PGDS)-7によるH-PGDSの分解に対するリガンド接続の必要性.を示す図である。H-PGDSのタンパク質量を内部標準であるβ-Actinのタンパク質量で補正し、DMSOコントロールを100とした相対値で評価した。実験は３回行い、棒グラフは平均±標準偏差を表す。図９に示されるように、TFC-007/Pomalidomide混合物の処理ではH-PGDSタンパク質の減少は観測されなかったことから、2つのリガンド(TFC-007とPomalidomide)をコンジュゲートしたキメラ化合物がH-PGDSタンパク質の分解にとって重要であることが明らかとなった。 Subsequently, the effects of TFC-007 and Pomalidomide on the H-PGDS protein were investigated. KU812 cells were treated with PROTAC (H-PGDS) -7 (100 pM) or TFC-007 / Pomalidomide mixture (100 pM each), cultured for 6 hours, and then evaluated for their effect on H-PGDS protein content. FIG. 9 is a diagram showing the necessity of ligand connection for the degradation of H-PGDS by PROTAC (H-PGDS) -7. The protein amount of H-PGDS was corrected by the protein amount of β-Actin, which is an internal standard, and evaluated by a relative value with DMSO control as 100. The experiment was performed 3 times and the bar graph shows the mean ± standard deviation. As shown in FIG. 9, no reduction in H-PGDS protein was observed in the treatment of the TFC-007 / Pomalidomide mixture, so the chimeric compound conjugated with two ligands (TFC-007 and Pomalidomide) was H-. It became clear that it is important for the degradation of PGDS protein.
またH-PGDSタンパク質の分解に関わるセレブロンをリクルートする必要性を検討した。KU812細胞をPROTAC(H-PGDS)-7(100 pM)に過剰量Pomalidomide(1 μM)を共添加することで処理し、6時間培養した後、H-PGDSタンパク質量への影響を評価した。図９は過剰量のポマリドミド(1 μM)を共添加した際のPROTAC(H-PGDS)-7によるH-PGDSタンパク質減少への影響を評価する図である。H-PGDSのタンパク質量を内部標準であるβ-Actinのタンパク質量で補正し、DMSOコントロールを100とした相対値で評価した。実験は３回行い、棒グラフは平均±標準偏差を表す。競合阻害アッセイの結果、過剰量のPomalidomide存在下においては、PROTAC(H-PGDS)-7によるH-PGDSタンパク質の減少は観測されなかった。H-PGDSのタンパク質分解にはPROTAC(H-PGDS)-7のPolmalidomide部位のセレブロンへの結合が重要であることが示唆された(図１０)。 We also investigated the need to recruit cereblon, which is involved in the degradation of H-PGDS protein. KU812 cells were treated by co-adding an excess amount of Pomalidomide (1 μM) to PROTAC (H-PGDS) -7 (100 pM), cultured for 6 hours, and then the effect on the amount of H-PGDS protein was evaluated. FIG. 9 is a diagram for evaluating the effect of PROTAC (H-PGDS) -7 on the reduction of H-PGDS protein when an excessive amount of pomalidomide (1 μM) is co-added. The protein amount of H-PGDS was corrected by the protein amount of β-Actin, which is an internal standard, and evaluated by a relative value with DMSO control as 100. The experiment was performed 3 times and the bar graph shows the mean ± standard deviation. As a result of competitive inhibition assay, no reduction of H-PGDS protein by PROTAC (H-PGDS) -7 was observed in the presence of an excess amount of Pomalidomide. It was suggested that the binding of PROTAC (H-PGDS) -7 to cereblon at the Polmaridomide site is important for the proteolysis of H-PGDS (Fig. 10).
