JP2015020966A

JP2015020966A - Akr1c3 inhibitor

Info

Publication number: JP2015020966A
Application number: JP2013149089A
Authority: JP
Inventors: 智史遠藤; Tomohito Endo; 俊之松永; Toshiyuki Matsunaga; 尚樹豊岡; Naoki Toyooka
Original assignee: Gifu City
Current assignee: Gifu City
Priority date: 2013-07-18
Filing date: 2013-07-18
Publication date: 2015-02-02

Abstract

PROBLEM TO BE SOLVED: To provide a AKR1C3 inhibitor having both strong inhibition effect and selectivity.SOLUTION: There is provided a AKR1C3 specific inhibitor as a baccharin derivative.

Description

本発明はAKR1C3（Aldo-Keto Reductase Family 1 Member C3）阻害剤及びその用途に関する。 The present invention relates to an AKR1C3 (Aldo-Keto Reductase Family 1 Member C3) inhibitor and use thereof.

最近、欧米・アジア諸国で化学療法済治療の転移性去勢抵抗性前立腺がん(CRPC)に対する薬剤として、アンドロゲン合成に関与するCYP17の選択的阻害剤アビラテロンが承認され、使用されている。しかし、本薬剤はグルココルチコイド合成も同時に阻害することから、グルココルチコイドの同時処方が必要となる。AKR1C3はアンドロゲン代謝におけるCYP17の下流に位置する。従って、AKR1C3阻害剤によればグルココルチコイドの合成を阻害することなしにアンドロゲン合成を阻害できる。また、AKR1C3はアンドロゲン合成のみならず、細胞増殖性のプロスタノイドやイソプレノイドの合成に関与することから、AKR1C3の阻害はホルモン依存性、非依存性に関わらず前立腺がん治療に有効と考えられる。AKR1C3には高次構造が類似するが、高いアンドロゲン作用を有する5α-ジヒドロアンドロステロンの不活性化に関わるAKR1C1とAKR1C2が存在する。したがって、AKR1C1とAKR1C2を阻害しないAKR1C3阻害剤の開発が望まれるが、未だ選択性と強力な阻害効果を兼ね備えたAKR1C3阻害剤に関する報告はない。 Recently, abiraterone, a selective inhibitor of CYP17 involved in androgen synthesis, has been approved and used as a drug for chemotherapy-treated metastatic castration-resistant prostate cancer (CRPC) in Western countries and Asian countries. However, since this drug also inhibits glucocorticoid synthesis at the same time, glucocorticoid co-prescription is required. AKR1C3 is located downstream of CYP17 in androgen metabolism. Therefore, according to the AKR1C3 inhibitor, androgen synthesis can be inhibited without inhibiting glucocorticoid synthesis. In addition, AKR1C3 is involved not only in androgen synthesis, but also in cell proliferative prostanoid and isoprenoid synthesis, so inhibition of AKR1C3 is considered to be effective in treating prostate cancer regardless of hormone dependence or independence. AKR1C3 is similar in conformation, but there are AKR1C1 and AKR1C2 involved in inactivation of 5α-dihydroandrosterone, which has a high androgenic action. Therefore, development of an AKR1C3 inhibitor that does not inhibit AKR1C1 and AKR1C2 is desired, but there is no report on an AKR1C3 inhibitor that has both selectivity and a strong inhibitory effect.

Endo S. et al. J. Nat. Prod. 2012, 75, 716-721.Endo S. et al. J. Nat. Prod. 2012, 75, 716-721.

AKR1C3に対する高い阻害効果に加えて、その他の構造類縁酵素(AKR1C1、AKR1C2)に対する高い選択性を兼ね備えたAKR1C3阻害剤は存在しない。そこで、本発明の課題は、強力な阻害効果と選択性を兼ね備えたAKR1C3阻害剤を提供することにある。 In addition to the high inhibitory effect on AKR1C3, there is no AKR1C3 inhibitor that combines high selectivity for other structurally related enzymes (AKR1C1, AKR1C2). Therefore, an object of the present invention is to provide an AKR1C3 inhibitor having a strong inhibitory effect and selectivity.

本発明者らの研究グループは、ブラジル産のプロポリスの成分に注目して研究し、バッカリンに選択的なAKR1C3阻害活性があることを報告した（非特許文献１）。本発明者らは、臨床応用を見据え、バッカリンよりも効果が高く且つ選択性の高いAKR1C3の創出を目指した。具体的には、バッカリンをリード化合物として種々の誘導体を合成し、特性を詳細に調べた。その結果、高い阻害効果と選択性を兼ね備えた新規化合物を見出すことに成功するとともに、阻害効果と選択性に重要な構造に関する知見が得られた。以下の発明は当該成果に基づく。
［１］以下の化学式１で表されるAKR1C3阻害剤：

但し、式中のR1は水酸基、アミノ基又は塩素原子であり、R2は以下の化学式２又は３で表され、

但し、式中のR3、R4、R5、R6、R7は、それぞれ独立して、水素原子、ヒドロキシ基、ハロゲン原子、又は置換基を有していてもよい炭化水素基である。
［２］以下の化学式４で表される、［１］に記載のAKR1C3阻害剤：

［３］［１］又は［２］に記載のAKR1C3阻害剤又はその薬理学的に許容可能な塩を有効成分として含有する、抗がん薬。
［４］前立腺がん、乳がん、肝細胞がん、非小細胞肺がん又は白血病の治療又は予防に使用される、［３］に記載の抗がん薬。
［５］［３］又は［４］に記載の抗がん薬を含有する食品組成物。
［６］がん患者に対して、［３］又は［４］に記載の抗がん薬を治療上有効量投与するステップを含む、がんの治療又は予防法。 The research group of the present inventors paid attention to the components of Brazilian propolis and reported that baccaline has selective AKR1C3 inhibitory activity (Non-patent Document 1). In anticipation of clinical application, the present inventors aimed to create AKR1C3 that is more effective and more selective than baccalin. Specifically, various derivatives were synthesized using baccalin as a lead compound, and the characteristics were examined in detail. As a result, the inventors succeeded in finding a novel compound having both a high inhibitory effect and selectivity, and obtained knowledge about structures important for the inhibitory effect and selectivity. The following invention is based on the results.
[1] AKR1C3 inhibitor represented by the following chemical formula 1:

However, R1 in a formula is a hydroxyl group, an amino group, or a chlorine atom, R2 is represented by the following

Chemical formula

2 or 3,

However, R 3, R 4, R 5, R 6 and R 7 in the formula are each independently a hydrogen atom, a hydroxy group, a halogen atom, or a hydrocarbon group which may have a substituent.
[2] The AKR1C3 inhibitor according to [1] represented by the following chemical formula 4:

[3] An anticancer drug comprising the AKR1C3 inhibitor according to [1] or [2] or a pharmacologically acceptable salt thereof as an active ingredient.
[4] The anticancer drug according to [3], which is used for treatment or prevention of prostate cancer, breast cancer, hepatocellular carcinoma, non-small cell lung cancer or leukemia.
[5] A food composition containing the anticancer drug according to [3] or [4].
[6] A method for treating or preventing cancer, comprising a step of administering a therapeutically effective amount of the anticancer drug according to [3] or [4] to a cancer patient.

新規化合物の合成スキーム１。Synthesis scheme 1 of a novel compound. 新規化合物の合成スキーム２。Synthesis scheme 2 of novel compound. バッカリン誘導体によるAKR1C3阻害効果。新規に合成した各種誘導体について比較評価した。下段はバッカリンの構造。AKR1C3 inhibitory effect by baccharin derivatives. Various newly synthesized derivatives were compared and evaluated. The bottom is the structure of baccaroin. バッカリンと化合物14の阻害選択性。括弧内は阻害活性の選択性（他の酵素（AKR1C1、AKR1C2、AKR1C4、AKR1B1又はAKR1B10）のIC₅₀／AKR1C3のIC₅₀）。Inhibition selectivity of baccharin and compound 14. In parentheses inhibitory activity selectivity (other enzymes (AKR1C1, AKR1C2, AKR1C4, AKR1B1 or AKR1B10 IC ₅₀ of IC ₅₀ / AKR1C3 in)). バッカリンと化合物14によるA549細胞内アンドロステロン代謝阻害。各種濃度のバッカリンと化合物14の存在下でA549細胞に50μM アンドロステロンを添加し、24時間後に培地中のアンドロステロン及びその代謝物5α-androstane-3α,17β-diolをLC/MSによって検出した。＊はバッカリンとの間で有意差（p < 0.05）があることを示す。Inhibition of androsterone metabolism in A549 cells by baccaline and compound 14. 50 μM androsterone was added to A549 cells in the presence of various concentrations of baccaline and compound 14, and after 24 hours, androsterone and its metabolite 5α-androstane-3α, 17β-diol were detected by LC / MS. * Indicates that there is a significant difference (p <0.05) from baccarain. 化合物14によるPC3及びU937細胞増殖抑制効果。AKR1C3発現ベクターを導入した細胞(AKR1C3 ; □)とベクターのみを導入した細胞(ベクター; ■)に化合物14とバッカリンを添加し、72時間後の生細胞数をカウントした。＊は、AKR1C3発現ベクターを導入した場合において、化合物（化合物14、バッカリン）の非添加と添加の間で有意差があることを示す。†は、化合物14とバッカリンの間で有意差があることを示す。PC3 and U937 cell growth inhibitory effect of Compound 14. Compound 14 and buccalin were added to cells into which the AKR1C3 expression vector was introduced (AKR1C3; □) and cells into which only the vector was introduced (vector; □), and the number of viable cells after 72 hours was counted. * Indicates that when the AKR1C3 expression vector is introduced, there is a significant difference between non-addition and addition of the compound (Compound 14, Baccalin). † indicates that there is a significant difference between compound 14 and baccalin. AKR1C3が関与する代謝経路。Metabolic pathways involving AKR1C3. AKRスーパーファミリーメンバー（AKR1B10 、AKR1B1、AKR1C1、AKR1C2、AKR1C3、AKR1C4）に対する各化合物の阻害活性。IC₅₀で比較した。括弧内は所定の濃度での阻害％。ndは「検出できず」を表す。Inhibitory activity of each compound against AKR superfamily members (AKR1B10, AKR1B1, AKR1C1, AKR1C2, AKR1C3, AKR1C4). Comparison was made with IC ₅₀ . In parentheses are% inhibition at a given concentration. nd represents “cannot be detected”. 図８の続き。Continuation of FIG.

