JP2012036124A - Hiv replication inhibitor - Google Patents
Hiv replication inhibitor Download PDFInfo
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- JP2012036124A JP2012036124A JP2010177176A JP2010177176A JP2012036124A JP 2012036124 A JP2012036124 A JP 2012036124A JP 2010177176 A JP2010177176 A JP 2010177176A JP 2010177176 A JP2010177176 A JP 2010177176A JP 2012036124 A JP2012036124 A JP 2012036124A
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- hiv replication
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Abstract
【課題】ＨＩＶの複製の仕組みや、ＬＳＤ１の機能を研究するために有用な研究ツールとなりうる、ＨＩＶ複製阻害剤を提供することを解決すべき課題とする。
【解決手段】下記一般式（１）（ただし、式中Ｒ１は水素又はフェニルアミド基を示し、Ｒ２は分枝を有することのあるアルキレン基を示し、シクロプロピルアミン置換基はメタ位又はパラ位に結合している）の化合物又はその薬学上許容される塩、水和物、溶媒和物若しくはプロドラッグからなることを特徴とするＨＩＶ複製阻害剤。
【選択図】図４An object of the present invention is to provide an HIV replication inhibitor that can be a useful research tool for studying the mechanism of HIV replication and the function of LSD1.
The following general formula (1) (wherein R 1 represents hydrogen or a phenylamide group, R 2 represents an alkylene group which may have a branch, and the cyclopropylamine substituent is in the meta position or Or a pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof.
[Selection] Figure 4
Description
本発明は、ＨＩＶのＤＮＡの複製を阻害することのできるＨＩＶ複製阻害剤に関する。 The present invention relates to an HIV replication inhibitor capable of inhibiting the replication of HIV DNA.
ヒストンは、真核生物においてＤＮＡを折りたたんでクロマチン構造を形成するタンパク質であり、遺伝子の発現に深くかかわっている。すなわち、ヒストンは様々な酵素の働きによって化学修飾され、これによりクロマチン構造が変化し、遺伝子の発現が制御されると考えられている。近年、こうしたエピジェネティックな遺伝子制御に関する様々な知見が発見されている。 Histone is a protein that forms a chromatin structure by folding DNA in eukaryotes, and is deeply involved in gene expression. That is, histones are chemically modified by the action of various enzymes, which are thought to change the chromatin structure and control gene expression. In recent years, various knowledge about such epigenetic gene regulation has been discovered.
Lysine-specific demethylase（以下「ＬＳＤ１」という）は、最初に発見されたヒストンの脱メチル化酵素であり、ヒストンH3の4番目のリシン残基のモノメチル化体及びジメチル化体(H3K4me1/2)の脱メチル化反応を触媒し、ホルムアルデヒドを副生する（非特許文献１参照）。また、ＬＳＤ１は補酵素の一種であるフラビンアデニンジヌクレオチド（以下「ＦＡＤ」という）と複合体をつくり、ＦＡＤが酸化還元のメディエーターとなって、酸素によるリジン残基の酸化が進行する（下記反応式参照）。 Lysine-specific demethylase (hereinafter referred to as “LSD1”) is the first discovered histone demethylase, which is a monomethylated and dimethylated form (H3K4me1 / 2) of the fourth lysine residue of histone H3. It catalyzes the demethylation reaction to formaldehyde as a by-product (see Non-Patent Document 1). In addition, LSD1 forms a complex with flavin adenine dinucleotide (hereinafter referred to as “FAD”), which is a type of coenzyme, and FAD serves as a redox mediator to promote oxidation of lysine residues by oxygen (the following reaction). See formula).
一方、ＬＳＤ１は前立腺がんにおいて過剰発現し、アンドロゲン受容体（AR）と相互作用してAR依存性の転写を活性化することが明らかになっている。また、ＬＳＤ１をノックダウンすると、アンドロゲンによって誘発された転写活性化が抑制され、細胞増殖が阻害されるということが報告されている（非特許文献２）。しかし、現在の段階ではＬＳＤ１が生体にどのような影響を与えるかなど、生物学的意義に関して不明な点が多い。また、ＬＳＤ１とＨＩＶの複製との関係についても、よく分かっていない。 On the other hand, LSD1 is overexpressed in prostate cancer and has been shown to interact with androgen receptor (AR) to activate AR-dependent transcription. Moreover, it has been reported that when LSD1 is knocked down, transcriptional activation induced by androgen is suppressed, and cell proliferation is inhibited (Non-patent Document 2). However, there are many unclear points regarding the biological significance such as how LSD1 affects the living body at the present stage. Also, the relationship between LSD1 and HIV replication is not well understood.
本発明のＨＩＶ複製阻害剤に用いられる、ＬＳＤ１阻害剤としての下記化合物（１ａ）は、本発明者らがその合成法及びＬＳＤ１の阻害作用について既に発表している（非特許文献３）。また、本発明者らは、ＨＩＶ−ＬＴＲ近傍のメチル化状態に関する研究発表を行っている（非特許文献４）。 The following compound (1a) as an LSD1 inhibitor used in the HIV replication inhibitor of the present invention has already been published by the present inventors on the synthesis method and the inhibitory action of LSD1 (Non-patent Document 3). In addition, the present inventors have made a research presentation on the methylation state in the vicinity of HIV-LTR (Non-Patent Document 4).
本発明は、上記従来の問題に鑑みなされたものであり、ＨＩＶの複製の仕組みや、ＬＳＤ１の機能を研究するために有用な研究ツールとなりうる、ＨＩＶ複製阻害剤を提供することを解決すべき課題とする。 The present invention has been made in view of the above-described conventional problems, and should provide an HIV replication inhibitor that can be a useful research tool for studying the mechanism of HIV replication and the function of LSD1. Let it be an issue.
本発明者らは、ＬＳＤ１が遺伝子の転写活性に関係していることから、ＨＩＶ複製とＬＳＤ１との関係について注目し、鋭意研究を行った。そして、ＬＳＤ１阻害剤によってＬＳＤ１の酵素活性を抑制すると、ＨＩＶの複製が阻害されるという事実を見出し、本発明を完成するに至った。すなわち、本発明のＨＩＶ複製阻害剤は、ＬＳＤ１阻害剤を有効成分として含有することを特徴とする。 Since the present inventors are related to the transcriptional activity of a gene, the present inventors paid attention to the relationship between HIV replication and LSD1 and conducted intensive studies. And when the enzyme activity of LSD1 was suppressed by the LSD1 inhibitor, the fact that HIV replication was inhibited was found, and the present invention was completed. That is, the HIV replication inhibitor of the present invention is characterized by containing an LSD1 inhibitor as an active ingredient.
前記ＬＳＤ１阻害剤としては、下記一般式（Ｉ）若しくは（II）（ただし、両式中Ｒ１は水素、置換基が結合していてもよいアルキル基、置換基が結合していてもよいフェニル基及び置換基が結合していてもよい複素環基のいずれかを示し、Ｒ２は分枝を有することがあり置換基が結合していてもよいアルキレン基を示し、Ｒ３は置換基が結合していてもよいアルキル基、置換基が結合していてもよいフェニル基、置換基が結合していてもよい複素環基及び置換基が結合していてもよいベンジル基のいずれかを示し、Ｒ４は置換基が結合していてもよいアルキル基、置換基が結合していてもよいフェニル基、置換基が結合していてもよい複素環基、置換基が結合していてもよいアルキルオキシ基、置換基が結合していてもよいフェニルオキシ基、置換基が結合していてもよいアルキルアミノ基及び置換基が結合していてもよいフェニルアミノ基のいずれかを示し、ＸはＯ、ＮＨ２、ＮＨＣＯ、ＣＯＮＨ、Ｓ又はＣＨ２を示す）のフェニルシクロプロピルアミン誘導体（I）及び（II）、又はその薬学上許容される塩、水和物、溶媒和物若しくはプロドラッグを用いることができる。
本発明者らの試験結果によれば、上記のフェニルシクロプロピルアミン誘導体（I）及び（II）は、ＬＳＤ１の阻害活性が高く、MAO-AやMAO-Bなどの阻害作用は、ＬＳＤ１阻害作用に比べて小さい。換言すれば、選択的にＬＳＤ１阻害を起こすことができる。そして、さらには、ＨＩＶの増殖を抑制する作用も有している。このため、ＬＳＤ１とＨＩＶの増殖との関係を調べる上で、MAO-AやMAO-Bなどの阻害作用の影響を避けることができる。このためＬＳＤ１とＨＩＶの増殖との関係を調べる研究ツールとして、特に好ましい。 According to the test results of the present inventors, the above-mentioned phenylcyclopropylamine derivatives (I) and (II) have high LSD1 inhibitory activity, and the inhibitory action such as MAO-A and MAO-B is LSD1 inhibitory action. Smaller than In other words, LSD1 inhibition can be selectively caused. Furthermore, it also has an action of suppressing the proliferation of HIV. For this reason, in examining the relationship between LSD1 and the proliferation of HIV, the influence of inhibitory action such as MAO-A and MAO-B can be avoided. Therefore, it is particularly preferable as a research tool for investigating the relationship between LSD1 and HIV proliferation.
なお、「プロドラッグ」とは、生体内で加水分解されてフェニルシクロプロピルアミン誘導体（I）又は（II）を再生する化合物をいい、例えばアミノ基の水素をアルカノイル基（アシル基）に置換した誘導体（すなわちアミド化した誘導体）、ヘミアミナールエーテル誘導体、アルコキシカルボニルオキシメチル基に置換した誘導体、N-オキシド誘導体等が挙げられる。
また、薬学上許容される塩としては、また、薬学上許容される塩としては、例えば塩酸塩、臭化水素酸塩、リン酸塩、硫酸塩、硝酸塩等の無機酸塩、ギ酸塩、酢酸塩、プロピオン酸塩、マレイン酸塩、フマル酸塩、コハク酸塩、乳酸塩、リンゴ酸塩、酒石酸塩、クエン酸塩、アスコルビン酸塩、マロン酸塩、シュウ酸塩、グリコール酸塩、フタル酸塩、ベンゼンスルホン酸塩等の有機酸塩が挙げられる。また、これらの塩を組み合わせて用いることもできる。
The “prodrug” refers to a compound that is hydrolyzed in vivo to regenerate the phenylcyclopropylamine derivative (I) or (II). For example, the amino group hydrogen is replaced with an alkanoyl group (acyl group). Derivatives (that is, amidated derivatives), hemiaminal ether derivatives, derivatives substituted with alkoxycarbonyloxymethyl groups, N-oxide derivatives and the like.
Examples of the pharmaceutically acceptable salt include pharmaceutically acceptable salts such as hydrochloride, hydrobromide, phosphate, sulfate, nitrate, and other inorganic acid salts, formate salts, acetic acid. Salt, propionate, maleate, fumarate, succinate, lactate, malate, tartrate, citrate, ascorbate, malonate, oxalate, glycolate, phthalic acid Examples thereof include organic acid salts such as salts and benzenesulfonate. Moreover, these salts can also be used in combination.
上記フェニルシクロプロピルアミン誘導体（I）又は（II）の中でも、下記一般式（１）（ただし、式中Ｒ１は水素又はフェニルアミド基を示し、Ｒ２は分枝を有することのあるアルキレン基を示し、シクロプロピルアミン置換基はメタ位又はパラ位に結合している）の化合物又はその薬学上許容される塩、水和物、溶媒和物若しくはプロドラッグからなることを特徴とするフェニルシクロプロピルアミン誘導体が好ましい。
また、Ｒ２はエチレン基とすることが特に好ましい。さらに好ましいのは、下記構造式（１ａ）に示すフェニルアミド基を有するフェニルシクロプロピルアミン誘導体（１ａ）又はその薬学上許容される塩、水和物、溶媒和物若しくはプロドラッグである。本発明者らの試験結果によれば、この化合物は特にＬＳＤ１阻害活性が大きく、MAO阻害との選択性にも優れている。このため、ＬＳＤ１とＨＩＶの増殖との関係を調べる上で、MAO-AやMAO-Bなどの阻害作用の影響を避けることができ、ＬＳＤ１とＨＩＶの増殖との関係を調べる研究ツールとして、特に好ましい。 R 2 is particularly preferably an ethylene group. More preferred is a phenylcyclopropylamine derivative (1a) having a phenylamide group represented by the following structural formula (1a), or a pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof. According to the test results of the present inventors, this compound has particularly large LSD1 inhibitory activity and is excellent in selectivity with MAO inhibition. For this reason, in examining the relationship between LSD1 and HIV proliferation, the influence of inhibitory action such as MAO-A and MAO-B can be avoided, and as a research tool to investigate the relationship between LSD1 and HIV proliferation, preferable.
また、前記ＬＳＤ１阻害剤は、下記一般式（２）の化合物又はその薬学上許容される塩、水和物、溶媒和物若しくはプロドラッグでもよい。 The LSD1 inhibitor may be a compound of the following general formula (2) or a pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof.