さらにPROTAC(H-PGDS)-7によるH-PGDSタンパク質の分解に対するUPSの関与を検討するために、プロテアソーム阻害剤(MG132)またはユビキチン活性化酵素阻害剤(MLN7243)を共添加した際のH-PGDSタンパク質の量を測定した。図１１は、KU812細胞にてプロテアソーム阻害剤(MG132，10 μM)またはユビキチン活性化酵素阻害剤(MLN7243，10 μM)共添加におけるPROTAC(H-PGDS)-7によるH-PGDSタンパク質減少への影響を評価する図である。H-PGDSのタンパク質量を内部標準であるβ-Actinのタンパク質量で補正し、DMSOコントロールを100とした相対値で評価した。実験は３回行い、棒グラフは平均±標準偏差を表す。各種阻害剤の添加により、PROTAC(H-PGDS)-7によって誘導されるH-PGDSタンパク質の分解が抑制されたことから、H-PGDSタンパク質の分解にはUPSが関与していることが示唆された(図１１)。PROTAC(H-PGDS)-7によるH-PGDSタンパク質分解は、これまで見出したPROTAC(H-PGDS)-1と同様にプロテアソーム、ユビキチン、ユビキチンリガーゼ（セレブロン）依存的である。 Furthermore, in order to investigate the involvement of UPS in the degradation of H-PGDS protein by PROTAC (H-PGDS) -7, H- when a proteasome inhibitor (MG132) or a ubiquitin activating enzyme inhibitor (MLN7243) was co-added. The amount of PGDS protein was measured. Figure 11 shows the effect of PROTAC (H-PGDS) -7 on H-PGDS protein reduction in KU812 cells with the addition of a proteasome inhibitor (MG132, 10 μM) or a ubiquitin activating enzyme inhibitor (MLN7243, 10 μM). It is a figure to evaluate. The protein amount of H-PGDS was corrected by the protein amount of β-Actin, which is an internal standard, and evaluated by a relative value with DMSO control as 100. The experiment was performed 3 times and the bar graph shows the mean ± standard deviation. The addition of various inhibitors suppressed the degradation of the H-PGDS protein induced by PROTAC (H-PGDS) -7, suggesting that UPS is involved in the degradation of the H-PGDS protein. (Fig. 11). H-PGDS proteolysis by PROTAC (H-PGDS) -7 is proteasome, ubiquitin, and ubiquitin ligase (cereblon) -dependent, similar to PROTAC (H-PGDS) -1 found so far.
6.PROTAC(H-PGDS)-7のH-PGDSへの結合能評価
PROTAC(H-PGDS)-7はUPSを介した強力なH-PGDSタンパク質分解誘導剤であることを明らかとしたが、PROTAC(H-PGDS)-7はH-PGDSの酵素活性を阻害するTFC-007構造を含むため、H-PGDSの酵素活性も阻害する可能性がある。今後PROTAC(H-PGDS)-7によるPGD2産生抑制効果を検討する上で、分解誘導活性の寄与を評価するために有用なTFC-007とN-Methylated pomalidomide(セレブロンへの結合活性のないPomalidomide誘導体)から構成されるPROTAC(H-PGDS)-8のH-PGDSへの結合活性、H-PGDSタンパク質の分解活性を評価した。図１２は、蛍光偏光アッセイによるH-PGDSへの結合評価を示す図である。各化合物のH-PGDSへの結合親和性を蛍光偏光アッセイにより評価した結果、TFC-007，PROTAC(H-PGDS)-7とPROTAC(H-PGDS)-8は、H-PGDSに対して同等の親和性(0.15 μM，0.14 μM，0.14 μM)を示した(図１２)。
6. Evaluation of the binding ability of PROTAC (H-PGDS) -7 to H-PGDS
PROTAC (H-PGDS) -7 was revealed to be a strong UPS-mediated H-PGDS proteolysis inducer, whereas PROTAC (H-PGDS) -7 is a TFC that inhibits the enzymatic activity of H-PGDS. Since it contains a -007 structure, it may also inhibit the enzymatic activity of H-PGDS. TFC-007 and N-Methylated pomalidomide (Pomalidomide derivative without binding activity to cereblon) useful for evaluating the contribution of degradation-inducing activity in investigating the effect of PROTAC (H-PGDS) -7 on suppressing PGD2 production in the future. ), The binding activity of PROTAC (H-PGDS) -8 to H-PGDS and the degrading activity of H-PGDS protein were evaluated. FIG. 12 is a diagram showing the evaluation of binding to H-PGDS by the fluorescence polarization assay. As a result of evaluating the binding affinity of each compound to H-PGDS by a fluorescence polarization assay, TFC-007, PROTAC (H-PGDS) -7 and PROTAC (H-PGDS) -8 are equivalent to H-PGDS. Affinities (0.15 μM, 0.14 μM, 0.14 μM) were shown (Fig. 12).
