JP2017137270A - Anticancer agent - Google Patents
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Abstract
Description
本発明は、がん細胞の増殖を抑制する抗がん剤に関する。 The present invention relates to an anticancer agent that suppresses the growth of cancer cells.
近年、分子レベルでの発がん機構の解明が進み、発がん機構に密接に関与しているシグナル伝達分子を標的とした、いわゆる分子標的薬剤の開発が盛んに行われている。シグナル伝達分子は、正常細胞では発現がほとんど認められないのに対して、がん細胞では発現が亢進しており、がん細胞の増殖過程において必須の役割を果たしていると言われている。この現象を利用し、シグナル伝達分子と強い相互作用を行う分子標的薬剤を見つけることにより、多くの抗がん剤が開発されてきた（例えば特許文献１）。 In recent years, elucidation of the mechanism of carcinogenesis at the molecular level has progressed, and so-called molecular target drugs targeting signal transduction molecules closely involved in the mechanism of carcinogenesis have been actively developed. Signal transduction molecules are hardly expressed in normal cells, but are highly expressed in cancer cells, and are said to play an essential role in the growth process of cancer cells. Many anticancer agents have been developed by using this phenomenon and finding molecular target drugs that interact strongly with signaling molecules (for example, Patent Document 1).
本発明は、がん細胞の増殖を抑制することができる、新たな抗がん剤を提供することを解決すべき課題としている。 This invention makes it the problem which should be solved to provide the new anticancer agent which can suppress the proliferation of a cancer cell.
Pygopusファミリーは，ヒストンのメチル化修飾部位に特異的に結合したり、ヒストンメチル化酵素と結合してヒストンのメチル化を促したりするタンパク質である。したがって、クロマチン修飾の“読み込み”および“書き込み”を同時に行うタンパク質であり、遺伝子の再生において重要な役割を担っている。その中でもPygopus2（Pygo2）は哺乳動物において機能的に重要な役割を果たしており，その全身性のノックアウトマウスは胎生致死である。またPygo2の発現レベルは正常細胞では低く、がん細胞では高い。これはPygoがβ-cateninを介して腫瘍形成を促進するためである。こうした事実から、本発明者らは、新たな抗がん剤を見出すための分子標的として、クロマチン制御タンパク質であるPygo2を選択し、その結合ポケットに焦点を当て、計算機によるドッキングミュレーションにより多数の化合物の効果を調べた。その結果、Pygo2の結合ポケットに強く結合する、一連のピペリジン誘導体を見出した。そして、さらに、それらのピペリジン誘導体が、がん細胞に対する抗がん作用を示すことを見出し、本発明を完成させるに至った。 The Pygopus family is a protein that specifically binds to histone methylation modification sites or promotes histone methylation by binding to histone methylases. Therefore, it is a protein that simultaneously “reads” and “writes” chromatin modifications, and plays an important role in gene regeneration. Among them, Pygopus2 (Pygo2) plays a functionally important role in mammals, and its systemic knockout mice are embryonic lethal. The expression level of Pygo2 is low in normal cells and high in cancer cells. This is because Pygo promotes tumor formation via β-catenin. Based on these facts, the present inventors selected Pygo2, a chromatin regulatory protein, as a molecular target for finding new anticancer agents, focused on its binding pocket, and a number of computer docking simulations. The effect of the compound was investigated. As a result, we found a series of piperidine derivatives that strongly bind to the binding pocket of Pygo2. Furthermore, the present inventors have found that these piperidine derivatives show an anticancer action against cancer cells, and have completed the present invention.
すなわち、本発明の抗がん剤は、下記化学式（１）で示される化合物（ただし、式中の置換基Ａはハロゲン元素、及びアルキル基のいずれか１種以上で修飾されていてもよいフェニル基、又はナフチル基を示し、置換基Ｂはアルキル基で修飾されていてもよい芳香族置換基を示す。）、又はその薬学上許容される塩、水和物、若しくは溶媒和物からなるピペリジン誘導体を有効成分として含むことを特徴とする。ここで、下記化学式（１）で示される化合物は、この化学式（１）で表現される限りおいて、ラセミ体のみならず光学異性体やその他の各種異性体も含む概念である。 That is, the anticancer agent of the present invention is a compound represented by the following chemical formula (1) (wherein the substituent A is phenyl optionally modified with one or more of a halogen element and an alkyl group). Or a naphthyl group, and the substituent B represents an aromatic substituent which may be modified with an alkyl group.), Or a piperidine comprising a pharmaceutically acceptable salt, hydrate or solvate thereof A derivative is contained as an active ingredient. Here, the compound represented by the following chemical formula (1) is a concept including not only a racemate but also an optical isomer and other various isomers as long as it is represented by this chemical formula (1).