本発明者らは、この化合物（２）によっても、ＨＩＶの複製が阻害されることを見出している。なお、この化合物（２）はMAO阻害剤としても知られているが、MAO阻害作用を受けないＬＳＤ１阻害剤との比較を行うことにより、新たな知見を得ることができることから、ＬＳＤ１とＨＩＶの増殖との関係を調べる研究ツールとして有用である。 The present inventors have found that this compound (2) also inhibits HIV replication. In addition, although this compound (2) is also known as a MAO inhibitor, since new knowledge can be obtained by comparing with LSD1 inhibitor which does not receive MAO inhibitory action, LSD1 and HIV It is useful as a research tool to investigate the relationship with proliferation.
以下、本発明を具体化した実施例について詳細に説明する。 Hereinafter, embodiments embodying the present invention will be described in detail.
（実施例１）
実施例１では、(S)-トランス-N-3-[3-(2-アミノシクロプロピル) フェノキシ]-1-ベンジルカルバモイルプロピルベンズアミド塩酸塩（NCL-1）を合成した（下記化学式参照）。詳細は以下のとおりである。
Example 1
In Example 1, (S) -trans-N-3- [3- (2-aminocyclopropyl) phenoxy] -1-benzylcarbamoylpropylbenzamide hydrochloride (NCL-1) was synthesized (see the following chemical formula). Details are as follows.
まず、NCL-1を合成するための一方の前駆体として、化合物（９）を以下の反応ルートにしたがって合成した。
以下に合成法の詳細を示す。
（ステップ１−１）
トランス-3- (3-ヒドロキシフェニル)アクリル酸メチルエステル（４）の合成
3- (3-ヒドロキシフェニル)アクリル酸（３）（25.0 g）をメタノール（82 mL）に溶解し、濃硫酸（2.0 mL）を加え、24時間還流した。反応液を濃縮し、残査を酢酸エチル（1000 mL）に溶解させ、水（500 mL）、飽和炭酸水素ナトリウム水溶液（500 mL）で洗浄した。有機層を飽和食塩水（500 mL）で洗浄して、無水硫酸ナトリウムで乾燥し、ろ過後、減圧濃縮し、化合物（４）（25.9 g, 収率96%）を白色固体として得た。化合物（４）の1H NMRのデータを以下に示す。
1H NMR (CDCl3, 500 MHz, δ; ppm)
δ：7.60 (1H, d, J = 15.8 Hz), 7.21 (1H, t, J = 8.0 Hz), 7.04 (1H, d, J = 8.0 Hz), 6.98 (1H, t, J =1.8 Hz), 6.82 (1H,dd, J =8.0, 1.8Hz), 6.44 (1H, d, J =15.8 Hz), 3.77 (3H, s).
Details of the synthesis method are shown below.
(Step 1-1)
Synthesis of trans-3- (3-hydroxyphenyl) acrylic acid methyl ester (4)
3- (3-hydroxyphenyl) acrylic acid (3) (25.0 g) was dissolved in methanol (82 mL), concentrated sulfuric acid (2.0 mL) was added, and the mixture was refluxed for 24 hours. The reaction mixture was concentrated, and the residue was dissolved in ethyl acetate (1000 mL) and washed with water (500 mL) and saturated aqueous sodium hydrogen carbonate solution (500 mL). The organic layer was washed with saturated brine (500 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give compound (4) (25.9 g, yield 96%) as a white solid. The 1 H NMR data of the compound (4) is shown below.
1 H NMR (CDCl 3 , 500 MHz, δ; ppm)
δ: 7.60 (1H, d, J = 15.8 Hz), 7.21 (1H, t, J = 8.0 Hz), 7.04 (1H, d, J = 8.0 Hz), 6.98 (1H, t, J = 1.8 Hz), 6.82 (1H, dd, J = 8.0, 1.8Hz), 6.44 (1H, d, J = 15.8 Hz), 3.77 (3H, s).
（ステップ１−２）
トランス-3-(3メトキシメトキシフェニル)アクリル酸メチルエステル（５）の合成
ステップ１−１で得られたトランス-3- (3-ヒドロキシフェニル)アクリル酸メチルエステル（４）（25.8 g）をアセトン（220 mL）に溶解し、炭酸カリウム（40.0 g）を加え、室温で20分攪拌した。その溶液にメトキシメチルクロリド（11 mL）をゆっくりと加え、室温で12時間攪拌した。炭酸カリウムをろ過後、ろ液を濃縮し、残渣をシリカゲルフラッシュカラムクロマトグラフィー（展開溶媒 n-ヘキサン：酢酸エチル＝10 ：1）で精製し、化合物（５）（27.2 g, 収率84%）を無色の油状物として得た。化合物（５）の1H NMRのデータを以下に示す。
1H NMR (CDCl3, 500 MHz, δ; ppm)
δ：7.66 (1H, d, J = 16.2 Hz), 7.30 (1H, t, J = 8.0 Hz), 7.20 (1H, s), 7.17 (1H, d, J = 8.0 Hz), 7.07 (1H, dd, J = 8.0, 2.4 Hz), 6.43 (1H, d, J = 16.0 Hz), 5.19 (2H, s), 3.81 (3H, s), 3.49 (3H, s).
(Step 1-2)
Synthesis of trans-3- (3methoxymethoxyphenyl) acrylic acid methyl ester (5) Trans-3- (3-hydroxyphenyl) acrylic acid methyl ester (4) (25.8 g) obtained in Step 1-1 was converted to acetone. (220 mL), potassium carbonate (40.0 g) was added, and the mixture was stirred at room temperature for 20 min. Methoxymethyl chloride (11 mL) was slowly added to the solution and stirred at room temperature for 12 hours. After filtration of potassium carbonate, the filtrate was concentrated, and the residue was purified by silica gel flash column chromatography (developing solvent n-hexane: ethyl acetate = 10: 1) to give compound (5) (27.2 g, yield 84%). Was obtained as a colorless oil. The 1 H NMR data of the compound (5) is shown below.
1 H NMR (CDCl 3 , 500 MHz, δ; ppm)
δ: 7.66 (1H, d, J = 16.2 Hz), 7.30 (1H, t, J = 8.0 Hz), 7.20 (1H, s), 7.17 (1H, d, J = 8.0 Hz), 7.07 (1H, dd , J = 8.0, 2.4 Hz), 6.43 (1H, d, J = 16.0 Hz), 5.19 (2H, s), 3.81 (3H, s), 3.49 (3H, s).
（ステップ１−３）
トランス-2-(3-メトキシメトキシフェニル)シクロプロパンカルボン酸メチルエステル（６）の合成
水素化ナトリウム（60%）（6.32 g）とトリメチルスルホニウムヨージド（34.8 g）の混合物に、ジメチルスルホキシド（137 mL）を室温でゆっくりと滴下した。その反応液を室温で1時間攪拌した後、ステップ１−３で得られたトランス-3-(3メトキシメトキシフェニル)アクリル酸メチルエステル（５）（27.0 g）のジメチルスルホキシド（137 mL）溶液を滴下した。反応液を室温で4時間攪拌した後、10%クエン酸水溶液（500 mL）を加え、酢酸エチル（500 mL）で抽出した。有機層を飽和食塩水（500 mL）で洗浄して、無水硫酸ナトリウムで乾燥し、ろ過後、残渣をシリカゲルフラッシュカラムクロマトグラフィー（展開溶媒 n-ヘキサン：酢酸エチル＝10 ：1）で精製し、化合物（６）（6.70 g, 収率23%）を無色の油状物として得た。化合物（６）の1H NMRのデータを以下に示す。
1H NMR (CDCl3, 500 MHz, δ; ppm)
δ：7.19 (1H, t, J = 8.0 Hz), 6.89 (1H, dd, J =8.0, 2.5 Hz), 6.78 (1H, t, J = 2.5 Hz), 6.74 (1H, d, J =8.0 Hz), 5.16 (2H, s), 3.71 (3H, s), 3.47 (3H, s), 2.51-2.49 (1H, m), 1.92-1.89 (1H, m), 1.61-1.57 (1H, m) 1.34-1.31 (1H, m).
(Step 1-3)
Synthesis of trans-2- (3-methoxymethoxyphenyl) cyclopropanecarboxylic acid methyl ester (6) To a mixture of sodium hydride (60%) (6.32 g) and trimethylsulfonium iodide (34.8 g), dimethyl sulfoxide (137 mL) was slowly added dropwise at room temperature. The reaction solution was stirred at room temperature for 1 hour, and then a solution of trans-3- (3methoxymethoxyphenyl) acrylic acid methyl ester (5) (27.0 g) obtained in Step 1-3 in dimethyl sulfoxide (137 mL) was added. It was dripped. The reaction mixture was stirred at room temperature for 4 hr, 10% aqueous citric acid solution (500 mL) was added, and the mixture was extracted with ethyl acetate (500 mL). The organic layer was washed with saturated brine (500 mL), dried over anhydrous sodium sulfate, filtered, and the residue was purified by silica gel flash column chromatography (developing solvent n-hexane: ethyl acetate = 10: 1). Compound (6) (6.70 g, yield 23%) was obtained as a colorless oil. The 1 H NMR data of the compound (6) is shown below.
1 H NMR (CDCl 3 , 500 MHz, δ; ppm)
δ: 7.19 (1H, t, J = 8.0 Hz), 6.89 (1H, dd, J = 8.0, 2.5 Hz), 6.78 (1H, t, J = 2.5 Hz), 6.74 (1H, d, J = 8.0 Hz) ), 5.16 (2H, s), 3.71 (3H, s), 3.47 (3H, s), 2.51-2.49 (1H, m), 1.92-1.89 (1H, m), 1.61-1.57 (1H, m) 1.34 -1.31 (1H, m).
（ステップ１−４）
トランス-2-(3-メトキシメトキシフェニル)シクロプロパンカルボン酸（７）の合成
ステップ１−３で得られたトランス-2-(3-メトキシメトキシフェニル)シクロプロパンカルボン酸メチルエステル（６）（6.70 g）をメタノール（56 mL）に溶解させ、氷冷下、水酸化カリウム（16.0 g）のメタノール溶液（130 mL）を加え、反応液を室温で15時間攪拌した。反応液を濃縮し、残査を水（100 mL）に懸濁させ、ジクロロメタン（100 mL）で洗浄した。水層に2N塩酸を加えて酸性（pH 1）にし、ジクロロメタン（300 mL）で抽出した。有機層を無水硫酸ナトリウムで乾燥し、ろ過後、減圧濃縮し、化合物（７）（6.00 g, 収率95%）を無色の油状物として得た。化合物（７）の1H NMRのデータを以下に示す。
1H NMR (CDCl3, 500 MHz, δ; ppm)
δ：7.20 (1H, t, J = 8.0 Hz), 6.90 (1H, dd, J = 8.0, 1.9 Hz), 6.79 (1H, t, J = 1.9 Hz), 6.75 (1H, d, J = 8.0 Hz), 5.16 (2H, s), 3.48 (3H, s), 2.60-2.56 (1H, m), 1.92-1.89 (1H, m), 1.65 (1H, quintet, J = 5.0 Hz)1.42-1.38 (1H, m).
(Step 1-4)
Synthesis of trans-2- (3-methoxymethoxyphenyl) cyclopropanecarboxylic acid (7) trans-2- (3-methoxymethoxyphenyl) cyclopropanecarboxylic acid methyl ester (6) (6.70) obtained in Step 1-3 g) was dissolved in methanol (56 mL), a solution of potassium hydroxide (16.0 g) in methanol (130 mL) was added under ice cooling, and the reaction mixture was stirred at room temperature for 15 hours. The reaction mixture was concentrated, and the residue was suspended in water (100 mL) and washed with dichloromethane (100 mL). The aqueous layer was acidified with 2N hydrochloric acid (pH 1) and extracted with dichloromethane (300 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give compound (7) (6.00 g, yield 95%) as a colorless oil. The 1 H NMR data of the compound (7) is shown below.
1 H NMR (CDCl 3 , 500 MHz, δ; ppm)
δ: 7.20 (1H, t, J = 8.0 Hz), 6.90 (1H, dd, J = 8.0, 1.9 Hz), 6.79 (1H, t, J = 1.9 Hz), 6.75 (1H, d, J = 8.0 Hz) ), 5.16 (2H, s), 3.48 (3H, s), 2.60-2.56 (1H, m), 1.92-1.89 (1H, m), 1.65 (1H, quintet, J = 5.0 Hz) 1.42-1.38 (1H , m).
（ステップ１−５）
トランス- 2-(3-メトキシメトキシフェニル)シクロプロピルtert-ブチルカルバメート（８）の合成
ステップ１−４で得られたトランス-2-(3-メトキシメトキシフェニル)シクロプロパンカルボン酸（７）（6.00 g）をシクロヘキサン（320 mL）に溶解させ、ジフェニルホスホリルアジド（6.7 mL）とトリエチルアミン（4.5 mL）をアルゴン雰囲気下、0 ℃で加えた。反応液を3時間還流後、tert-ブタノール（52 mL）を加え、さらに11時間還流した。反応液を濃縮後、残渣をシリカゲルフラッシュカラムクロマトグラフィー（展開溶媒 n-ヘキサン：酢酸エチル＝ 7 ：1）で精製し、化合物（８）（3.70 g, 収率47%）を白色固体として得た。化合物（７）の1H NMRのデータを以下に示す。
1H NMR (CDCl3, 500 MHz, δ; ppm)
δ：7.17 (1H, t, J = 8.0 Hz), 6.85 (1H, d, J = 9.2 Hz), 6.78 (1H, s), 6.76 (1H, s), 5.15 (2H, s),4.82 (1H, broad s), 3.47 (3H, s)2.72-2.76 (1H, m), 1.98-2.02 (1H, m), 1.45 (9H, s), 1.74-1.16 (2H, m).
