JP2021138671A - PPM1D inhibitor - Google Patents
PPM1D inhibitor Download PDFInfo
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- JP2021138671A JP2021138671A JP2020040300A JP2020040300A JP2021138671A JP 2021138671 A JP2021138671 A JP 2021138671A JP 2020040300 A JP2020040300 A JP 2020040300A JP 2020040300 A JP2020040300 A JP 2020040300A JP 2021138671 A JP2021138671 A JP 2021138671A
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Images
Abstract
Description
本発明は、ＰＰＭ１ファミリーに属するＰＰＭ１Ｄ（Ｐｒｏｔｅｉｎ Ｐｈｏｓｐｈａｔａｓｅ Ｍａｇｎｅｓｉｕｍ‐Ｄｅｐｅｎｄｅｎｔ １、 Ｄｅｌｔａ）に対する新規のプロテインホスファターゼ阻害剤に関する。 The present invention relates to a novel protein phosphatase inhibitor for PPM1D (Protein Phosphatase Magnesium-Dependent 1, Delta) belonging to the PPM1 family.
プロテインホスファターゼはリン酸化タンパク質を脱リン酸化する酵素であり、生体内で糖代謝、平滑筋収縮、細胞周期、ＤＮＡ複製、転写、翻訳、細胞接着、細胞活性化・分化などの制御、生体の免疫系や神経系の維持に作用しているといわれている。従って、プロテインホスファターゼ阻害剤は種々の医薬になる可能性があることから広く探索されている。プロテインホスファターゼの中でもセリン／スレオニンホスファターゼ（Ｓｅｒ／Ｔｈｒホスファターゼ）は、化学的性質及び遺伝子構造よりＰＰ１、ＰＰ２Ａ、ＰＰ２Ｂ、ＰＰ２Ｃ（ＰＰＭ１）ファミリーの４種に分類されている。このＰＰＭ１ファミリーは、特にＤＮＡ修復機構、ストレス応答、シグナル伝達、細胞増殖などの細胞機能の制御に関与していることから、特にその阻害剤の開発は注目されている。 Protein phosphatase is an enzyme that dephosphorylates phosphorylated proteins, and controls glucose metabolism, smooth muscle contraction, cell cycle, DNA replication, transcription, translation, cell adhesion, cell activation / differentiation, etc. in the body, and immunity of the body. It is said to act on the maintenance of the system and nervous system. Therefore, protein phosphatase inhibitors have been widely sought because they have the potential to be a variety of pharmaceuticals. Among protein phosphatases, serine / threonine phosphatase (Ser / Threonine phosphatase) is classified into four types of PP1, PP2A, PP2B, and PP2C (PPM1) families according to their chemical properties and gene structure. Since this PPM1 family is particularly involved in the regulation of cell functions such as DNA repair mechanism, stress response, signal transduction, and cell proliferation, the development of its inhibitor has attracted particular attention.
ＰＰＭ１Ｄ／Ｗｉｐ１は、癌抑制タンパク質ｐ５３によって発現誘導されるＳｅｒ／Ｔｈｒホスファターゼとして同定された（例えば、非特許文献１参照。）。ＰＰＭ１Ｄは、ｐ５３経路やＤＮＡ損傷応答に関与するタンパク質を脱リン酸化するネガティブフィードバック機構を介して細胞周期を制御している。ＰＰＭ１Ｄは、多様な悪性腫瘍で変異や過剰発現が見つかっており、癌化学療法の標的として注目を浴びている（例えば、非特許文献２〜４参照。）。さらに、ＰＰＭ１Ｄは生体内において恒常的に発現していること、また、ノックアウトマウスを用いた研究から、ＰＰＭ１Ｄが、精子形成や老化、免疫応答にも関与していることが示唆されている（非特許文献５）。
PPM1D / Wip1 was identified as a Ser / Thr phosphatase whose expression is induced by the tumor suppressor protein p53 (see, eg, Non-Patent Document 1). PPM1D regulates the cell cycle via the p53 pathway and a negative feedback mechanism that dephosphorylates proteins involved in the DNA damage response. Mutations and overexpressions of PPM1D have been found in various malignant tumors, and it is attracting attention as a target for cancer chemotherapy (see, for example, Non-Patent
現在まで、数種の有力なＰＰＭ１Ｄ阻害剤が発表されている。ＰＰＭ１Ｄ阻害剤としては、例えば、非特許文献６に記載の多環式化合物、ＧＳＫ２８３０３７１（CAS No.1404456-53-6）（非特許文献７）、非特許文献８、９、特許文献１に記載の第一級アルコールを有するケイ素化合物等が報告されている。
To date, several leading PPM1D inhibitors have been announced. Examples of the PPM1D inhibitor include polycyclic compounds described in Non-Patent Document 6, GSK2830371 (CAS No. 1404456-53-6) (Non-Patent Document 7), Non-Patent
非特許文献６〜９、特許文献１等に記載されているＰＰＭ１Ｄ阻害剤は、癌細胞内で阻害効果を発揮し、また優れたがん細胞増殖抑制効果を発揮することから、悪性腫瘍治療薬として有用である。しかし、細胞透過性が不十分であり、より細胞への透過性が高く、ＰＰＭ１Ｄ阻害活性も十分であるＰＰＭ１Ｄ阻害剤が求められている。すなわち、本発明は、細胞透過性が高いＰＰＭ１Ｄ阻害剤を提供することを目的とする。 The PPM1D inhibitors described in Non-Patent Documents 6 to 9, Patent Document 1, etc. exert an inhibitory effect in cancer cells and also exert an excellent cancer cell growth inhibitory effect, and thus are therapeutic agents for malignant tumors. It is useful as. However, there is a need for a PPM1D inhibitor that has insufficient cell permeability, has higher cell permeability, and has sufficient PPM1D inhibitory activity. That is, an object of the present invention is to provide a PPM1D inhibitor having high cell permeability.
本発明者らは、非特許文献８、９、又は特許文献１に記載のケイ素化合物のうち、カルボキシ基を有するケイ素化合物について、カルボキシ基をアミノアルキルエステル化することにより、ＰＰＭ１Ｄ阻害活性を低下させ、かつ細胞透過性を高められること、この誘導体は、細胞内に取り込まれると、分解されて元のカルボキシ基となり、高いＰＰＭ１Ｄ阻害活性を示すことを見出し、本発明を完成させた。
The present inventors reduce the PPM1D inhibitory activity of a silicon compound having a carboxy group among the silicon compounds described in Non-Patent
すなわち、本発明は、以下の、ＰＰＭ１Ｄ阻害剤等を提供するものである。
［１］ 下記一般式（１）
That is, the present invention provides the following PPM1D inhibitors and the like.
[1] The following general formula (1)
［式中、Ｒ１、Ｒ２及びＲ３は、それぞれ独立して、炭素数１〜１２の炭化水素基であり；ｎは１又は２であり；ｐは１〜６の整数であり；Ｘは、置換基を有していてもよい炭素数３〜３６の炭化水素基又は置換基を有していてもよい複素環式基である。］
で表される化合物からなる、ＰＰＭ１Ｄ阻害剤。
［２］ 前記Ｘが、デカヒドロナフタレン環から誘導された脂環式基である、前記［１］のＰＰＭ１Ｄ阻害剤。
［３］ 細胞内のＰＰＭ１Ｄ活性を阻害するために用いられる、前記［１］又は［２］のＰＰＭ１Ｄ阻害剤。
［４］ 前記［１］〜［３］のいずれかのＰＰＭ１Ｄ阻害剤を有効成分とする、医薬用組成物。
［５］ 癌治療に用いられる、前記［４］の医薬用組成物。
[In the formula, R 1 , R 2 and R 3 are independently hydrocarbon groups with 1 to 12 carbon atoms; n is 1 or 2; p is an integer of 1 to 6; X. Is a hydrocarbon group having 3 to 36 carbon atoms which may have a substituent or a heterocyclic group which may have a substituent. ]
A PPM1D inhibitor comprising a compound represented by.
[2] The PPM1D inhibitor of [1] above, wherein X is an alicyclic group derived from a decahydronaphthalene ring.
[3] The PPM1D inhibitor of the above [1] or [2], which is used to inhibit intracellular PPM1D activity.
[4] A pharmaceutical composition containing the PPM1D inhibitor according to any one of [1] to [3] as an active ingredient.
[5] The pharmaceutical composition according to the above [4], which is used for cancer treatment.
本発明に係るＰＰＭ１Ｄ阻害剤は、ＰＰＭ１Ｄ阻害活性に重要なカルボキシ基が、アミノアルキルエステル化された化合物からなる。アミノアルキルエステル化によってカルボキシ基がブロックされたことにより、ＰＰＭ１Ｄ阻害活性は低下し、かつ細胞透過性が向上する。このため、当該ＰＰＭ１Ｄ阻害剤は、in vivoにおけるＰＰＭ１Ｄ阻害剤として好適であり、ＰＰＭ１Ｄの阻害により治療又は予防効果が期待される疾患の治療に用いられる医薬用組成物の有効成分として非常に有用である。 The PPM1D inhibitor according to the present invention comprises a compound in which a carboxy group important for PPM1D inhibitory activity is aminoalkyl esterified. Blocking the carboxy group by aminoalkyl esterification reduces PPM1D inhibitory activity and improves cell permeability. Therefore, the PPM1D inhibitor is suitable as a PPM1D inhibitor in vivo, and is very useful as an active ingredient of a pharmaceutical composition used for the treatment of diseases in which inhibition of PPM1D is expected to have a therapeutic or preventive effect. be.
本発明及び本願明細書において、「Ｃｙ−ｚ（ｙ及びｚは、ｙ＜ｚを充たす正の整数である。）」は、炭素数がｙ以上ｚ以下であることを意味する。 In the present invention and the present specification, " Cyz (y and z are positive integers satisfying y <z)" means that the number of carbon atoms is y or more and z or less.
本発明に係るＰＰＭ１Ｄ阻害剤は、下記一般式（１）で表される化合物（以下、「化合物（１）」ということがある。）からなる。当該化合物中のアミノアルキルエステル部分、すなわち、ＮＨ２−（ＣＨ２）ｐ−Ｏ−ＣＯ−基がカルボキシ基である化合物は、in vitroの反応系において、ＰＰＭ１Ｄ阻害活性を示す化合物である。なお、一般式（１）中、ｐは１〜６の整数である。 The PPM1D inhibitor according to the present invention comprises a compound represented by the following general formula (1) (hereinafter, may be referred to as “compound (1)”). The aminoalkyl ester moiety in the compound, that is, the compound in which the NH 2- (CH 2 ) p-O-CO- group is a carboxy group, is a compound that exhibits PPM1D inhibitory activity in an in vitro reaction system. In the general formula (1), p is an integer of 1 to 6.