また、前記置換基Ａとしては、フェニル基、クロロフェニル基、フルオロフェニル基、トリル基、クロロトリル基、キシリル基、及びナフチル基のいずれかとすることができる。 The substituent A can be any of a phenyl group, a chlorophenyl group, a fluorophenyl group, a tolyl group, a chlorotolyl group, a xylyl group, and a naphthyl group.
さらに、前記置換基Ｂとしては、フェニル基、アルキルフェニル基、アルコキシフェニル基、ナフチル基、並びに下記構造式（ａ）,（ｂ）,（ｃ）及び（ｄ）で示される置換基のいずれかとすることができる。
本発明において、好ましい抗がん剤は、下記化学式（２）〜（１８）のいずれかで示される化合物、又はその薬学上許容される塩、水和物、若しくは溶媒和物を有効成分として含む抗がん剤である。これらの中でも、さらに好ましいのは下記化学式（２）〜（７）、（９）〜（１２）及び（１７）（さらに、さらに好ましいのは下記化学式（２）、（３）、（５）、（１０）、（１１）及び（１２））のいずれかで示される化合物、又はその薬学上許容される塩、水和物、若しくは溶媒和物を有効成分として含む抗がん剤である。特に化学式（２）で示される化合物は、ヒト由来の肺がん細胞やヒト由来の結腸がん細胞に対して優れた増殖抑制効果を奏しており、肺がんや結腸がんに対する抗がん剤として好適である。
本発明者らが、新たな抗がん剤を見出すための分子標的として選択したPygo2の立体構造を図１に示す。このPygo2において、ヒストンのメチル化修飾を制御しているのはPHD1ドメインであるため、この部位をターゲットとして、計算機によるドッキングミュレーションにより多数の化合物の効果を調べた。計算を行うためのプログラムとしては、ドッキングシミュレーションプログラムであるAutoDock Vina、分子動力学計算プログラムであるAmber、量子化学計算プログラムであるPAICSを実装した、
統合創薬プログラム「NAGARA」を用いた（どちらも岐阜大学人獣感染防御研究センターで開発されたプログラムである（MOLECULAR SCIENCE 5, NP0015 (2011)参照。）。「PAICS」ではフラグメント分子軌道（FMO）法を利用しており、これによりタンパク質のような巨大分子の量子化学計算を行うことができる。また、「NAGARA」では，粗視化モデル計算や分子動力学計算を用いてターゲットの座標を準備し，PAICSを使って量子化学計算を実行するという一連の作業を，それぞれのタスクを繋いでワークフローを構築するという形で，簡便かつ統一的に行うことができる。
The three-dimensional structure of Pygo2 selected by the present inventors as a molecular target for finding a new anticancer agent is shown in FIG. In Pygo2, the methylation modification of histone is controlled by the PHD1 domain, and the effect of a large number of compounds was investigated by computer docking simulation using this site as a target. As a program for performing calculations, AutoDock Vina, a docking simulation program, Amber, a molecular dynamics calculation program, and PAICS, a quantum chemistry calculation program, were implemented.
The integrated drug discovery program “NAGARA” was used (both were developed at Gifu University Research Center for Animal Protection (see MOLECULAR SCIENCE 5, NP0015 (2011)). ) Method, which enables quantum chemistry calculations of macromolecules such as proteins, and “NAGARA” uses coarse-grained model calculations and molecular dynamics calculations to calculate target coordinates. A series of operations of preparing and executing quantum chemical calculations using PAICS can be performed simply and uniformly in the form of building a workflow by connecting each task.
スクリーニングにはin silicoバーチャルリガンドスクリーニングシステムを用いた。検索するリガンドのデータベースとしては、LigandBoxデータベースのAsinexサブセット（データ蓄積量：360,000種類の化合物）を用いた。Pygo2のPHDフィンガードメインを標的とし、2XB1（PDBコード）のＣ鎖をドッキング部位として選択した。格子のサイズは36 angstrom×35 angstrom×33 angstrom、格子の中心は-17.5、-19.6、6.7とした。Auto Dock Vinaのパラメータは以下のように設定した：exhaustiveness=8、最大結合モード数=20、エネルギーレンジ=4 kcal/mol。その他のパラメータはデフォルト値に設定した。その結果、Pygo2のPHD1ドメインに結合する化合物として、下記の実施例１〜実施例１７のピペリジン誘導体が見出された。
＜各ピペリジン誘導体の調製＞
実施例１〜実施例１７のピペリジン誘導体は、ASINEX社の販売経路によって入手した市販品をそのまま用いた。
<Preparation of each piperidine derivative>
As the piperidine derivatives of Examples 1 to 17, commercially available products obtained by the sales channels of ASINEX were used as they were.