(Step 1-5)
Synthesis of trans-2- (3-methoxymethoxyphenyl) cyclopropyl tert-butylcarbamate (8) trans-2- (3-methoxymethoxyphenyl) cyclopropanecarboxylic acid (7) (6.00) obtained in Step 1-4 g) was dissolved in cyclohexane (320 mL), and diphenylphosphoryl azide (6.7 mL) and triethylamine (4.5 mL) were added at 0 ° C. under an argon atmosphere. The reaction mixture was refluxed for 3 hours, tert-butanol (52 mL) was added, and the mixture was further refluxed for 11 hours. After concentration of the reaction solution, the residue was purified by silica gel flash column chromatography (developing solvent n-hexane: ethyl acetate = 7: 1) to obtain compound (8) (3.70 g, yield 47%) as a white solid. . The 1 H NMR data of the compound (7) is shown below.
1 H NMR (CDCl 3 , 500 MHz, δ; ppm)
δ: 7.17 (1H, t, J = 8.0 Hz), 6.85 (1H, d, J = 9.2 Hz), 6.78 (1H, s), 6.76 (1H, s), 5.15 (2H, s), 4.82 (1H , broad s), 3.47 (3H, s) 2.72-2.76 (1H, m), 1.98-2.02 (1H, m), 1.45 (9H, s), 1.74-1.16 (2H, m).
（ステップ１−６）
トランス-2-(3-ヒドロキシフェニル)シクロプロピルtert-ブチルカルバメート（９）の合成
ステップ１−５で得られたトランス-2-(3-メトキシメトキシフェニル)シクロプロピルtert-ブチルカルバメート（８）（3.70 g）をジクロロメタン（60 mL）に溶解させ、4N塩酸酢酸エチル溶液（68 mL）を加え、室温で4時間攪拌した。反応液を濃縮し、残渣を1,4-ジオキサン（33 mL） と水 (33 mL)に溶解させ、トリエチルアミン (20 mL) と Boc2O (4.6 mL)を加えた。反応液を室温で12時間攪拌後、10%クエン酸溶液（200 mL）に注ぎ、酢酸エチル（300 mL）で抽出した。有機層を飽和食塩水（300 mL）で洗浄して、無水硫酸ナトリウムで乾燥し、ろ過後、残渣をシリカゲルフラッシュカラムクロマトグラフィー（展開溶媒 n-ヘキサン：酢酸エチル＝ 5 ：1）で精製し、化合物（９）（1.59 g, 収率35%）を無色の油状物として得た。化合物（９）の1H NMRのデータを以下に示す。
1H NMR (CDCl3, 500 MHz, δ; ppm)
δ：7.10 (1H, d, J = 7.6 Hz), 6.67 (1H, d, J =8.0 Hz), 6.64 (1H, dd, J = 8.0, 2.4 Hz), 6.59 (1H, s), 5.28 (1H, broad s), 4.86 (1H, broad s), 2.69-2.72 (1H, m), 1.97-2.01 (1H, m), 1.46 (9H, s), 1.15-1.12 (2H, m).
(Step 1-6)
Synthesis of trans-2- (3-hydroxyphenyl) cyclopropyl tert-butylcarbamate (9) trans-2- (3-methoxymethoxyphenyl) cyclopropyl tert-butylcarbamate (8) obtained in Step 1-5 ( 3.70 g) was dissolved in dichloromethane (60 mL), 4N hydrochloric acid ethyl acetate solution (68 mL) was added, and the mixture was stirred at room temperature for 4 hr. The reaction mixture was concentrated, the residue was dissolved in 1,4-dioxane (33 mL) and water (33 mL), and triethylamine (20 mL) and Boc2O (4.6 mL) were added. The reaction solution was stirred at room temperature for 12 hours, poured into a 10% citric acid solution (200 mL), and extracted with ethyl acetate (300 mL). The organic layer was washed with saturated brine (300 mL), dried over anhydrous sodium sulfate, filtered, and the residue was purified by silica gel flash column chromatography (developing solvent n-hexane: ethyl acetate = 5: 1) Compound (9) (1.59 g, yield 35%) was obtained as a colorless oil. The 1 H NMR data of the compound (9) is shown below.
1 H NMR (CDCl 3 , 500 MHz, δ; ppm)
δ: 7.10 (1H, d, J = 7.6 Hz), 6.67 (1H, d, J = 8.0 Hz), 6.64 (1H, dd, J = 8.0, 2.4 Hz), 6.59 (1H, s), 5.28 (1H , broad s), 4.86 (1H, broad s), 2.69-2.72 (1H, m), 1.97-2.01 (1H, m), 1.46 (9H, s), 1.15-1.12 (2H, m).
さらに、NCL-1を合成するための他方の前駆体として、化合物（１３）を以下の反応ルートにしたがって合成した。
以下に合成法の詳細を示す。
（ステップ１−７）
(S)-1-ベンジルカルバモイル-3-ヒドロキシプロピルtert-ブチルカルバメート（１１）の合成
N- tert-ブトキシカルボニル(S) -ホモセリン（１０）（5.50 g）、ベンジルアミン（2.8 mL）、PyBOP（13.1 g）、トリエチルアミン（7.0 mL）をN,N-ジメチルホルムアミド（55 mL）に溶解させ、室温で10時間攪拌した。反応液を水（300 mL）で希釈し、クロロホルム（300 mL）で抽出した。有機層を飽和食塩水（300 mL）で洗浄して、無水硫酸ナトリウムで乾燥し、ろ過後、残渣をシリカゲルフラッシュカラムクロマトグラフィー（展開溶媒 n-ヘキサン：酢酸エチル＝ 1 ：2）で精製し、化合物（１１）（5.46 g, 収率70%）を白色固体として得た。化合物（１１）の1H NMRのデータを以下に示す。
1H NMR (CDCl3, 500 MHz, δ; ppm)
δ：7.35-7.32 (2H, m), 7.29-7.25 (3H, m), 6.71 (1H, broad s), 5.58 (1H, d, J = 7.3 Hz), 4.45 (2H, s), 4.35 (1H, s), 3.71 (2H, s), 3.27 (1H, broad s), 2.04-1.98 (1H, m), 1.72-1.85 (1H, m), 1.43 (9H, s)
Details of the synthesis method are shown below.
(Step 1-7)
Synthesis of (S) -1-benzylcarbamoyl-3-hydroxypropyl tert-butylcarbamate (11)
N-tert-butoxycarbonyl (S) -homoserine (10) (5.50 g), benzylamine (2.8 mL), PyBOP (13.1 g), triethylamine (7.0 mL) dissolved in N, N-dimethylformamide (55 mL) And stirred at room temperature for 10 hours. The reaction mixture was diluted with water (300 mL) and extracted with chloroform (300 mL). The organic layer was washed with saturated brine (300 mL), dried over anhydrous sodium sulfate, filtered, and the residue was purified by silica gel flash column chromatography (developing solvent n-hexane: ethyl acetate = 1: 2). Compound (11) (5.46 g, yield 70%) was obtained as a white solid. The 1 H NMR data of the compound (11) is shown below.
1 H NMR (CDCl 3 , 500 MHz, δ; ppm)
δ: 7.35-7.32 (2H, m), 7.29-7.25 (3H, m), 6.71 (1H, broad s), 5.58 (1H, d, J = 7.3 Hz), 4.45 (2H, s), 4.35 (1H , s), 3.71 (2H, s), 3.27 (1H, broad s), 2.04-1.98 (1H, m), 1.72-1.85 (1H, m), 1.43 (9H, s)
（ステップ１−８）
(S)-2-アミノ-N-ベンジル-4-ヒドロキシブタンアミド塩酸塩（１２）の合成
ステップ１−７で得られた(S)-1-ベンジルカルバモイル-3-ヒドロキシプロピルtert-ブチルカルバメート（１１）（5.40 g）をジクロロメタン（60 mL）に溶解させ、4N塩酸1,4-ジオキサン溶液（87 mL）を加え、室温で5時間攪拌した。反応液を濃縮し、化合物（4.27 g, 収率100%）を白色固体として得た。化合物（１２）の1H NMRのデータを以下に示す。
δ：1H NMR (CD3OD, 500 MHz, δ; ppm)
7.44-7.41 (2H, m), 7.34-7.30 (3H, m), 7.27 (1H, broad s), 4.43 (2H, s), 3.94 (1H, d, J = 7.0 Hz) 2.03 (2H, s)
(Step 1-8)
Synthesis of (S) -2-amino-N-benzyl-4-hydroxybutanamide hydrochloride (12) (S) -1-benzylcarbamoyl-3-hydroxypropyl tert-butylcarbamate (12) obtained in Step 1-7 11) (5.40 g) was dissolved in dichloromethane (60 mL), 4N hydrochloric acid 1,4-dioxane solution (87 mL) was added, and the mixture was stirred at room temperature for 5 hr. The reaction mixture was concentrated to give the compound (4.27 g, yield 100%) as a white solid. The 1 H NMR data of the compound (12) is shown below.
δ: 1 H NMR (CD 3 OD, 500 MHz, δ; ppm)
7.44-7.41 (2H, m), 7.34-7.30 (3H, m), 7.27 (1H, broad s), 4.43 (2H, s), 3.94 (1H, d, J = 7.0 Hz) 2.03 (2H, s)
（ステップ１−９）
(S)-N-(1-ベンジルカルバモイル-3-ヒドロキシプロピル)ベンズアミド（１３）の合成
ステップ１−８で得られた(S)-2-アミノ-N-ベンジル-4-ヒドロキシブタンアミド塩酸塩（１２）（118 mg）、安息香酸（60 mg）、PyBOP（303 mg）、トリエチルアミン（0.2 mL）をN,N-ジメチルホルムアミド（1 mL）に溶解させ、室温で20時間攪拌した。反応液を水（100 mL）で希釈し、ジクロロメタン（100 mL）で抽出した。有機層を飽和食塩水（100 mL）で洗浄して、無水硫酸ナトリウムで乾燥し、ろ過後、残渣をシリカゲルフラッシュカラムクロマトグラフィー（展開溶媒 ｎ-ヘキサン：酢酸エチル＝2：3）で精製し、化合物（１３）（72 mg, 収率47%）を白色固体として得た。化合物（１３）の1H NMRのデータを以下に示す。
1H NMR (CDCl3, 500 MHz, δ; ppm)
δ：7.79 (2H, d, J = 7.3 Hz), 7.53 (1H, t, J = 7.3 Hz), 7.44 (2H, t, J = 8.0 Hz), 7.33-7.25 (5H, m), 7.06 (1H, d, J = 7.3 Hz), 6.89 (1H, broad s), 4.77 (1H, q, J = 6.4 Hz), 4.47-4.45 (2H, m), 4.32-4.28 (1H, m), 4.22-4.18 (1H, m), 2.28-2.19 (2H, m), 2.03 (3H, s).
(Step 1-9)
Synthesis of (S) -N- (1-benzylcarbamoyl-3-hydroxypropyl) benzamide (13) (S) -2-amino-N-benzyl-4-hydroxybutanamide hydrochloride obtained in step 1-8 (12) (118 mg), benzoic acid (60 mg), PyBOP (303 mg) and triethylamine (0.2 mL) were dissolved in N, N-dimethylformamide (1 mL), and the mixture was stirred at room temperature for 20 hours. The reaction was diluted with water (100 mL) and extracted with dichloromethane (100 mL). The organic layer was washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and the residue was purified by silica gel flash column chromatography (developing solvent n-hexane: ethyl acetate = 2: 3). Compound (13) (72 mg, 47% yield) was obtained as a white solid. The 1 H NMR data of the compound (13) is shown below.
1 H NMR (CDCl 3 , 500 MHz, δ; ppm)
δ: 7.79 (2H, d, J = 7.3 Hz), 7.53 (1H, t, J = 7.3 Hz), 7.44 (2H, t, J = 8.0 Hz), 7.33-7.25 (5H, m), 7.06 (1H , d, J = 7.3 Hz), 6.89 (1H, broad s), 4.77 (1H, q, J = 6.4 Hz), 4.47-4.45 (2H, m), 4.32-4.28 (1H, m), 4.22-4.18 (1H, m), 2.28-2.19 (2H, m), 2.03 (3H, s).