本発明の第１の局面はAKR1C3阻害剤に関する。AKR1C3は別名17β-ヒドロキシステロイド脱水素酵素5型とも呼ばれ、アンドロゲン合成及びプロスタグランジン（PG）代謝に重要な役割を果たす（図７を参照）。AKR1C3は白血病細胞、ホルモン依存性がんである前立腺がんと乳がんなどで著しく高発現し（Kurkela, R. Li, Y. Patrikainen, L. Pulkka, A. Soronen, P. Torn, S. J. Steroid Biochem. Mol. Biol. 2005, 93, 277-283.; Byrns, M. C. Penning, T. M. Chem.-Biol. Interact. 2009, 178, 221-227.; Penning, T. M. Curr. Opin. Endocrinol. Diabetes Obes. 2010, 17, 233-239.）、また本酵素の発現上昇は肝細胞がんや非小細胞肺がんを含む様々ながんにおいても認められてきた（Guise, C. P. Abbattista, M. R. Singleton, R. S. Holford, S. D. Connolly, J. Dachs, G. U. Fox, S. B. Pollock, R. Harvey, J. Guilford,P. Donate, F. Wilson, W. R. Patterson, A. V. Cancer Res. 2010, 70,1573-1584.）。AKR1C3遺伝子をノックダウンすると前立腺がん細胞の増殖は阻害され、逆に本酵素の人為的過剰発現は前立腺がん細胞および乳がん細胞の増殖を促進する（Downs, T. M. Burton, D. W. Araiza, F. L. Hastings, R. H. Deftos, L. J. Cancer Lett. 2011, 306, 52-59.; Byrns, M. C. Duan, L. Lee, S. H. Blair, I. A. Penning, T. M. J. Steroid Biochem. Mol. Biol. 2010, 118, 177-187.; Dozmorov, M. G. Azzarello, J. T. Wren, J. D. Fung, K. M. Yang, Q. Davis, J. S. Hurst, R. E. Culkin, D. J. Penning, T. M.; Lin, H. K. BMC Cancer 2010, 10, 672.）。 The first aspect of the present invention relates to an AKR1C3 inhibitor. AKR1C3, also called 17β-hydroxysteroid dehydrogenase type 5, plays an important role in androgen synthesis and prostaglandin (PG) metabolism (see FIG. 7). AKR1C3 is highly expressed in leukemia cells, hormone-dependent cancers such as prostate cancer and breast cancer (Kurkela, R. Li, Y. Patrikainen, L. Pulkka, A. Soronen, P. Torn, SJ Steroid Biochem. Mol) Biol. 2005, 93, 277-283 .; Byrns, MC Penning, TM Chem.-Biol. Interact. 2009, 178, 221-227 .; Penning, TM Curr. Opin. Endocrinol. Diabetes Obes. 2010, 17, 233-239.) In addition, increased expression of this enzyme has been observed in various cancers including hepatocellular carcinoma and non-small cell lung cancer (Guise, CP Abbattista, MR Singleton, RS Holford, SD Connolly, J Dachs, GU Fox, SB Pollock, R. Harvey, J. Guilford, P. Donate, F. Wilson, WR Patterson, AV Cancer Res. 2010, 70, 1573-1584.). Knockdown of the AKR1C3 gene inhibits prostate cancer cell growth, whereas artificial overexpression of the enzyme promotes proliferation of prostate and breast cancer cells (Downs, TM Burton, DW Araiza, FL Hastings, RH Deftos, LJ Cancer Lett. 2011, 306, 52-59 .; Byrns, MC Duan, L. Lee, SH Blair, IA Penning, TMJ Steroid Biochem. Mol. Biol. 2010, 118, 177-187 .; Dozmorov, MG Azzarello, JT Wren, JD Fung, KM Yang, Q. Davis, JS Hurst, RE Culkin, DJ Penning, TM; Lin, HK BMC Cancer 2010, 10, 672.).

「AKR1C3阻害剤」とは、AKR1C3の活性を阻害ないし抑制する剤である。本発明のAKR1C3阻害剤は、AKR1C3の阻害を介して、アンドロゲンや細胞増殖性のプロスタノイドやイソプレノイドの合成を抑制するため、がん増殖の抑制に用いることができる。 An “AKR1C3 inhibitor” is an agent that inhibits or suppresses the activity of AKR1C3. The AKR1C3 inhibitor of the present invention suppresses the synthesis of androgens, cell proliferative prostanoids and isoprenoids through the inhibition of AKR1C3, and thus can be used to suppress cancer growth.

ここで、AKR1C3に高次構造が類似するAKR1C1とAKR1C2が存在する。AKR1C1とAKR1C2は高いアンドロゲン作用を有する5α-ジヒドロアンドロステロンの不活性化に関わる。本発明のAKR1C3阻害剤は、AKR1C3に対する特異性が高い。本明細書において「AKR1C3に対する特異性が高い」とは、AKR1C1、AKR1C2及びAKR1C3の中で、選択的にAKR1C3を阻害することを意味する。 Here, there are AKR1C1 and AKR1C2 having higher-order structures similar to AKR1C3. AKR1C1 and AKR1C2 are involved in inactivation of 5α-dihydroandrosterone, which has a high androgenic action. The AKR1C3 inhibitor of the present invention has high specificity for AKR1C3. As used herein, “high specificity for AKR1C3” means that AKR1C3 is selectively inhibited among AKR1C1, AKR1C2, and AKR1C3.

本発明のAKR1C3阻害剤は以下の化学式１で表される。尚、本発明のAKR1C3阻害剤はバッカリンをリード化合物として創出されたものであり、バッカリンに構造が類似する。 The AKR1C3 inhibitor of the present invention is represented by the following chemical formula 1. The AKR1C3 inhibitor of the present invention was created using baccaline as a lead compound and has a structure similar to that of baccaline.

但し、構造解析の結果より、R1の位置において水素結合又は水素結合類似の結合を形成できることが好ましいことから、式中のR1は水酸基、アミノ基又は塩素原子である。一方、R2は以下の化学式２又は３で表される。

However, from the result of structural analysis, it is preferable that a hydrogen bond or a hydrogen bond-like bond can be formed at the position of R1, and therefore R1 in the formula is a hydroxyl group, an amino group, or a chlorine atom. On the other hand, R2 is represented by the following

chemical formula

2 or 3.

但し、式中のR3、R4、R5、R6、R7は、それぞれ独立して、水素原子、ヒドロキシ基、ハロゲン原子、又は置換基を有していてもよい炭化水素基である。「置換基を有していても良い炭化水素基」の「炭化水素基」としては、例えばＣ１〜Ｃ６の直鎖状、分岐状、環状のアルキル基などが挙げられる。本発明において「Ｃ１〜Ｃ６アルキル基」とは、炭素数１乃至６個の直鎖、分枝鎖または環状アルキル基を示し、例えば、メチル基、エチル基、プロピル基、イソプロピル基、ブチル基、イソブチル基、ｔｅｒｔ−ブチル基、ペンチル基、イソペンチル基、２−メチルブチル基、ネオペンチル基、１−エチルプロピル基、ヘキシル基、４−メチルペンチル基、３−メチルペンチル基、２−メチルペンチル基、１−メチルペンチル基、３，３−ジメチルブチル基、２，２−ジメチルブチル基、１，１−ジメチルブチル基、１，２−ジメチルブチル基、１，３−ジメチルブチル基、２，３−ジメチルブチル基、２−エチルブチル基、シクロプロピル基、シクロペンチル基およびシクロヘキシル基を挙げることができる。好ましくはメチル基、エチル基、プロピル基、イソプロピル基、ブチル基、イソブチル基またはｔｅｒｔ−ブチル基であり、より好ましくは、メチル基、エチル基、プロピル基またはイソプロピル基である。尚、アルキル基の炭素数が多すぎると、立体障害による活性の低下のおそれがある。

However, R 3, R 4, R 5, R 6 and R 7 in the formula are each independently a hydrogen atom, a hydroxy group, a halogen atom, or a hydrocarbon group which may have a substituent. Examples of the “hydrocarbon group” of the “hydrocarbon group optionally having substituent (s)” include C1-C6 linear, branched, and cyclic alkyl groups. In the present invention, the “C1 to C6 alkyl group” means a linear, branched or cyclic alkyl group having 1 to 6 carbon atoms, such as a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, Isobutyl group, tert-butyl group, pentyl group, isopentyl group, 2-methylbutyl group, neopentyl group, 1-ethylpropyl group, hexyl group, 4-methylpentyl group, 3-methylpentyl group, 2-methylpentyl group, 1 -Methylpentyl group, 3,3-dimethylbutyl group, 2,2-dimethylbutyl group, 1,1-dimethylbutyl group, 1,2-dimethylbutyl group, 1,3-dimethylbutyl group, 2,3-dimethyl Mention may be made of a butyl group, a 2-ethylbutyl group, a cyclopropyl group, a cyclopentyl group and a cyclohexyl group. A methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group or a tert-butyl group is preferable, and a methyl group, an ethyl group, a propyl group or an isopropyl group is more preferable. If the alkyl group has too many carbon atoms, the activity may be reduced due to steric hindrance.

本願が提供する化合物の具体例を以下に示す。

Specific examples of the compounds provided by the present application are shown below.

上記化合物（化４）の選択性は極めて高く、AKR1C1とAKR1C2と比べてAKR1C3に約3000倍の選択性（IC₅₀で比較）を示す(後述の実施例に示した実験ではIC₅₀ = 28 nM)。 The selectivity of the above compound (Chemical Formula 4) is extremely high, and the AKR1C3 is approximately 3000 times more selective than the AKR1C1 and AKR1C2 (compared by IC ₅₀ ) (IC ₅₀ = 28 nM in the experiment described in the examples below) ).

上記の通り、様々ながんにおいてAKR1C3の発現上昇が認められる。また、がん細胞の増殖・成長へのAKR1C3の関与が報告されている。これらの事実に鑑みれば、AKR1C3阻害剤は、がんの治療や予防に有効である。換言すれば、抗がん薬の有効成分としてAKR1C3阻害剤が有用である。そこで本発明の第２の局面は、本発明のAKR1C3阻害剤又はその薬理学的に許容可能な塩を有効成分として含有する抗がん薬を提供する。 As described above, increased expression of AKR1C3 is observed in various cancers. In addition, the involvement of AKR1C3 in the proliferation and growth of cancer cells has been reported. In view of these facts, AKR1C3 inhibitors are effective in the treatment and prevention of cancer. In other words, an AKR1C3 inhibitor is useful as an active ingredient of an anticancer drug. Therefore, a second aspect of the present invention provides an anticancer drug containing the AKR1C3 inhibitor of the present invention or a pharmacologically acceptable salt thereof as an active ingredient.