一般式（１）中、Ｒ１、Ｒ２及びＲ３は、それぞれ独立して、炭素数１〜１２の炭化水素基である。当該炭化水素基としては、直鎖状、分岐状又は環状の飽和又は不飽和の炭化水素基が挙げられ、さらに直鎖状、分岐状又は環状のアルキル基、アラルキル基、又は芳香族炭化水素基が好ましい。さらに、炭素数１〜８の直鎖状又は分岐状のアルキル基、炭素数６〜１４の芳香族炭化水素基、又はＣ６−１０アリール−Ｃ１−６アルキル基（炭素数６〜１０のアリール基で置換された炭素数１〜６のアルキル基）が好ましい。より詳細には、メチル基、エチル基、ｎ−プロピル基、イソプロピル基、ｎ−ブチル基、ｓｅｃ−ブチル基、イソブチル基、ｔｅｒｔ−ブチル基等の炭素数１〜６のアルキル基が好ましい。また、フェニル基、ナフチル基等の炭素数６〜１０のアリール基が好ましい。さらに、ベンジル基、フェネチル基等のフェニル−Ｃ１−４アルキル基（フェニル基で置換された炭素数１〜４のアルキル基）が好ましい。これらのシリル基上の置換基Ｒ１、Ｒ２及びＲ３は、同一でもよく、互いに異なっていてもよい。 In the general formula (1), R 1 , R 2 and R 3 are each independently a hydrocarbon group having 1 to 12 carbon atoms. Examples of the hydrocarbon group include linear, branched or cyclic saturated or unsaturated hydrocarbon groups, and further linear, branched or cyclic alkyl groups, aralkyl groups, or aromatic hydrocarbon groups. Is preferable. Further, a linear or branched alkyl group having 1 to 8 carbon atoms, an aromatic hydrocarbon group having 6 to 14 carbon atoms, or a C 6-10 aryl-C 1-6 alkyl group (having 6 to 10 carbon atoms). Alkyl groups having 1 to 6 carbon atoms substituted with aryl groups) are preferable. More specifically, alkyl groups having 1 to 6 carbon atoms such as methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, sec-butyl group, isobutyl group and tert-butyl group are preferable. Further, an aryl group having 6 to 10 carbon atoms such as a phenyl group and a naphthyl group is preferable. Further, a phenyl-C 1-4 alkyl group (alkyl group having 1 to 4 carbon atoms substituted with a phenyl group) such as a benzyl group and a phenethyl group is preferable. Substituents R 1 , R 2 and R 3 on these silyl groups may be the same or different from each other.
一般式（１）中、(Ｒ１)(Ｒ２)(Ｒ３)Ｓｉ−の具体例としては、トリメチルシリル基、トリエチルシリル基、トリ（n−プロピル）シリル基、トリイソプロピルシリル基、トリ（ｎ−ブチル）シリル基、トリ（ｓｅｃ−ブチル）シリル基、トリイソブチルシリル基、ｔｅｒｔ−ブチルジメチルシリル基、ジメチルフェニルシリル基、メチルジフェニルシリル基、トリフェニルシリル基、ｔｅｒｔ−ブチルジフェニルシリル基等が挙げられる。 Specific examples of (R 1 ) (R 2 ) (R 3 ) Si- in the general formula (1) include a trimethylsilyl group, a triethylsilyl group, a tri (n-propyl) silyl group, a triisopropylsilyl group, and a tri (tri (n-propyl) silyl group. n-butyl) silyl group, tri (sec-butyl) silyl group, triisobutylsilyl group, tert-butyldimethylsilyl group, dimethylphenylsilyl group, methyldiphenylsilyl group, triphenylsilyl group, tert-butyldiphenylsilyl group, etc. Can be mentioned.
一般式（１）中、Ｘは、置換基を有していてもよい炭素数３〜３６の炭化水素基又は置換基を有していてもよい複素環式基を示す。ここで、炭素数３〜３６の炭化水素基としては、直鎖、分岐鎖又は環状の炭化水素いずれでもよく、また飽和でも不飽和でもよい。より好ましくは、炭素数３〜２４の直鎖、分岐鎖又は環状の炭化水素基であり、さらに好ましくは、炭素数６〜２４の環状の脂肪族炭化水素基（脂環式基）である。 In the general formula (1), X represents a hydrocarbon group having 3 to 36 carbon atoms which may have a substituent or a heterocyclic group which may have a substituent. Here, the hydrocarbon group having 3 to 36 carbon atoms may be a straight chain, a branched chain, or a cyclic hydrocarbon, and may be saturated or unsaturated. A linear, branched or cyclic hydrocarbon group having 3 to 24 carbon atoms is more preferable, and a cyclic aliphatic hydrocarbon group (alicyclic group) having 6 to 24 carbon atoms is more preferable.
当該炭化水素基の例としては、Ｃ３−２４アルキル基、Ｃ３−２４アルケニル基、Ｃ３−２４アルキニル基、炭素数３〜２４の環状構造を有する飽和又は不飽和炭化水素基が挙げられる。Ｃ３−２４アルキル基の例としては、ｎ−プロピル基、イソプロピル基、ｎ−ブチル基、ｓｅｃ−ブチル基、イソブチル基、ｎ−ペンチル基、ｎ−ヘキシル基等のＣ３−６アルキル基が好ましい。Ｃ３−２４アルケニル基としては、１−プロペン−１−イル基、２−プロペン−１−イル基、イソプロペニル基、１−ブテン−１−イル基、２−ブテン−１−イル基、３−ブテン−１−イル基、１−ブテン−２−イル基、２−ブテン−２−イル基、３−ブテン−２−イル基等のＣ３−８アルケニル基が好ましい。Ｃ３−２４アルキニル基としては、プロピニル基、ペンチニル基、ヘキシニル基、オクチニル基等のＣ３−８アルキニル基が好ましい。 Examples of the hydrocarbon group include a C 3-24 alkyl group, a C 3-24 alkenyl group, a C 3-24 alkynyl group, and a saturated or unsaturated hydrocarbon group having a cyclic structure having 3 to 24 carbon atoms. .. Examples of C 3-24 alkyl groups include C 3-6 alkyl groups such as n-propyl group, isopropyl group, n-butyl group, sec-butyl group, isobutyl group, n-pentyl group and n-hexyl group. preferable. C 3-24 alkenyl groups include 1-propene-1-yl group, 2-propen-1-yl group, isopropenyl group, 1-butene-1-yl group, 2-butene-1-yl group, 3 C 3-8 alkenyl groups such as −butene-1-yl group, 1-buten-2-yl group, 2-butene-2-yl group and 3-buten-2-yl group are preferable. As the C 3-24 alkynyl group, a C 3-8 alkynyl group such as a propynyl group, a pentynyl group, a hexynyl group, or an octynyl group is preferable.
炭素数３〜２４の環状構造を有する飽和又は不飽和炭化水素基としては、例えば、５又は６員環状の飽和又は不飽和の炭化水素を１〜３個有する環状構造を有する炭化水素基が挙げられる。当該炭化水素基としては、脂環式基であってもよく、アリール基であってもよい。脂環式基としては、例えば、シクロペンタン、シクロヘキサン、シクロペンテン、シクロヘキセン、ビシクロウンデカン、デカヒドロナフタレン（デカリン）、ノルボルナン、ピネン、ノルボルネン、ノルボルナジエン、リモネン、テトラヒドロナフタレン等の環化合物から誘導される脂環式基が挙げられる。アリール基としては、例えば、フェニル基、ナフチル基、ビフェニル基等の炭素数６〜１０のアリール基が挙げられる。 Examples of the saturated or unsaturated hydrocarbon group having a cyclic structure having 3 to 24 carbon atoms include a hydrocarbon group having a cyclic structure having 1 to 3 saturated or unsaturated hydrocarbons having a 5- or 6-membered cyclic structure. Be done. The hydrocarbon group may be an alicyclic group or an aryl group. Examples of the alicyclic group include an alicyclic derived from a ring compound such as cyclopentane, cyclohexane, cyclopentene, cyclohexene, bicycloundecane, decahydronaphthalene (decalin), norbornane, pinen, norbornene, norbornadiene, limonene, and tetrahydronaphthalene. The formula group is mentioned. Examples of the aryl group include an aryl group having 6 to 10 carbon atoms such as a phenyl group, a naphthyl group and a biphenyl group.
これらの炭化水素基には、ヒドロキシ基、ニトリル基、カルボキシル基、アルコキシカルボニル基、シアノ基、アルコキシ基、アルキルチオ基、置換スルホニル基、ハロゲン原子、トリ置換シリル基及びトリ置換シリルオキシ基から選ばれる１〜４個の置換基を有していてもよい。ここで、アルコキシ基及びアルキルチオ基の炭素数は１〜６が好ましい。アルコキシカルボニル基の炭素数は２〜７が好ましい。また、トリ置換シリルオキシ基としては、前記の(Ｒ１)(Ｒ２)(Ｒ３)Ｓｉ−Ｏ−基が好ましい。置換スルホニル基としては、メタンスルホニル基等のＣ 1-4アルカンスルホニル基、ベンゼンスルホニル基、トルエンスルホニル基等が挙げられる。これらの置換基は１〜４個有していてもよい。 These hydrocarbon groups are selected from hydroxy group, nitrile group, carboxyl group, alkoxycarbonyl group, cyano group, alkoxy group, alkylthio group, substituted sulfonyl group, halogen atom, tri-substituted silyl group and tri-substituted silyloxy group. It may have ~ 4 substituents. Here, the number of carbon atoms of the alkoxy group and the alkylthio group is preferably 1 to 6. The alkoxycarbonyl group preferably has 2 to 7 carbon atoms. Further, as the tri-substituted silyloxy group, the above-mentioned (R 1 ) (R 2 ) (R 3 ) Si—O— group is preferable. Examples of the substituted sulfonyl group include a C 1-4 alkanesulfonyl group such as a methanesulfonyl group, a benzenesulfonyl group, and a toluenesulfonyl group. These substituents may have 1 to 4 groups.
Ｘで表される複素環式基としては、窒素原子又は酸素原子を含む５又は６員の複素環式基が挙げられる。具体的にはピロリル基、ピロリジニル基、ピリジル基、フラニル基、テトラヒドロフラニル基、ピラニル基、テトラヒドロピラニル基等が挙げられる。 Examples of the heterocyclic group represented by X include a 5- or 6-membered heterocyclic group containing a nitrogen atom or an oxygen atom. Specific examples thereof include a pyrrolyl group, a pyrrolidinyl group, a pyridyl group, a furanyl group, a tetrahydrofuranyl group, a pyranyl group, a tetrahydropyranyl group and the like.