−評 価−
上記実施例１のピペリジン誘導体について、以下に示す「がん細胞を用いた増殖抑制試験」、「正常繊維芽細胞に対する細胞毒試験」及び「ヌードマウスを用いた異種移植試験」を行った。なお、「がん細胞を用いた増殖抑制試験」については、比較例１として「crizotinib」、比較例２として「ICG001」、比較例３として「staurosuporine」の各抗がん剤についても試験を行った。以下、詳述する。
-Evaluation-
The piperidine derivative of Example 1 was subjected to the following “proliferation inhibition test using cancer cells”, “cytotoxicity test against normal fibroblasts”, and “xenotransplantation test using nude mice”. In addition, regarding the “proliferation inhibition test using cancer cells”, tests were also conducted for each anticancer agent of “crizotinib” as Comparative Example 1, “ICG001” as Comparative Example 2, and “staurosuporine” as Comparative Example 3. It was. Details will be described below.
＜がん細胞を用いた増殖抑制試験＞
ヒト由来の肺がん細胞であるA549細胞を、10%ウシ胎児血清を加えたDulbecco’s modified Eagle’s Medium（DMEM）培地中、37℃、5%CO2条件下で培養した。細胞は96ウェルプレートに播種し24時間前培養した。実施例１及び比較例１〜３（A549細胞では比較例１及び２、HCT116細胞では比較例２及び３）の各化合物を加え、72時間培養後、Cell Counting Kit-8（Dojindo）を用いて細胞数を測定した。ネガティブコントロールとしてDimehtyl sulfoxide（DMSO）を、ポジティブコントロールとしてStaurosporine、Crizotinib、ICG001を使用した。また、IC50についてはGraph Pad Prism 6.0 for windows（Graph Pad Software, Inc., La Jolla, CA., USA）を使用して計算した。
同様の試験をヒト由来の結腸がん細胞であるHCT116細胞についても行った。
<Proliferation inhibition test using cancer cells>
A549 cells, which are human lung cancer cells, were cultured in a Dulbecco's modified Eagle's Medium (DMEM) medium supplemented with 10% fetal bovine serum under conditions of 37 ° C. and 5% CO 2. Cells were seeded in 96-well plates and pre-cultured for 24 hours. Each compound of Example 1 and Comparative Examples 1 to 3 (Comparative Examples 1 and 2 for A549 cells, Comparative Examples 2 and 3 for HCT116 cells) was added, cultured for 72 hours, and then used with Cell Counting Kit-8 (Dojindo). Cell number was measured. Diimehtyl sulfoxide (DMSO) was used as a negative control, and Staurosporine, Crizotinib, and ICG001 were used as positive controls. IC 50 was calculated using Graph Pad Prism 6.0 for windows (Graph Pad Software, Inc., La Jolla, CA., USA).
A similar test was performed on HCT116 cells, which are human-derived colon cancer cells.
その結果、肺がん細胞A549に対する抗がん効果については、図２に示すように、IC50については、市販の抗がん剤である比較例１（crizotinib）では9.664、比較例２（ICG001）では8.031であるのに対し、実施例１では4.868となり、優れた細胞増殖の抑制効果を奏することが分かった。また、結腸がん細胞HCT116に対する抗がん効果についても、図３に示すように、実施例１では2.345、比較例２（ICG001）では3.812、比較例３（staurosporine）では0.01631となり、優れた細胞増殖の抑制効果を奏することが分かった。なお、比較例３（staurosporine）のIC50は0.01631と極めて低い値であるが、IC50以上の濃度においては増殖抑制効果がそれほど増大せず、実施例１のピペリジン誘導体の方が優れていた。 As a result, as shown in FIG. 2, as for the anticancer effect against lung cancer cell A549, as for IC 50 , in Comparative Example 1 (crizotinib), which is a commercially available anticancer agent, in 9.664, in Comparative Example 2 (ICG001) In contrast to 8.031, it was 4.868 in Example 1, indicating that the cell growth was excellently suppressed. In addition, as shown in FIG. 3, the anticancer effect against colon cancer cell HCT116 was 2.345 in Example 1, 3.812 in Comparative Example 2 (ICG001), and 0.01631 in Comparative Example 3 (staurosporine). It has been found that there is an effect of suppressing proliferation. The IC 50 of Comparative Example 3 (staurosporine) is a very low value of 0.01631, but the growth inhibitory effect did not increase so much at a concentration of IC 50 or higher, and the piperidine derivative of Example 1 was superior.