本発明において用語「がん」は広義に解釈され、用語「悪性腫瘍」と互換的に使用される。また、病理学的に診断が確定される前の段階、すなわち腫瘍としての良性、悪性のどちらかが確定される前には、良性腫瘍、良性悪性境界病変、悪性腫瘍を総括的に含む場合もあり得る。一般に、がんはその発生の母体となった臓器の名、もしくは発生母組織の名で呼ばれ、主なものを列記すると、舌癌、歯肉癌、咽頭癌、上顎癌、喉頭癌、唾液腺癌、食道癌、胃癌、小腸癌、大腸癌、直腸癌、肝臓癌、胆道癌、胆嚢癌、膵臓癌、肺癌、乳癌、甲状腺癌、副腎癌、脳下垂体腫瘍、松果体腫瘍、子宮癌、卵巣癌、膣癌、膀胱癌、腎臓癌、前立腺癌、尿道癌、網膜芽細胞腫、結膜癌、神経芽腫、神経膠腫、神経膠芽細胞腫、皮膚癌、髄芽種、白血病、悪性リンパ腫、睾丸腫瘍、骨肉腫、横紋筋肉腫、平滑筋肉腫、血管肉腫、脂肪肉腫、軟骨肉腫、ユーイング肉腫などである。そして、さらに発生臓器の部位の特徴によって、上・中・下咽頭癌、上部・中部・下部食道癌、胃噴門癌、胃幽門癌、子宮頚癌、子宮体癌などと細分類されているが、これらが限定的ではなく本発明の「がん」としての記載に含まれる。 In the present invention, the term “cancer” is interpreted broadly and used interchangeably with the term “malignant tumor”. It may also include benign tumors, benign malignant border lesions, and malignant tumors before the pathological diagnosis is confirmed, that is, before any benign or malignant tumor is confirmed. possible. In general, cancer is called the name of the organ that developed it, or the name of the developing mother tissue, and the main ones are listed: tongue cancer, gingival cancer, pharyngeal cancer, maxillary cancer, laryngeal cancer, salivary gland cancer , Esophageal cancer, stomach cancer, small intestine cancer, colon cancer, rectal cancer, liver cancer, biliary tract cancer, gallbladder cancer, pancreatic cancer, lung cancer, breast cancer, thyroid cancer, adrenal cancer, pituitary tumor, pineal tumor, uterine cancer, Ovarian cancer, vaginal cancer, bladder cancer, kidney cancer, prostate cancer, urethral cancer, retinoblastoma, conjunctival cancer, neuroblastoma, glioma, glioblastoma, skin cancer, medulloblastoma, leukemia, malignant Examples include lymphoma, testicular tumor, osteosarcoma, rhabdomyosarcoma, leiomyosarcoma, angiosarcoma, liposarcoma, chondrosarcoma, and Ewing sarcoma. And it is subdivided into upper, middle and lower pharyngeal cancer, upper / middle / lower esophageal cancer, gastric cardia cancer, gastric pyloric cancer, cervical cancer, endometrial cancer, etc. These are not limiting and are included in the description of “cancer” of the present invention.

AKR1C3はアンドロゲン代謝におけるCYP17の下流に位置する。従って、AKR1C3阻害剤によればグルココルチコイドの合成を阻害することなしにアンドロゲン合成を阻害できる。また、AKR1C3はアンドロゲン合成のみならず、細胞増殖性のプロスタノイドやイソプレノイドの合成に関与することからAKR1C3の阻害はホルモン依存性、非依存性に関わらず前立腺がん治療に有効と考えられる。この観点から、本発明の抗がん薬の典型的な標的の一つは、前立腺がんである。 AKR1C3 is located downstream of CYP17 in androgen metabolism. Therefore, according to the AKR1C3 inhibitor, androgen synthesis can be inhibited without inhibiting glucocorticoid synthesis. AKR1C3 is involved not only in androgen synthesis, but also in the synthesis of cell proliferative prostanoids and isoprenoids. Therefore, inhibition of AKR1C3 is considered to be effective in treating prostate cancer regardless of hormone dependency or independence. From this point of view, one of the typical targets of the anticancer drug of the present invention is prostate cancer.

「抗がん薬」とは、標的の疾病ないし病態である、がんに対する治療的又は予防的効果を示す医薬のことをいう。治療的効果には、がんに特徴的な症状又は随伴症状を緩和すること（軽症化）、症状の悪化を阻止ないし遅延すること等が含まれる。後者については、重症化を予防するという点において予防的効果の一つと捉えることができる。このように、治療的効果と予防的効果は一部において重複する概念であり、明確に区別して捉えることは困難であり、またそうすることの実益は少ない。尚、予防的効果の典型的なものは、がんに特徴的な症状の再発発現（発症）を阻止ないし遅延することである。尚、がんに対して何らかの治療的効果又は予防的効果、或いはこの両者を示す限り、抗がん薬に該当する。 “Anticancer drug” refers to a drug that exhibits a therapeutic or prophylactic effect against cancer, which is a target disease or condition. The therapeutic effect includes alleviation of symptoms or associated symptoms characteristic of cancer (mildness), prevention or delay of deterioration of symptoms, and the like. The latter can be regarded as one of the preventive effects in terms of preventing the seriousness. In this way, the therapeutic effect and the preventive effect are partially overlapping concepts, and it is difficult to clearly distinguish them from each other, and there is little benefit in doing so. A typical preventive effect is to prevent or delay the recurrence (onset) of symptoms characteristic of cancer. In addition, as long as it shows some therapeutic effect or preventive effect with respect to cancer, or both, it corresponds to an anticancer drug.

本明細書における「薬理学的に許容される塩」の例として塩酸、リン酸、硫酸、硝酸、ホウ酸等との塩（無機酸塩）や、ギ酸、酢酸、乳酸、フマル酸、マレイン酸、酒石酸、クエン酸、コハク酸、マロン酸等との塩（有機酸塩）を挙げることができる。これらの塩の調製は慣用手段によって行なうことができる。尚、以上の例示は、「薬理学的に許容される塩」が限定解釈されるために用いられるべきではない。即ち、「薬理学的に許容される塩」は、広義に解釈されるべきであり、各種の塩を含む用語である。 Examples of “pharmacologically acceptable salts” in this specification include salts (inorganic acid salts) with hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, boric acid, etc., formic acid, acetic acid, lactic acid, fumaric acid, maleic acid And salts (organic acid salts) with tartaric acid, citric acid, succinic acid, malonic acid and the like. These salts can be prepared by conventional means. In addition, the above illustration should not be used because “pharmacologically acceptable salt” is limitedly interpreted. That is, “pharmacologically acceptable salt” is to be interpreted broadly and is a term that includes various salts.

本発明の抗がん薬の製剤化は常法に従って行うことができる。製剤化する場合には、製剤上許容される他の成分（例えば、担体、賦形剤、崩壊剤、緩衝剤、乳化剤、懸濁剤、無痛化剤、安定剤、保存剤、防腐剤、界面活性剤、滑沢剤、稀釈剤、被覆剤、糖衣剤、矯味矯臭剤、乳化・可溶化・分散剤、pH調製剤、等張剤、可溶化剤、香料、着色剤、溶解補助剤、生理食塩水など）を含有させることができる。製剤化する場合の剤形も特に限定されない。剤形の例は錠剤、散剤、細粒剤、顆粒剤、カプセル剤、シロップ剤、液剤、懸濁剤、乳剤、ゼリー剤、注射剤、外用剤、吸入剤、点鼻剤、点眼剤及び座剤である。本発明の抗がん薬には、期待される治療効果（又は予防効果）を得るために必要な量（即ち治療上有効量）の有効成分が含有される。本発明の抗がん薬中の有効成分量は一般に剤形によって異なるが、所望の投与量を達成できるように有効成分量を例えば約0.01重量％〜約95重量％の範囲内で設定する。 The anticancer drug of the present invention can be formulated according to a conventional method. When formulating, other pharmaceutically acceptable ingredients (for example, carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, preservatives, interfaces) Activators, lubricants, diluents, coating agents, sugar coatings, flavoring agents, emulsifying / solubilizing / dispersing agents, pH adjusting agents, isotonic agents, solubilizers, fragrances, coloring agents, solubilizing agents, physiological Saline solution and the like). The dosage form for formulation is not particularly limited. Examples of dosage forms are tablets, powders, fine granules, granules, capsules, syrups, solutions, suspensions, emulsions, jellies, injections, external preparations, inhalants, nasal drops, eye drops and suppositories. It is an agent. The anticancer drug of the present invention contains an active ingredient in an amount necessary for obtaining an expected therapeutic effect (or preventive effect) (that is, a therapeutically effective amount). The amount of the active ingredient in the anticancer drug of the present invention generally varies depending on the dosage form, but the amount of the active ingredient is set, for example, within a range of about 0.01 wt% to about 95 wt% so that a desired dose can be achieved.

本発明の抗がん薬はその剤形に応じて経口投与又は非経口投与（静脈内、動脈内、皮下、皮内、筋肉内、又は腹腔内注射、経皮、経鼻、経粘膜など）によって対象に適用される。これらの投与経路は互いに排他的なものではなく、任意に選択される二つ以上を併用することもできる（例えば、経口投与と同時に又は所定時間経過後に静脈注射等を行う等）。全身投与によらず、局所投与することにしてもよい。ドラッグデリバリーシステム（DDS）を利用して標的組織特異的に有効成分が送達されるように投与してもよい。ここでの「対象」は特に限定されず、ヒト及びヒト以外の哺乳動物（ペット動物、家畜、実験動物を含む。具体的には例えばマウス、ラット、モルモット、ハムスター、サル、ウシ、ブタ、ヤギ、ヒツジ、イヌ、ネコ、ニワトリ、ウズラ等である）を含む。好ましい一態様では、適用対象はヒトである。 The anticancer drug of the present invention is administered orally or parenterally (intravenous, intraarterial, subcutaneous, intradermal, intramuscular or intraperitoneal injection, transdermal, nasal, transmucosal, etc.) according to the dosage form. Applied to the subject. These administration routes are not mutually exclusive, and two or more arbitrarily selected can be used in combination (for example, intravenous injection or the like is performed simultaneously with oral administration or after a predetermined time has elapsed). Local administration may be used instead of systemic administration. A drug delivery system (DDS) may be used to administer the active ingredient in a target tissue-specific manner. The “subject” here is not particularly limited, and includes humans and non-human mammals (including pet animals, domestic animals, laboratory animals. Specifically, for example, mice, rats, guinea pigs, hamsters, monkeys, cows, pigs, goats. , Sheep, dogs, cats, chickens, quails, etc.). In a preferred embodiment, the application subject is a human.

本発明の抗がん薬の使用形態の一つとして、本発明の抗がん薬を含有する食品組成物が提供される。本発明での「食品組成物」の例として一般食品（穀類、野菜、食肉、各種加工食品、菓子類（例えばクッキー、ビスケット、ゼリー、飴）、牛乳、清涼飲料水、アルコール飲料等）、栄養補助食品（サプリメント、栄養ドリンク等）、食品添加物を挙げることができる。栄養補助食品又は食品添加物の場合、粉末、顆粒末、タブレット、ペースト、液体等の形状で提供することができる。食品組成物の形態で提供することによって、本発明の抗がん薬を日常的に摂取したり、継続的に摂取したりすることが容易となる。本発明の食品組成物には、治療的又は予防的効果が期待できる量の有効成分が含有されることが好ましい。添加量は、それが使用される対象となる者の病状、健康状態、年齢、性別、体重などを考慮して定めることができる。 As one of the usage forms of the anticancer drug of the present invention, a food composition containing the anticancer drug of the present invention is provided. Examples of the “food composition” in the present invention include general foods (cereals, vegetables, meat, various processed foods, confectionery (for example, cookies, biscuits, jelly, rice cakes), milk, soft drinks, alcoholic beverages, etc.), nutrition Examples include supplements (supplements, energy drinks, etc.) and food additives. In the case of a dietary supplement or food additive, it can be provided in the form of powder, granule powder, tablet, paste, liquid or the like. By providing it in the form of a food composition, it becomes easy to ingest the anticancer drug of the present invention on a daily basis or continuously. The food composition of the present invention preferably contains an active ingredient in an amount that can be expected to have a therapeutic or prophylactic effect. The amount added can be determined in consideration of the medical condition, health status, age, sex, weight, etc. of the person to whom it is used.