<カップリング反応>
ステップ１−１〜ステップ１−６で合成したカップリング前駆体である化合物（９）と、ステップ１−７〜ステップ１−９で合成したカップリング前駆体（１３）とを用いて、化１０に示す合成ルートに従い、光延反応を用いたカップリング反応を行った。
Using the compound (9), which is the coupling precursor synthesized in Step 1-1 to Step 1-6, and the coupling precursor (13) synthesized in Step 1-7 to Step 1-9, The coupling reaction using Mitsunobu reaction was performed according to the synthesis route shown in FIG.
以下にカップリング反応の詳細を示す。
（ステップ１−１０）
(S)-トランス-[2-[3-(3-ベンゾイルアミノ-3-ベンジルカルバモイルプロポキシ)フェニル] シクロプロピルtert-ブチルカルバメート（１４）の合成
ステップ１−９で得られた(S)-N-(1-ベンジルカルバモイル-3-ヒドロキシプロピル)ベンズアミド（１３）（200 mg）と、ステップ１−６で得られたトランス-2-(3-ヒドロキシフェニル)シクロプロピルtert-ブチルカルバメート（９）（230 mg）と、トリフェニルホスフィン（504 mg）とを無水テトラヒドロフラン（3 mL）に溶解させ、氷冷下、ジイソプロピルアゾジカルボキシレート（1mL）を加えた。反応液を室温で5時間攪拌後、濃縮し、残渣をシリカゲルフラッシュカラムクロマトグラフィー（展開溶媒 n-ヘキサン：酢酸エチル＝ 1 ：1）で精製し、化合物（１４）（64 mg, 収率18%）を淡黄色固体として得た。化合物（１４）の1H NMRのデータを以下に示す。
1H NMR (CDCl3, 500 MHz, δ; ppm)
δ：7.82 (2H, d, J = 8.5 Hz), 7.52 (1H, t, J = 8.5 Hz), 7.46 (2H, t, J = 8.5 Hz), 7.26-7.21 (5H, m), 7.16 (1H, t, J = 8.0 Hz), 6.92 (1H, s), 6.75 (1H, d, J = 8.0 Hz), 6.63 (1H, d, J = 8.0 Hz), 6.60 (1H, broad s), 4.88 (1H, quartet, J = 6.0 Hz), 4.47 (2H, d, J = 6.0 Hz), 4.32-4.29 (1H, m), 4.13-4.10 (1H, m), 2.65-2.70 (1H, m), 2.43-2.37 (2H, m), 1.99-1.97 (1H, m), 1.45 (9H, s), 1.14-1.10 (2H, m).
The details of the coupling reaction are shown below.
(Step 1-10)
Synthesis of (S) -trans- [2- [3- (3-benzoylamino-3-benzylcarbamoylpropoxy) phenyl] cyclopropyl tert-butylcarbamate (14) (S) -N obtained in step 1-9 -(1-Benzylcarbamoyl-3-hydroxypropyl) benzamide (13) (200 mg) and trans-2- (3-hydroxyphenyl) cyclopropyl tert-butylcarbamate (9) obtained in Step 1-6 (9) 230 mg) and triphenylphosphine (504 mg) were dissolved in anhydrous tetrahydrofuran (3 mL), and diisopropyl azodicarboxylate (1 mL) was added under ice cooling. The reaction mixture was stirred at room temperature for 5 hours and concentrated. The residue was purified by silica gel flash column chromatography (developing solvent n-hexane: ethyl acetate = 1: 1) to give compound (14) (64 mg, 18% yield). ) Was obtained as a pale yellow solid. The 1 H NMR data of the compound (14) is shown below.
1 H NMR (CDCl 3 , 500 MHz, δ; ppm)
δ: 7.82 (2H, d, J = 8.5 Hz), 7.52 (1H, t, J = 8.5 Hz), 7.46 (2H, t, J = 8.5 Hz), 7.26-7.21 (5H, m), 7.16 (1H , t, J = 8.0 Hz), 6.92 (1H, s), 6.75 (1H, d, J = 8.0 Hz), 6.63 (1H, d, J = 8.0 Hz), 6.60 (1H, broad s), 4.88 ( 1H, quartet, J = 6.0 Hz), 4.47 (2H, d, J = 6.0 Hz), 4.32-4.29 (1H, m), 4.13-4.10 (1H, m), 2.65-2.70 (1H, m), 2.43 -2.37 (2H, m), 1.99-1.97 (1H, m), 1.45 (9H, s), 1.14-1.10 (2H, m).
（ステップ１−１１）
(S)-トランス-N-3-[3-(2-アミノシクロプロピル) フェノキシ]-1-ベンジルカルバモイルプロピルベンズアミド塩酸塩（NCL-1）の合成
ステップ１−１０で得られた(S)-トランス-[2-[3-(3-ベンゾイルアミノ-3-ベンジルカルバモイルプロポキシ)フェニル] シクロプロピルtert-ブチルカルバメート（１４）（70 mg）をジクロロメタン（1 mL）に溶解させ、4N塩酸酢酸エチル溶液（0.8 mL）を加え、室温で4時間攪拌した。反応液を濃縮し、残渣をクロロホルム-メタノールから再結晶し、実施例１の化合物（NCL-1）（22 mg, 収率36%）を淡黄色固体として得た。NCL-1の融点、1H NMR、13C NMR、MS (FAB)のデータを示す。
融点 134.8-139.1 ℃
1H NMR (CD3OD, 500 MHz, δ; ppm)
δ：7.84 (2H, d, J = 8.5 Hz), 7.54 (1H, t, J = 8.5 Hz), 7.46 (2H, t, J = 8.5 Hz), 7.26-7.17 (6H, m), 6.77 (1H, dd, J = 8.0, 1.5 Hz), 6.74-6.72 (1H, m), 6.67 (1H, d, J = 1.5 Hz), 4.47-4.37 (2H, m), 4.08 (2H, t, J = 6.0 Hz), 2.28-2.24 (2H, m), 1.35-1.33 (1H, m), 1.29-1.26 (1H, m)
13C NMR (CD3OD, 500 MHz, δ; ppm)
δ：173.93, 170.34, 160.52, 141.32, 139.79, 135.21, 132.96, 130.81, 129.55, 128.54, 128.46, 128.19, 120.00, 114.15, 114.05, 113.84, 65.92, 53.13, 44.15, 32.68, 31.96, 22.58, 13.84
MS (FAB) m/z = 444 (M+)
Anal. Calcd. for C27H30ClN3O3 3/2H2O: C, 63.96; H, 6.56; N, 8.29.
Found: C, 63.83; H, 6.29; N, 7.98. 3/2
(Step 1-11)
Synthesis of (S) -trans-N-3- [3- (2-aminocyclopropyl) phenoxy] -1-benzylcarbamoylpropylbenzamide hydrochloride (NCL-1) (S)-obtained in step 1-10 Trans- [2- [3- (3-Benzoylamino-3-benzylcarbamoylpropoxy) phenyl] cyclopropyl tert-butylcarbamate (14) (70 mg) was dissolved in dichloromethane (1 mL) and 4N hydrochloric acid ethyl acetate solution (0.8 mL) was added, and the mixture was stirred at room temperature for 4 hours. The reaction mixture was concentrated, and the residue was recrystallized from chloroform-methanol to obtain the compound of Example 1 (NCL-1) (22 mg, yield 36%) as a pale yellow solid. The melting point, 1 H NMR, 13 C NMR, and MS (FAB) data of NCL-1 are shown.
Melting point 134.8-139.1 ℃
1 H NMR (CD 3 OD, 500 MHz, δ; ppm)
δ: 7.84 (2H, d, J = 8.5 Hz), 7.54 (1H, t, J = 8.5 Hz), 7.46 (2H, t, J = 8.5 Hz), 7.26-7.17 (6H, m), 6.77 (1H , dd, J = 8.0, 1.5 Hz), 6.74-6.72 (1H, m), 6.67 (1H, d, J = 1.5 Hz), 4.47-4.37 (2H, m), 4.08 (2H, t, J = 6.0 Hz), 2.28-2.24 (2H, m), 1.35-1.33 (1H, m), 1.29-1.26 (1H, m)
13 C NMR (CD3OD, 500 MHz, δ; ppm)
δ: 173.93, 170.34, 160.52, 141.32, 139.79, 135.21, 132.96, 130.81, 129.55, 128.54, 128.46, 128.19, 120.00, 114.15, 114.05, 113.84, 65.92, 53.13, 44.15, 32.68, 31.96, 22.58, 13.84
MS (FAB) m / z = 444 (M + )
Anal.Calcd.for C27H30ClN 3 O 3 3 / 2H 2 O: C, 63.96; H, 6.56; N, 8.29.
Found: C, 63.83; H, 6.29; N, 7.98. 3/2
（実施例２）
実施例２では、(S)-トランス-N-3-[4-(2-アミノシクロプロピル)フェノキシ]-1-ベンジルカルバモイルプロピルベンズアミド塩酸塩（NCL-2）を合成した（下記化学式参照）。詳細は以下のとおりである。
(Example 2)
In Example 2, (S) -trans-N-3- [4- (2-aminocyclopropyl) phenoxy] -1-benzylcarbamoylpropylbenzamide hydrochloride (NCL-2) was synthesized (see the following chemical formula). Details are as follows.
まず、NCL-2を合成するための一方の前駆体として、化合物（２１）を以下の反応ルートにしたがって合成した。 First, compound (21) was synthesized according to the following reaction route as one precursor for synthesizing NCL-2.
以下に合成法の詳細を示す。
（ステップ２−１）
トランス-3- (4-ヒドロキシフェニル)アクリル酸メチルエステル（１６）の合成
実施例１のステップ１−１と同様の方法により、3-(4-ヒドロキシフェニル)アクリル酸（１５）（20.0 g）を出発原料とし、化合物（１６）（21.2 g, 収率99%）を白色固体として得た。化合物（１６）の1H NMRのデータを以下に示す。
1H NMR (CDCl3, 500 MHz, δ; ppm)
δ：7.64 (1H, d, J = 15.8 Hz), 7.43 (2H, d, J = 8.5 Hz), 6.84 (2H, d, J = 8.5 Hz), 6.31 (1H, d, J =15.8 Hz), 5.21 (1H,s), 3.80 (3H, s).
Details of the synthesis method are shown below.
(Step 2-1)
Synthesis of trans-3- (4-hydroxyphenyl) acrylic acid methyl ester (16) In the same manner as in Step 1-1 of Example 1, 3- (4-hydroxyphenyl) acrylic acid (15) (20.0 g) Was used as a starting material to obtain Compound (16) (21.2 g, yield 99%) as a white solid. The 1 H NMR data of the compound (16) is shown below.
1 H NMR (CDCl 3 , 500 MHz, δ; ppm)
δ: 7.64 (1H, d, J = 15.8 Hz), 7.43 (2H, d, J = 8.5 Hz), 6.84 (2H, d, J = 8.5 Hz), 6.31 (1H, d, J = 15.8 Hz), 5.21 (1H, s), 3.80 (3H, s).
（ステップ２−２）
トランス-3-(4-メトキシメトキシフェニル)アクリル酸メチルエステル（１７）の合成
実施例１のステップ１−２と同様の方法により、ステップ２−１より得られたトランス-3- (4-ヒドロキシフェニル)アクリル酸メチルエステル（１６）（10.0 g）より、化合物（１７）（11.1 g, 収率89%）を無色油状物として得た。化合物（１７）の1H NMRのデータを以下に示す。
1H NMR (CDCl3, 500 MHz, δ; ppm)
δ：7.65 (1H, d, J = 6.2 Hz), 7.47 (2H, d, J = 8.8 Hz), 7.04 (2H, d, J = 8.8 Hz), 6.33 (1H, d, J = 6.2 Hz), 5.20 (2H, s), 3.80 (3H, s), 3.48 (3H, s).
(Step 2-2)
Synthesis of trans-3- (4-methoxymethoxyphenyl) acrylic acid methyl ester (17) The trans-3- (4-hydroxy) obtained from Step 2-1 was prepared in the same manner as in Step 1-2 of Example 1. From phenyl) acrylic acid methyl ester (16) (10.0 g), compound (17) (11.1 g, yield 89%) was obtained as a colorless oil. The 1 H NMR data of the compound (17) is shown below.
1 H NMR (CDCl 3 , 500 MHz, δ; ppm)
δ: 7.65 (1H, d, J = 6.2 Hz), 7.47 (2H, d, J = 8.8 Hz), 7.04 (2H, d, J = 8.8 Hz), 6.33 (1H, d, J = 6.2 Hz), 5.20 (2H, s), 3.80 (3H, s), 3.48 (3H, s).
（ステップ２−３）
トランス-2-(4-メトキシメトキシフェニル)シクロプロパンカルボン酸メチルエステル（１８）の合成
実施例１のステップ１−３と同様の方法により、ステップ２−２より得られたトランス-3-(4-メトキシメトキシフェニル)アクリル酸メチルエステル（１５）（11.0 g）より、化合物（１８）（1.68 g, 収率14%）を無色油状物として得た。化合物（１８）の1H NMRのデータを以下に示す。
1H NMR (CDCl3, 500 MHz, δ; ppm)
δ：7.03 (2H, d, J = 8.5 Hz), 6.95 (2H, d, J =8.5 Hz), 5.14 (2H, s), 3.71 (3H, s), 3.46 (3H, s), 2.51-2.47 (1H, m), 1.85-1.81 (1H, m), 1.58-1.54 (1H, m) 1.29-1.25 (1H, m).