当該複素環には、アリール基、アラルキル基、アルキル基、アミノ基、アルキルアミノ基、アルケニルアミノ基、アシルオキシ基及びアラルキルオキシ基から選ばれる１〜５個の置換基を有していてもよい。アリール基としては、フェニル基、ナフチル基等が挙げられる。アラルキル基としてはベンジル基、フェネチル基等が挙げられる。アルキル基としては、メチル基、エチル基等の炭素数１〜６のアルキル基が挙げられる。アルキルアミノ基としてはメチルアミノ基、エチルアミノ基等の炭素数１〜６のアルキルアミノ基が挙げられる。アルケニルアミノ基としては、プロペニルアミノ基、２−メチレン−プロピルアミノ基等が挙げられる。アシルオキシ基としてはアセトキシ基、プロピオニルオキシ基等が挙げられる。アラルキルオキシ基としてはベンジルオキシ基等が挙げられる。 The heterocycle may have 1 to 5 substituents selected from an aryl group, an aralkyl group, an alkyl group, an amino group, an alkylamino group, an alkenylamino group, an acyloxy group and an aralkyloxy group. Examples of the aryl group include a phenyl group and a naphthyl group. Examples of the aralkyl group include a benzyl group and a phenethyl group. Examples of the alkyl group include an alkyl group having 1 to 6 carbon atoms such as a methyl group and an ethyl group. Examples of the alkylamino group include an alkylamino group having 1 to 6 carbon atoms such as a methylamino group and an ethylamino group. Examples of the alkenylamino group include a propenylamino group and a 2-methylene-propylamino group. Examples of the acyloxy group include an acetoxy group and a propionyloxy group. Examples of the aralkyloxy group include a benzyloxy group.
一般式（１）中、ｎは１又は２である。すなわち、化合物（１）は、炭素数３〜３６の炭化水素又は複素環が、アミノアルキルエステル基に加えて、１個又は２個の(Ｒ１)(Ｒ２)(Ｒ３)Ｓｉ−Ｏ−基で置換された化合物である。ｎが２の場合、２個の(Ｒ１)(Ｒ２)(Ｒ３)Ｓｉ−Ｏ−基は、同種の基であってもよく、異種の基であってもよい。 In the general formula (1), n is 1 or 2. That is, in compound (1), a hydrocarbon or heterocycle having 3 to 36 carbon atoms has one or two (R 1 ) (R 2 ) (R 3 ) Si—O in addition to the aminoalkyl ester group. -A compound substituted with a group. When n is 2, the two (R 1 ) (R 2 ) (R 3 ) Si—O− groups may be homologous groups or dissimilar groups.
化合物（１）としては、Ｒ１、Ｒ２及びＲ３がそれぞれ独立して、炭素数１〜８の直鎖状又は分岐状のアルキル基、炭素数６〜１４の芳香族炭化水素基、又はＣ６−１０アリール−Ｃ１−６アルキル基であり、Ｘが、炭素数３〜２４の環状構造を有する飽和又は不飽和炭化水素基である化合物が好ましく；(Ｒ１)(Ｒ２)(Ｒ３)Ｓｉ−が、トリメチルシリル基、トリエチルシリル基、トリ（ｎ−プロピル）シリル基、トリイソプロピルシリル基、トリ（ｎ−ブチル）シリル基、トリ（ｓｅｃ−ブチル）シリル基、トリイソブチルシリル基、ｔｅｒｔ−ブチルジメチルシリル基、ジメチルフェニルシリル基、メチルジフェニルシリル基、トリフェニルシリル基、又はｔｅｒｔ−ブチルジフェニルシリル基であり、Ｘが炭素数６〜２４の脂環式基又はアリール基であり、ｎが１又は２であり、ｐが１〜６の整数である化合物がより好ましく；(Ｒ１)(Ｒ２)(Ｒ３)Ｓｉ−が、トリメチルシリル基、トリエチルシリル基、トリ（ｎ−プロピル）シリル基、トリイソプロピルシリル基、トリ（ｎ−ブチル）シリル基、トリ（ｓｅｃ−ブチル）シリル基、トリイソブチルシリル基、ｔｅｒｔ−ブチルジメチルシリル基、ジメチルフェニルシリル基、メチルジフェニルシリル基、トリフェニルシリル基、又はｔｅｒｔ−ブチルジフェニルシリル基であり、Ｘが炭素数６〜２４の脂環式基であり、ｎが１又は２であり、ｐが１〜６の整数である化合物がさらに好ましく；(Ｒ１)(Ｒ２)(Ｒ３)Ｓｉ−が、トリメチルシリル基、トリエチルシリル基、トリ（n−プロピル）シリル基、トリイソプロピルシリル基、トリ（ｎ−ブチル）シリル基、トリ（ｓｅｃ−ブチル）シリル基、トリイソブチルシリル基、ｔｅｒｔ−ブチルジメチルシリル基、ジメチルフェニルシリル基、メチルジフェニルシリル基、トリフェニルシリル基、又はｔｅｒｔ−ブチルジフェニルシリル基であり、Ｘがデカヒドロナフタレン基（デカヒドロナフタレンから誘導された脂環式基）又はテトラデカヒドロフェナントレン基（テトラデカヒドロフェナントレンから誘導された脂環式基）であり、ｎが１又は２であり、ｐが１〜６の整数である化合物がよりさらに好ましい。 As the compound (1), R 1 , R 2 and R 3 are independently each of a linear or branched alkyl group having 1 to 8 carbon atoms, an aromatic hydrocarbon group having 6 to 14 carbon atoms, or an aromatic hydrocarbon group having 6 to 14 carbon atoms. A compound which is a C 6-10 aryl-C 1-6 alkyl group and X is a saturated or unsaturated hydrocarbon group having a cyclic structure having 3 to 24 carbon atoms is preferable; (R 1 ) (R 2 ) ( R 3 ) Si- is a trimethylsilyl group, a triethylsilyl group, a tri (n-propyl) silyl group, a triisopropylsilyl group, a tri (n-butyl) silyl group, a tri (sec-butyl) silyl group, and a triisobutylsilyl group. , A tert-butyl dimethyl silyl group, a dimethyl phenyl silyl group, a methyl diphenyl silyl group, a triphenyl silyl group, or a tert-butyl diphenyl silyl group, and X is an alicyclic group or an aryl group having 6 to 24 carbon atoms. , N is 1 or 2 and p is an integer of 1 to 6; (R 1 ) (R 2 ) (R 3 ) Si-is a trimethylsilyl group, a triethylsilyl group, a tri (n-). Propyl) silyl group, triisopropylsilyl group, tri (n-butyl) silyl group, tri (sec-butyl) silyl group, triisobutylsilyl group, tert-butyldimethylsilyl group, dimethylphenylsilyl group, methyldiphenylsilyl group, Further compounds are a triphenylsilyl group or a tert-butyldiphenylsilyl group, X is an alicyclic group having 6 to 24 carbon atoms, n is 1 or 2, and p is an integer of 1 to 6. Preferably; (R 1 ) (R 2 ) (R 3 ) Si- is a trimethylsilyl group, a triethylsilyl group, a tri (n-propyl) silyl group, a triisopropylsilyl group, a tri (n-butyl) silyl group, a tri (n-butyl) silyl group. sec-butyl) silyl group, triisobutylsilyl group, tert-butyldimethylsilyl group, dimethylphenylsilyl group, methyldiphenylsilyl group, triphenylsilyl group, or tert-butyldiphenylsilyl group, where X is a decahydronaphthalene group. (Alicyclic group derived from decahydronaphthalene) or tetradecahydrophenantrene group (alicyclic group derived from tetradecahydrophenantrene), n is 1 or 2, and p is 1 to 6. Compounds that are integers are even more preferred.
本発明の化合物としては、例えば、下記一般式（２−１）で表される化合物が挙げられる。一般式（２−１）中、(Ｒ１)(Ｒ２)(Ｒ３)Ｓｉ−基及びｐは、それぞれ、一般式（１）と同じである。一分子中、２個ある(Ｒ１)(Ｒ２)(Ｒ３)Ｓｉ−基は、同種の基であってもよく、異種の基であってもよい。 Examples of the compound of the present invention include compounds represented by the following general formula (2-1). In the general formula (2-1), the (R 1 ) (R 2 ) (R 3 ) Si− groups and p are the same as those in the general formula (1), respectively. The two (R 1 ) (R 2 ) (R 3 ) Si− groups in one molecule may be the same type of group or different types of groups.
一般式（２−１）中、(Ｒ１)(Ｒ２)(Ｒ３)Ｓｉ−基は、トリメチルシリル基、トリエチルシリル基、トリ（ｎ−プロピル）シリル基、トリイソプロピルシリル基、トリ（ｎ−ブチル）シリル基、トリ（ｓｅｃ−ブチル）シリル基、トリイソブチルシリル基、ｔｅｒｔ−ブチルジメチルシリル基、ジメチルフェニルシリル基、メチルジフェニルシリル基、トリフェニルシリル基、又はｔｅｒｔ−ブチルジフェニルシリル基が好ましい。 In the general formula (2-1), the (R 1 ) (R 2 ) (R 3 ) Si- group is a trimethylsilyl group, a triethylsilyl group, a tri (n-propyl) silyl group, a triisopropylsilyl group, or a tri (n). -Butyl) silyl group, tri (sec-butyl) silyl group, triisobutylsilyl group, tert-butyldimethylsilyl group, dimethylphenylsilyl group, methyldiphenylsilyl group, triphenylsilyl group, or tert-butyldiphenylsilyl group preferable.
化合物（１）は、例えば、少なくとも第３級カルボキシ基と１又は２個のヒドロキシ基を備える炭素数３〜３６の炭化水素又は複素環に対して、当該ヒドロキシ基をシリル化して(Ｒ１)(Ｒ２)(Ｒ３)Ｓｉ−基を導入した後、当該第３級カルボキシ基をアミノアルキルエステル化することにより合成できる。炭素数３〜３６の炭化水素又は複素環のヒドロキシ基への(Ｒ１)(Ｒ２)(Ｒ３)Ｓｉ−基の導入は、例えば、非特許文献８、９、又は特許文献１に記載の方法で合成できる。シリル化後の第３級カルボキシ基を有するケイ素化合物のアミノアルキルエステル化反応は、常法により行うことができる。
Compound (1) has, for example, silylated the hydroxy group with respect to a hydrocarbon or heterocycle having 3 to 36 carbon atoms having at least a tertiary carboxy group and 1 or 2 hydroxy groups (R 1 ). It can be synthesized by introducing the (R 2 ) (R 3 ) Si- group and then aminoalkyl esterifying the tertiary carboxy group. The introduction of the (R 1 ) (R 2 ) (R 3 ) Si− group into the hydroxy group of a hydrocarbon or heterocycle having 3 to 36 carbon atoms is described in, for example,
化合物（１）は、塩を形成してもよく、その塩を形成する酸又は塩基としては、塩酸、硫酸などの鉱酸；酢酸、コハク酸、クエン酸等の有機酸；ナトリウム、カリウム等のアルカリ金属；カルシウム、マグネシウム等のアルカリ土類金属等が挙げられる。また、本発明のアミノアルキルエステル化合物は、水和物等の形態であってもよい。 The compound (1) may form a salt, and the acid or base forming the salt includes mineral acids such as hydrochloric acid and sulfuric acid; organic acids such as acetic acid, succinic acid and citric acid; sodium and potassium and the like. Alkaline metal; Examples thereof include alkaline earth metals such as calcium and magnesium. Further, the aminoalkyl ester compound of the present invention may be in the form of a hydrate or the like.