本発明の更なる局面は、本発明の抗がん薬を使用した、がんに対する治療方法又は予防方法（以下、これら二つの方法をまとめて「治療方法等」という）が提供される。本発明の治療方法等は、上記本発明の抗がん薬を、がんを罹患する又はがんの兆候を認める患者に投与するステップを含む。投与経路は特に限定されず例えば経口、静脈内、動脈内、皮内、皮下、筋肉内、腹腔内、経皮、経鼻、経粘膜などを挙げることができる。これらの投与経路は互いに排他的なものではなく、任意に選択される二つ以上を併用することもできる。抗がん薬の投与量は一般に、患者の症状、年齢、性別、及び体重などによって変動し得るが、当業者であれば適宜適当な投与量を設定することが可能である。投与スケジュールとしては例えば一日一回〜数回、二日に一回、或いは三日に一回などを採用できる。投与スケジュールの設定においては、患者の症状や有効成分の効果持続時間などを考慮することができる。 A further aspect of the present invention provides a method for treating or preventing cancer (hereinafter, these two methods are collectively referred to as “therapeutic methods”) using the anticancer drug of the present invention. The treatment method of the present invention includes a step of administering the anticancer drug of the present invention to a patient suffering from cancer or showing signs of cancer. The administration route is not particularly limited, and examples thereof include oral, intravenous, intraarterial, intradermal, subcutaneous, intramuscular, intraperitoneal, transdermal, nasal, and transmucosal. These administration routes are not mutually exclusive, and two or more arbitrarily selected can be used in combination. In general, the dose of an anticancer drug may vary depending on the patient's symptoms, age, sex, weight, etc., but those skilled in the art can appropriately set an appropriate dose. As the administration schedule, for example, once to several times a day, once every two days, or once every three days can be adopted. In setting the administration schedule, it is possible to consider patient symptoms, duration of effect of active ingredients, and the like.

バッカリンよりも効果が高く且つ選択性の高いAKR1C3阻害剤の創出を目指し検討した。具体的には、分子ドッキング技術とアミノ酸部位特異的変異法から得られる構造情報をもとに種々のバッカリン誘導体を合成し、各誘導体の特性を比較評価した。 The aim was to create an AKR1C3 inhibitor that is more effective and more selective than baccharin. Specifically, various buccalin derivatives were synthesized based on structural information obtained from molecular docking techniques and amino acid site-specific mutation methods, and the properties of each derivative were compared and evaluated.

１．方法
（１）バッカリン誘導体の合成
図１及び図２に示すスキームに従って、様々な化合物を合成した。以下、スキーム２に関して詳細に説明する。 1. Method (1) Synthesis of Baccalin Derivatives Various compounds were synthesized according to the schemes shown in FIGS. Hereinafter, scheme 2 will be described in detail.

3,4-Dihydroxybenzaldehyde(8, 1 mmol)のDMSO(8 mL)溶液にNaH(2.2 mmol)を0℃にて加え３０分間攪拌した。反応液にBenzyl chloride(1 mmol)を加え室温にて１２時間攪拌した。反応を10% HClにて停止した反応混合物をEtOAc(5 mL x 3)で抽出した有機層をNa₂SO₄で乾燥し溶媒を留去した残渣をシリカゲルカラムクロマトグラフィー(15 g, ヘキサン：アセトン＝10：1〜50：1)にて精製し以下の化合物を得た。 NaH (2.2 mmol) was added to a DMSO (8 mL) solution of 3,4-Dihydroxybenzaldehyde (8, 1 mmol) at 0 ° C. and stirred for 30 minutes. Benzyl chloride (1 mmol) was added to the reaction solution and stirred at room temperature for 12 hours. The reaction mixture was quenched with 10% HCl and the reaction mixture was extracted with EtOAc (5 mL x 3). The organic layer was dried over Na ₂ SO ₄ and the solvent was distilled off. The residue was subjected to silica gel column chromatography (15 g, hexane: acetone). = 10: 1 to 50: 1) to obtain the following compound.

4-hydroxy-3-{3-(methoxymethoxy)benzyloxy}benzaldehyde. (9a) Yiled: 92%; ¹H-NMR (400 MHz, CDCl₃): δ 3.49 (3H, s), 5.16 (2H, s), 5.21 (2H, s), 6.22 (1H, br), 7.06 (2H, d, J = 8.3 Hz), 7.11 (1H, s), 7.34 (1H, t, J = 7.9 Hz), 7.44 (1H, t, J = 6.8, 1.3 Hz), 7.49 (1H, d, J = 1.7 Hz); ¹³C-NMR (125 MHz, CDCl3): δ 56.0, 71.0, 94.3, 110.3, 114.7, 115.8, 116.4, 121.4, 127.6, 129.8, 129.9, 137.0, 146.3, 151.8, 157.5, 190.8; IR (neat): 1684, 1508, 1288, 1151 cm^-1; MS (EI): m/z 288 (M⁺); HRMS: Calcd for C₁₆H₁₆O₅288.0998, Found: 288.0999.

4-hydroxy-3- {3- (methoxymethoxy) benzyloxy} benzaldehyde. (9a) Yiled: 92%; ¹ H-NMR (400 MHz, CDCl ₃ ): δ 3.49 (3H, s), 5.16 (2H, s) , 5.21 (2H, s), 6.22 (1H, br), 7.06 (2H, d, J = 8.3 Hz), 7.11 (1H, s), 7.34 (1H, t, J = 7.9 Hz), 7.44 (1H, t, J = 6.8, 1.3 Hz), 7.49 (1H, d, J = 1.7 Hz); ¹³ C-NMR (125 MHz, CDCl3): δ 56.0, 71.0, 94.3, 110.3, 114.7, 115.8, 116.4, 121.4, 127.6, 129.8, 129.9, 137.0, 146.3, 151.8, 157.5, 190.8; IR (neat): 1684, 1508, 1288, 1151 cm ^-1 ; MS (EI): m / z 288 (M ⁺ ); HRMS: Calcd for C ₁₆ H ₁₆ O ₅ 288.0998, Found: 288.0999.

4-hydroxy-3-{(3-methoxybenzyl)oxy}benzaldehyde. (9b) Yield: 92%; mp: 92-93 ^oC; ¹H-NMR (500 MHz, CDCl₃): δ 3.83 (3H, s), 5.15 (2H, s), 6.22 (1H, br), 6.92 (H, dd, J = 5.7, 2.6 Hz), 6.93 (1H, s), 7.01 (1H, d, J = 7.5 Hz), 7.06 (1H, d, J = 8.0 Hz), 7.34 (1H, t, J = 8.0 Hz), 7.45 (1H, dd, J = 6.3, 1.7 Hz), 7.50 (1H, d, J = 1.7 Hz); ¹³C-NMR (125 MHz, CDCl₃): δ 55.2, 71.0, 110.3, 113.5, 113.9, 114.7, 120.1, 127.5, 129.7, 129.8, 136.9, 146.3, 151.9, 159.8, 190.8; IR (KBr): 3368, 1686, 1597, 1292, 1271 cm^-1; MS (EI): m/z 258 (M⁺); HRMS: Calcd for C₁₅H₁₄O₄258.0892, Found: 258.0892.

4-hydroxy-3-{(3-methoxybenzyl) oxy} benzaldehyde. (9b) Yield: 92%; mp: 92-93 ^o C; ¹ H-NMR (500 MHz, CDCl ₃ ): δ 3.83 (3H, s ), 5.15 (2H, s), 6.22 (1H, br), 6.92 (H, dd, J = 5.7, 2.6 Hz), 6.93 (1H, s), 7.01 (1H, d, J = 7.5 Hz), 7.06 (1H, d, J = 8.0 Hz), 7.34 (1H, t, J = 8.0 Hz), 7.45 (1H, dd, J = 6.3, 1.7 Hz), 7.50 (1H, d, J = 1.7 Hz); ¹³ C-NMR (125 MHz, CDCl ₃ ): δ 55.2, 71.0, 110.3, 113.5, 113.9, 114.7, 120.1, 127.5, 129.7, 129.8, 136.9, 146.3, 151.9, 159.8, 190.8; IR (KBr): 3368, 1686 , 1597, 1292, 1271 cm ^-1 ; MS (EI): m / z 258 (M ⁺ ); HRMS: Calcd for C ₁₅ H ₁₄ O ₄ 258.0892, Found: 258.0892.

4-Hydroxy-3-benzylbenzaldehyde(9, 1 mmol)のCH₂Cl₂(4 mL)溶液に3.4-Dihydro-2H-pyran(10 mmol)とPPTS(0.2 mmol)を加え１２時間加熱還流した。冷後反応を飽和NaHCO₃水溶液で停止した混合物をCH₂Cl₂(5 mL x 3)で抽出した有機層をNa₂SO₄で乾燥し溶媒を留去した残渣をシリカゲルカラムクロマトグラフィー(15 g, ヘキサン：アセトン＝40：1〜20：1)にて精製し以下の化合物を得た。 3.4-Dihydro-2H-pyran (10 mmol) and PPTS (0.2 mmol) were added to a CH ₂ Cl ₂ (4 mL) solution of 4-Hydroxy-3-benzylbenzaldehyde (9, 1 mmol), and the mixture was heated to reflux for 12 hours. After cooling, the reaction was stopped with saturated aqueous NaHCO ₃ and the mixture was extracted with CH ₂ Cl ₂ (5 mL x 3) .The organic layer was dried over Na ₂ SO ₄ and the solvent was distilled off. , Hexane: acetone = 40: 1 to 20: 1) to obtain the following compounds.

3-((3-(methoxymethoxy)benzyl)oxy)-4-((tetrahydro-2H-pyran-2-yl)oxy)benzaldehyde. (10a) Yield: 61%; ¹H-NMR (400 MHz, CDCl₃): δ 1.60-2.06 (6H, m), 3.64 (1H, d, J = 11.0 Hz), 3.90 (1H, t, J = 8.1 Hz), 5.16 (2H, s), 5.19 (2H, s), 5.60 (1H, t, J = 5.6 Hz), 6.99 (1H, dd, J = 4.9, 2.7 Hz), 7.10 (1H, d, J = 7.6 Hz), 7.18 (1H, s), 7.27-7.32 (2H, m), 7.45 (1H, dd, J = 6.3, 2.0 Hz), 7.49 (1H, d, J = 7.8 Hz); ¹³C-NMR (125 MHz, CDCl₃): δ 18.1, 24.8, 29.8, 54.8, 61.6, 70.4, 96.5, 97.1, 112.1, 112.5, 113.2, 115.8, 118.9, 126.2, 129.5, 130.5, 138.7, 149.1, 152.2, 159.5, 190.6; IR (neat): 1689, 1514, 1267, 1153 cm^-1; MS (EI): m/z 288 (M⁺-84); HRMS: Calcd for C₁₆H₁₆O₅288.0998, Found: 288.1000.

