JP2019167302A - Akr1c3 selective inhibitor and use therefor - Google Patents

Abstract

To provide a novel, potent and selective AKR1C3 inhibitor and an application thereof.SOLUTION: The present invention provides a novel AKR1C3 inhibitor composed of a 8-hydroxy chromene derivative. The AKR1C3 can be used for e.g. treating castration resistant prostate cancer.SELECTED DRAWING: None

Description

  The present invention relates to an AKR1C3 (Aldo-Keto Reductase Family 1 Member C3) inhibitor. Specifically, it relates to highly selective AKR1C3 inhibitors and their uses (anticancer drugs, cancer treatment, etc.).

  Recently, abiraterone (Zytiga (registered trademark)) and androgen receptor, selective inhibitors of CYP17 involved in androgen synthesis, as drugs for metastatic castration-resistant prostate cancer (CRPC) that have been treated with chemotherapy in Western countries and Asia The antagonist enzalutamide (XTANDI® has been approved and used. However, since abiraterone also inhibits glucocorticoid synthesis at the same time, co-prescription of glucocorticoid is required, and dietary AUC changes The need for fasting prescriptions has made it difficult to use in the clinic, and problems with Extange include high blood pressure, fatigue, loss of appetite, and vomiting.

On the other hand, since AKR1C3 is located downstream of CYP17 in androgen metabolism, targeting AKR1C3 can inhibit androgen synthesis without inhibiting glucocorticoid synthesis. In addition, AKR1C3 is involved not only in androgen synthesis, but also in the synthesis of cell proliferative prostanoids and isoprenoids. Therefore, AKR1C3 is a promising target for prostate cancer treatment regardless of whether it is hormone-dependent or independent. Many AKR1C3 inhibitors have been reported so far, and compounds exhibiting nM-level IC ₅₀ values have also been reported (see, for example, Non-Patent Document 1), but the effects on other AKR isoforms have been investigated. Absent. In addition, the research group of the present inventors has reported that baccharin has selective AKR1C3 inhibitory activity (Non-patent Document 2), and has also found a baccaline derivative as a new AKR1C3 inhibitor (Patent Document 1). .

Japanese Patent Laying-Open No. 2015-020966

Endo S. et al. Bioorg. Med. Chem. 2014, 22, 5220-5233. Endo S. et al. J. Nat. Prod. 2012, 75, 716-721.

  AKR1C1 and AKR1C2 are involved in the inactivation of 5α-dihydroandrosterone, which is similar to AKR1C3 in higher order structure but has high androgenic activity. Therefore, development of an AKR1C3 inhibitor that does not inhibit AKR1C1 and AKR1C2 is desired, but there are still few reports on an AKR1C3 inhibitor that has both selectivity and a strong inhibitory effect. Thus, an object of the present invention is to provide a powerful and selective novel AKR1C3 inhibitor and to apply it (for example, application to cancer treatment).

但し、式中のR1は水素原子、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］前記式中のR1が水素原子、フルオロ基、クロロ基、メチル基、エチル基、ヒドロキシ基（但し、2位及び4位を除く）、ジフルオロ基、トリフルオロ基、トリフルオロメチル基、ジトリフルオロメチル基又はイソプロピル基である、［１］に記載のAKR1C3阻害剤。
［３］前記式中のR1が水素原子、2-フルオロ、3-フルオロ、4-フルオロ、2-クロロ、3-クロロ、4-クロロ、2-メチル、3-メチル、4-メチル、2-エチル、3-エチル、4-エチル、3-ヒドロキシ、3,4-ジフルオロ、3,4,5-トリフルオロ、2-トリフルオロメチル、3-トリフルオロメチル、4-トリフルオロメチル、2,4-ジトリフルオロメチル、2-イソプロピル又は3-イソプロピルである、［１］に記載のAKR1C3阻害剤。
［４］以下の化学式２〜４のいずれかで表される、［１］に記載のAKR1C3阻害剤：