化合物（１）は、第３級カルボキシ基がアミノアルキルエステル化されていることにより、細胞膜への親和性が高く、細胞透過性に優れている。加えて、当該第３級カルボキシ基はＰＰＭ１Ｄ阻害活性に重要な部位であり、アミノアルキルエステル化されてブロックされることによって、ブロックされる前の化合物よりもＰＰＭ１Ｄ阻害活性は低下している。当該化合物が細胞内へ取り込まれると、恐らくは細胞内の酵素によりアミノアルキルエステル部分が加水分解され、第３級カルボキシ基を有するケイ素化合物となる。このケイ素化合物は非常に高いＰＰＭ１Ｄ阻害活性を有する。つまり、化合物（１）は、細胞外ではＰＰＭ１Ｄ阻害活性は低いが、細胞透過性に優れており、細胞内に取り込まれると高いＰＰＭ１Ｄ阻害活性を示す。このため、細胞内のＰＰＭ１Ｄ活性を阻害するために用いられるＰＰＭ１Ｄ阻害剤として非常に有用である。 Compound (1) has a high affinity for cell membranes and excellent cell permeability because the tertiary carboxy group is aminoalkyl esterified. In addition, the tertiary carboxy group is an important site for PPM1D inhibitory activity, and by being blocked by aminoalkyl esterification, the PPM1D inhibitory activity is lower than that of the compound before being blocked. When the compound is taken up into the cell, the aminoalkyl ester moiety is probably hydrolyzed by the intracellular enzyme to obtain a silicon compound having a tertiary carboxy group. This silicon compound has a very high PPM1D inhibitory activity. That is, compound (1) has low PPM1D inhibitory activity extracellularly, but is excellent in cell permeability, and exhibits high PPM1D inhibitory activity when taken up into cells. Therefore, it is very useful as a PPM1D inhibitor used to inhibit intracellular PPM1D activity.
化合物（１）は、ＰＰＭ１Ｄを阻害すること及び癌細胞の増殖を抑制することから、悪性腫瘍治療薬に代表される医薬の有効成分として有用である。また、化合物（１）又はその塩は、低分子化合物であることから、免疫原性等の問題がない。また、経口投与も可能であり、投与経路もあまり制限されないため、特に、ヒトを含む哺乳類に対する医薬の有効成分として有用である。 Compound (1) is useful as an active ingredient of a drug represented by a therapeutic agent for malignant tumors because it inhibits PPM1D and suppresses the growth of cancer cells. Further, since the compound (1) or a salt thereof is a small molecule compound, there is no problem such as immunogenicity. In addition, oral administration is possible, and the route of administration is not so restricted, so that it is particularly useful as an active ingredient of a drug for mammals including humans.
化合物（１）又はその塩を医薬組成物に含有せしめる場合、必要に応じて薬学的に許容される担体と配合し、予防又は治療目的に応じて各種の投与形態を採用可能である。該形態としては、例えば、経口剤、注射剤、坐剤、軟膏剤、貼付剤等が挙げられるが、経口剤が好ましい。これらの投与形態は、各々当業者に公知慣用の製剤方法により製造できる。 When compound (1) or a salt thereof is contained in a pharmaceutical composition, it can be blended with a pharmaceutically acceptable carrier as necessary, and various administration forms can be adopted depending on the purpose of prevention or treatment. Examples of the form include oral preparations, injections, suppositories, ointments, patches and the like, but oral preparations are preferable. Each of these dosage forms can be produced by a conventional formulation method known to those skilled in the art.
薬学的に許容される担体としては、固形製剤における賦形剤、結合剤、崩壊剤、滑沢剤、着色剤；液状製剤における溶剤、溶解補助剤、懸濁化剤、等張化剤、緩衝剤、無痛化剤等が用いられる。また、必要に応じて防腐剤、抗酸化剤、着色剤、甘味剤、安定化剤等の製剤添加物を用いることもできる。 Pharmaceutically acceptable carriers include excipients, binders, disintegrants, lubricants, colorants in solid formulations; solvents, solubilizers, suspending agents, isotonic agents, buffers in liquid formulations. Agents, pain-relieving agents, etc. are used. Further, if necessary, pharmaceutical additives such as preservatives, antioxidants, colorants, sweeteners and stabilizers can be used.
経口用固形製剤を調製する場合は、化合物（１）に賦形剤、必要に応じて、結合剤、崩壊剤、滑沢剤、着色剤、矯味・矯臭剤等を加えた後、常法により錠剤、被覆錠剤、顆粒剤、散剤、カプセル剤等を製造することができる。 When preparing an oral solid preparation, add an excipient, if necessary, a binder, a disintegrant, a lubricant, a colorant, a flavoring / odorant, etc. to compound (1), and then use a conventional method. Tablets, coated tablets, granules, powders, capsules and the like can be produced.
経口用液体製剤を調製する場合は、化合物（１）に、矯味剤、緩衝剤、安定化剤、矯臭剤等を加えて、常法により内服液剤、シロップ剤、エリキシル剤等を製造することができる。 When preparing an oral liquid preparation, it is possible to add a flavoring agent, a buffering agent, a stabilizer, a odorant, etc. to compound (1) to produce an oral liquid preparation, a syrup agent, an elixir agent, etc. by a conventional method. can.
注射剤を調製する場合は、化合物（１）に、ｐＨ調節剤、緩衝剤、安定化剤、等張化剤、局所麻酔剤等を添加し、常法により皮下、筋肉内及び静脈内用注射剤を製造することができる。 When preparing an injection, add a pH regulator, buffer, stabilizer, isotonic agent, local anesthetic, etc. to compound (1), and inject subcutaneously, intramuscularly, and intravenously by a conventional method. The agent can be manufactured.
坐剤を調製する場合は、化合物（１）に当業界において公知の製剤用担体、例えば、ポリエチレングリコール、ラノリン、カカオ脂、脂肪酸トリグリセリド等を加えた後、常法により製造することができる。
軟膏剤を調製する場合は、化合物（１）に通常使用される基剤、安定剤、湿潤剤、保存剤等が必要に応じて配合され、常法により混合、製剤化される。
貼付剤を調製する場合は、通常の支持体に前記軟膏、クリーム、ゲル、ペースト等を常法により塗布すればよい。
When preparing a suppository, it can be produced by a conventional method after adding a pharmaceutical carrier known in the art such as polyethylene glycol, lanolin, cocoa butter, fatty acid triglyceride, etc. to compound (1).
When preparing an ointment, a base, a stabilizer, a wetting agent, a preservative and the like usually used for compound (1) are blended as necessary, and mixed and formulated by a conventional method.
When preparing a patch, the ointment, cream, gel, paste or the like may be applied to a normal support by a conventional method.
前記の各製剤中の化合物（１）の含有量は、患者の症状、その剤形等により一定ではないが、一般に経口剤では約０．０５〜１０００ｍｇ、注射剤では約０．０１〜５００ｍｇ、坐剤では約１〜１０００ｍｇ程度である。 The content of compound (1) in each of the above-mentioned preparations is not constant depending on the patient's symptoms, dosage form, etc., but is generally about 0.05 to 1000 mg for oral preparations and about 0.01 to 500 mg for injections. The suppository is about 1 to 1000 mg.
また、これらの製剤の１日あたりの投与量は、患者の症状、体重、年齢、性別等によって異なり一概には決定できないが、通常成人（体重６０ｋｇ）１日あたり約０．０５〜５０００ｍｇ程度であり、０．１〜１０００ｍｇが好ましく、これを１日１回又は２〜３回程度に分けて投与するのが好ましい。
The daily dose of these preparations varies depending on the patient's symptoms, body weight, age, gender, etc. and cannot be unconditionally determined, but is usually about 0.05 to 5000 mg per day for an adult (
化合物（１）は、ＰＰＭ１Ｄの阻害により治療又は予防効果が期待される疾患の治療に用いられる医薬用組成物の有効成分として非常に有用である。当該疾患としては、例えば、悪性腫瘍（癌）が挙げられ、例えば、頭頚部癌、食道癌、胃癌、結腸癌、直腸癌、肝臓癌、胆嚢・胆管癌、膵臓癌、肺癌、乳癌、卵巣癌、子宮頚癌、子宮体癌、腎癌、膀胱癌、前立腺癌、精巣腫瘍、骨・軟部肉腫、白血病、悪性リンパ腫、多発性骨髄腫、皮膚癌、脳腫瘍等が挙げられる。 Compound (1) is very useful as an active ingredient of a pharmaceutical composition used for the treatment of diseases that are expected to have a therapeutic or preventive effect by inhibiting PPM1D. Examples of the disease include malignant tumors (cancer), such as head and neck cancer, esophageal cancer, gastric cancer, colon cancer, rectal cancer, liver cancer, bile sac / bile duct cancer, pancreatic cancer, lung cancer, breast cancer, and ovarian cancer. , Cervical cancer, uterine body cancer, renal cancer, bladder cancer, prostate cancer, testis tumor, bone / soft sarcoma, leukemia, malignant lymphoma, multiple myeloma, skin cancer, brain tumor and the like.
化合物（１）は、免疫抑制剤や抗ホルモン薬の有効成分としても有用である。例えば、化合物（１）は臓器移植などにおける免疫抑制剤としても有用である。 Compound (1) is also useful as an active ingredient of immunosuppressants and antihormonal agents. For example, compound (1) is also useful as an immunosuppressant in organ transplantation and the like.
化合物（１）を有効成分とするＰＰＭ１Ｄ阻害剤及び医薬用組成物が投与される動物は、特に限定されるものではなく、ヒトであってもよく、ヒト以外の動物であってもよい。非ヒト動物としては、ウシ、ブタ、ウマ、ヒツジ、ヤギ、サル、イヌ、ネコ、ウサギ、マウス、ラット、ハムスター、モルモット等の哺乳動物や、ニワトリ、ウズラ、カモ等の鳥類等が挙げられる。 The animal to which the PPM1D inhibitor and the pharmaceutical composition containing the compound (1) as an active ingredient is administered is not particularly limited, and may be a human or a non-human animal. Examples of non-human animals include mammals such as cows, pigs, horses, sheep, goats, monkeys, dogs, cats, rabbits, mice, rats, hamsters and guinea pigs, and birds such as chickens, quails and ducks.
次に実施例を示して本発明をさらに詳細に説明するが、本発明は以下の実施例に限定されるものではない。 Next, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to the following Examples.