(Step 2-3)
Synthesis of trans-2- (4-methoxymethoxyphenyl) cyclopropanecarboxylic acid methyl ester (18) In the same manner as in Step 1-3 of Example 1, trans-3- (4 Compound (18) (1.68 g, 14% yield) was obtained as a colorless oil from (methoxymethoxyphenyl) acrylic acid methyl ester (15) (11.0 g). The 1 H NMR data of the compound (18) is shown below.
1 H NMR (CDCl 3 , 500 MHz, δ; ppm)
δ: 7.03 (2H, d, J = 8.5 Hz), 6.95 (2H, d, J = 8.5 Hz), 5.14 (2H, s), 3.71 (3H, s), 3.46 (3H, s), 2.51-2.47 (1H, m), 1.85-1.81 (1H, m), 1.58-1.54 (1H, m) 1.29-1.25 (1H, m).
（ステップ２−４）
トランス-2-(4-メトキシメトキシフェニル)シクロプロパンカルボン酸（１９）の合成
実施例１のステップ１−４と同様の方法により、ステップ２−３より得られたトランス-2-(4-メトキシメトキシフェニル)シクロプロパンカルボン酸メチルエステル（１８）（1.68 g）より、化合物（１９）（1.50 g, 収率95%）を白色固体として得た。化合物（１９）の1H NMRのデータを以下に示す。
1H NMR (CDCl3, 500 MHz, δ; ppm)
δ：7.05 (2H, d, J = 6.7 Hz), 6.94 (2H, d, J = 6.7 Hz), 5.12 (2H, s), 3.42 (3H, s), 2.43-2.39 (1H, m), 1.76-1.73 (1H, m), 1.49-1.46 (1H, m), 1.33-1.28 (1H, m).
(Step 2-4)
Synthesis of trans-2- (4-methoxymethoxyphenyl) cyclopropanecarboxylic acid (19) The trans-2- (4-methoxy) obtained from Step 2-3 was prepared in the same manner as in Step 1-4 of Example 1. From methoxyphenyl) cyclopropanecarboxylic acid methyl ester (18) (1.68 g), compound (19) (1.50 g, yield 95%) was obtained as a white solid. The 1 H NMR data of the compound (19) is shown below.
1 H NMR (CDCl 3 , 500 MHz, δ; ppm)
δ: 7.05 (2H, d, J = 6.7 Hz), 6.94 (2H, d, J = 6.7 Hz), 5.12 (2H, s), 3.42 (3H, s), 2.43-2.39 (1H, m), 1.76 -1.73 (1H, m), 1.49-1.46 (1H, m), 1.33-1.28 (1H, m).
（ステップ２−５）
トランス- 2-(4-メトキシメトキシフェニル)シクロプロピルtert-ブチルカルバメート（２０）の合成
実施例１のステップ１−５と同様の方法により、ステップ２−４より得られたトランス-2-(4-メトキシメトキシフェニル)シクロプロパンカルボン酸（１９）（1.05 g）より、化合物（２０）（788 mg, 収率56%）を淡黄色固体として得た。化合物（２０）の1H NMRのデータを以下に示す。
1H NMR (CDCl3, 500 MHz, δ; ppm)
δ：7.07 (2H, d, J = 8.2 Hz), 6.94 (2H, d, J = 8.2 Hz), 5.13 (2H, s), 4.89 (1H, broad s), 3.46 (3H, s), 2.64-2.69 (1H, m), 2.02-1.98 (1H, m), 1.46 (9H, s), 1.12-1.09 (2H, m).
(Step 2-5)
Synthesis of trans-2- (4-methoxymethoxyphenyl) cyclopropyl tert-butylcarbamate (20) Trans-2- (4) obtained from Step 2-4 in the same manner as in Step 1-5 of Example 1 Compound (20) (788 mg, yield 56%) was obtained as a pale yellow solid from -methoxymethoxyphenyl) cyclopropanecarboxylic acid (19) (1.05 g). The 1 H NMR data of the compound (20) is shown below.
1 H NMR (CDCl 3 , 500 MHz, δ; ppm)
δ: 7.07 (2H, d, J = 8.2 Hz), 6.94 (2H, d, J = 8.2 Hz), 5.13 (2H, s), 4.89 (1H, broad s), 3.46 (3H, s), 2.64- 2.69 (1H, m), 2.02-1.98 (1H, m), 1.46 (9H, s), 1.12-1.09 (2H, m).
（ステップ２−６）
トランス-2-(4-ヒドロキシフェニル)シクロプロピルtert-ブチルカルバメート（２１）の合成
実施例１のステップ１−６と同様の方法により、ステップ２−５より得られたトランス- 2-(4-メトキシメトキシフェニル)シクロプロピルtert-ブチルカルバメート（２０）（476 mg）より、化合物（２１）（300 mg, 収率74%）を淡黄色油状物として得た。化合物（２１）の1H NMRのデータを以下に示す。
1H NMR (CDCl3, 500 MHz, δ; ppm)
δ：6.94 (2H, d, J = 8.5 Hz), 6.72 (2H, d, J =8.5 Hz), 5.13 (1H, broad s), 4.84 (1H, broad s), 2.60-2.70 (1H, m), 1.98 (1H, m), 2.09-1.96 (1H, m), 1.10-1.05 (2H, m).
(Step 2-6)
Synthesis of trans-2- (4-hydroxyphenyl) cyclopropyl tert-butylcarbamate (21) In the same manner as in Step 1-6 of Example 1, trans-2- (4- From methoxymethoxyphenyl) cyclopropyl tert-butylcarbamate (20) (476 mg), compound (21) (300 mg, 74% yield) was obtained as a pale yellow oil. The 1 H NMR data of the compound (21) is shown below.
1 H NMR (CDCl 3 , 500 MHz, δ; ppm)
δ: 6.94 (2H, d, J = 8.5 Hz), 6.72 (2H, d, J = 8.5 Hz), 5.13 (1H, broad s), 4.84 (1H, broad s), 2.60-2.70 (1H, m) , 1.98 (1H, m), 2.09-1.96 (1H, m), 1.10-1.05 (2H, m).
NCL-2を合成するための他方の前駆体として、実施例１のステップ１−７〜ステップ１−９で合成した化合物（１３）を用いた。 As the other precursor for synthesizing NCL-2, the compound (13) synthesized in Step 1-7 to Step 1-9 of Example 1 was used.
<カップリング反応>
上記ステップ２−１〜ステップ２−６で合成したカップリング前駆体である化合物（２１）と、化合物（１３）とを用いて、光延反応によるたカップリング反応によって、実施例２の化合物NCL-2を合成した。
<Coupling reaction>
Using the compound (21), which is the coupling precursor synthesized in Steps 2-1 to 2-6, and the compound (13), the compound NCL- of Example 2 was subjected to a coupling reaction by Mitsunobu reaction. 2 was synthesized.
以下に合成法の詳細を示す。
（ステップ２−７）
(S)-トランス-[2-[4-(3-ベンゾイルアミノ-3-ベンジルカルバモイルプロポキシ)フェニル] シクロプロピルtert-ブチルカルバメート（２２）の合成
実施例１のステップ１−７と同様の方法により、ステップ２−６より得られたトランス-2-(4-ヒドロキシフェニル)シクロプロピルtert-ブチルカルバメート（２１）（220 mg）と
実施例１のステップ１−７〜１−９で合成したカップリング前駆体（１３）のカップリングを行い、化合物（２２）（83 mg, 収率25%）を淡黄色固体として得た。化合物（２２）の1H NMRのデータを以下に示す。
1H NMR (CDCl3, 500 MHz, δ; ppm)
δ：7.79 (2H, d, J = 8.5 Hz), 7.53-7.49 (1H, m), 7.41 (2H, t, J = 8.5 Hz), 7.26-7.13 (5H, m), 7.04 (2H, d, J = 8.5 Hz), 6.71 (2H, d, J = 8.5 Hz), 4.94-4.89 (2H, m), 4.46-4.38 (2H, m), 4.25-4.21 (1H, m), 4.08-4.04 (1H, m), 2.55-2.59 (1H, m), 2.43-2.37 (2H, m), 1.95-2.01 (1H, m), 1.45 (9H, s), 1.08 (2H, t, J = 7.0 Hz).
Details of the synthesis method are shown below.
(Step 2-7)
Synthesis of (S) -trans- [2- [4- (3-Benzoylamino-3-benzylcarbamoylpropoxy) phenyl] cyclopropyl tert-butylcarbamate (22) In the same manner as in Step 1-7 of Example 1 Coupling synthesized in steps 1-7 to 1-9 of Example 1 with trans-2- (4-hydroxyphenyl) cyclopropyl tert-butylcarbamate (21) (220 mg) obtained from Step 2-6 The precursor (13) was coupled to obtain the compound (22) (83 mg, yield 25%) as a pale yellow solid. The 1 H NMR data of the compound (22) is shown below.
1 H NMR (CDCl 3 , 500 MHz, δ; ppm)
δ: 7.79 (2H, d, J = 8.5 Hz), 7.53-7.49 (1H, m), 7.41 (2H, t, J = 8.5 Hz), 7.26-7.13 (5H, m), 7.04 (2H, d, J = 8.5 Hz), 6.71 (2H, d, J = 8.5 Hz), 4.94-4.89 (2H, m), 4.46-4.38 (2H, m), 4.25-4.21 (1H, m), 4.08-4.04 (1H , m), 2.55-2.59 (1H, m), 2.43-2.37 (2H, m), 1.95-2.01 (1H, m), 1.45 (9H, s), 1.08 (2H, t, J = 7.0 Hz).
（ステップ２−８）
(S)-トランス-N-3-[4-(2-アミノシクロプロピル) フェノキシ]-1-ベンジルカルバモイルプロピルベンズアミド塩酸塩（NCL-2）の合成
実施例１のステップ１−１１と同様の方法により、ステップ２−７より得られた、(S)-トランス-[2-[4-(3-ベンゾイルアミノ-3-ベンジルカルバモイルプロポキシ)フェニル] シクロプロピルtert-ブチルカルバメート（２２）（83 mg）より、化合物（NCL-2）（18 mg, 収率25%）を淡黄色固体として得た。NCL-2の融点、1H NMR、13C NMR、MS (FAB)のデータを示す。
融点：141.2-144.5 ℃
1H NMR (CD3OD, 500 MHz, δ; ppm)
δ：7.83 (2H, d, J = 7.0 Hz), 7.54 (1H, t, J = 7.0 Hz), 7.45 (2H, t, J = 7.0 Hz), 7.27-7.19 (6H, m), 7.05 (2H, dd, J = 9.0 Hz), 6.84 (2H, d, J = 9.0 Hz), 4.40 (2H, d, J = 8.0 Hz), 4.06 (2H, t, J = 8.0 Hz), 2.74-2.72 (1H, m), 2.42-2.36 (1H, m), 2.29-2.21 (1H, m), 1.34-1.29 (1H, m), 1.26-1.22 (1H, m)
13C NMR (CD3OD, 500 MHz, δ; ppm)
δ：173.91, 170.32, 159.23, 139.80, 135.21, 132.93, 131.84, 129.55, 129.52, 128.62, 128.52, 128.48, 128.17, 115.89, 65.95, 53.13, 44.16, 32.70, 31.73, 21.96, 13.41
MS (FAB) m/z = 444 (M+);
Anal. Calcd For C27H30ClN3O3 2H2O: C, 62.84; H, 6.64; N, 8.14.
Found: C, 62.47; H, 6.08; N, 8.21.
(Step 2-8)
Synthesis of (S) -trans-N-3- [4- (2-aminocyclopropyl) phenoxy] -1-benzylcarbamoylpropylbenzamide hydrochloride (NCL-2) Method similar to step 1-11 of Example 1 (S) -trans- [2- [4- (3-benzoylamino-3-benzylcarbamoylpropoxy) phenyl] cyclopropyl tert-butylcarbamate (22) (83 mg) obtained from step 2-7 From the above, compound (NCL-2) (18 mg, yield 25%) was obtained as a pale yellow solid. The melting point, 1 H NMR, 13 C NMR and MS (FAB) data of NCL-2 are shown.