［５］［１］〜［４］のいずれか一項に記載の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。
［６］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、［５］に記載の抗がん薬。
［７］前立腺がんの治療又は予防に使用される、［５］に記載の抗がん薬。
［８］前立腺がんが、去勢抵抗性前立腺がんである、［７］に記載の抗がん薬。
［９］抗アンドロゲン薬と併用される、［７］又は［８］に記載の抗がん薬。
［１０］がん患者に対して、［５］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。
［１１］［１］〜［４］のいずれか一項に記載のAKR1C3阻害剤を含む研究用試薬。 Based on the results of previous studies, we focused on compounds with a chromene skeleton and attempted to optimize the structure using these compounds as lead compounds. Specifically, various derivatives were synthesized, and their AKR1C3 inhibitory activity, selectivity, effectiveness as anticancer drugs, etc. were examined. As a result, it was revealed that 8-hydroxychromene derivatives show potent and selective AKR1C3 inhibitory activity and are promising as anticancer drugs targeting AKR1C3 or candidates thereof. It should be noted that the 8-hydroxychromene derivative also exhibits an AR antagonistic action and can be expected to have a potent PSA lowering action compared to conventional AKR1C3 inhibitors. On the other hand, as a result of detailed studies, anti-cancer activity against prostate cancer in vivo (model animal experiment) was confirmed for representative compounds, and as an anti-cancer drug or candidate for prostate cancer (especially CRPC) In addition to supporting the effectiveness of 8-hydroxychromene derivatives, it is possible to enhance the therapeutic effect (inhibition of cancer cell growth) in combination with existing drugs and to obtain information useful for clinical application such as the mechanism of action. It was. The following invention is mainly based on the above results and considerations.
[1] AKR1C3 inhibitor represented by the following chemical formula 1:

However, R1 in the formula is a hydrogen atom, a hydroxy group, a halogen atom, or a hydrocarbon group which may have a substituent.
[2] R1 in the above formula is a hydrogen atom, a fluoro group, a chloro group, a methyl group, an ethyl group, a hydroxy group (except for the 2nd and 4th positions), a difluoro group, a trifluoro group, a trifluoromethyl group, The AKR1C3 inhibitor according to [1], which is a ditrifluoromethyl group or an isopropyl group.
[3] R1 in the above formula is a hydrogen atom, 2-fluoro, 3-fluoro, 4-fluoro, 2-chloro, 3-chloro, 4-chloro, 2-methyl, 3-methyl, 4-methyl, 2- Ethyl, 3-ethyl, 4-ethyl, 3-hydroxy, 3,4-difluoro, 3,4,5-trifluoro, 2-trifluoromethyl, 3-trifluoromethyl, 4-trifluoromethyl, 2,4 -The AKR1C3 inhibitor according to [1], which is ditrifluoromethyl, 2-isopropyl or 3-isopropyl.
[4] The AKR1C3 inhibitor according to [1], represented by any one of the following chemical formulas 2 to 4:

[5] An anticancer drug comprising the AKR1C3 inhibitor according to any one of [1] to [4] or a pharmacologically acceptable salt thereof as an active ingredient.
[6] The anticancer drug according to [5], which is used for treatment or prevention of prostate cancer, breast cancer, hepatocellular carcinoma, non-small cell lung cancer or leukemia.
[7] The anticancer drug according to [5], which is used for treatment or prevention of prostate cancer.
[8] The anticancer drug according to [7], wherein the prostate cancer is castration resistant prostate cancer.
[9] The anticancer drug according to [7] or [8], which is used in combination with an antiandrogen drug.
[10] A method for treating or preventing cancer, comprising a step of administering a therapeutically effective amount of the anticancer drug according to [5] to a cancer patient.
[11] A research reagent comprising the AKR1C3 inhibitor according to any one of [1] to [4].

The effect on the proliferative capacity of prostate cancer 22Rv1 cells. Effect on prostate androgen signal of prostate cancer 22Rv1 cells. Effect on androgen-independent prostate cancer PC3 cell proliferation. Assessment of androgen receptor agonist activity (A) and antagonist activity (B). The combined effect of an AKR1C3 inhibitor on the antiproliferative ability of CRPC therapeutics. Apoptosis-inducing effect by combined use of AKR1C3 inhibitor and enzalutamide. The enhancement effect of abiraterone-induced apoptosis by AKR1C3 inhibitor. Effect of AKR1C3 inhibitor 2l on CRPC therapeutic drug-induced apoptosis. In vivo antitumor effect by AKR1C3 inhibitor 2l.