3-((3- (methoxymethoxy) benzyl) oxy) -4-((tetrahydro-2H-pyran-2-yl) oxy) benzaldehyde. (10a) Yield: 61%; ¹ H-NMR (400 MHz, CDCl ₃ ): δ 1.60-2.06 (6H, m), 3.64 (1H, d, J = 11.0 Hz), 3.90 (1H, t, J = 8.1 Hz), 5.16 (2H, s), 5.19 (2H, s), 5.60 (1H, t, J = 5.6 Hz), 6.99 (1H, dd, J = 4.9, 2.7 Hz), 7.10 (1H, d, J = 7.6 Hz), 7.18 (1H, s), 7.27-7.32 (2H , m), 7.45 (1H, dd, J = 6.3, 2.0 Hz), 7.49 (1H, d, J = 7.8 Hz); ¹³ C-NMR (125 MHz, CDCl ₃ ): δ 18.1, 24.8, 29.8, 54.8 , 61.6, 70.4, 96.5, 97.1, 112.1, 112.5, 113.2, 115.8, 118.9, 126.2, 129.5, 130.5, 138.7, 149.1, 152.2, 159.5, 190.6; IR (neat): 1689, 1514, 1267, 1153 cm ^-1 ; MS (EI): m / z 288 (M ⁺ -84); HRMS: Calcd for C ₁₆ H ₁₆ O ₅ 288.0998, Found: 288.1000.

3-((3-methoxybenzyl)oxy)-4-((tetrahydro-2H-pyran-2-yl)oxy)benzaldehyde. (10b) Yield: 63%; mp:73-77 ^oC; ¹H-NMR (500 MHz, CDCl₃): δ 1.55-2.17 (6H, m), 3.64 (1H, d, J = 7.1 Hz), 3.82 (3H, s), 3.90 (1H, t, J = 8.0 Hz), 5.17 (2H, s), 5.61 (1H, t, J = 6.1 Hz), 6.86 (1H, dd, J = 5.7, 2.6 Hz), 7.03 (1H, d, J = 6.3 Hz), 7.05 (1H, s), 7.27 (1H, m), 7.30 (1H, t, J = 7.5 Hz), 7.45 (1H, dd, J = 6.3, 2.0 Hz), 7.50 (1H, d, J = 2.0 Hz); ¹³C-NMR (125 MHz, CDCl₃): δ 18.1, 24.8, 29.8, 54.8, 61.6, 70.2, 96.5, 112.1, 112.5, 113.2, 115.8, 118.9, 126.2, 129.3, 130.5, 138.1, 149.1, 152.2, 159.5, 190.6; IR (KBr): 1684, 1508, 1273, 1123 cm^-1; MS (EI): m/z 258 (M⁺-84); HRMS: Calcd for C₁₅H₁₄O₄258.0892, Found: 258.0892.

3-((3-methoxybenzyl) oxy) -4-((tetrahydro-2H-pyran-2-yl) oxy) benzaldehyde. (10b) Yield: 63%; mp: 73-77 ^o C; ¹ H-NMR ( _{500 MHz, CDCl 3): δ} 1.55-2.17 (6H, m), 3.64 (1H, d, J = 7.1 Hz), 3.82 (3H, s), 3.90 (1H, t, J = 8.0 Hz), 5.17 ( 2H, s), 5.61 (1H, t, J = 6.1 Hz), 6.86 (1H, dd, J = 5.7, 2.6 Hz), 7.03 (1H, d, J = 6.3 Hz), 7.05 (1H, s), 7.27 (1H, m), 7.30 (1H, t, J = 7.5 Hz), 7.45 (1H, dd, J = 6.3, 2.0 Hz), 7.50 (1H, d, J = 2.0 Hz); ¹³ C-NMR ( 125 MHz, CDCl ₃ ): δ 18.1, 24.8, 29.8, 54.8, 61.6, 70.2, 96.5, 112.1, 112.5, 113.2, 115.8, 118.9, 126.2, 129.3, 130.5, 138.1, 149.1, 152.2, 159.5, 190.6; IR ( (KBr): 1684, 1508, 1273, 1123 cm ^-1 ; MS (EI): m / z 258 (M ⁺ -84); HRMS: Calcd for C ₁₅ H ₁₄ O ₄ 258.0892, Found: 258.0892.

Horner-Wadsworth-Emmons (HWE)反応の一般的方法（スキーム１と同様）で以下の化合物を得た。 The following compounds were obtained by the general method of Horner-Wadsworth-Emmons (HWE) reaction (similar to Scheme 1).

(E)-ethyl3-(3-((3-(methoxymethoxy)benzyl)oxy)-4-((tetrahydro-2H-pyran-2-yl)oxy)phenyl)acrylate. (11a) Yield: 82%; ¹H-NMR (400 MHz, CDCl₃): δ 1.33 (3H, t, J = 7.1 Hz), 1.63-1.96 (6H, m), 3.47 (3H, s), 3.61 (1H, d, J = 11.1 Hz), 3.95 (1H, t, J = 7.8 Hz), 4.24 (2H, quint, J = 7.1 Hz), 5.11 (2H, s), 5.18 (2H, s), 5.51 (1H, t, J = 5.9 Hz), 6.27 (1H, d, J = 15.9 Hz), 6.98 (1H, dd, J = 5.6, 2.7 Hz), 7.08-7.16 (5H, m), 7.30 (1H, t, J = 7.8 Hz), 7.58 (1H, d, J = 15.9 Hz); ¹³C-NMR (125 MHz, CDCl₃): δ 14.8, 18.3, 25.0, 30.0, 55.8, 60.2, 61.8, 70.9, 94.2, 96.9, 114.0, 114.8, 115.6, 116.2, 117.3, 122.7, 128.5, 129.4, 138.6, 144.2, 149.0, 149.1, 157.3, 167.0; IR (neat): 1684, 1508, 1121 cm^-1; MS (EI): m/z 358 (M⁺-84); HRMS: Calcd for C₂₀H₂₀O₆358.1416, Found: 358.1419.

(E) -ethyl3- (3-((3- (methoxymethoxy) benzyl) oxy) -4-((tetrahydro-2H-pyran-2-yl) oxy) phenyl) acrylate. (11a) Yield: 82%; ¹ H-NMR (400 MHz, CDCl ₃ ): δ 1.33 (3H, t, J = 7.1 Hz), 1.63-1.96 (6H, m), 3.47 (3H, s), 3.61 (1H, d, J = 11.1 Hz ), 3.95 (1H, t, J = 7.8 Hz), 4.24 (2H, quint, J = 7.1 Hz), 5.11 (2H, s), 5.18 (2H, s), 5.51 (1H, t, J = 5.9 Hz) ), 6.27 (1H, d, J = 15.9 Hz), 6.98 (1H, dd, J = 5.6, 2.7 Hz), 7.08-7.16 (5H, m), 7.30 (1H, t, J = 7.8 Hz), 7.58 (1H, d, J = 15.9 Hz); ¹³ C-NMR (125 MHz, CDCl ₃ ): δ 14.8, 18.3, 25.0, 30.0, 55.8, 60.2, 61.8, 70.9, 94.2, 96.9, 114.0, 114.8, 115.6, 116.2, 117.3, 122.7, 128.5, 129.4, 138.6, 144.2, 149.0, 149.1, 157.3, 167.0; IR (neat): 1684, 1508, 1121 cm ^-1 ; MS (EI): m / z 358 (M ⁺ -84 ); HRMS: Calcd for C ₂₀ H ₂₀ O ₆ 358.1416, Found: 358.1419.

(E)-ethyl-3-(3-((3-methoxybenzyl)oxy)-4-((tetrahydro-2H-pyran-2-yl)oxy)phenyl)acrylate. (11b) Yield: 96%; ¹H-NMR (500 MHz, CDCl₃): δ 1.33 (3H, t, J = 7.2 Hz)1.56-2.17 (6H, m), 3.59-3.63 (1H, m), 3.82 (3H, s), 3.94 (1H, t, J = 8.0 Hz), 4.24 (2H, quin, J = 7.2 Hz), 5.12 (2H, s), 5.51 (1H, m), 6.28 (1H, d, J = 15.9 Hz), 6.85 (1H, dd, J = 6.3, 2.3 Hz), 7.02 (1H, d, J = 7.8 Hz), 7.05 (1H, s), 7.10-7.16 (3H, m), 7.29 (1H, t, J = 7.8 Hz), 7.59 (1H, d, J = 15.9 Hz); ¹³C-NMR (125 MHz, CDCl₃): δ 14.2, 18.3, 25.0, 30.0, 54.9, 60.1, 61.7, 70.8, 96.8, 112.2, 113.2, 113.8, 116.2, 117.7, 119.0, 122.6, 128.5, 129.3, 138.5, 144.2, 148.9, 149.1, 159.6, 166.9; IR (neat): 1707, 1508, 1258, 1165 cm^-1; MS (EI): m/z 328(M⁺-84); HRMS: Calcd for C₁₉H₂₀O₅328.1311, Found: 328.1309.

(E) -ethyl-3- (3-((3-methoxybenzyl) oxy) -4-((tetrahydro-2H-pyran-2-yl) oxy) phenyl) acrylate. (11b) Yield: 96%; ¹ H -NMR (500 MHz, CDCl ₃ ): δ 1.33 (3H, t, J = 7.2 Hz) 1.56-2.17 (6H, m), 3.59-3.63 (1H, m), 3.82 (3H, s), 3.94 (1H , t, J = 8.0 Hz), 4.24 (2H, quin, J = 7.2 Hz), 5.12 (2H, s), 5.51 (1H, m), 6.28 (1H, d, J = 15.9 Hz), 6.85 (1H , dd, J = 6.3, 2.3 Hz), 7.02 (1H, d, J = 7.8 Hz), 7.05 (1H, s), 7.10-7.16 (3H, m), 7.29 (1H, t, J = 7.8 Hz) , 7.59 (1H, d, J = 15.9 Hz); ¹³ C-NMR (125 MHz, CDCl ₃ ): δ 14.2, 18.3, 25.0, 30.0, 54.9, 60.1, 61.7, 70.8, 96.8, 112.2, 113.2, 113.8, 116.2, 117.7, 119.0, 122.6, 128.5, 129.3, 138.5, 144.2, 148.9, 149.1, 159.6, 166.9; IR (neat): 1707, 1508, 1258, 1165 cm ^-1 ; MS (EI): m / z 328 ( M ⁺ -84); HRMS: Calcd for C ₁₉ H ₂₀ O ₅ 328.1311, Found: 328.1309.

エステル(11, 1 mmol)のMeOH:H₂O (3:1)溶液にLiOH-H₂O(2 mmol)を加え２時間加熱還流した。冷後反応を10% HClで停止し酸性とした後EtOAc(5 mL x 3)で抽出した有機層を Na₂SO₄で乾燥し溶媒を留去した残渣をシリカゲルカラムクロマトグラフィー(15 g, CH₂Cl₂：MeOH＝80：1〜70：1)にて精製し以下の化合物を得た。 LiOH-H ₂ O (2 mmol) was added to a solution of the ester (11, 1 mmol) in MeOH: H ₂ O (3: 1), and the mixture was heated to reflux for 2 hours. After cooling, the reaction was quenched with 10% HCl, acidified, and the organic layer extracted with EtOAc (5 mL x 3) was dried over Na ₂ SO ₄ and the solvent was distilled off.The residue was purified by silica gel column chromatography (15 g, CH _The following compounds were obtained by purification with 2Cl ₂ : MeOH = 80: 1 to 70: 1).