Melting point: 141.2-144.5 ℃
1 H NMR (CD 3 OD, 500 MHz, δ; ppm)
δ: 7.83 (2H, d, J = 7.0 Hz), 7.54 (1H, t, J = 7.0 Hz), 7.45 (2H, t, J = 7.0 Hz), 7.27-7.19 (6H, m), 7.05 (2H , dd, J = 9.0 Hz), 6.84 (2H, d, J = 9.0 Hz), 4.40 (2H, d, J = 8.0 Hz), 4.06 (2H, t, J = 8.0 Hz), 2.74-2.72 (1H , m), 2.42-2.36 (1H, m), 2.29-2.21 (1H, m), 1.34-1.29 (1H, m), 1.26-1.22 (1H, m)
13 C NMR (CD 3 OD, 500 MHz, δ; ppm)
δ: 173.91, 170.32, 159.23, 139.80, 135.21, 132.93, 131.84, 129.55, 129.52, 128.62, 128.52, 128.48, 128.17, 115.89, 65.95, 53.13, 44.16, 32.70, 31.73, 21.96, 13.41
MS (FAB) m / z = 444 (M + );
Anal.Calcd For C27H 30 ClN 3 O 3 2H 2 O: C, 62.84; H, 6.64; N, 8.14.
Found: C, 62.47; H, 6.08; N, 8.21.
（実施例３）
実施例３では、トランス-4-[3-(2-アミノシクロプロピル)フェノキシ]-N-ベンジルブチルアミド塩酸塩（NCL-3）を合成した（下記化学式参照）。詳細は以下のとおりである。
(Example 3)
In Example 3, trans-4- [3- (2-aminocyclopropyl) phenoxy] -N-benzylbutyramide hydrochloride (NCL-3) was synthesized (see the following chemical formula). Details are as follows.
<カップリング前駆体>
実施例３では、NCL-3を合成するための前駆体として、実施例１（ステップ１−１〜１−６）で合成した、化合物（９）を用いた。また、NCL-3を合成するためのもう一方の前駆体として、化合物（２４）を次の反応によって合成した。
In Example 3, the compound (9) synthesized in Example 1 (Steps 1-1 to 1-6) was used as a precursor for synthesizing NCL-3. Moreover, the compound (24) was synthesized by the following reaction as another precursor for synthesizing NCL-3.
（ステップ３−１）
N-ベンジル-4-クロロブチルアミド（２４）の合成
ベンジルアミン（1.1 mL）とトリエチルアミン（4.5 mL）をジクロロメタン（26 mL）に溶解し、氷冷下、4-クロロブチリルクロリド（２３）（1.5 mL）のジクロロメタン（15 mL）溶液を滴下した。0°Cで30分攪拌後、反応液を水（100 mL）に注ぎ、酢酸エチル（200 mL）で抽出した。有機層を飽和食塩水（100 mL）で洗浄して、無水硫酸ナトリウムで乾燥し、ろ過後、残渣をシリカゲルフラッシュカラムクロマトグラフィー（展開溶媒 n-ヘキサン：酢酸エチル＝ 3 ：1）で精製し、化合物（２４）（1.26 g, 収率59%）を淡黄色油状物として得た。化合物（２４）の1H NMRのデータを以下に示す。
1H NMR (CDCl3, 500 MHz, δ; ppm)
δ：7.36-7.26 (5H, m), 5.84 (1H, broad s), 4.44 (2H, d, J = 5.8 Hz), 3.62 (2H, t, J = 6.0 Hz), 2.40 (2H, t, J = 7.0 Hz), 2.14 (2H, quintet, J = 7.0 Hz).
(Step 3-1)
Synthesis of N-benzyl-4-chlorobutyramide (24) Benzylamine (1.1 mL) and triethylamine (4.5 mL) were dissolved in dichloromethane (26 mL), and 4-chlorobutyryl chloride (23) (23) ( 1.5 mL) in dichloromethane (15 mL) was added dropwise. After stirring at 0 ° C for 30 minutes, the reaction mixture was poured into water (100 mL) and extracted with ethyl acetate (200 mL). The organic layer was washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and the residue was purified by silica gel flash column chromatography (developing solvent n-hexane: ethyl acetate = 3: 1) Compound (24) (1.26 g, yield 59%) was obtained as a pale yellow oil. The 1 H NMR data of the compound (24) is shown below.
1 H NMR (CDCl 3 , 500 MHz, δ; ppm)
δ: 7.36-7.26 (5H, m), 5.84 (1H, broad s), 4.44 (2H, d, J = 5.8 Hz), 3.62 (2H, t, J = 6.0 Hz), 2.40 (2H, t, J = 7.0 Hz), 2.14 (2H, quintet, J = 7.0 Hz).
NCL-3を合成するための他方の前駆体として、実施例１のステップ１−１〜ステップ１−６で合成した化合物（９）を用いた。 As the other precursor for synthesizing NCL-3, the compound (9) synthesized in Step 1-1 to Step 1-6 of Example 1 was used.
<カップリング反応>
上記のようにして合成した化合物（２４）と化合物（９）とを光延反応によってカップリングさせて実施例３の化合物NCL-3を得た。
Compound (24) synthesized as described above and compound (9) were coupled by Mitsunobu reaction to obtain compound NCL-3 of Example 3.
以下に合成法の詳細を示す。
（ステップ３−２）
トランス-2-[3-(3-ベンジルカルバモイルプロポキシ)フェニル]シクロプロピル tert-ブチルカルバメート（２５）の合成
実施例1のステップ１−６で得られたトランス-2-(3-ヒドロキシフェニル)シクロプロピルtert-ブチルカルバメート（９）（400 mg）をアセトニトリル（7.5 mL）に溶解させ、炭酸セシウム（1.54 g）、ヨウ化ナトリウム（70 mg）を加えて、室温で15分間攪拌した。反応液にステップ３−１で得られたN-ベンジル-4-クロロブチルアミド（２５）（900 mg）のアセトニトリル（10 mL）溶液を添加し、9時間加熱還流した。反応液をろ過し、不溶物を取り除き、ろ液を濃縮後、残渣をシリカゲルフラッシュカラムクロマトグラフィー（展開溶媒 n-ヘキサン：酢酸エチル＝ 1：1）で精製し、化合物（２４）（140 mg, 収率21%）を淡黄色油状物として得た。化合物（２５）の1H NMRのデータを以下に示す。
1H NMR (CDCl3, 500 MHz, δ; ppm)
δ：7.30-7.24 (5H, m), 7.15 (1H, t, J = 7.0 Hz), 6.71 (1H, d, J = 7.0 Hz), 6.66 (1H, d, J = 7.0 Hz), 6.62 (1H, s), 5.85 (1H, broad s), 4.84 (1H, broad s), 4.44 (2H, d, J = 5.8 Hz), 3.99 (2H, t, J = 5.8 Hz), 2.67-2.75 (1H, m), 2.43 (2H, t, J = 7.2 Hz), 2.16-2.13 (2H, m), 2.01-1.96 (1H, m), 1.45 (9H, s), 1.12-1.15 (2H, m).
Details of the synthesis method are shown below.
(Step 3-2)
Synthesis of trans-2- [3- (3-benzylcarbamoylpropoxy) phenyl] cyclopropyl tert-butylcarbamate (25) trans-2- (3-hydroxyphenyl) cyclo obtained in Step 1-6 of Example 1 Propyl tert-butyl carbamate (9) (400 mg) was dissolved in acetonitrile (7.5 mL), cesium carbonate (1.54 g) and sodium iodide (70 mg) were added, and the mixture was stirred at room temperature for 15 minutes. To the reaction solution, a solution of N-benzyl-4-chlorobutyramide (25) (900 mg) obtained in Step 3-1 in acetonitrile (10 mL) was added, and the mixture was heated to reflux for 9 hours. The reaction mixture was filtered to remove insolubles, the filtrate was concentrated, and the residue was purified by silica gel flash column chromatography (developing solvent n-hexane: ethyl acetate = 1: 1) to give compound (24) (140 mg, 140 mg, (21% yield) was obtained as a pale yellow oil. The 1 H NMR data of the compound (25) is shown below.
1 H NMR (CDCl 3 , 500 MHz, δ; ppm)
δ: 7.30-7.24 (5H, m), 7.15 (1H, t, J = 7.0 Hz), 6.71 (1H, d, J = 7.0 Hz), 6.66 (1H, d, J = 7.0 Hz), 6.62 (1H , s), 5.85 (1H, broad s), 4.84 (1H, broad s), 4.44 (2H, d, J = 5.8 Hz), 3.99 (2H, t, J = 5.8 Hz), 2.67-2.75 (1H, m), 2.43 (2H, t, J = 7.2 Hz), 2.16-2.13 (2H, m), 2.01-1.96 (1H, m), 1.45 (9H, s), 1.12-1.15 (2H, m).
（ステップ３−３）
トランス-4-[3-(2-アミノシクロプロピル)フェノキシ]-N-ベンジルブチルアミド塩酸塩（NCL-3）の合成
実施例１のステップ１−１１と同様の方法により、ステップ３−２より得られた、トランス-2-[3-(3-ベンジルカルバモイルプロポキシ)フェニル]シクロプロピル tert-ブチルカルバメート（２５）（140 mg）より、実施例３のフェニルシクロプロピルアミン誘導体である化合物（NCL-3）（82 mg, 収率69%）を白色固体として得た。NCL-3の融点、1H NMR、13C NMR、MS (ELC)のデータを示す。
融点：83.3-86.7 °C
1H NMR (CD3OD, 500 MHz, δ; ppm)
δ：7.26-7.17 (6H, m), 6.77 (1H, dd, J = 7.6, 2.5 Hz), 6.72 (1H, d, J = 7.6 Hz), 6.69 (1H, s), 4.36 (2H, s), 3.98 (2H, t, J = 6.4 Hz), 2.82-2.80 (1H, m), 2.43 (2H, t, J = 7.2 Hz), 2.32-2.30 (1H, m), 2.10-2.06 (2H, m), 1.39-1.35 (1H, m), 1.32-1.28 (1H, m)
13C NMR (CD3OD, 500 MHz, δ; ppm)
δ：175.37, 160.71, 141.30, 140.02, 130.78, 129.53, 128.52, 128.18, 119.68, 113.94 113.81, 68.06, 33.46, 31.96, 26.57, 22.62, 13.84
MS (ELC) m/z: 325 (M+)
Anal. Calcd For C20H25ClN2O2 2/3H2O: C, 64.42; H, 7.12; N, 7.51.
Found: C, 64.44; H, 6.87; N, 7.80.
(Step 3-3)
Synthesis of trans-4- [3- (2-aminocyclopropyl) phenoxy] -N-benzylbutyramide hydrochloride (NCL-3) In the same manner as in Step 1-11 of Example 1, from Step 3-2 From the obtained trans-2- [3- (3-benzylcarbamoylpropoxy) phenyl] cyclopropyl tert-butylcarbamate (25) (140 mg), the compound (NCL- 3) (82 mg, 69% yield) was obtained as a white solid. The melting point, 1 H NMR, 13 C NMR, and MS (ELC) data of NCL-3 are shown.
Melting point: 83.3-86.7 ° C
1 H NMR (CD 3 OD, 500 MHz, δ; ppm)
δ: 7.26-7.17 (6H, m), 6.77 (1H, dd, J = 7.6, 2.5 Hz), 6.72 (1H, d, J = 7.6 Hz), 6.69 (1H, s), 4.36 (2H, s) , 3.98 (2H, t, J = 6.4 Hz), 2.82-2.80 (1H, m), 2.43 (2H, t, J = 7.2 Hz), 2.32-2.30 (1H, m), 2.10-2.06 (2H, m ), 1.39-1.35 (1H, m), 1.32-1.28 (1H, m)
13 C NMR (CD 3 OD, 500 MHz, δ; ppm)
δ: 175.37, 160.71, 141.30, 140.02, 130.78, 129.53, 128.52, 128.18, 119.68, 113.94 113.81, 68.06, 33.46, 31.96, 26.57, 22.62, 13.84
MS (ELC) m / z: 325 (M + )
Anal.Calcd For C 20 H 25 ClN 2 O 2 2 / 3H 2 O: C, 64.42; H, 7.12; N, 7.51.
Found: C, 64.44; H, 6.87; N, 7.80.
（実施例４）
実施例４では、トランス-4-[4-(2-アミノシクロプロピル)フェノキシ]-N-ベンジルブチルアミド塩酸塩（NCL-4）を合成した（下記化学式参照）。詳細は以下のとおりである。
Example 4
In Example 4, trans-4- [4- (2-aminocyclopropyl) phenoxy] -N-benzylbutyramide hydrochloride (NCL-4) was synthesized (see the following chemical formula). Details are as follows.
<カップリング前駆体>
実施例４では、カップリング前駆体は、実施例２におけるステップ２−１〜ステップ２−６で合成した、化合物（２１）を用いた。また、もう一方のカップリング前駆体としては、実施例３におけるステップ３−１で合成した化合物（２４）を用いた。
<Coupling precursor>
In Example 4, the compound (21) synthesized in Step 2-1 to Step 2-6 in Example 2 was used as the coupling precursor. Further, as the other coupling precursor, the compound (24) synthesized in Step 3-1 in Example 3 was used.
<カップリング反応>
上記のようにして合成した化合物（２４）と化合物（２１）とを光延反応によってカップリングさせて実施例４の化合物NCL-4を得た。
<Coupling reaction>
Compound (24) synthesized as described above and compound (21) were coupled by Mitsunobu reaction to obtain compound NCL-4 of Example 4.