  The first aspect of the present invention relates to an AKR1C3 inhibitor. An “AKR1C3 inhibitor” is an agent that inhibits or suppresses the activity of AKR1C3. AKR1C3, also known as 17β-hydroxysteroid dehydrogenase type 5, plays an important role in androgen synthesis and prostaglandin (PG) metabolism. AKR1C3 is highly expressed in cancerous lesions of patients with leukemia cells, hormone-dependent cancers (prostate cancer (including CRPC), breast cancer, etc.) (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.), Is involved in the promotion of cancer cell growth through steroid, prostanoid and isoprenoid metabolism. Increased expression of AKR1C3 has also 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.). Knocking down the AKR1C3 gene inhibits prostate cancer cell proliferation, whereas artificial overexpression of AKR1C3 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 exhibits 17β-hydroxysteroid dehydrogenase (HSD) activity, is expressed in prostate and adrenal glands and is involved in androgen synthesis, and is also expressed in estrogen sensitive tissues such as uterus and mammary gland and is also involved in estrogen synthesis. Therefore, the AKR1C3 inhibitor can be expected to have an effect on breast cancer, which is an estrogen-dependent disease, in addition to prostate cancer, which is an androgen-dependent disease. AKR1C3 is involved in androgen-independent cell growth by catalyzing prostaglandin (PG) metabolism and isoprenoid metabolism, as well as cancer cell survival by detoxifying and metabolizing reactive aldehydes produced during oxidative stress. Also involved. AKR1C3 has been attracting attention as a new target for leukemia because PGF2α produced from PGD2 by AKR1C3 is known to suppress differentiation and promote proliferation of leukemia cells (Trippier et al., Future Med. Chem. , 9, 1453-1456 (2017)). Therefore, the AKR1C3 inhibitor of the present invention inhibits androgen-dependent / independent cell growth, and thus becomes an active ingredient of an anticancer drug that can be applied to many cancer types.

Here, there are AKR1C1, AKR1C2 and AKR1C4 whose higher order structure is similar to AKR1C3. The AKR1C3 inhibitor of the present invention has higher specificity for AKR1C3 than these structurally similar enzymes. As used herein, “high specificity for AKR1C3” means that AKR1C3 is selectively inhibited among AKR1C1, AKR1C2, AKR1C3, and AKR1C4. Among these AKR1C subfamily, AKR1C1 exhibits 20α-hydroxysteroid dehydrogenase activity and is also involved in inactivation of progesterone, progesterone, inactivation of neurosteroids such as allopregnenolone in the brain and catabolism of precursors . In addition, AKR1C2 has 3α-hydroxysteroid dehydrogenase activity, generation of neurosteroid allopregnenolone from 5α-dihydroprogesterone and 5α-pregnane-17α-ol-3,20-dione to 5α-pregnane-3α, 17α- Involved in androgen production by reduction to diol-20-one. Therefore, in addition to controlling androgen levels in prostate cancer treatment, not having inhibitory activity against these similar enzymes is important from the viewpoint of reducing side effects by not inhibiting these physiological functions. is there. When compared with IC ₅₀ , the inhibitory activity against the AKR1C3 of the AKR1C3 inhibitor of the present invention is, for example, 100 times or more, preferably 200 times or more, more preferably 300 times or more, and still more preferably 500 times that of AKR1C1 or AKR1C2. It is more than twice, and more preferably more than 1,000 times.

但し、式中のR1は水素原子、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。 The AKR1C3 inhibitor of the present invention is represented by the following chemical formula 1.

However, R1 in the formula is 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.

  Preferably, R1 in the formula is a hydrogen atom, a fluoro group, a chloro group, a methyl group, an ethyl group, a hydroxy group (except for the 2-position and 4-position), a difluoro group, a trifluoro group, a trifluoromethyl group, a ditri A fluoromethyl group and an isopropyl group; If the alkyl group has too many carbon atoms, the activity may decrease due to steric hindrance, and a hydrophilic side chain is not desirable at the 2- and 4-positions.

  More preferably, R1 in the formula is a hydrogen atom, 2-fluoro, 3-fluoro, 4-fluoro, 2-chloro, 3-chloro, 4-chloro, 2-methyl, 3-methyl, 4-methyl, 2-methyl Ethyl, 3-ethyl, 4-ethyl, 3-hydroxy, 3,4-difluoro, 3,4,5-trifluoro, 2-trifluoromethyl, 3-trifluoromethyl, 4-trifluoromethyl, 2,4 -Ditrifluoromethyl, 2-isopropyl or 3-isopropyl.

Particularly preferred AKR1C3 inhibitors include the following three compounds (chemical formulas 2 to 4).

(Order in shown in the Examples below _{experiments, IC 50 = 25nM, IC 50} = 9.1nM, IC 50 = 21nM) These compounds strongly on to inhibit AKR1C3, high AKR1C3 compared to AKR1C1 or AKR1C2 Selectivity (in comparison with IC ₅₀ , approximately 240 times (vs. AKR1C1); 400 times or more (vs. AKR1C2), 1,100 times or more (vs. AKR1C1); 1,100 times or more (vs. AKR1C2); 480 times or more (vs. AKR1C2)).