また、H-PGDSタンパク質の分解を検討した。KU812細胞をPROTAC(H-PGDS)-7(10 pM-10000 pM)またはPROTAC(H-PGDS)-8(10 pM-10000 pM)で処理し、6時間培養した後、H-PGDSタンパク質量への影響を評価した。H-PGDSのタンパク質量を内部標準であるβ-Actinのタンパク質量で補正し、DMSOコントロールを100とした相対値で評価した。実験は３回行い、棒グラフは平均±標準偏差を表す。図１３はPROTAC(H-PGDS)-8によるH-PGDS分解活性評価を示す図である。PROTAC(H-PGDS)-8はH-PGDSタンパク質の量を減少させなかった(図１３)。PROTAC(H-PGDS)-7はH-PGDSタンパク質分解誘導活性と酵素阻害活性がある一方、PROTAC(H-PGDS)-8はH-PGDS酵素阻害活性のみ持つと考えられる。 We also investigated the degradation of H-PGDS protein. KU812 cells were treated with PROTAC (H-PGDS) -7 (10 pM-10000 pM) or PROTAC (H-PGDS) -8 (10 pM-10000 pM), cultured for 6 hours, and then to the amount of H-PGDS protein. The effect of was evaluated. The protein amount of H-PGDS was corrected by the protein amount of β-Actin, which is an internal standard, and evaluated by a relative value with DMSO control as 100. The experiment was performed 3 times and the bar graph shows the mean ± standard deviation. FIG. 13 is a diagram showing the evaluation of H-PGDS decomposition activity by PROTAC (H-PGDS) -8. PROTAC (H-PGDS) -8 did not reduce the amount of H-PGDS protein (Fig. 13). PROTAC (H-PGDS) -7 has H-PGDS proteolysis-inducing activity and enzyme inhibitory activity, while PROTAC (H-PGDS) -8 has only H-PGDS enzyme inhibitory activity.
以上よりPROTAC(H-PGDS)-8はH-PGDSの酵素阻害活性を持つが分解誘導活性のない化合物であることが示唆され、PROTAC(H-PGDS)-7によるPGD2産生抑制効果を検討する際のネガティブコントロール化合物になると考えられる。図１４は、蛍光偏光アッセイによる H-PGDSへの結合評価を示す図である。図１５は、PROTAC(H-PGDS)-9によるH-PGDS分解活性評価を示す図である。KU812細胞をPROTAC(H-PGDS)-9(10 pM-10000 pM)で処理し、6時間培養した後、H-PGDSタンパク質量への影響を評価した。H-PGDSのタンパク質量を内部標準であるβ-Actinのタンパク質量で補正し、DMSOコントロールを100とした相対値で評価した。実験は３回行い、棒グラフは平均±標準偏差を表す。PROTAC(H-PGDS)-9およびそのリガンド部位である化合物11は、H-PGDSに対してTFC-007と同等の親和性を示すものの(図１４)、PROTAC(H-PGDS)-9はH-PGDSタンパク質の量を減少させなかった(図１５)。PROTAC(H-PGDS)-9はPROTAC(H-PGDS)-7のピペラジン部位を除いた構造を含むため、PROTAC(H-PGDS)-7のピペラジン部位が分解活性に必要であることが明らかとなった。 From the above, it is suggested that PROTAC (H-PGDS) -8 is a compound that has the enzyme inhibitory activity of H-PGDS but not the degradation-inducing activity, and the effect of PROTAC (H-PGDS) -7 on suppressing PGD2 production will be investigated. It is considered to be a negative control compound. FIG. 14 is a diagram showing the evaluation of binding to H-PGDS by the fluorescence polarization assay. FIG. 15 is a diagram showing the evaluation of H-PGDS degradation activity by PROTAC (H-PGDS) -9. KU812 cells were treated with PROTAC (H-PGDS) -9 (10 pM-10000 pM), cultured for 6 hours, and then the effect on the amount of H-PGDS protein was evaluated. The protein amount of H-PGDS was corrected by the protein amount of β-Actin, which is an internal standard, and evaluated by a relative value with DMSO control as 100. The experiment was performed 3 times and the bar graph shows the mean ± standard deviation. Although PROTAC (H-PGDS) -9 and its ligand site, compound 11, have the same affinity for H-PGDS as TFC-007 (Fig. 14), PROTAC (H-PGDS) -9 is H. -No reduction in the amount of PGDS protein (Fig. 15). Since PROTAC (H-PGDS) -9 contains a structure excluding the piperazine site of PROTAC (H-PGDS) -7, it is clear that the piperazine site of PROTAC (H-PGDS) -7 is required for degradation activity. became.