＜細胞毒性試験＞
実施例１のピペリジン誘導体について正常細胞に対する毒性テストを行うために、線維芽細胞を用いた増殖抑制試験を行った。すなわち、ヒト繊維芽細胞（2F0-C75）を、10%ウシ胎児血清を加えたDMEMで培養した。細胞を96ウェルプレートに播種し80%〜90%コンフルエントになるまで前培養した。ここに実施例１のピペリジン誘導体を加え72時間培養後、Cell Counting Kit-8（Dojindo）を用いて細胞数を測定した。
<Cytotoxicity test>
In order to conduct a toxicity test on normal cells of the piperidine derivative of Example 1, a growth inhibition test using fibroblasts was performed. That is, human fibroblasts (2F0-C75) were cultured in DMEM supplemented with 10% fetal bovine serum. Cells were seeded in 96-well plates and pre-cultured until 80% -90% confluent. The piperidine derivative of Example 1 was added here, and after culturing for 72 hours, the number of cells was measured using Cell Counting Kit-8 (Dojindo).
その結果、図４に示すように、正常繊維芽細胞の増殖を抑制する効果は小さく、毒性は極めて低いことが分かった。このことから、実施例１のピペリジン誘導体の生体に対する安全性は高いことが示唆された。 As a result, as shown in FIG. 4, it was found that the effect of suppressing the growth of normal fibroblasts was small and the toxicity was extremely low. From this, it was suggested that the piperidine derivative of Example 1 is highly safe for living bodies.
＜ヌードマウスを用いた異種移植試験＞
異種移植試験を行うために、6週齢のメスのBALB/cヌードマウス（日本エスエルシー株式会社）を使用した。マウスの腹側部に肺がん細胞であるA549細胞（又は結腸がん細胞であるHCT116細胞）を接種し、腫瘍体積が平均230 mm3になったところで実施例１のピペリジン誘導体の投与を開始した。投与は1日1回、14日間行った。腫瘍の体積はtumor volume (mm3) = π/6 (length x height x width)として計算した。試験終了時にマウスを安楽殺し、腫瘍を摘出して病理組織標本を作成した。すべての試験は岐阜大学動物実験倫理審査委員会の許可のもとで行った。
また、比較例２（ICG001）についても、同様の異種移植試験を行った。
<Xenotransplantation test using nude mice>
To conduct the xenograft test, 6-week-old female BALB / c nude mice (Japan SLC, Inc.) were used. A549 cells (or HCT116 cells, which are colon cancer cells), which are lung cancer cells, were inoculated into the ventral part of mice, and administration of the piperidine derivative of Example 1 was started when the tumor volume reached an average of 230 mm 3 . Administration was performed once a day for 14 days. Tumor volume was calculated as tumor volume (mm 3 ) = π / 6 (length × height × width). At the end of the study, the mice were euthanized, and the tumors were excised to make histopathological specimens. All tests were conducted with the permission of the Gifu University Animal Experiment Ethics Committee.
Moreover, the similar xenograft test was done also about the comparative example 2 (ICG001).
その結果、肺がん細胞A549を用いた異種移植試験では、図５に示すように、実施例１のピペリジン誘導体を投与した場合には、腫瘍の体積が顕著に小さくなった。これに対して比較例２（ICG001）では、腫瘍の体積は当初は僅かに小さくなったものの、その後はほとんど変化がなかった。また、コントロールでは、腫瘍の体積が徐々に大きくなった。以上の結果から、実施例１のピペリジン誘導体は肺がんに対する抗がん剤として優れた効果を示すことが分かった。 As a result, in the xenograft test using lung cancer cells A549, as shown in FIG. 5, when the piperidine derivative of Example 1 was administered, the tumor volume was significantly reduced. On the other hand, in Comparative Example 2 (ICG001), the tumor volume was slightly smaller at the beginning, but there was little change thereafter. In the control, the tumor volume gradually increased. From the above results, it was found that the piperidine derivative of Example 1 showed an excellent effect as an anticancer agent against lung cancer.