  AKR1C3 is an important target for cancer treatment and is a target for the development of cancer treatments. The AKR1C3 inhibitor of the present invention is also useful as a tool (research reagent) in such research and development.

  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.

  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 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 for prostate cancer treatment regardless of whether it is hormone-dependent or independent. One of the typical targets of the anticancer drug of the present invention is prostate cancer, and particularly CRPC is a suitable target. The fact that the AKR1C3 inhibitor of the present invention has an AR antagonistic action and can also exert a powerful PSA lowering action indicates that it is extremely useful as a therapeutic agent for prostate cancer.

  “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.

  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.

  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.

  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.

  As shown in Examples described later, when the AKR1C3 inhibitor of the present invention was used in combination with an existing antiandrogen, its anticancer activity was enhanced. Based on this fact, in one aspect, the anticancer drug of the present invention is used in combination with an antiandrogen drug to enhance its therapeutic effect. The antiandrogen used in combination is not particularly limited. Examples of antiandrogens include bicalutamide, flutamide, chlormadinone acetate, enzalutamide, and abiraterone. Enzalutamide and abiraterone are approved drugs for CRPC, and combined use with the drug is effective as a treatment for CRPC.

  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.

<Method>
1. Synthesis of compounds

General synthesis of cyanoacetamide 1
Under Ar atmosphere, cyanoacetic acid (100 mg, 1.176 mmol) at room temperature CH ₂ Cl ₂ (5 mL) solution of aniline ^{(1.176 mmol), EDC. HCl} (450 mg, 2.352 mmol), DMAP (29 mg, 0.235 mmol) was sequentially added, and the mixture was stirred at room temperature for 24 hours. After completion of the reaction, the solvent was distilled off, and the resulting residue was purified by silica gel column chromatography (SiO ₂ : 10 g, hexane: acetone = 5: 1 to 1: 1) to obtain white crystals (1a-g ¹⁾ , 1h ²⁾ , 1i ³⁾ , 1j-k ²⁾ , 1l ⁴⁾ , 1m ⁵⁾ , 1n-p ¹⁾ , 1q ⁶⁾ , 1r ⁷⁾ , 1s ⁶⁾ , 1t ⁸⁾ , 1u ²⁾ , 1v , 1w ⁶⁾ , 1x ⁹⁾ ).
References: 1) Org. Biomol. Chem. 2015, 13, 7487. 2) J. Med. Chem. 2017, 60, 4626. 3) Bioorg. Med. Chem. Lett. 2009, 19, 1861. 4) Eur J. Chem. 2012, 3, 228. 5) ChemCatChem 2016, 8, 3420. 6) J. Med. Chem. 2012, 55, 7378. 7) Commercially available: Aurora Building Blocks, A02.108.441. 8) Arch Pharm. Res. 2003, 26, 197. 9) Commercially available: Aurora Building Blocks, A00.271.854.

N- (2,4-Bis-trifluoromethylphenyl) -2-cyanoacetamide (1v)
Yield: 51%; ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 4.01 (2H, s), 7.85 (1H, d, J = 8.4 Hz), 8.08 (1H, s), 8.12 (1H, d, J = 8.4 Hz), 10.26 (1H, s)

General synthesis of chromene 2
Under an Ar atmosphere, 2,3-dihydroxybenzaldehyde (0.432 mmol) and piperidine (3 drops) were added sequentially to a solution of the amide compound (1a-x, 0.432 mmol) in EtOH (3 mL) at room temperature. Stir for hours. After completion of the reaction, the precipitated crystals were collected by filtration and then air-dried until the next day to obtain pale yellow crystals 2a-x.

8-Hydroxy-2-imino-2H-chromene-3-carboxylic acid phenylamide (2a)
Yield: 82%; mp: 235-237 ° C; ¹ H-NMR (500 MHz, DMSO-d ₆ ) δ 7.04-7.14 (3H, m), 7.23 (1H, dd, J = 4.0, 3.0 Hz), 7.37 (1H, t, J = 8.5 Hz), 7.67 (2H, dd, J = 7.5, 1.0 Hz), 8.50 (1H, d, J = 1.5 Hz), 9.09 (1H, s), 10.23 (1H, br ); ¹³ C-NMR (125 MHz, DMSO-d ₆ ) δ 119.9, 120.1, 120.2, 120.4, 120.5, 124.5, 124.6, 129.6, 138.8, 142.5, 142.6, 144.5, 156.5, 160.3; IR (KBr): 3298 , 1674, 1598, 1473 cm ^-1 ; MS (EI): m / z 280 (M ⁺ ); HRMS: Calcd for C ₁₆ H ₁₂ N ₂ O ₃ 280.0848, Found 280.0847.