KU812細胞にPROTAC(H-PGDS)-7，PROTAC(H-PGDS)-8或いはHPGDS阻害薬のTFC-007を6時間作用させ、その後培養液中の化合物を取り除いた後に、KU812細胞をカルシウムイオノフォア(A23187)刺激によりプロスタグランジン産生を惹起した。KU812細胞をA23187刺激すると、刺激していない細胞に比べて明らかなPGD2，PGE2，PGF2α産生が増強された。TFC-007(10-100 nM)を6時間前処置させた後、培養液からTFC-007を取り除いた細胞をA23187刺激すると、PGD2，PGE2，PGF2α産生は、溶媒で前処置した細胞からの産生量と変わらなかった。一方PROTAC(H-PGDS)-7(10-100 nM)を6時間前処置させた後、培養液からPROTAC(H-PGDS)-7を取り除いた細胞をA23187刺激すると、いずれの用量でもPGD2産生は有意に抑制された。一方、PGE2，PGF2α産生は有意に増強された(図１６)。実験は３回行い、棒グラフは平均±標準偏差を表す。 KU812 cells were allowed to act on PROTAC (H-PGDS) -7, PROTAC (H-PGDS) -8 or the HPGDS inhibitor TFC-007 for 6 hours, after which the compounds in the culture medium were removed, and then the KU812 cells were calcium ionophore. (A23187) Stimulation induced prostaglandin production. Stimulation of KU812 cells with A23187 enhanced PGD 2 , PGE 2 , and PGF 2α production, which was apparent compared to unstimulated cells. When TFC-007 (10-100 nM) was pretreated for 6 hours and then the cells from which TFC-007 was removed from the culture medium were stimulated with A23187, PGD 2 , PGE 2 , and PGF 2α production were produced by the cells pretreated with a solvent. It was the same as the amount produced from. On the other hand, after pretreatment with PROTAC (H-PGDS) -7 (10-100 nM) for 6 hours, cells from which PROTAC (H-PGDS) -7 had been removed from the culture medium were stimulated with A23187, and PGD 2 at any dose. Production was significantly suppressed. On the other hand, PGE 2 and PGF 2 α production was significantly enhanced (Fig. 16). The experiment was performed 3 times and the bar graph shows the mean ± standard deviation.
PROTAC(H-PGDS)-8(10-100 nM)を6時間前処置させた後、培養液からPROTAC(H-PGDS)-8を取り除いた細胞をA23187刺激すると、30と100nMで前処置するとPGD2産生は有意に抑制され、PGE2，PGF2α産生は増強された(図１６)。 After pretreatment with PROTAC (H-PGDS) -8 (10-100 nM) for 6 hours, cells from which PROTAC (H-PGDS) -8 has been removed from the culture medium are stimulated with A23187 and pretreated with 30 and 100 nM. PGD 2 production was significantly suppressed, and PGE 2 and PGF 2 α production was enhanced (Fig. 16).