以下に合成法の詳細を示す。
（ステップ４−１）
トランス-2-[4-(3-ベンジルカルバモイルプロポキシ)フェニル]シクロプロピル tert-ブチルカルバメート（２６）の合成
実施例３のステップ３−２と同様の方法により、化合物（２１）（500 mg）と化合物（２４）とをカップリングさせ、実施例４のフェニルシクロプロピルアミン誘導体である化合物（２６）（NCL-3）（82 mg, 収率69%）を白色固体として得た。化合物（２６）の1H NMRのデータを以下に示す。
1H NMR (CDCl3, 500 MHz, δ; ppm)
δ：7.32-7.24 (5H, m), 7.05 (2H, d, J = 8.2 Hz), 6.75 (2H, d, J = 8.2 Hz), 5.82 (1H, broad s), 4.84 (1H, broad s), 4.44 (2H, d, J = 5.8 Hz), 3.97 (2H, t, J = 5.8 Hz), 2.60-2.70 (1H, m), 2.42 (2H, t, J = 7.2 Hz), 2.16-2.12 (2H, m), 1.97-2.04(1H, m), 1.46 (9H, s), 1.02-1.15 (2H, m).
Details of the synthesis method are shown below.
(Step 4-1)
Synthesis of trans-2- [4- (3-benzylcarbamoylpropoxy) phenyl] cyclopropyl tert-butylcarbamate (26) In the same manner as in Step 3-2 of Example 3, compound (21) (500 mg) was synthesized. Compound (24) was coupled to obtain Compound (26) (NCL-3) (82 mg, 69% yield), which is a phenylcyclopropylamine derivative of Example 4, as a white solid. The 1 H NMR data of the compound (26) is shown below.
1 H NMR (CDCl 3 , 500 MHz, δ; ppm)
δ: 7.32-7.24 (5H, m), 7.05 (2H, d, J = 8.2 Hz), 6.75 (2H, d, J = 8.2 Hz), 5.82 (1H, broad s), 4.84 (1H, broad s) , 4.44 (2H, d, J = 5.8 Hz), 3.97 (2H, t, J = 5.8 Hz), 2.60-2.70 (1H, m), 2.42 (2H, t, J = 7.2 Hz), 2.16-2.12 ( 2H, m), 1.97-2.04 (1H, m), 1.46 (9H, s), 1.02-1.15 (2H, m).
（ステップ４−２）
トランス-4-[4-(2-アミノシクロプロピル)フェノキシ]-N-ベンジルブチルアミド塩酸塩（NCL-4）の合成
実施例１のステップ１−１１と同様の方法により、ステップ４−１より得られた、化合物（２６）（140 mg）より、実施例４のフェニルシクロプロピルアミン誘導体である化合物（NCL-4）（82 mg, 収率69%）を白色固体として得た。NCL-4の融点、1H NMR、13C NMR、MS (ELC)のデータを示す。
融点：156.3-159.4 °C; 1H NMR (CD3OD, 500 MHz, δ; ppm)
δ：7.27-7.21 (5H, m), 7.06 (2H, d, J = 8.5 Hz), 6.83 (2H, d, J = 8.5 Hz), 4.35 (2H, s), 3.95 (2H, t, J = 6.4 Hz), 2.75-2.74 (1H, m), 2.42 (2H, t, J = 7.2 Hz), 2.30-2.27 (1H, m), 2.09-2.05 (2H, m), 1.35-1.32 (1H, m), 1.27-1.24 (1H, m)
δ：13C NMR (CD3OD, 500 MHz, δ; ppm) 175.34, 159.44, 140.06, 131.58, 129.51, 128.61, 128.53, 128.16, 115.81, 68.17, 44.11, 33.48, 31.73, 26.59, 21.96, 13.40
MS (ELC) m/z: 325 (M+)
Anal. Calcd For C20H25ClN2O2 H2O: C, 63.40; H, 7.18; N, 7.39.
Found: C, 63.50; H, 6.68; N, 7.44.
(Step 4-2)
Synthesis of trans-4- [4- (2-aminocyclopropyl) phenoxy] -N-benzylbutyramide hydrochloride (NCL-4) In the same manner as in Step 1-11 of Example 1, from Step 4-1. From the obtained compound (26) (140 mg), compound (NCL-4) (82 mg, yield 69%) which is a phenylcyclopropylamine derivative of Example 4 was obtained as a white solid. The melting point, 1 H NMR, 13 C NMR, and MS (ELC) data of NCL-4 are shown.
Melting point: 156.3-159.4 ° C; 1 H NMR (CD3OD, 500 MHz, δ; ppm)
δ: 7.27-7.21 (5H, m), 7.06 (2H, d, J = 8.5 Hz), 6.83 (2H, d, J = 8.5 Hz), 4.35 (2H, s), 3.95 (2H, t, J = 6.4 Hz), 2.75-2.74 (1H, m), 2.42 (2H, t, J = 7.2 Hz), 2.30-2.27 (1H, m), 2.09-2.05 (2H, m), 1.35-1.32 (1H, m ), 1.27-1.24 (1H, m)
δ: 13 C NMR (CD 3 OD, 500 MHz, δ; ppm) 175.34, 159.44, 140.06, 131.58, 129.51, 128.61, 128.53, 128.16, 115.81, 68.17, 44.11, 33.48, 31.73, 26.59, 21.96, 13.40
MS (ELC) m / z: 325 (M + )
Anal.Calcd For C 20 H 25 ClN 2 O 2 H 2 O: C, 63.40; H, 7.18; N, 7.39.
Found: C, 63.50; H, 6.68; N, 7.44.
−評 価−
以上のようにして得られた実施例１〜実施例４の化合物（NCL-1〜NCL-4）について、ＬＳＤ１阻害活性試験、モノアミンオキシダーゼ阻害活性試験及びＨＩＶ複製に対する阻害活性試験を行った。
-Evaluation-
The compounds of Examples 1 to 4 (NCL-1 to NCL-4) obtained as described above were subjected to an LSD1 inhibitory activity test, a monoamine oxidase inhibitory activity test, and an inhibitory activity test against HIV replication.
＜ＬＳＤ１阻害試験＞
ＬＳＤ１酵素は以下のようにして調製した。
全長のＬＳＤ１（1-851aa）のN末端にヒスチジン5残基を付加した組換えタンパク質をコードするプラスミドを調製し、このプラスミドで形質転換した組換え大腸菌を用いてＬＳＤ１を発現させた。その後、組換え大腸菌を超音波処理で溶解し、その可溶性画分をHisTrapクロマトグラフィーで精製し、ＬＳＤ１酵素溶液を得た。ＬＳＤ１の酵素活性は、ＬＳＤ１の脱メチル化反応の際に生成する過酸化水素をペルオキシダーゼと試薬によって発色させ、吸光光度法で定量することにより測定した。すなわち、384ウエルマイクロタイタープレート内で、50mM Hepes-NaOH buffer（pH7.5）、0.1mM 4-アミノアンチピリン、1mM 3,5-ジクロロ-2-ヒドロキシベンゼンスルホン酸、20μM histone H3- lysine 4 dimethyl peptide、0.05μM ＬＳＤ１、0.35μM horseradish peroxidaseからなる20μlの溶液を25℃で30分間、経時的に酵素反応を測定した。測定にはSpectra Max M2e(Molecular Devices社)を用いて、生成物の515nmでの吸光度を測定して求めた。また、阻害活性については、ジメチルスルホキシド（dimethyl sulfoxide）添加時の酵素活性を100%とし、フェニルシクロプロピルアミン誘導体の添加濃度を様々に変えて残存活性を測定し、50％の活性を阻害する濃度（IC50）を求めた。
<LSD1 inhibition test>
The LSD1 enzyme was prepared as follows.
A plasmid encoding a recombinant protein in which 5 residues of histidine were added to the N-terminus of full-length LSD1 (1-851aa) was prepared, and LSD1 was expressed using recombinant Escherichia coli transformed with this plasmid. Thereafter, the recombinant E. coli was lysed by sonication, and the soluble fraction was purified by HisTrap chromatography to obtain an LSD1 enzyme solution. The enzymatic activity of LSD1 was measured by coloring the hydrogen peroxide produced during the demethylation reaction of LSD1 with peroxidase and a reagent and quantifying it by absorptiometry. That is, in a 384-well microtiter plate, 50 mM Hepes-NaOH buffer (pH 7.5), 0.1 mM 4-aminoantipyrine, 1 mM 3,5-dichloro-2-hydroxybenzenesulfonic acid, 20 μM histone H3-lysine 4 dimethyl peptide 20 μl of a solution consisting of 0.05 μM LSD1, 0.35 μM horseradish peroxidase was assayed for 30 minutes at 25 ° C. over time. The measurement was performed by measuring the absorbance of the product at 515 nm using Spectra Max M2e (Molecular Devices). As for inhibitory activity, the enzyme activity when dimethyl sulfoxide was added was taken as 100%, the residual activity was measured by varying the addition concentration of phenylcyclopropylamine derivatives, and the concentration that inhibited 50% of the activity. (IC 50 ) was determined.
ＬＳＤ１阻害試験の結果を図１に示す。この図に示すように、実施例１〜４の化合物（NCL-1〜４）は、いずれもtranylcypromineより高いＬＳＤ１阻害活性を示した。また、実施例１〜４のの化合物（NCL-1〜４）中でも、アミノ酸構造を有する実施例１のNCL-1及び実施例２のNCL-2では、極めて高いＬＳＤ１阻害活性を示した。 The results of the LSD1 inhibition test are shown in FIG. As shown in this figure, the compounds of Examples 1 to 4 (NCL-1 to 4) all showed higher LSD1 inhibitory activity than tranylcypromine. In addition, among the compounds of Examples 1 to 4 (NCL-1 to 4), NCL-1 of Example 1 having an amino acid structure and NCL-2 of Example 2 showed extremely high LSD1 inhibitory activity.
＜モノアミンオキシダーゼ阻害活性測定試験＞
モノアミンオキシダーゼA（MAO-A）及びモノアミンオキシダーゼB（MAO-B）阻害活性の測定を、Promega社のMAO-GloアッセイキットとSigma-Aldrich社から購入したMAO-AおよびMAO-Bを用いて以下のように行った。
12.5 μLの4倍MAO基質（最終濃度40 μM）、12.5 μLの4倍阻害剤溶液（最終濃度0.1〜1000 μM）、25 μLのMAO-A（最終濃度 9 unit/mL）あるいは25 μLのMAO-B（最終濃度2.3 unit/mL）を混合し、室温で1時間反応させた。この反応液に50 μLのルシフェリン検出試薬を添加し、室温で20分反応させ、蛍光プレートリーダーも用いて蛍光強度（蛍光測定波長：562 nm）を測定し、IC50値（酵素を50%阻害する阻害剤濃度）を求めた。
<Monoamine oxidase inhibitory activity measurement test>
Measurement of monoamine oxidase A (MAO-A) and monoamine oxidase B (MAO-B) inhibitory activity using MAO-Glo assay kit from Promega and MAO-A and MAO-B purchased from Sigma-Aldrich Went like that.
12.5 μL 4X MAO substrate (final concentration 40 μM), 12.5 μL 4X inhibitor solution (final concentration 0.1-1000 μM), 25 μL MAO-A (final concentration 9 unit / mL) or 25 μL MAO -B (final concentration 2.3 unit / mL) was mixed and reacted at room temperature for 1 hour. Add 50 μL of luciferin detection reagent to this reaction, react at room temperature for 20 minutes, measure fluorescence intensity (fluorescence measurement wavelength: 562 nm) using a fluorescence plate reader, and determine IC 50 value (enzyme inhibition by 50% Inhibitor concentration).
MAO-Aに対する阻害活性の結果を図２に示す。この図に示すように、実施例１〜４の化合物（NCL-1〜４）は、いずれもtranylcypromineやpargylineより低いMAO A阻害活性を示した。その中でも、アミノ酸構造を有する実施例１のNCL-1及び実施例２のNCL-2では、特に阻害活性が低かった。
また、MAO-Bに対する阻害活性についても、図３に示すように、実施例１〜４の化合物（NCL-1〜４）は、いずれもtranylcypromineやpargyline（下記化学式参照）より低いMAO-B阻害活性を示し、その中でも、実施例１のNCL-1及び実施例２のNCL-2では、特に阻害活性が低かった。
The results of the inhibitory activity against MAO-A are shown in FIG. As shown in this figure, the compounds of Examples 1 to 4 (NCL-1 to 4) all showed MAO A inhibitory activity lower than that of tranylcypromine or pargyline. Among these, NCL-1 of Example 1 and NCL-2 of Example 2 having an amino acid structure had particularly low inhibitory activity.
Moreover, also about the inhibitory activity with respect to MAO-B, as shown in FIG. 3, as for the compound (NCL-1-4) of Examples 1-4, all are MAO-B inhibition lower than tranylcypromine and pargyline (refer following Chemical formula). In particular, NCL-1 of Example 1 and NCL-2 of Example 2 showed particularly low inhibitory activity.