一方、結腸がん細胞HCT116を用いた異種移植試験では、図６に示すように、実施例１のピペリジン誘導体及び比較例１（ICG001）を投与した場合には、腫瘍の体積はほとんど変わらず、結腸がん細胞HCT116の細胞増殖を抑制していることが分かった。これに対して、コントロールでは、腫瘍の体積が徐々に大きくなった。以上の結果から、実施例１のピペリジン誘導体は結腸がんに対する抗がん剤としても、比較例１（ICG001）と同程度の抗がん作用を奏することが示唆された。 On the other hand, in the xenograft test using colon cancer cells HCT116, as shown in FIG. 6, when the piperidine derivative of Example 1 and Comparative Example 1 (ICG001) were administered, the volume of the tumor was hardly changed. It was found that the cell growth of colon cancer cell HCT116 was suppressed. In contrast, in the control, the tumor volume gradually increased. From the above results, it was suggested that the piperidine derivative of Example 1 exhibited the same anticancer activity as Comparative Example 1 (ICG001) as an anticancer agent against colon cancer.
＜がん細胞を用いた増殖抑制試験＞
実施例２〜１７のピペリジン誘導体についても実施例１と同様の方法により、結腸がん細胞HCT116及び肺がん細胞A549を用いた増殖抑制試験を行った。増殖抑制試験の結果から得られたIC50の値を実施例１の場合も含めて表１及び表２に示す。
The piperidine derivatives of Examples 2 to 17 were subjected to a growth inhibition test using colon cancer cells HCT116 and lung cancer cells A549 by the same method as in Example 1. The values of IC 50 obtained from the results of the growth inhibition test are shown in Tables 1 and 2 including those in Example 1.
上記表１から、実施例１〜１７のピペリジン誘導体は結腸がん細胞HTC116に対する増殖抑制効果を奏することが分かった。このなかでも高い増殖抑制効果を示したのは実施例１〜３、５、６、８〜１０及び１５のピペリジン誘導体であり、特に優れた増殖抑制効果を示したのは実施例１、２、１０及び１１であり、最も高い増殖抑制効果を示したのは、実施例１のピペリジン誘導体であった。
また、上記表２から、実施例１〜１７のピペリジン誘導体は肺がん細胞A549に対する増殖抑制効果も奏することが分かった。このなかでも高い増殖抑制効果を示したのは実施例１、２及び４のピペリジン誘導体であり、特に優れた増殖抑制効果を示したのは実施例１及び実施例４であり、最も高い増殖抑制効果を示したのは、実施例１のピペリジン誘導体であった。
From Table 1 above, it was found that the piperidine derivatives of Examples 1 to 17 exhibited a growth inhibitory effect on colon cancer cells HTC116. Among them, the piperidine derivatives of Examples 1 to 3, 5, 6, 8 to 10 and 15 showed a high growth inhibitory effect, and Examples 1, 2 and 1 showed a particularly excellent growth inhibitory effect. It was the piperidine derivative of Example 1 that showed the highest growth inhibitory effect, which was 10 and 11.
Moreover, from the said Table 2, it turned out that the piperidine derivative of Examples 1-17 also has the growth inhibitory effect with respect to lung cancer cell A549. Among these, the piperidine derivatives of Examples 1, 2 and 4 showed a high growth inhibitory effect, and Examples 1 and 4 showed the most excellent growth inhibitory effect, and the highest growth inhibitory effect. It was the piperidine derivative of Example 1 that showed the effect.
この発明は上記発明の実施の態様及び実施例の説明に何ら限定されるものではない。特許請求の範囲を逸脱せず、当業者が容易に想到できる範囲で種々の変形態様もこの発明に含まれる。 The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications are also included in the present invention as long as those skilled in the art can easily conceive without departing from the scope of the claims.
本発明の抗がん剤は、肺がん細胞A549や結腸がん細胞HCT116の増殖を抑制することから、抗がん剤あるいはそのリード化合物として利用できる。 The anticancer agent of the present invention can be used as an anticancer agent or a lead compound thereof because it suppresses the growth of lung cancer cells A549 and colon cancer cells HCT116.
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| JP2011517317A (en) * | 2008-02-28 | 2011-06-02 | ライフ テクノロジーズ コーポレーション | Fluorescence polarization hERG assay |
| US20130310389A1 (en) * | 2008-01-30 | 2013-11-21 | Cephalon, Inc. | Substituted spirocyclic piperidine derivatives as histamine-3 (h3) receptor ligands |
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| US20130310389A1 (en) * | 2008-01-30 | 2013-11-21 | Cephalon, Inc. | Substituted spirocyclic piperidine derivatives as histamine-3 (h3) receptor ligands |
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