＜Ｈｉｓ−ＰＰＭ１Ｄ（１−４２０）の調製＞
ＰＰＭ１Ｄタンパク質（Ｐｒｏｔｅｉｎ ＩＤ：Ｏ１５２９７／ｃＤＮＡ ＩＤ：Ｕ７０３８５）は、触媒ドメイン（１位から４２０位）をコードしたｃＤＮＡをｐＣｏｌｄＩ ベクター（ＴａＫａＲａ社）に導入し、Ｈｉｓタグ融合タンパク質として大腸菌を用いて発現させた。精製はＢＤ−ＴＡＬＯＮ樹脂（ＣＬＯＮＴＥＣＨ社）を用いた金属アフィニティークロマトグラフィーによって行い、溶出バッファー（１５０ｍＭ イミダゾール、ＰＢＳ（ｐＨ７．５）、５００ｍＭ ＮａＣｌ、１０% グリセロール、０．２% エタノール、１ｍＭ ２−メルカプトエタノール）を用いて目的タンパク質の溶出を行った。溶出されたＰＰＭ１Ｄタンパク質は透析バッファー（５０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ７．５）、０．１ｍＭ ＥＧＴＡ、０．０２% ２−メルカプトエタノール）に対して１６時間透析した後、５０% グリセロールストック（酵素濃度１μＭ）として−８０℃で保存した。
このＰＰＭ１Ｄの触媒ドメインのＨｉｓタグ融合タンパク質（Ｈｉｓ−ＰＰＭ１Ｄ（１−４２０））を、ＰＰＭ１Ｄ活性の測定に用いた。
<Preparation of His-PPM1D (1-420)>
For the PPM1D protein (Protein ID: O15297 / cDNA ID: U70385), a cDNA encoding a catalytic domain (positions 1 to 420) was introduced into a pColdI vector (TaKaRa) and expressed using Escherichia coli as a His tag fusion protein. rice field. Purification is performed by metal affinity chromatography using BD-TALON resin (CLONTECH), elution buffer (150 mM imidazole, PBS (pH 7.5), 500 mM NaCl, 10% glycerol, 0.2% ethanol, 1 mM 2-mercapto). The target protein was eluted using ethanol). The eluted PPM1D protein was dialyzed against dialysis buffer (50 mM Tris-HCl (pH 7.5), 0.1 mM EGTA, 0.02% 2-mercaptoethanol) for 16 hours and then 50% glycerol stock (enzyme concentration 1 μM). ), Stored at −80 ° C.
The His-tag fusion protein of the catalytic domain of PPM1D (His-PPM1D (1-420)) was used to measure PPM1D activity.
＜ＰＰＭ１Ｄ活性（ホスファターゼ活性）のin vitroアッセイ＞
基質には、ｐ５３の１５位リン酸化Ｓｅｒ含有ペプチド（Ａｃ−Ｖａｌ−Ｇｌｕ−Ｐｒｏ−Ｐｒｏ−Ｌｅｕ−Ｓｅｒ（Ｐ）−Ｇｌｎ−Ｇｌｕ−Ｔｈｒ−Ｐｈｅ−Ｓｅｒ−Ａｓｐ−Ｌｅｕ−Ｔｒｐ−ＮＨ２：Ｓｅｒ（Ｐ）はリン酸化Ｓｅｒを示す）を用いた。当該ペプチドは、Ｆｍｏｃアミノ酸（Novabiochem社製）を用いたＦｍｏｃ固相法によって、固相ペプチド合成した。ペプチド画分は、Vydac C8（２２×２５０ｍｍ）を使用した逆相ＨＰＬＣにより手動で精製した。ＨＰＬＣピークは、ＭＡＬＤＩ−ＴＯＦ−ＭＳで分析した。
<In vitro assay of PPM1D activity (phosphatase activity)>
As a substrate, a peptide containing 15-position phosphorylated Ser of p53 (Ac-Val-Glu-Pro-Pro-Leu-Ser (P) -Gln-Glu-Thr-Phe-Ser-Asp-Leu-Trp-NH 2 : Ser (P) indicates phosphorylated Ser) was used. The peptide was synthesized as a solid-phase peptide by the Fmoc solid-phase method using Fmoc amino acid (manufactured by Novabiochem). The peptide fraction was manually purified by reverse phase HPLC using Vydac C8 (22 x 250 mm). HPLC peaks were analyzed by MALDI-TOF-MS.
ホスファターゼ活性は、放出された遊離リン酸塩を、BIOMOL GREEN Reagent（Enzo life sciences社製）を使用して測定することにより実施した。全てのアッセイは、２ｎＭ Ｈｉｓ−ＰＰＭ１Ｄ（１−４２０）を含む反応用バッファー（５０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ７．５）、５０ｍＭ ＮａＣｌ、３０ｍＭ ＭｇＣｌ２、０．１ｍＭ ＥＧＴＡ、０．０２％ ２−メルカプトエタノール、０．５％ ＤＭＳＯ）中で、３０℃、６分間実施した。 Phosphatase activity was performed by measuring the released free phosphate using BIOMOL GREEN Reagent (manufactured by Enzo life sciences). All assays included a reaction buffer containing 2 nM His-PPM1D (1-420) (50 mM Tris-HCl (pH 7.5), 50 mM NaCl, 30 mM MgCl 2 , 0.1 mM EGTA, 0.02% 2-mercaptoethanol). , 0.5% DMSO) at 30 ° C. for 6 minutes.
＜細胞生存性アッセイ＞
ヒト乳癌細胞株ＭＣＦ７及びヒト非小細胞肺癌細胞株Ｈ１２９９（いずれも、ＡＴＣＣから入手）を用いた。Ｈ１２９９にＨＡ−ＰＰＭ１Ｄを過剰発現させた形質転換細胞Ｈ１２９９（ＰＭＤ−９）株は、非特許文献９に記載の方法で樹立した。
まず、９６ウェルの平底プレートに、１×１０４個／ウェルの細胞を播種し、２４時間インキュベートした。２４時間インキュベート後の細胞を被験化合物で処理した。処理から３日後に、細胞の生存率を、ＭＴＳアッセイ又はビルケルチュルク計算盤（Erma社製）を用いて調べた。データのフィッティングは、KaleidaGraph4.0（HUKINKS）によって実施した。
<Cell viability assay>
Human breast cancer cell line MCF7 and human non-small cell lung cancer cell line H1299 (both obtained from ATCC) were used. The transformed cell H1299 (PMD-9) strain in which HA-PPM1D was overexpressed in H1299 was established by the method described in Non-Patent Document 9.
First, 1 × 10 4 cells / well cells were seeded on a 96-well flat bottom plate and incubated for 24 hours. Cells after incubation for 24 hours were treated with the test compound. Three days after treatment, cell viability was examined using an MTS assay or a Birkel Turkic calculator (Erma). Data fitting was performed by KaleidaGraph4.0 (HUKINKS).
＜蛍光プローブを使用した細胞内取り込み率アッセイ＞
まず、ＭＣＦ７細胞を３５ｍｍディッシュに播種し、２４時間インキュベートした。２４時間インキュベート後、細胞を０．１μＭ ５−ＣＦＤＡ又は５−ＡＢＦＤＡで処理した。細胞は、蛍光顕微鏡（BZ-9000、キーエンス社製）で観察した。
<Intracellular uptake rate assay using fluorescent probe>
First, MCF7 cells were seeded on a 35 mm dish and incubated for 24 hours. After incubation for 24 hours, cells were treated with 0.1 μM 5-CFDA or 5-ABFDA. The cells were observed with a fluorescence microscope (BZ-9000, manufactured by KEYENCE CORPORATION).
＜代謝分析＞
代謝産物抽出物は、被験化合物で６時間処理した後に、１０ｃｍディッシュで９０％コンフルエンシーであるＭＣＦ７細胞から、１０％のＰＢＳを含む１０ｍＬの氷冷アセトンを使用して調製した。当該代謝産物抽出物を、室温で一晩インキュベートした後、上澄みを乾燥させた。乾燥した抽出物を、１００μＬのアセトンで再懸濁させたものを、超音波処理した後、１５，０００ｒｐｍで５分間遠心処理して不溶性画分を沈殿除去した。得られた蒸発濃縮上清を、ＬＣ−ＭＳ／ＭＳシステムに注入して分析した。当該システムでは、１０×１ｍｍ ガードカラムを備えたInertSustainSwift C18（１００×１ｍｍ、３μｍ）を用いてクロマトグラフィーを実施した。ＦＴ−ＭＳ及びＦＴ−ＭＳ／ＭＳは、ネガティブＥＳＩモードで、LTQ-Orbitrap XLにより３００−１２００ｍ／ｚのスキャンで実施した。
<Metabolism analysis>
Metabolite extracts were prepared using 10 mL ice-cold acetone containing 10% PBS from MCF7 cells that were 90% confluent in a 10 cm dish after treatment with the test compound for 6 hours. The metabolite extract was incubated overnight at room temperature and then the supernatant was dried. The dried extract was resuspended in 100 μL of acetone, sonicated, and then centrifuged at 15,000 rpm for 5 minutes to precipitate and remove the insoluble fraction. The obtained evaporated concentrated supernatant was injected into an LC-MS / MS system for analysis. Chromatography was performed in the system using an Inert Sustain Swift C18 (100 x 1 mm, 3 μm) equipped with a 10 x 1 mm guard column. FT-MS and FT-MS / MS were performed in negative ESI mode with LTQ-Orbitrap XL scanning at 300-1200 m / z.
［実施例１］
カルボキシ基がブロックされていない化合物ＳＬ−１７６、化合物ＳＬ−１７６のアルキルシリルエーテルがヒドロキシ基に置換された化合物ＳＬ−１８８、化合物ＳＬ−１７６のカルボキシ基がアミノアルキル基に置換された化合物ＳＬ−１７７、及び化合物ＳＬ−１７６のカルボキシ基がアミノアルキルエステル基に置換された化合物ＳＬ−１８０、ＳＬ−１８３を合成した。化合物Ａ〜Ｉ、及び化合物ＳＬ−１７６は、非特許文献１０に記載の方法で合成した。
[Example 1]
Compound SL-176 in which the carboxy group is not blocked, compound SL-188 in which the alkylsilyl ether of compound SL-176 is substituted with a hydroxy group, and compound SL- in which the carboxy group of compound SL-176 is substituted with an aminoalkyl group. 177 and compounds SL-180 and SL-183 in which the carboxy group of compound SL-176 was replaced with an aminoalkyl ester group were synthesized. Compounds A to I and SL-176 were synthesized by the method described in
上記式中、ＴＥＳはトリエチルシリル基であり、ＴＢＳはｔｅｒｔ−ブチルジメチルシリル基であり、Ｐｈｔｈはフタロイル基であり、Ｂｏｃはｔｅｒｔ−ブトキシカルボニル基である。 In the above formula, TES is a triethylsilyl group, TBS is a tert-butyldimethylsilyl group, Phth is a phthaloyl group, and Boc is a tert-butoxycarbonyl group.
５ｍＬ容試験管に化合物ＳＬ−１７６（１６．６ｍｇ、３６．３μｍｏｌ）と炭酸セシウム（２３．７ｍｇ、７２．６μｍｏｌ）を入れて、当該試験管内の空気をアルゴンで置換した後、乾燥ジメチルホルムアミド（０．２４ｍＬ）に溶解させた。当該試験管に、遮光下でＮ−（２−ブロモエチル）フタルイミド（１９．４ ｍｇ、７６．３μｍｏｌ）を加え、室温で５．５時間撹拌した。その後、氷冷して飽和塩化アンモニウム水溶液を加えることによって、反応を停止させた。次いで、反応液をジエチルエーテルで３回抽出し、有機層を水洗した後、無水硫酸マグネシウムで乾燥した。除媒後、粗生成物をフラッシュカラムクロマトグラフィー（シリカゲル、ヘキサン／酢酸エチル＝４／１）で精製して、不純物を含有する化合物Ｋが得られた。これを再度、プレパラティブ薄層クロマトグラフィー（ヘキサン／ジエチルエーテル＝４／１）精製し、純粋な化合物Ｋ（２１．１ｍｇ、９２％）を淡黄色液体として得た。化合物Ｋの分析結果は以下の通りであった。 Compound SL-176 (16.6 mg, 36.3 μmol) and cesium carbonate (23.7 mg, 72.6 μmol) were placed in a 5 mL test tube, the air in the test tube was replaced with argon, and then dried dimethylformamide (dry dimethylformamide) ( It was dissolved in 0.24 mL). N- (2-Bromoethyl) phthalimide (19.4 mg, 76.3 μmol) was added to the test tube under shading, and the mixture was stirred at room temperature for 5.5 hours. Then, the reaction was stopped by cooling with ice and adding a saturated aqueous solution of ammonium chloride. Then, the reaction solution was extracted three times with diethyl ether, the organic layer was washed with water, and then dried over anhydrous magnesium sulfate. After removing the medium, the crude product was purified by flash column chromatography (silica gel, hexane / ethyl acetate = 4/1) to obtain Compound K containing impurities. This was purified again by preparative thin layer chromatography (hexane / diethyl ether = 4/1) to give pure compound K (21.1 mg, 92%) as a pale yellow liquid. The analysis results of compound K were as follows.