(E)-3-(4-hydroxy-3-((3-(methoxymethoxy)benzyl)oxy)phenyl)acrylic acid. (12a) Yield: 99 %; ¹H-NMR (400 MHz, CDCl₃): δ 3.51 (3H, s), 5.12 (2H, s), 5.20 (2H, s), 6.26 (1H, d, J = 15.9 Hz), 6.95 (1H, d, J = 8.1 Hz), 7.05-7.14 (5H, m), 7.34 (1H, t, J = 8.1 Hz), 7.67 (1H, d, J = 15.9 Hz); ¹³C-NMR (125 MHz, CDCl₃): δ 56.0, 71.0, 94.3, 111.3, 114.7, 115.1, 115.6, 116.4, 121.2, 123.7, 126.6, 129.9, 137.3, 146.0, 146.9, 148.6, 157.5, 172.3; IR (neat): 2361, 1684, 1508, 1271 cm^-1; MS (EI): m/z 330 (M⁺); HRMS: Calcd for C₁₈H₁₈O₆330.1103, Found: 330.1106.

(E) -3- (4-hydroxy-3-((3- (methoxymethoxy) benzyl) oxy) phenyl) acrylic acid. (12a) Yield: 99%; ¹ H-NMR (400 MHz, CDCl ₃ ): δ 3.51 (3H, s), 5.12 (2H, s), 5.20 (2H, s), 6.26 (1H, d, J = 15.9 Hz), 6.95 (1H, d, J = 8.1 Hz), 7.05-7.14 (5H , m), 7.34 (1H, t, J = 8.1 Hz), 7.67 (1H, d, J = 15.9 Hz); ¹³ C-NMR (125 MHz, CDCl ₃ ): δ 56.0, 71.0, 94.3, 111.3, 114.7 , 115.1, 115.6, 116.4, 121.2, 123.7, 126.6, 129.9, 137.3, 146.0, 146.9, 148.6, 157.5, 172.3; IR (neat): 2361, 1684, 1508, 1271 cm ^-1 ; MS (EI): m / z 330 (M ⁺ ); HRMS: Calcd for C ₁₈ H ₁₈ O ₆ 330.1103, Found: 330.1106.

(E)-3-(4-hydroxy-3-((3-methoxybenzyl)oxy)phenyl)acrylic acid. (12b) Yield: 89%; mp: 167-169 ^oC; ¹H-NMR (500 MHz, CDCl₃): δ 3.83 (3H, s), 5.13 (2H, s), 6.26 (1H, d, J = 15.9 Hz), 6.91-6.97 (3H, m); 7.01 (1H, d, J = 8.0 Hz), 7.12 (2H, m), 7.34 (1H, t, J = 7.7 Hz), 7.67 (1H, d, J = 15.9 Hz); ¹³C-NMR (125 MHz, DMSO-d₆): δ 55.6, 70.2, 113.5, 113.7, 113.8, 116.2, 116.4, 120.4, 123.7, 126.3, 130.0, 139.3, 145.0, 147.3, 150.0, 159.9, 168.6; IR (KBr): 2939, 1684, 1514, 1271 cm^-1; MS (EI): m/z 300 (M⁺); HRMS: Calcd for C₁₇H₁₆O₅300.0998, Found: 300.1000 .

(E) -3- (4-hydroxy-3-((3-methoxybenzyl) oxy) phenyl) acrylic acid. (12b) Yield: 89%; mp: 167-169 ^o C; ¹ H-NMR (500 MHz, CDCl ₃ ): δ 3.83 (3H, s), 5.13 (2H, s), 6.26 (1H, d, J = 15.9 Hz), 6.91-6.97 (3H, m); 7.01 (1H, d, J = 8.0 Hz ), 7.12 (2H, m), 7.34 (1H, t, J = 7.7 Hz), 7.67 (1H, d, J = 15.9 Hz); ¹³ C-NMR (125 MHz, DMSO-d ₆ ): δ 55.6, 70.2, 113.5, 113.7, 113.8, 116.2, 116.4, 120.4, 123.7, 126.3, 130.0, 139.3, 145.0, 147.3, 150.0, 159.9, 168.6; IR (KBr): 2939, 1684, 1514, 1271 cm ^-1 ; MS ( EI): m / z 300 (M ⁺ ); HRMS: Calcd for C ₁₇ H ₁₆ O ₅ 300.0998 , Found: 300.1000.

化合物 (6a-z)と同様に以下の化合物の合成を行った。 The following compounds were synthesized in the same manner as compound (6a-z).

(E)-3-(3-((3-methoxybenzyl)oxy)-4-((3-phenylpropanoyl)oxy)phenyl)acrylic acid. (13b) Yield: 54%; mp: 97-99 ^oC; ¹H-NMR (500 MHz, CDCl₃): δ 2.79 (2H, t, J = 7.4 Hz), 3.02 (2H, t, J = 7.4 Hz), 3.80 (3H, s), 5.08 (2H, s), 6.34 (1H, d, J = 15.9 Hz), 6.86 (1H, dd, J = 5.3, 2.3 Hz), 6.95 (1H, s), 6.96 (1H, d, J = 8.0 Hz), 7.03 (1H, d, J = 8.0 Hz), 7.14-7.18 (2H, m), 7.20-7.24 (3H, m), 7.28-7.31 (3H, m), 7.68 (1H, d, J = 15.9 Hz), ¹³C-NMR (125 MHz, CDCl₃): δ 30.9, 35.6, 55.2, 70.6, 112.8, 113.0, 113.6, 117.3, 119.3, 121.8, 123.3, 126.4, 128.3, 128.5, 129.7, 132.9, 137.7, 140.2, 142.2, 146.2, 150.5, 159.9, 170.7, 171.4; IR (KBr): 2934, 1763, 1690, 1265, 1122 cm^-1; MS (EI): m/z 432 (M⁺); HRMS: Calcd for C₂₆H₂₄O₆423.1573, Found: 432.1572.

(E) -3- (3-((3-methoxybenzyl) oxy) -4-((3-phenylpropanoyl) oxy) phenyl) acrylic acid. (13b) Yield: 54%; mp: 97-99 ^o C; ¹ H-NMR (500 MHz, CDCl ₃ ): δ 2.79 (2H, t, J = 7.4 Hz), 3.02 (2H, t, J = 7.4 Hz), 3.80 (3H, s), 5.08 (2H, s), 6.34 (1H, d, J = 15.9 Hz), 6.86 (1H, dd, J = 5.3, 2.3 Hz), 6.95 (1H, s), 6.96 (1H, d, J = 8.0 Hz), 7.03 (1H, d , J = 8.0 Hz), 7.14-7.18 (2H, m), 7.20-7.24 (3H, m), 7.28-7.31 (3H, m), 7.68 (1H, d, J = 15.9 Hz), ¹³ C-NMR (125 MHz, CDCl ₃ ): δ 30.9, 35.6, 55.2, 70.6, 112.8, 113.0, 113.6, 117.3, 119.3, 121.8, 123.3, 126.4, 128.3, 128.5, 129.7, 132.9, 137.7, 140.2, 142.2, 146.2, 150.5 , 159.9, 170.7, 171.4; IR (KBr): 2934, 1763, 1690, 1265, 1122 cm ^-1 ; MS (EI): m / z 432 (M ⁺ ); HRMS: Calcd for C ₂₆ H ₂₄ O ₆ 423.1573 , Found: 432.1572.

(E)-3-(3-((3-hydroxybenzyl)oxy)-4-((3-phenylpropanoyl)oxy)phenyl)acrylic acid. (14)
Yield: 52%; mp: 150-152 ^oC; ¹H-NMR (400 MHz, CDCl₃): δ 2.89 (2H, t, J = 7.6 Hz), 3.03 (2H, t, J = 7.6 Hz), 5.05 (2H, s), 6.31 (1H, d, J = 15.7 Hz), 6.78 (1H, dd, J = 6.1, 1.9 Hz), 6.83 (1H, s), 6.91 (1H, d, J = 7.8 Hz), 7.01 (1H, d, J = 8.1 Hz), 7.09 (1H, s), 7.12 (1H, d, J = 8.1 Hz), 7.19-7.31 (6H, m), 7.66 (1H, d, J = 15.9 Hz); ¹³C-NMR (125 MHz, CDCl₃): δ 31.0, 35.7, 70.4, 113.1, 114.1, 115.3, 115.7, 119.2, 121.9, 123.3, 126.5, 128.4, 128.6, 133.0, 140.2, 142.2, 146.2, 150.5, 156.2, 171.3, 171.4; IR (KBr): 3030, 1686, 1508, 1263, 1121 cm^-1; MS (EI): m/z 418 (M⁺); HRMS: Calcd for C₂₅H₂₂O₆418.1416, Found: 418.1413.

(E) -3- (3-((3-hydroxybenzyl) oxy) -4-((3-phenylpropanoyl) oxy) phenyl) acrylic acid. (14)
Yield: 52%; mp: 150-152 ^o C; ¹ H-NMR (400 MHz, CDCl ₃ ): δ 2.89 (2H, t, J = 7.6 Hz), 3.03 (2H, t, J = 7.6 Hz), 5.05 (2H, s), 6.31 (1H, d, J = 15.7 Hz), 6.78 (1H, dd, J = 6.1, 1.9 Hz), 6.83 (1H, s), 6.91 (1H, d, J = 7.8 Hz ), 7.01 (1H, d, J = 8.1 Hz), 7.09 (1H, s), 7.12 (1H, d, J = 8.1 Hz), 7.19-7.31 (6H, m), 7.66 (1H, d, J = 15.9 Hz); ¹³ C-NMR (125 MHz, CDCl ₃ ): δ 31.0, 35.7, 70.4, 113.1, 114.1, 115.3, 115.7, 119.2, 121.9, 123.3, 126.5, 128.4, 128.6, 133.0, 140.2, 142.2, 146.2 , 150.5, 156.2, 171.3, 171.4; IR (KBr): 3030, 1686, 1508, 1263, 1121 cm ^-1 ; MS (EI): m / z 418 (M ⁺ ); HRMS: Calcd for C ₂₅ H ₂₂ O ₆ 418.1416, Found: 418.1413.