デュシェンヌ型筋ジストロフィーモデルマウス(mdxマウス)に甲状腺ホルモンの3,4,3'-トリヨード-L-チロニン(T3)を2mg/kg/dayで2週間皮下投与すると、T3を投与していないmdxマウスに比べて明らかな心肥大を発症した。心肥大は、各マウスの心臓の湿重量を体重で除した値で比較した。実験は３～５回行い、棒グラフは平均±標準偏差を表す。PROTAC(H-PGDS)-7(78mg/kg/day)或いは、TFC-007(30 mg/kg/day)をT3投与開始時から2週間皮下投与すると溶媒を投与したマウスに比べて心肥大を抑制し、PROTAC(H-PGDS)-7の抑制作用は、TFC-007と同等かそれ以上であった(図１７)。
Subcutaneous administration of the
mdxマウスにT3(2mg/kg/day)を2週間の投与し心筋炎症を発症させた。心筋障害マーカーの1つである、血漿中の心筋トロポニンI (Cardiac Troponin I, cTnI)を測定したところ、T3を投与していないmdxマウスに比べてT3を2mg/kg/dayを投与したマウスは、明らかな血漿中cTnI値の上昇を示した。PROTAC(H-PGDS)-7(78mg/kg/day)或いは、TFC-007 (30 mg/kg/day)をT3投与開始時から2週間皮下投与すると溶媒を投与したマウスに比べて、PROTAC(H-PGDS)-7は、血漿中cTnI値を低下させた(図１８)。実験は３～５回行い、棒グラフは平均±標準偏差を表す。 T3 (2 mg / kg / day) was administered to mdx mice for 2 weeks to cause myocardial inflammation. Plasma myocardial troponin I (Cardiac Troponin I, cTnI), which is one of the markers of myocardial damage, was measured. , Showed a clear increase in plasma cTnI levels. When PROTAC (H-PGDS) -7 (78 mg / kg / day) or TFC-007 (30 mg / kg / day) is subcutaneously administered for 2 weeks from the start of T3 administration, PROTAC ( H-PGDS) -7 reduced plasma cTnI levels (Fig. 18). The experiment was performed 3 to 5 times, and the bar graph shows the mean ± standard deviation.
T3を2mg/kg/dayで投与して、心肥大を発症させたmdxマウスの心臓中の炎症性サイトカインのTNF-α，線維化を促進するTGF-β，或いはマクロファージや単球の浸潤マーカーであるCD11bのmRNA量を定量PCR法で調べた。 T3 is administered at 2 mg / kg / day to TNF-α, an inflammatory cytokine in the heart of mdx mice that develop cardiac hypertrophy, TGF-β that promotes fibrosis, or macrophage or monocyte infiltration markers. The amount of mRNA in a certain CD11b was examined by a quantitative PCR method.
mdxマウスにT3を2mg/kg/dayで2週間投与すると、T3非投与のmdxマウスに比べて明らかに、TNF-α，TGF-β，CD11bのmRNAの発現が上昇し、心筋で単球やマクロファージの浸潤を伴う炎症や線維化が進行していることが強く示唆された。PROTAC(H-PGDS)-7(78mg/kg/day)或いは、TFC-007 (30 mg/kg/day)をT3投与開始時から2週間皮下投与すると、これらのmRNAの発現量が抑制され、その作用はPROTAC(H-PGDS)-7の方が強かった(図１９)。実験は３～５回行い、棒グラフは平均±標準偏差を表す。 When T3 was administered to mdx mice at 2 mg / kg / day for 2 weeks, the expression of TNF-α, TGF-β, and CD11b mRNA was clearly increased compared to mdx mice not treated with T3, and monocytes and monocytes in the myocardium were observed. It was strongly suggested that inflammation and fibrosis accompanied by macrophage infiltration were progressing. Subcutaneous administration of PROTAC (H-PGDS) -7 (78 mg / kg / day) or TFC-007 (30 mg / kg / day) for 2 weeks from the start of T3 administration suppressed the expression level of these mRNAs. The effect was stronger with PROTAC (H-PGDS) -7 (Fig. 19). The experiment was performed 3 to 5 times, and the bar graph shows the mean ± standard deviation.
以上の結果から、mdxマウスにT3を連続投与して発症させた筋ジストロフィー性の心筋症に対して、PROTAC(H-PGDS)-7は病態の進行を抑制することが明らかとなった。前述のようにPROTAC(H-PGDS)-7のみならずPROTAC(H-PGDS)-10もH-PGDS分解誘導活性を示すことが判明しており、PROTAC(H-PGDS)-10も筋ジストロフィー症に対し、特にデュシェンヌ型筋ジストロフィー症に対して有効である。 From the above results, it was clarified that PROTAC (H-PGDS) -7 suppresses the progression of the pathological condition for muscular dystrophy cardiomyopathy caused by continuous administration of T3 to mdx mice. As mentioned above, not only PROTAC (H-PGDS) -7 but also PROTAC (H-PGDS) -10 has been found to show H-PGDS degradation-inducing activity, and PROTAC (H-PGDS) -10 also has muscular dystrophy. In contrast, it is particularly effective against Duchenne muscular dystrophy.
アレルギー疾患や炎症性疾患の治療薬として利用できる。 It can be used as a therapeutic agent for allergic diseases and inflammatory diseases.
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