IR (ATR): ν 2950, 2932, 2876, 2854, 1777, 1716, 1468, 1428, 1391, 1227, 1202, 1068, 1026, 836, 717 cm-1;
1H NMR (500 MHz, CDCl3): δ 0.00 (6H, s), 0.54 (6H, q, J = 8.1 Hz), 0.86 (9H, s), 0.92 (9H, t, J = 7.5 Hz), 0.95-1.04 (1H, m), 1.19 (3H, s), 1.20-1.34 (5H, m), 1.52-1.68 (4H, m), 1.73 (1H, dd, J = 13.8, 2.9 Hz), 1.96 (1H, td, J = 11.5, 2.9 Hz), 3.71 (1H, brs), 3.93 (1H, ddd, J = 14.3, 6.3, 4.6 Hz), 3.98 (1H, ddd, J = 14.3, 6.3, 4.6 Hz), 4.07-4.12 (1H, m), 4.28 (1H, ddd, J = 11.5, 5.7, 4.6 Hz), 4.35 (1H, ddd, J = 11.5, 6.3, 4.6 Hz), 7.70-7.74 (2H, m), 7.83-7.87 (2H, m);
13C NMR (125 MHz, CDCl3): δ -5.01, 5.04, 7.00, 17.97, 18.28, 19.86, 25.83, 27.87, 34.42, 35.57, 37.02, 37.36, 39.31, 42.41, 46.32, 61.07, 67.49, 71.18, 123.28, 131.99, 133.96, 167.94, 178.25;
HRMS (FD): Calcd for C34H56NO6Si2 (M+H)+: 630.3646, found: 630.3627.
IR (ATR): ν 2950, 2932, 2876, 2854, 1777, 1716, 1468, 1428, 1391, 1227, 1202, 1068, 1026, 836, 717 cm -1 ;
1 H NMR (500 MHz, CDCl 3 ): δ 0.00 (6H, s), 0.54 (6H, q, J = 8.1 Hz), 0.86 (9H, s), 0.92 (9H, t, J = 7.5 Hz), 0.95-1.04 (1H, m), 1.19 (3H, s), 1.20-1.34 (5H, m), 1.52-1.68 (4H, m), 1.73 (1H, dd, J = 13.8, 2.9 Hz), 1.96 ( 1H, td, J = 11.5, 2.9 Hz), 3.71 (1H, brs), 3.93 (1H, ddd, J = 14.3, 6.3, 4.6 Hz), 3.98 (1H, ddd, J = 14.3, 6.3, 4.6 Hz) , 4.07-4.12 (1H, m), 4.28 (1H, ddd, J = 11.5, 5.7, 4.6 Hz), 4.35 (1H, ddd, J = 11.5, 6.3, 4.6 Hz), 7.70-7.74 (2H, m) , 7.83-7.87 (2H, m);
13 C NMR (125 MHz, CDCl 3 ): δ -5.01, 5.04, 7.00, 17.97, 18.28, 19.86, 25.83, 27.87, 34.42, 35.57, 37.02, 37.36, 39.31, 42.41, 46.32, 61.07, 67.49, 71.18, 123.28 , 131.99, 133.96, 167.94, 178.25;
HRMS (FD): Calcd for C 34 H 56 NO 6 Si 2 (M + H) + : 630.3646, found: 630.3627.
５ｍＬ容試験管に化合物Ｋ（２１．１ｍｇ、３３．５μｍｏｌ）を入れて、当該試験管内の空気をアルゴンで置換した後、無水エタノール（０．６７ｍＬ）に溶解させた。当該試験管に、エチレンジアミン（２２μＬ、３３．５μｍｏｌ）を加え、５０℃で２時間撹拌した。その後、放冷して、減圧下で揮発性成分を除去した後、残渣をベンゼンで希釈して減圧濃縮する操作を３回行った。得られた粗生成物をフラッシュカラムクロマトグラフィー（シリカゲル、ヘキサン／酢酸エチル＝５／１の後、塩化メチレン／メタノール／トリエチルアミン＝１：０：０〜２０：１：０〜４０：２：１）で精製し、化合物ＳＬ−１８０（１４．９ ｍｇ、８８％）を無色液体として得た。化合物ＳＬ−１８０の分析結果は以下の通りであった。 Compound K (21.1 mg, 33.5 μmol) was placed in a 5 mL test tube, the air in the test tube was replaced with argon, and then dissolved in absolute ethanol (0.67 mL). Ethylenediamine (22 μL, 33.5 μmol) was added to the test tube, and the mixture was stirred at 50 ° C. for 2 hours. Then, the mixture was allowed to cool to remove volatile components under reduced pressure, and then the residue was diluted with benzene and concentrated under reduced pressure three times. The obtained crude product was subjected to flash column chromatography (silica gel, hexane / ethyl acetate = 5/1, followed by methylene chloride / methanol / triethylamine = 1: 0: 0 to 20: 1: 0 to 40: 2: 1). The compound SL-180 (14.9 mg, 88%) was obtained as a colorless liquid. The analysis results of compound SL-180 were as follows.
IR (ATR): ν 2950, 2934, 2876, 2858, 2360, 1726, 1462, 1229, 1068, 1007, 860, 773 cm-1;
1H NMR (500 MHz, CDCl3): δ 0.02 (6H, s), 0.58 (6H, q, J = 8.1 Hz), 0.88 (9H, s), 0.96 (9H, t, J = 8.0 Hz), 1.05-1.16 (1H, m), 1.29 (3H, s), 1.25-1.45 (5H, m), 1.57-1.76 (4H, m), 1.83 (1H, dd, J = 13.8, 3.5 Hz), 2.08 (1H, td, J = 11.5, 3.5 Hz), 2.92 (2H, t, J = 5.8 Hz), 3.76 (1H, brs), 4.02-4.11 (2H, m), 4.12-4.16 (1H, m);
13C NMR (125 MHz, CDCl3): δ -5.01, 5.08, 7.01, 17.97, 18.46, 20.04, 25.82, 28.02, 34.43, 35.66, 37.37, 39.48, 41.12, 42.61, 46.49, 66.68, 67.43, 71.16, 178.52;
HRMS (FD): Calcd for C26H54NO4Si2 (M+H)+: 500.3591, found: 500.3613.
IR (ATR): ν 2950, 2934, 2876, 2858, 2360, 1726, 1462, 1229, 1068, 1007, 860, 773 cm -1 ;
1 1 H NMR (500 MHz, CDCl 3 ): δ 0.02 (6H, s), 0.58 (6H, q, J = 8.1 Hz), 0.88 (9H, s), 0.96 (9H, t, J = 8.0 Hz), 1.05-1.16 (1H, m), 1.29 (3H, s), 1.25-1.45 (5H, m), 1.57-1.76 (4H, m), 1.83 (1H, dd, J = 13.8, 3.5 Hz), 2.08 ( 1H, td, J = 11.5, 3.5 Hz), 2.92 (2H, t, J = 5.8 Hz), 3.76 (1H, brs), 4.02-4.11 (2H, m), 4.12-4.16 (1H, m);
13 C NMR (125 MHz, CDCl 3 ): δ -5.01, 5.08, 7.01, 17.97, 18.46, 20.04, 25.82, 28.02, 34.43, 35.66, 37.37, 39.48, 41.12, 42.61, 46.49, 66.68, 67.43, 71.16, 178.52 ;
HRMS (FD): Calcd for C 26 H 54 NO 4 Si 2 (M + H) + : 500.3591, found: 500.3613.
５０ｍＬ容ナスフラスコに、化合物ＳＬ−１７６（２１５ ｍｇ、０．４７１ ｍｍｏｌ）と炭酸セシウム（３０７ ｍｇ、０．９４２ ｍｍｏｌ）を入れて、当該ナスフラスコ内の空気をアルゴンで置換した後、乾燥ジメチルホルムアミド（４．０ｍＬ）に溶解させた。当該ナスフラスコに、ｔｅｒｔ−ブチルＮ−（４−ブロモブチル）カルバマート（２３８ ｍｇ、０．９４２ ｍｍｏｌ）（非特許文献１０）の乾燥ジメチルホルムアミド（３．４ｍＬ）溶液を加え、乾燥ジメチルホルムアミド（２．０ｍＬ）で洗い込んだ後、室温で１９時間撹拌した。その後、飽和塩化アンモニウム水溶液を加えることによって、反応を停止させた。次いで、反応液をジエチルエーテルで３回抽出し、有機層を水洗した後、無水硫酸マグネシウムで乾燥した。除媒後、粗生成物をフラッシュカラムクロマトグラフィー（シリカゲル、ヘキサン／酢酸エチル＝５／１）で精製し、化合物Ｌ（２８７ｍｇ、９７％）を無色液体として得た。化合物Ｌの分析結果は以下の通りであった。 Compound SL-176 (215 mg, 0.471 mmol) and cesium carbonate (307 mg, 0.942 mmol) were placed in a 50 mL eggplant flask, the air in the eggplant flask was replaced with argon, and then dried dimethyl was used. It was dissolved in formamide (4.0 mL). To the eggplant flask, a solution of tert-butyl N- (4-bromobutyl) carbamate (238 mg, 0.942 mmol) (Non-Patent Document 10) in dry dimethylformamide (3.4 mL) was added, and dry dimethylformamide (2. After washing with 0 mL), the mixture was stirred at room temperature for 19 hours. Then, the reaction was stopped by adding a saturated aqueous solution of ammonium chloride. Then, the reaction solution was extracted three times with diethyl ether, the organic layer was washed with water, and then dried over anhydrous magnesium sulfate. After removing the medium, the crude product was purified by flash column chromatography (silica gel, hexane / ethyl acetate = 5/1) to obtain Compound L (287 mg, 97%) as a colorless liquid. The analysis results of compound L were as follows.