（参考文献）
1. Rene, F.; Candice, M.; Angelique, S.; Caroline, M.; Karin K.; Heike, S.; Alan C.; Gary, W.; Denis, B. Org. Biomol. Chem. 2010,8, 5199-5211..
2. Yun, S. L.; Hye, Y. K.; Young, S. K.; Jae, H. S.; Eun, J. R.; Hogyu, H. K.; Jung, S. Bioorg. Med. Chem. 2012, 15, 4921-4935.
3. Reddy, S. V.; Rao, R. J.; Kumar, U. S.; Rao, J. M. Chem. Lett. 2003, 32, 1038-1039.
4. Veldhoven, J. P. D.; Blad, C. C.; Artsen, M.; Klopman, C.; Wolfram, D. R.; Abdelkadir, M. J.; Lane, J. R.; Brussee, J.; IJzerman A. P. Bioorg. Med. Chem. Lett. 2011, 1, 2736-2739. (References)
1. Rene, F .; Candice, M .; Angelique, S .; Caroline, M .; Karin K .; Heike, S .; Alan C .; Gary, W .; Denis, B. Org. Biomol. Chem. 2010,8, 5199-5211 ..
2. Yun, SL; Hye, YK; Young, SK; Jae, HS; Eun, JR; Hogyu, HK; Jung, S. Bioorg. Med. Chem. 2012, 15, 4921-4935.
3. Reddy, SV; Rao, RJ; Kumar, US; Rao, JM Chem. Lett. 2003, 32, 1038-1039.
4. Veldhoven, JPD; Blad, CC; Artsen, M .; Klopman, C .; Wolfram, DR; Abdelkadir, MJ; Lane, JR; Brussee, J .; IJzerman AP Bioorg. Med. Chem. Lett. 2011, 1 , 2736-2739.

（２）AKR1C3タンパクの調製
AKR1C3 cDNAを組み込んだpkk223-3ベクターのプラスミドによって形質転換された大腸菌JM109を50 μg/mLアンピシリンを含むLB培養液中に懸濁して37℃で一晩培養した。その大腸菌を1 LのLB培養液に植菌し、600 nmにおける濁度が0.4〜0.6になるまで37℃で培養した後、isopropyl-β-D-galactopyranoside (IPTG)を終濃度が1 mMになるように添加し、37℃にてさらに8時間培養した。リコンビナント酵素の発現を誘導した大腸菌は、遠心分離(5,000 x g、15分間、4℃)により集菌し、0.5 mM EDTAと5 mM 2-メルカプトエタノール(2-ME)を含む10 mM Tris-HCl(pH 8.0)に懸濁した。この懸濁液を氷冷下超音波処理（150 W、5分間）した後、遠心分離(12,000 x g、15分間、4℃)し、その上清を大腸菌粗抽出液とした。粗抽出液を0.15 M NaCl、20 % グリセロールを添加したbuffer A(10 mM Tris-HCl、0.5 mM EDTA、5 mM 2-ME; pH 8.0)で平衡化したSephadex G-100カラムを用いてゲル濾過を行った。溶出した酵素画分をYM-10限外濾過膜を用いて濃縮後、buffer Aに対して透析し、buffer Aで平衡化したQ-Sepharose カラムに添加した。未吸着タンパク質をbuffer Aで洗浄後、吸着した酵素を0〜0.2 M NaClまでグラジエントにより溶出した。酵素活性画分をYM-10限外濾過膜を用いて濃縮後、buffer Aで平衡化したRed A-Sepharoseカラムに添加した。Buffer Aでカラムを洗浄後、酵素画分を0.5 mM NADP⁺を含むbuffer Aにより溶出した。AKR1C3精製標品はsodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) 分析後のクマジーブリリアントブルー(CBB)R250染色において、単一のバンドを示した。 (2) Preparation of AKR1C3 protein
E. coli JM109 transformed with the pkk223-3 vector plasmid incorporating AKR1C3 cDNA was suspended in an LB culture solution containing 50 μg / mL ampicillin and cultured at 37 ° C. overnight. The E. coli was inoculated into 1 L of LB culture and cultured at 37 ° C until the turbidity at 600 nm reached 0.4 to 0.6, and then isopropyl-β-D-galactopyranoside (IPTG) was added to a final concentration of 1 mM. And then further cultured at 37 ° C. for 8 hours. E. coli that induced the expression of the recombinant enzyme was collected by centrifugation (5,000 xg, 15 minutes, 4 ° C), and 10 mM Tris-HCl (0.5 mM EDTA and 5 mM 2-mercaptoethanol (2-ME)) suspended in pH 8.0). The suspension was sonicated under ice-cooling (150 W, 5 minutes) and then centrifuged (12,000 × g, 15 minutes, 4 ° C.), and the supernatant was used as a crude E. coli extract. Gel filtration of the crude extract using Sephadex G-100 column equilibrated with buffer A (10 mM Tris-HCl, 0.5 mM EDTA, 5 mM 2-ME; pH 8.0) supplemented with 0.15 M NaCl and 20% glycerol Went. The eluted enzyme fraction was concentrated using a YM-10 ultrafiltration membrane, dialyzed against buffer A, and added to a Q-Sepharose column equilibrated with buffer A. After washing unadsorbed protein with buffer A, the adsorbed enzyme was eluted with a gradient from 0 to 0.2 M NaCl. The enzyme active fraction was concentrated using a YM-10 ultrafiltration membrane and then added to a Red A-Sepharose column equilibrated with buffer A. After washing the column with Buffer A, the enzyme fraction was eluted with buffer A containing 0.5 mM NADP ⁺ . The purified AKR1C3 sample showed a single band in Coomassie brilliant blue (CBB) R250 staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis.

（３）AKR1C1、AKR1C2、AKR1C4タンパクの調製
AKR1C1、AKR1C4はMatsuuraらの方法（Matsuura et al., Biochem. J., 336, 429-436. (1998)）、AKR1C2はShiraishiらの方法（Shiraishi et al., Biochem. J., 334, 399-405）に従って均一に精製した。 (3) Preparation of AKR1C1, AKR1C2, and AKR1C4 proteins
AKR1C1 and AKR1C4 are the methods of Matsuura et al. (Matsuura et al., Biochem. J., 336, 429-436. (1998)), and AKR1C2 is the method of Shirishi et al. (Shiraishi et al., Biochem. J., 334, 399). -405).

（４）バッカリン誘導体によるAKR1C3阻害
AKR1Cアイソフォーム (AKR1C1、AKR1C2、AKR1C3)の脱水素酵素活性は、以下の反応系におけるNADPHの生成速度を蛍光分光学的 (Ex. 340 nm、Em. 455 nm)に測定した。標準反応系は、0.1 M リン酸カリウム緩衝液(pH 7.4)、0.25 mM NADP⁺、S-(+)-1,2,3,4-tetrahydro-1-naphthol(S-tetralol)および酵素を含む全量2.0 mLとした。酵素活性1 unit(U)は、25℃において1分間に1μmolのNADPHを生成する酵素量とした。阻害剤のIC₅₀値は、0.1 M リン酸カリウム緩衝液(pH 7.4)、0.25 mM NADP⁺、S-tetralol (AKR1C1の場合は0.1 mM、他のAKR1Cアイソフォームでは1 mM)および酵素を含む全量2.0 mLの反応系にて、5点の異なる濃度の阻害剤をに添加した時の阻害率から算出した。これらの阻害定数は少なくとも3回以上の測定の平均値±標準偏差で表した。 (4) AKR1C3 inhibition by baccaline derivatives
The dehydrogenase activity of AKR1C isoforms (AKR1C1, AKR1C2, AKR1C3) was measured spectrophotometrically (Ex. 340 nm, Em. 455 nm) for the rate of NADPH production in the following reaction system. Standard reaction system includes 0.1 M potassium phosphate buffer (pH 7.4), 0.25 mM NADP ⁺ , S-(+)-1,2,3,4-tetrahydro-1-naphthol (S-tetralol) and enzyme The total volume was 2.0 mL. Enzyme activity 1 unit (U) was defined as the amount of enzyme that produced 1 μmol of NADPH per minute at 25 ° C. IC ₅₀ value of the inhibitor is 0.1 M potassium phosphate buffer (pH 7.4), 0.25 mM NADP ⁺ , S-tetralol (0.1 mM for AKR1C1, 1 mM for other AKR1C isoforms) and total amount of enzyme It was calculated from the inhibition rate when 5 different concentrations of inhibitors were added to the 2.0 mL reaction system. These inhibition constants were expressed as an average value ± standard deviation of at least three measurements.

（５）バッカリン誘導体によるA549細胞内アンドロステロン代謝阻害
ヒト肺癌A549細胞は、37℃、5% CO₂条件下で培養し、2日毎に培地を交換して4〜6日毎に継代維持した。培地として10% FBS、100 U/mLぺニシリンGカリウムおよび100μg/mL硫酸ストレプトマイシンを含むDMEM(pH 7.4)を用いた。細胞の剥離には0.25%トリプシンおよび0.02% ethylenediaminetetraacetic acid (EDTA)を含むDPBS(pH 7.4)を用い、トリプシン処理の停止は同量の培地を添加した。 (5) Inhibition of A549 intracellular androsterone metabolism by baccaline derivatives Human lung cancer A549 cells were cultured under conditions of 37 ° C. and 5% CO ₂ , and the culture medium was changed every 2 days and maintained every 4 to 6 days. As a medium, DMEM (pH 7.4) containing 10% FBS, 100 U / mL potassium penicillin G and 100 μg / mL streptomycin sulfate was used. For detachment of cells, DPBS (pH 7.4) containing 0.25% trypsin and 0.02% ethylenediaminetetraacetic acid (EDTA) was used, and the same amount of medium was added to stop the trypsin treatment.

A549細胞を2 x 10⁴ cells/mLずつ播種し、90%コンフルエントに達したところで、FBS不含培地に交換して2時間培養した。様々な濃度の阻害剤を培地に添加して2時間培養後、50μMアンドロステロンを加え、それぞれ24 時間培養した。その培地を遠心分離(1,500 x g、 10分間)により回収した。培地に等量の蒸留水を混合し、このまま2容量の酢酸エチルを添加し、ステロイドおよびその代謝物を抽出した。有機溶媒層を蒸発乾固させ、得られた残渣をイソプロピルアルコール(IPA)に溶解させた。ステロイドおよびその代謝物の定量は、溶媒(n-hexane : IPA = 90 : 10)とDaicel Chiralcel OJ-H 5 μm (4.6 x 250 mm)カラムを用いたLC/MSにより定量した。アンドロステロン(m/z ＝ 274.5)とその代謝物5α-androstane-3α,17β-diol (m/z = 276.5)の分子イオンピークは小さく、それぞれからH₂Oが脱離したイオンピーク(それぞれm/z = 265.5とm/z = 258.5)が高いので、このピーク面積により定量した。なお、両ステロイドの保持時間はそれぞれ24分と27分であった。両ステロイドの代謝における代謝率とバッカリン及び化合物14による阻害率は2回の実験の平均値として表した。 A549 cells were seeded at 2 × 10 ⁴ cells / mL, and when they reached 90% confluence, they were replaced with a medium not containing FBS and cultured for 2 hours. Inhibitors of various concentrations were added to the medium and cultured for 2 hours, 50 μM androsterone was added, and each was cultured for 24 hours. The medium was collected by centrifugation (1,500 × g, 10 minutes). An equal amount of distilled water was mixed with the medium, and 2 volumes of ethyl acetate were added as it was to extract steroids and their metabolites. The organic solvent layer was evaporated to dryness and the resulting residue was dissolved in isopropyl alcohol (IPA). Steroids and their metabolites were quantified by LC / MS using a solvent (n-hexane: IPA = 90: 10) and a Daicel Chiralcel OJ-H 5 μm (4.6 × 250 mm) column. The molecular ion peaks of androsterone (m / z = 274.5) and its metabolite 5α-androstane-3α, 17β-diol (m / z = 276.5) are small, and the ion peaks from which H ₂ O is eliminated (each m Since /z=265.5 and m / z = 258.5) were high, it was determined by this peak area. The retention times for both steroids were 24 and 27 minutes, respectively. The metabolic rate and the inhibition rate by baccaline and compound 14 in the metabolism of both steroids were expressed as the average of two experiments.