＜ＨＩＶ複製に対する阻害活性試験＞
上記ＬＳＤ１阻害試験において阻害活性を示したNCL-2及びpargylineについて、ＨＩＶ複製に対する阻害活性試験を行った。詳細を以下に示す。
<Inhibitory activity test against HIV replication>
NCL-2 and pargyline that showed inhibitory activity in the LSD1 inhibition test were tested for inhibitory activity against HIV replication. Details are shown below.
（ＨＩＶ複製に対するNCL-2の阻害活性）
HIV潜伏感染細胞Ach2にNCL-2を0-10μM／Ｌとなるように加え、24時間後の細胞培養上清中のHIVのコア蛋白p24の濃度をp24antigen assay法により測定し、HIV複製の指標とした。
また、上記試験においてNCL-2とともにTNF (1μg/ml)を加えて潜伏感染細胞を刺激し、24時間後の細胞培養上清中のHIVのコア蛋白p24を測定し、HIV複製の指標とした。
(Inhibitory activity of NCL-2 against HIV replication)
NCL-2 was added to HIV latently infected cells Ach2 at 0-10 μM / L, and the concentration of HIV core protein p24 in the cell culture supernatant 24 hours later was measured by p24antigen assay, indicating HIV replication. It was.
In the above test, TNF (1 μg / ml) was added together with NCL-2 to stimulate latently infected cells, and the HIV core protein p24 in the cell culture supernatant after 24 hours was measured and used as an index of HIV replication. .
その結果、TNFを加えなかった場合には、図４（ａ）左側のグラフに示すように、NCL-2の添加量が0-10μM／Ｌの範囲において、HIVのコア蛋白p24はいずれも検出感度ぎりぎりで増減は認められず、ＨＩＶ複製が阻害されることはなかった。
これに対して、TNFを加えた場合には、図４（ａ）右側のグラフに示すように、NCL-2の濃度の増加とともにHIVのコア蛋白p24が減少し（ｐ値＝0.055)、ＨＩＶ複製が阻害されることが分かった。
また、上記ＨＩＶ複製に対する阻害活性試験における細胞毒性効果をWST-1 assay法によって調べた結果、図４（ｂ）に示すように、TNFの添加の有無にかかわらず、NCL-2の添加量が0-10μM／Ｌの範囲において、細胞毒性は20％を超えることはなかった。このことから、ＨＩＶ複製の阻害が、細胞毒性によるものではないことが分かった。
As a result, when TNF was not added, as shown in the graph on the left side of FIG. 4 (a), the HIV core protein p24 was detected in the range where the amount of NCL-2 added was 0-10 μM / L. No increase or decrease was observed at the limit of sensitivity, and HIV replication was not inhibited.
On the other hand, when TNF was added, as shown in the graph on the right side of FIG. 4 (a), the HIV core protein p24 decreased with increasing NCL-2 concentration (p value = 0.055). It was found that replication was inhibited.
Further, as a result of examining the cytotoxic effect in the inhibitory activity test against HIV replication by the WST-1 assay, as shown in FIG. 4 (b), the amount of NCL-2 added was increased regardless of whether TNF was added or not. In the range of 0-10 μM / L, cytotoxicity did not exceed 20%. This indicates that inhibition of HIV replication is not due to cytotoxicity.
（ＨＩＶ複製に対するPargylineの阻害活性）
HIV潜伏感染細胞Ach2にPargylineを0-3 mM／Ｌとなるように加え、24時間後の細胞培養上清中のHIVのコア蛋白p24の濃度をp24antigen assay法により測定し、HIV複製の指標とした。
また、上記試験においてPargylineとともにTNF (1μg/ml)を加えて潜伏感染細胞を刺激し、24時間後の細胞培養上清中のHIVのコア蛋白p24を測定し、HIV複製の指標とした。
(Inhibitory activity of Pargyline against HIV replication)
Pargyline was added to HIV latently infected cells Ach2 at 0-3 mM / L, and the concentration of HIV core protein p24 in the cell culture supernatant after 24 hours was measured by p24antigen assay. did.
In the above test, TNF (1 μg / ml) was added together with Pargyline to stimulate latently infected cells, and the HIV core protein p24 in the cell culture supernatant after 24 hours was measured and used as an index of HIV replication.
その結果、Pargylineを加えなかった場合には、図５（ａ）左側のグラフに示すように、Pargylineの添加量が0-3 mM／Ｌの範囲において、HIVのコア蛋白p24はいずれも検出感度ぎりぎりで増減は認められず、ＨＩＶ複製が阻害されることはなかった。
これに対して、Pargylineを加えた場合には、図５（ａ）右側のグラフに示すように、Pargylineの濃度の増加とともにHIVのコア蛋白p24が減少し（ｐ値＝0.139)、ＨＩＶ複製が阻害されることが分かった。
また、上記ＨＩＶ複製に対する阻害活性試験における細胞毒性効果をWST-1 assay法によって調べた結果、図５（ｂ）に示すように、TNFの添加の有無にかかわらず、Pargylineの添加量が0-3 mM／Ｌの範囲において、細胞毒性は20％を超えることはなかった。このことから、ＨＩＶ複製の阻害が、細胞毒性によるものではないことが分かった。
As a result, when Pargyline was not added, as shown in the graph on the left side of FIG. 5 (a), the detection sensitivity of all HIV core protein p24 was within the range of Pargyline addition amount of 0-3 mM / L. There was no increase or decrease at the last minute, and HIV replication was not inhibited.
On the other hand, when Pargyline was added, as shown in the graph on the right side of FIG. 5 (a), the HIV core protein p24 decreased with increasing Pargyline concentration (p value = 0.139), and HIV replication occurred. It was found to be inhibited.
Further, as a result of examining the cytotoxic effect in the inhibitory activity test against HIV replication by the WST-1 assay, as shown in FIG. 5 (b), the amount of Pargyline added was 0--regardless of whether TNF was added or not. In the range of 3 mM / L, cytotoxicity did not exceed 20%. This indicates that inhibition of HIV replication is not due to cytotoxicity.
＜ＨＩＶ−ＬＴＲ近傍のメチル化状態の解析＞
本発明者らは、ＨＩＶ−ＬＴＲ近傍のメチル化状態について、以下に示す研究発表を既に行っている（非特許文献４）。
すなわち、HIV (AIDSウイルス)の転写を調節するLTRと呼ばれるプロウイルスDNAの「転写スイッチ」領域のメチル化状態とヒストンメチル化酵素G9aおよびRNA合成酵素II型(RNAPII)の結合状態を、ヒストンメチル化酵素G9a阻害剤BIX01294を加えた場合と加えない場合とについて、クロマチン免疫沈降(ChIP)アッセイで調べた。
<Analysis of methylation state near HIV-LTR>
The present inventors have already made the following research presentation on the methylation state in the vicinity of HIV-LTR (Non-patent Document 4).
That is, the methylation state of the “transcription switch” region of proviral DNA called LTR that regulates HIV (AIDS virus) transcription and the binding state of histone methyltransferase G9a and RNA synthase type II (RNAPII) The chromatin immunoprecipitation (ChIP) assay was examined with and without the addition of the oxidase G9a inhibitor BIX01294.
その結果、図６の左側のグラフが示すように、ヒストンメチル化酵素G9a阻害剤であるBIX阻害剤を加えない場合には、G9aはHIVプロウイルスLTRと結合していて、その部位のH3K9のジメチル化が十分に起こっている。これに対して、ヒストンメチル化酵素G9a阻害剤であるBIXを加えるとG9aは離脱し、しかもH3K9のジメチル化も著しく低下している。このことはG9aが離脱しただけでは説明ができず、ある種のヒストン脱メチル化酵素がG9aの代わりに新たに結合したことを示唆している。ただし、この事実のみからは、ＬＳＤ１がＨＩＶ複製と関係しているか否かについては不明である。 As a result, as shown in the graph on the left side of FIG. 6, when no BIX inhibitor, which is a histone methylase G9a inhibitor, was added, G9a was bound to the HIV provirus LTR and H3K9 Dimethylation has occurred sufficiently. On the other hand, when BIX, which is a histone methylase G9a inhibitor, was added, G9a was released, and H3K9 dimethylation was significantly reduced. This cannot be explained only by the withdrawal of G9a, suggesting that some histone demethylase was newly bound in place of G9a. However, from this fact alone, it is unclear whether LSD1 is associated with HIV replication.
しかしながら、前述したように、NCL-2及びpargylineがＨＩＶ複製の阻害作用を示すこと、及び、NCL-2やpargylineがＬＳＤ１阻害剤作用を示すことを考え合わせれば、ＬＳＤ１阻害剤作用を有する化合物はＨＩＶ複製の阻害作用を示すことを強く示唆しており、今回始めて明らかにされたことである。 However, as described above, considering that NCL-2 and pargyline show an inhibitory action on HIV replication and that NCL-2 and pargyline show an LSD1 inhibitory action, compounds having an LSD1 inhibitory action are This strongly suggests that it has an inhibitory action on HIV replication, and this is the first time that it has been revealed.
また、図６の右側のグラフは、潜伏感染HIVからの転写を促進する作用のあるTNFαを加えていない対照（左）と、TNFαを加えた場合（右）の、ChIPアッセイの結果を示す。このグラフから、TNFαを加えることによってもG9aの離脱は引き起こされるが、BIX01294を加えた場合の効果よりも小さいことが分かる。 Moreover, the graph on the right side of FIG. 6 shows the results of the ChIP assay in the case where TNFα having an effect of promoting transcription from latently infected HIV is not added (left) and in the case where TNFα is added (right). From this graph, it can be seen that G9a detachment is also caused by adding TNFα, but the effect is smaller than when BIX01294 is added.
さらに、図７のグラフは、HIVが潜伏感染していて細胞からはほとんどウイルスの産生が起こっていないAch-2細胞（HIV感染ヒトT細胞株）にG9a阻害剤BIX01294を加えた場合の、細胞培養上清中のHIVのコア蛋白p24の濃度をp24antigen assay法により測定した結果である。この図から、G9a阻害剤BIX01294の添加量が多いほど、HIVのコア蛋白p24の量も多くなり、ＨＩＶ複製が活性化されることが分かる。ただし、このことのみからは、ＬＳＤ１とＨＩＶ複製との関係についての知見を得ることはできない。 Furthermore, the graph of FIG. 7 shows that cells obtained when the G9a inhibitor BIX01294 was added to Ach-2 cells (HIV-infected human T cell line) in which HIV was latently infected and almost no virus was produced from the cells. It is the result of having measured the density | concentration of the core protein p24 of HIV in a culture supernatant by p24antigen assay method. From this figure, it can be seen that the greater the amount of G9a inhibitor BIX01294 added, the greater the amount of HIV core protein p24, which activates HIV replication. However, from this alone, knowledge about the relationship between LSD1 and HIV replication cannot be obtained.
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.
本発明のフェニルシクロプロピルアミン誘導体及びＬＳＤ１阻害剤は、ＬＳＤ１の機能を調べるための生物学的ツールとして用いたり、抗がん剤として用いたりすることができると期待される。 It is expected that the phenylcyclopropylamine derivative and LSD1 inhibitor of the present invention can be used as a biological tool for examining the function of LSD1 or as an anticancer agent.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2014058071A1 (en) * | 2012-10-12 | 2014-04-17 | 武田薬品工業株式会社 | Cyclopropanamine compound and use thereof |
| US9487511B2 (en) | 2014-04-11 | 2016-11-08 | Takeda Pharmaceutical Company Limited | Cyclopropanamine compound and use thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2014058071A1 (en) * | 2012-10-12 | 2014-04-17 | 武田薬品工業株式会社 | Cyclopropanamine compound and use thereof |
| JPWO2014058071A1 (en) * | 2012-10-12 | 2016-09-05 | 武田薬品工業株式会社 | Cyclopropanamine compounds and uses thereof |
| US9751885B2 (en) | 2012-10-12 | 2017-09-05 | Takeda Pharmaceutical Company Limited | Cyclopropanamine compound and use thereof |
| US9487511B2 (en) | 2014-04-11 | 2016-11-08 | Takeda Pharmaceutical Company Limited | Cyclopropanamine compound and use thereof |
| US9714241B2 (en) | 2014-04-11 | 2017-07-25 | Takeda Pharmaceutical Company Limited | Cyclopropanamine compound and use thereof |
| US9718814B2 (en) | 2014-04-11 | 2017-08-01 | Takeda Pharmaceutical Company Limited | Cyclopropanamine compound and use thereof |
| US9920047B2 (en) | 2014-04-11 | 2018-03-20 | Takeda Pharmaceutical Company Limited | Cyclopropanamine compound and use thereof |
| US10053456B2 (en) | 2014-04-11 | 2018-08-21 | Takeda Pharmaceutical Company Limited | Cyclopropanamine compound and use thereof |
| US10414761B2 (en) | 2014-04-11 | 2019-09-17 | Takeda Pharmaceutical Company Limited | Cyclopropanamine compound and use thereof |
| US10968213B2 (en) | 2014-04-11 | 2021-04-06 | Takeda Pharmaceutical Company Limited | Cyclopropanamine compound and use thereof |
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