IR (ATR): ν 2935, 2876, 1718, 1365, 1230, 1178, 1133, 1068, 1025, 1007, 908, 773, 733 cm-1;
1H NMR (500 MHz, CDCl3): δ 0.02 (6H, s), 0.59 (6H, q, J = 8.0 Hz), 0.88 (9H, s), 0.96 (9H, t, J = 8.0 Hz), 1.04-1.14 (1H, m), 1.26 (3H, s), 1.28-1.46 (5H, m), 1.43 (9H, s), 1.50-1.73 (8H, m), 1.80 (1H, dd, J = 13.2, 2.9 Hz), 2.08 (1H, td, J = 11.5, 2.9 Hz), 3.14 (2H, d, J = 6.3 Hz), 3.74-3.78 (1H, brs), 4.05 (2H, q, J = 5.7 Hz), 4.12-4.15 (1H, m);
13C NMR (125 MHz, CDCl3): δ -5.01, 5.09, 7.01, 17.97, 18.43, 20.01, 25.82, 26.01, 26.03, 26.67, 27.98, 28.37, 28.54, 34.45, 35.66, 37.39, 39.38, 40.16, 42.55, 46.35, 63.82, 67.47, 71.19, 79.11, 155.90, 178.53;
HRMS (FD): Calcd for C33H66NO6Si2 (M+H)+: 628.4429, found: 628.4413.
IR (ATR): ν 2935, 2876, 1718, 1365, 1230, 1178, 1133, 1068, 1025, 1007, 908, 773, 733 cm -1 ;
1 1 H NMR (500 MHz, CDCl 3 ): δ 0.02 (6H, s), 0.59 (6H, q, J = 8.0 Hz), 0.88 (9H, s), 0.96 (9H, t, J = 8.0 Hz), 1.04-1.14 (1H, m), 1.26 (3H, s), 1.28-1.46 (5H, m), 1.43 (9H, s), 1.50-1.73 (8H, m), 1.80 (1H, dd, J = 13.2) , 2.9 Hz), 2.08 (1H, td, J = 11.5, 2.9 Hz), 3.14 (2H, d, J = 6.3 Hz), 3.74-3.78 (1H, brs), 4.05 (2H, q, J = 5.7 Hz) ), 4.12-4.15 (1H, m);
13 C NMR (125 MHz, CDCl 3 ): δ -5.01, 5.09, 7.01, 17.97, 18.43, 20.01, 25.82, 26.01, 26.03, 26.67, 27.98, 28.37, 28.54, 34.45, 35.66, 37.39, 39.38, 40.16, 42.55 , 46.35, 63.82, 67.47, 71.19, 79.11, 155.90, 178.53;
HRMS (FD): Calcd for C 33 H 66 NO 6 Si 2 (M + H) + : 628.4429, found: 628.4413.
５０ｍＬ容ナスフラスコに、化合物Ｌ（２８７ ｍｇ、０．４５６ｍｍｏｌ）と２，６−ルチジン（０．４８ｍＬ、４．１ｍｍｏｌ）を入れて、当該ナスフラスコ内の空気をアルゴンで置換した後、乾燥ジクロロメタン（９．９ｍＬ）に溶解させた。当該ナスフラスコを−５０℃に冷却した後、トリメチルシリルトリフルオロメタンスルホナート（０．４９６ ｍＬ、２．７４ｍｍｏｌ）を加え、０℃で２時間撹拌した。その後、飽和炭酸水素ナトリウム水溶液を加えることによって、反応を停止させた。次いで、反応液を酢酸エチルで３回抽出し、有機層を水洗した後、無水硫酸マグネシウムで乾燥した。除媒後、粗生成物をフラッシュカラムクロマトグラフィー（富士シリシア製NH Silica Gel (DM 1020)、 ヘキサン／酢酸エチル＝１／１〜０／１の後、クロロホルム／メタノール＝４０：１〜２０：１〜１０：１〜３：１）で精製し、化合物ＳＬ−１８３（２０４ｍｇ、８５％）を無色液体として得た。化合物ＳＬ−１８３の分析結果は以下の通りであった。 Compound L (287 mg, 0.456 mmol) and 2,6-lutidine (0.48 mL, 4.1 mmol) were placed in a 50 mL eggplant flask, the air in the eggplant flask was replaced with argon, and then dried dichloromethane. It was dissolved in (9.9 mL). After cooling the eggplant flask to −50 ° C., trimethylsilyltrifluoromethanesulfonate (0.496 mL, 2.74 mmol) was added, and the mixture was stirred at 0 ° C. for 2 hours. Then, the reaction was stopped by adding a saturated aqueous solution of sodium hydrogen carbonate. Then, the reaction solution was extracted three times with ethyl acetate, the organic layer was washed with water, and then dried over anhydrous magnesium sulfate. After removal of the medium, the crude product was subjected to flash column chromatography (NH Silica Gel (DM 1020) manufactured by Fuji Silysia Chemical Ltd., hexane / ethyl acetate = 1/1 to 0/1, and then chloroform / methanol = 40: 1 to 20: 1). Purification with 10: 1-3: 1) gave compound SL-183 (204 mg, 85%) as a colorless liquid. The analysis results of compound SL-183 were as follows.
IR (ATR): ν 2932, 2875, 2857, 1724, 1463, 1230, 1180, 1134, 1068, 1025, 961, 835, 742, 723 cm-1;
1H NMR (500 MHz, CDCl3): δ 0.02 (6H, s), 0.59 (6H, q, J = 8.0 Hz), 0.88 (9H, s), 0.96 (9H, t, J = 8.0 Hz), 1.04-1.14 (1H, m), 1.27 (3H, s), 1.28-1.44 (5H, m), 1.48-1.74 (8H, m), 1.81 (1H, dd, J = 13.8, 2.9 Hz), 2.08 (1H, td, J = 11.5, 2.9 Hz), 2.72 (2H, m), 3.76 (1H, brs), 4.00-4.09 (2H, m), 4.13-4.16 (1H, m);
13C NMR (125 MHz, CDCl3): δ -5.07, 5.03, 6.95, 14.05, 17.91, 18.38, 19.97, 22.58, 25.76, 25.98, 27.93, 30.10, 31.52, 34.40, 35.61, 37.35, 39.30, 41.70, 42.51, 46.29, 64.06, 67.43, 71.15, 178.52;
HRMS (FD): Calcd for C28H58NO4Si2 (M+H)+: 528.3904, found: 528.3923.
IR (ATR): ν 2932, 2875, 2857, 1724, 1463, 1230, 1180, 1134, 1068, 1025, 961, 835, 742, 723 cm -1 ;
1 1 H NMR (500 MHz, CDCl 3 ): δ 0.02 (6H, s), 0.59 (6H, q, J = 8.0 Hz), 0.88 (9H, s), 0.96 (9H, t, J = 8.0 Hz), 1.04-1.14 (1H, m), 1.27 (3H, s), 1.28-1.44 (5H, m), 1.48-1.74 (8H, m), 1.81 (1H, dd, J = 13.8, 2.9 Hz), 2.08 ( 1H, td, J = 11.5, 2.9 Hz), 2.72 (2H, m), 3.76 (1H, brs), 4.00-4.09 (2H, m), 4.13-4.16 (1H, m);
13 C NMR (125 MHz, CDCl 3 ): δ -5.07, 5.03, 6.95, 14.05, 17.91, 18.38, 19.97, 22.58, 25.76, 25.98, 27.93, 30.10, 31.52, 34.40, 35.61, 37.35, 39.30, 41.70, 42.51 , 46.29, 64.06, 67.43, 71.15, 178.52;
HRMS (FD): Calcd for C 28 H 58 NO 4 Si 2 (M + H) + : 528.3904, found: 528.3923.
［参考例１］
第３級カルボン酸を有する下記式で表される化合物について、in vitroにおけるホスファターゼ活性と細胞生存性を調べた（ｎ＝３）。ＣｌｏｇＤ（分布係数の計算値）は、親油性の最も一般的に使用される測定パラメーターであり、オンラインプラットフォーム「chemicalize」（ChemAxon社製）を用いて求めた。測定結果を表１に示す。表中のＩＣ５０は、平均±S.D.である。
[Reference example 1]
The in vitro phosphatase activity and cell viability of the compound represented by the following formula having a tertiary carboxylic acid were examined (n = 3). ClogD (calculated value of partition coefficient) is the most commonly used measurement parameter of lipophilicity and was determined using the online platform "chemicalize" (manufactured by ChemAxon). The measurement results are shown in Table 1. IC 50 in the table is mean ± SD.
デカヒドロナフタレン基に結合している−ＯＲ１０１及び−ＯＲ１０２が、水素である化合物ＳＬ−１８８や嵩の小さいｔｅｒｔ−ブチル基である化合物ＳＬ−１８９は、in vitroのホスファターゼ活性アッセイの結果得られたＩＣ５０が大きく、ＰＰＭ１Ｄ阻害活性が非常に小さかった。また、細胞生存アッセイで得られたＩＣ５０も大きく、細胞増殖に対する阻害活性も比較的小さかった。これに対して、第３級カルボン酸を有する化合物のうち、−ＯＲ１０１及び−ＯＲ１０２が疎水性の嵩高い基である化合物ＳＬ−１７６、ＳＬ−１９４、ＳＬ−１９５、及びＳＬ−１９６は、ＰＰＭ１Ｄ阻害活性が高く、細胞増殖に対する阻害活性が高かった。特に、−ＯＲ１０１及び−ＯＲ１０２の両方がトリアルキルシリル基である化合物ＳＬ−１７６及びＳＬ−１９６は、in vitro においてＰＰＭ１Ｄ特異的に脱リン酸化活性を抑制し、癌由来の細胞の細胞生存率を大きく低下させた。これらの結果から、第３級カルボン酸を有し、かつ立体的に嵩高い疎水性分子であることが、ＰＰＭ１Ｄ阻害活性に重要であることがわかった。 Compounds SL-188 in which -OR 101 and -OR 102 attached to the decahydronaphthalene group are hydrogen and compound SL-189, which is a small tert-butyl group, are obtained as a result of an in vitro phosphatase activity assay. was IC 50 of greater, PPM1D inhibitory activity was very small. Further, IC 50 is large obtained in cell viability assays, inhibitory activity against cell proliferation was relatively small. On the other hand, among the compounds having a tertiary carboxylic acid, the compounds SL-176, SL-194, SL-195, and SL-196 in which -OR 101 and -OR 102 are bulky hydrophobic groups are used. , PPM1D inhibitory activity was high, and inhibitory activity on cell proliferation was high. In particular, the compounds SL-176 and SL-196 in which both -OR 101 and -OR 102 are trialkylsilyl groups suppress PPM1D-specific dephosphorylation activity in vitro, and cell survival of cancer-derived cells. The rate was greatly reduced. From these results, it was found that having a tertiary carboxylic acid and being a sterically bulky hydrophobic molecule is important for PPM1D inhibitory activity.
［実施例２］
ＳＬ−１７６等の第３級カルボン酸をアミノアルキルエステル化した化合物について、in vitroにおけるホスファターゼ活性と細胞生存性を調べた（ｎ＝９）。測定結果を表２、図１及び図２に示す。表中のＩＣ５０は、平均±S.D.であり、「構造」欄には、ＳＬ−１９６中の第一級カルボン酸に相当する部分の構造を示す。
[Example 2]
In vitro phosphatase activity and cell viability were examined for compounds obtained by aminoalkyl esterifying a tertiary carboxylic acid such as SL-176 (n = 9). The measurement results are shown in Table 2, FIG. 1 and FIG. IC 50 in the table has an average of ± SD, and the “Structure” column shows the structure of the portion corresponding to the primary carboxylic acid in SL-196.