（６）バッカリン誘導体による癌細胞増殖抑制効果
ヒト白血病U937細胞とヒト前立腺癌PC3細胞を48穴プレートに3 x 10⁴ cells/mLで播種し、24時間後に抗生物質を不含培地に交換した。接着細胞PC3細胞においては、2時間培養後、AKR1C3のcDNAを含有するpGW1ベクターをLipofectamine 2000を用いて導入した。導入効果はAKR1C3抗体を用いたウエスタンブロット分析により確認した。ベクター導入後に抗生物質を含む培地に交換し、バッカリンを培地に添加し、その0、24、48および72時間後に5 mM WST-1および0.2 mM 1-methoxy PMSを含む20 mM HEPES-NaOH緩衝液(pH 7.4) 10 μL を添加した。37℃、3時間培養した後、マイクロプレートリーダーModel 680 (Bio Rad)を用いて測定した吸光度から生細胞数を算出した。浮遊細胞U937細胞においては、AKR1C3過剰発現細胞あるいはコントロール細胞を48穴プレートに3 x 10⁴ cells/mLずつ播種し、阻害剤を添加し、トリパンブルー色素排除試験法を用いて可視化した生細胞数を測定した。その結果は少なくとも3回以上の測定の平均値±標準偏差で表した。統計解析は、対応のないStudent t-testおよびANOVA、Fisher’s testにより行った。p < 0.05で有意差ありと判定した。 (6) Cancer Cell Growth Inhibitory Effect by Baccaline Derivative Human leukemia U937 cells and human prostate cancer PC3 cells were seeded at 3 × 10 ⁴ cells / mL in a 48-well plate, and 24 hours later, the antibiotics were replaced with a medium containing no antibiotics. In the adherent cell PC3 cell, after culturing for 2 hours, the pGW1 vector containing AKR1C3 cDNA was introduced using Lipofectamine 2000. The effect of introduction was confirmed by Western blot analysis using AKR1C3 antibody. After vector introduction, change to medium containing antibiotics, add buccalin to medium, 0, 24, 48 and 72 hours later, 20 mM HEPES-NaOH buffer containing 5 mM WST-1 and 0.2 mM 1-methoxy PMS (pH 7.4) 10 μL was added. After culturing at 37 ° C. for 3 hours, the number of living cells was calculated from the absorbance measured using a microplate reader Model 680 (Bio Rad). In suspension cells U937 cells, AKR1C3 overexpressing cells or control cells are seeded at 3 x 10 ⁴ cells / mL in 48-well plates, inhibitors are added, and the number of viable cells visualized using the trypan blue dye exclusion test method Was measured. The result was expressed as an average value ± standard deviation of at least three measurements. Statistical analysis was performed by unsupported Student t-test, ANOVA, and Fisher's test. A significant difference was determined at p <0.05.

２．結果
AKR1C3の選択的阻害剤として見出してきたバッカリンをリード化合物として種々の誘導体を合成し、その阻害効果を評価した（図３）。多様な化合物の合成を可能にするため、バッカリン(図３の下段)のイソプレニル部分（R2）はエーテルに変更した。これら18化合物はすべて新規化合物である。その結果、化合物では阻害効果は若干低下したが、エーテルを有する誘導体の中からバッカリンと同程度の阻害効果を示す化合物6m、6nを見出した。さらに、化合物6mのAKR1C3へのドッキングモデルを作成し、そのモデルから得られた結合様式に基づき、バッカリンよりも強力な阻害効果を示す化合物14（ (E)-3-(3-((3-hydroxybenzyl)oxy)-4-((3-phenylpropanoyl)oxy)phenyl)acrylic acid）を得た。化合物14は他のAKRアイソフォームに対して約3000倍の高い選択性を示した（図４）。また、化合物14はAKR1C3を高発現している肺がんA549細胞におけるステロイド代謝を有意に阻害し、その効果はバッカリンよりも強かった（図５）。さらに、化合物14はAKR1C3過剰発現細胞において、バッカリンと比較し72時間後の生細胞数を有意に減少させたことより、化合物14のバッカリンよりも強い細胞増殖抑制効果が示された（図６）。これまでにバッカリンがAKR1C3阻害活性を介してがん細胞増殖および浸潤に関与することを明らかにしている。化合物14は現時点で最も高い選択性を示すAKR1C3阻害剤であり、高い細胞増殖抑制効果を示すため、CRPCを含めたAKR1C3を高発現しているがんの治療薬の有効成分として有用といえる。 2. result
Various derivatives were synthesized using baccharin, which has been found as a selective inhibitor of AKR1C3, as a lead compound, and its inhibitory effect was evaluated (FIG. 3). In order to enable the synthesis of various compounds, the isoprenyl moiety (R2) of baccaroin (bottom of FIG. 3) was changed to ether. These 18 compounds are all novel compounds. As a result, compounds 6m and 6n were found out of the derivatives having ether, which showed the same inhibitory effect as baccalin, although the inhibitory effect was slightly reduced. Furthermore, a docking model of compound 6m to AKR1C3 was created, and based on the binding mode obtained from the model, compound 14 ((E) -3- (3-((3- hydroxybenzyl) oxy) -4-((3-phenylpropanoyl) oxy) phenyl) acrylic acid). Compound 14 showed about 3000 times higher selectivity for other AKR isoforms (FIG. 4). Compound 14 significantly inhibited steroid metabolism in lung cancer A549 cells that highly expressed AKR1C3, and the effect was stronger than that of baccalin (FIG. 5). Furthermore, Compound 14 significantly reduced the number of viable cells after 72 hours in AKR1C3 overexpressing cells compared to baccarain, indicating that the compound 14 had a stronger cell growth inhibitory effect than baccharin (FIG. 6). . So far, it has been clarified that baccalin is involved in cancer cell proliferation and invasion through AKR1C3 inhibitory activity. Compound 14 is an AKR1C3 inhibitor exhibiting the highest selectivity at the present time, and exhibits a high cell growth inhibitory effect. Therefore, it can be said that compound 14 is useful as an active ingredient of a therapeutic agent for cancers that highly express AKR1C3 including CRPC.

尚、新規に合成した各化合物について、AKRスーパーファミリーメンバー（AKR1B10、AKR1B1、AKR1C1、AKR1C2、AKR1C3、AKR1C4）に対する阻害活性を図８及び９に比較して示した。 In addition, about each newly synthesize | combined compound, the inhibitory activity with respect to AKR superfamily member (AKR1B10, AKR1B1, AKR1C1, AKR1C2, AKR1C3, AKR1C4) was shown compared with FIG.

世界保健機構の報告によると、全世界で新たながん患者は毎年1400万人以上増え、年間760万人が死亡している。中でもホルモン依存性がんである前立腺がんの日本における罹患率は欧米の10％程度と低いが、食の欧米化などに伴い増加している。これらホルモン依存性がんの治療にはホルモン療法が有効であるが、先天的に若しくは治療の過程でホルモン感受性が消失した去勢抵抗性前立腺がん(CRPC)についてはホルモン療法は全く効果がなく、新規抗がん剤の開発が必要である。近年、治療奏効率の優れた抗がん剤が上市されているが、その大半は抗体医薬である。抗体医薬はがんの原因タンパクに直接作用するために高い効果が得られる反面、大量生産が困難などの原因によるその高い薬価が問題となり、金銭上の理由から治療を選択できない患者も多く存在する。そのため、大量生産が容易であり、比較的安価である低分子医薬の開発が望まれる。 According to a World Health Organization report, there are more than 14 million new cancer patients every year, with 7.6 million deaths annually. In particular, the incidence of prostate cancer, which is a hormone-dependent cancer, in Japan is as low as 10% in Europe and the United States, but it is increasing with the westernization of food. Hormone therapy is effective for the treatment of these hormone-dependent cancers, but hormone therapy has no effect on castration-resistant prostate cancer (CRPC), which has lost hormone sensitivity either congenitally or in the course of treatment, New anticancer drugs need to be developed. In recent years, anticancer agents with excellent therapeutic response have been put on the market, most of which are antibody drugs. Antibody drugs can be highly effective because they act directly on cancer-causing proteins, but there are many patients who are unable to choose treatment due to financial reasons due to their high price due to difficulties in mass production. . Therefore, it is desired to develop a low molecular weight drug that is easy to mass-produce and relatively inexpensive.

本発明のAKR1C3阻害剤はAKR1C3を強力かつ特異的に阻害できるものであり、上記の現状を打開する新規がん治療を可能にする。本発明のAkR1C3阻害剤は化学合成も容易であり、大量生産にも適する。 The AKR1C3 inhibitor of the present invention can potently and specifically inhibit AKR1C3, and enables novel cancer treatment that overcomes the above-mentioned current situation. The AkR1C3 inhibitor of the present invention is easy to chemically synthesize and is suitable for mass production.

この発明は、上記発明の実施の形態及び実施例の説明に何ら限定されるものではない。特許請求の範囲の記載を逸脱せず、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。本明細書の中で明示した論文、公開特許公報、及び特許公報などの内容は、その全ての内容を援用によって引用することとする。 The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims. The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.

Claims

以下の化学式１で表されるAKR1C3阻害剤：

但し、式中のR3、R4、R5、R6、R7は、それぞれ独立して、水素原子、ヒドロキシ基、ハロゲン原子、又は置換基を有していてもよい炭化水素基である。 AKR1C3 inhibitor represented by the following chemical formula 1:

However, R1 in a formula is a hydroxyl group, an amino group, or a chlorine atom, R2 is represented by the following Chemical formula 2 or 3,

However, R 3, R 4, R 5, R 6 and R 7 in the formula are each independently a hydrogen atom, a hydroxy group, a halogen atom, or a hydrocarbon group which may have a substituent.

以下の化学式４で表される、請求項１に記載のAKR1C3阻害剤：

The AKR1C3 inhibitor according to claim 1, which is represented by the following chemical formula 4:

請求項１又は２に記載のAKR1C3阻害剤又はその薬理学的に許容可能な塩を有効成分として含有する、抗がん薬。 An anticancer drug comprising the AKR1C3 inhibitor according to claim 1 or 2 or a pharmacologically acceptable salt thereof as an active ingredient.

前立腺がん、乳がん、肝細胞がん、非小細胞肺がん又は白血病の治療又は予防に使用される、請求項３に記載の抗がん薬。 The anticancer drug according to claim 3, which is used for treatment or prevention of prostate cancer, breast cancer, hepatocellular carcinoma, non-small cell lung cancer or leukemia.

請求項３又は４に記載の抗がん薬を含有する食品組成物。 A food composition containing the anticancer drug according to claim 3 or 4.

がん患者に対して、請求項３又は４に記載の抗がん薬を治療上有効量投与するステップを含む、がんの治療又は予防法。 A method for treating or preventing cancer, comprising a step of administering a therapeutically effective amount of the anticancer drug according to claim 3 or 4 to a cancer patient.