図１及び表１に示すように、第３級カルボキシ基がアミノアルキルエステル化又はアミノアルキル化した化合物ＳＬ−１７７、ＳＬ−１８０及びＳＬ−１８３は、in vitroのホスファターゼ活性はほとんどなかった。当該結果から、ＰＰＭ１Ｄ阻害剤ＳＬ−１７６の第３級カルボキシ基をアミノアルキルエステル化した化合物は、それ自身のＰＰＭ１Ｄ阻害活性は失われており、このアミノアルキルエステル化はＰＰＭ１Ｄ阻害活性をマスクすることが明らかとなった。 As shown in FIGS. 1 and 1, the compounds SL-177, SL-180 and SL-183 in which the tertiary carboxy group was aminoalkyl esterified or aminoalkylated had almost no in vitro phosphatase activity. From the results, the compound obtained by aminoalkyl esterifying the tertiary carboxy group of the PPM1D inhibitor SL-176 loses its own PPM1D inhibitory activity, and this aminoalkyl esterification masks the PPM1D inhibitory activity. Became clear.
一方で、化合物ＳＬ−１７７、ＳＬ−１８０及びＳＬ−１８３の細胞生存アッセイで得られたＩＣ５０は、化合物ＳＬ−１７６と同様に小さく、細胞増殖抑制能を有することが示された。特に、アミノアルキルエステル化した化合物ＳＬ−１８０及びＳＬ−１８３は、化合物ＳＬ−１７６よりも細胞増殖に対する阻害活性は高かった。また、図２に示すように、化合物ＳＬ−１８０及びＳＬ−１８３は、用量反応曲線の閾値の濃度範囲が狭く、シャープな細胞増殖抑制効果を示した。当該結果から、これらの化合物は、狭い濃度範囲で効果的に作用可能な癌細胞増殖抑制能をもつＰＰＭ１Ｄ阻害剤であることがわかった。 On the other hand, Compound SL-177, SL-180 and IC 50 obtained in cell survival assays of SL-183, like the compounds SL-176 small, it was shown to have a cytostatic capacity. In particular, the aminoalkyl esterified compounds SL-180 and SL-183 had higher inhibitory activity on cell proliferation than the compound SL-176. Further, as shown in FIG. 2, the compounds SL-180 and SL-183 showed a sharp cell proliferation inhibitory effect because the concentration range of the threshold value of the dose-response curve was narrow. From the results, it was found that these compounds are PPM1D inhibitors having a cancer cell proliferation inhibitory ability that can act effectively in a narrow concentration range.
ＭＣＦ７細胞を、０．１μＭの５−ＣＦＤＡ（５−カルボキシフルオレセインジアセテート）又は５−ＡＢＦＤＡ（５−アミノブチルフルオレセインジアセテート）で処理し、処理後２、５、又は１５分後に、蛍光顕微鏡を用いて蛍光イメージングを観察した。結果を図３に示す。この結果、５−ＡＢＦＤＡは処理直後に細胞に取り込まれ、５−ＡＢＦＤＡは５−ＣＦＤＡよりも非常に速い取り込みを示した。当該結果から、アミノアルキルエステル基を持つ化合物は、カルボキシ基を持つ化合物よりも、細胞への取り込み効率が高いことが示された。アミノアルキルエステル化した化合物ＳＬ−１８０及びＳＬ−１８３が化合物ＳＬ−１７６よりも高い細胞増殖抑制能を示したのは、アミノアルキルエステル基部分によって細胞透過性が高くなり、細胞への取り込み効率が向上したためと推察された。 MCF7 cells were treated with 0.1 μM 5-CFDA (5-carboxyfluorescein diacetate) or 5-ABFDA (5-aminobutylfluorescein diacetate), and 2, 5, or 15 minutes after treatment, fluorescence microscopy was performed. Fluorescence imaging was observed using. The results are shown in FIG. As a result, 5-ABFDA was taken up by cells immediately after treatment, and 5-ABFDA showed much faster uptake than 5-CFDA. From the results, it was shown that the compound having an aminoalkyl ester group has a higher efficiency of uptake into cells than the compound having a carboxy group. The aminoalkyl esterified compounds SL-180 and SL-183 showed higher cell proliferation inhibitory ability than the compound SL-176 because the aminoalkyl ester group moiety increased cell permeability and increased the efficiency of cell uptake. It was presumed that it was improved.
化合物ＳＬ−１８３の細胞内代謝物を調べた。ＭＣＦ７細胞を化合物ＳＬ−１８３で６時間処理し、得られた細胞懸濁液をアセトンに加えて抽出物を得た。得らえた抽出物を、ＬＣ−ＭＳ／ＭＳで直接分析した。比較対象として、化合物ＳＬ−１７６についても同様にして細胞内代謝物を調べた。図４に、化合物ＳＬ−１７６で処理した細胞の抽出物のＬＣ−ＭＳ／ＭＳクロマトグラムを示し、図５に、化合物ＳＬ−１８３で処理した細胞の抽出物のＬＣ−ＭＳ／ＭＳクロマトグラムを示す。 The intracellular metabolites of compound SL-183 were examined. MCF7 cells were treated with compound SL-183 for 6 hours and the resulting cell suspension was added to acetone to give an extract. The obtained extract was directly analyzed by LC-MS / MS. For comparison, the intracellular metabolites of compound SL-176 were examined in the same manner. FIG. 4 shows an LC-MS / MS chromatogram of an extract of cells treated with compound SL-176, and FIG. 5 shows an LC-MS / MS chromatogram of an extract of cells treated with compound SL-183. show.
図４に示すように、化合物ＳＬ−１７６処理細胞の抽出物では、リテンション時間が１９．６６分であり、ＭＳスペクトルは、ｍ／ｚ ４５５の質量を示した。ＭＳ／ＭＳスペクトルでは、２つのイオン、ｍ／ｚ １３１．０８及び２０９．１１が検出された。一方で、図５に示すように、化合物ＳＬ−１８３処理細胞の抽出物では、リテンション時間が１８．９２分であり、ＭＳスペクトルは、ｍ／ｚ ４５５の質量を示した。ＭＳ／ＭＳスペクトルでは、2つのイオン、ｍ／ｚ １３１．０８及び２０９．１１が検出された。つまり、化合物ＳＬ−１８３処理細胞の抽出物のＭＳスペクトル及びＭＳ／ＭＳスペクトルでは、化合物ＳＬ−１７６処理細胞の抽出物と同じＭＳフラグメントが観察された。当該結果から、細胞内に取り込まれた化合物ＳＬ−１８３は、化合物ＳＬ−１７６に変換されて高いＰＰＭ１Ｄ阻害機能を発揮することがわかった。すなわち、化合物ＳＬ−１７６等の第３級カルボン酸をもつＰＰＭ１Ｄ阻害剤を、そのカルボキシ基をアミノアルキルエステル化した化合物（１）は、細胞内に取り込まれる前にはＰＰＭ１Ｄ阻害活性を示さず、標的細胞内に取り込まれると高いＰＰＭ１Ｄ阻害活性を示すプロドラッグとして好適であることが示された。 As shown in FIG. 4, in the extract of compound SL-176 treated cells, the retention time was 19.66 minutes and the MS spectrum showed a mass of m / z 455. Two ions, m / z 131.08 and 209.11, were detected in the MS / MS spectrum. On the other hand, as shown in FIG. 5, in the extract of the compound SL-183 treated cells, the retention time was 18.92 minutes, and the MS spectrum showed a mass of m / z 455. Two ions, m / z 131.08 and 209.11, were detected in the MS / MS spectrum. That is, in the MS spectrum and MS / MS spectrum of the extract of compound SL-183 treated cells, the same MS fragment as that of the extract of compound SL-176 treated cells was observed. From the results, it was found that the compound SL-183 taken up into the cells was converted to the compound SL-176 and exhibited a high PPM1D inhibitory function. That is, the compound (1) obtained by aminoalkyl esterifying a PPM1D inhibitor having a tertiary carboxylic acid such as compound SL-176 and its carboxy group did not show PPM1D inhibitory activity before being taken up into cells. It has been shown to be suitable as a prodrug that exhibits high PPM1D inhibitory activity when incorporated into target cells.
さらに、化合物ＳＬ−１８３で処理したＨ１２９９細胞及びＨ１２９９（ＰＭＤ−９）細胞の細胞生存性を調べた。比較対象として、化合物ＳＬ−１７６又は抗癌剤エトポシドを用いて同様に細胞生存性を調べた。Ｈ１２９９細胞及びＨ１２９９（ＰＭＤ−９）細胞を、それぞれ、１０ｍｇ／ｍＬのエトポシド、１０μＭの化合物ＳＬ−１７６、又は１０μＭの化合物ＳＬ−１８３で処理し、処理から３日後の細胞の生存率を調べた。結果を図６に示す（ｎ＝３、平均値±標準偏差）。図中、「＊＊＊」は、ｐ＜０．００１（ｔ検定）を示す。 Furthermore, the cell viability of H1299 cells and H1299 (PMD-9) cells treated with compound SL-183 was examined. For comparison, the compound SL-176 or the anticancer drug etoposide was used in the same manner to examine cell viability. H1299 cells and H1299 (PMD-9) cells were treated with 10 mg / mL etoposide, 10 μM compound SL-176, or 10 μM compound SL-183, respectively, and the viability of the cells 3 days after treatment was examined. .. The results are shown in FIG. 6 (n = 3, mean ± standard deviation). In the figure, "***" indicates p <0.001 (t-test).
図６に示すように、抗癌剤エトポシドは、Ｈ１２９９（ＰＭＤ−９）細胞よりもＨ１２９９細胞の生存率を有意に抑制した。これに対して、化合物ＳＬ−１７６とＳＬ−１８３はいずれも、Ｈ１２９９細胞よりもＨ１２９９（ＰＭＤ−９）細胞に対して、強力な細胞増殖阻害を示した。この細胞増殖阻害能は、化合物ＳＬ−１８３のほうが化合物ＳＬ−１７６よりも強力であった。当該結果は、化合物ＳＬ−１８３が、細胞レベルでＰＰＭ１Ｄ特異性を発揮することを示す。 As shown in FIG. 6, the anticancer drug etoposide significantly suppressed the viability of H1299 cells more than that of H1299 (PMD-9) cells. In contrast, both compounds SL-176 and SL-183 showed stronger cell proliferation inhibition on H1299 (PMD-9) cells than on H1299 cells. This cell proliferation inhibitory ability was stronger in compound SL-183 than in compound SL-176. The results show that compound SL-183 exerts PPM1D specificity at the cellular level.
Claims (5)
で表される化合物からなる、ＰＰＭ１Ｄ阻害剤。 The following general formula (1)
A PPM1D inhibitor comprising a compound represented by.
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