8-Hydroxy-2-imino-2H-chromene-3-carboxylic acid (2-fluorophenyl) amide (2b)
Yield: 82%; mp: 247-249 ° C; ¹ H-NMR (500 MHz, DMSO-d ₆ ) δ 7.10-7.17 (3H, m), 7.22-7.27 (2H, m), 7.31-7.35 (1H , m), 8.48 (1H, td, J = 3.5, 1.5 Hz), 8.55 (1H, d, J = 1.5 Hz), 9.14 (1H, br); ¹³ C-NMR (125 MHz, DMSO-d ₆ ) δ 115.6 (d, J = 23.1 Hz), 119.8, 120.2 (d, J = 23.1 Hz), 120.7, 121.9, 124.6, 125.0 (d, J = 8.5 Hz), 125.2 (d, J = 3.6 Hz), 127.0 (d, J = 8.5 Hz), 142.5, 143.1, 144.5, 153.0 (d, J = 241.8 Hz), 156.3, 160.6; IR (KBr): 3310, 1607, 1569, 1473 cm ^-1 ; MS (EI): m / z 298 (M ⁺ ); HRMS: Calcd for C ₁₆ H ₁₁ FN ₂ O ₃ 298.0754, Found 298.0754.

8-Hydroxy-2-imino-2H-chromene-3-carboxylic acid (3-fluorophenyl) amide (2c)
Yield: 51%; mp: 225-227 ° C; ¹ H-NMR (500 MHz, DMSO-d ₆ ) δ 6.96 (1H, m), 7.06-7.11 (2H, m), 7.22 (1H, dd, J = 4.5, 2.5 Hz), 7.28 (1H, d, J = 9.0 Hz), 7.39 (1H, q, J = 7.0 Hz), 7.74 (1H, d, J = 9.0 Hz), 8.50 (1H, s), 9.10 (1H, br), 10.22 (1H, br); ¹³ C-NMR (125 MHz, DMSO-d ₆ ) δ 107.0 (d, J = 23.1 Hz), 110.0 (d, J = 23.1 Hz), 116.0, 119.8, 120.1, 120.3, 120.6, 124.6 (d, J = 1.3 Hz), 131.1 (d, J = 10.3 Hz), 140.4 (d, J = 10.3 Hz), 142.5, 142.9, 144.5, 156.4, 160.7, 162.8 ( d, J = 239.4 Hz); IR (KBr): 3305, 1609, 1579, 1473 cm ^-1 ; MS (EI): m / z 298 (M ⁺ ); HRMS: Calcd for C ₁₆ H ₁₁ FN ₂ O ₃ 298.0754, Found 298.0754.

8-Hydroxy-2-imino-2H-chromene-3-carboxylic acid (4-fluorophenyl) amide (2d)
Yield: 65%; mp: 240-242 ° C; ¹ H-NMR (500 MHz, DMSO-d ₆ ) δ 7.09-7.10 (2H, m), 7.22-7.24 (3H, m), 7.71 (1H, q , J = 5.0 Hz), 8.50 (1H, s), 9.09 (1H, br); ¹³ C-NMR (125 MHz, DMSO-d ₆ ) δ 116.1 (d, J = 23.1 Hz), 119.8, 120.2, 120.3 , 120.5, 121.9 (d, J = 7.4 Hz), 124.6 (d, J = 3.6 Hz), 135.2, 142.4, 142.6, 144.5, 156.4, 158.9 (d, J = 239.0 Hz), 160.3; IR (KBr): 3298, 1674, 1511,1218 cm ^-1 ; MS (EI): m / z 298 (M ⁺ ); HRMS: Calcd for C ₁₆ H ₁₁ FN ₂ O ₃ 298.0754, Found 298.0754.

8-Hydroxy-2-imino-2H-chromene-3-carboxylic acid (2-chlorophenyl) amide (2e)
Yield: 67%; mp: 282-283 ° C; ¹ H-NMR (500 MHz, DMSO-d ₆ ) δ; 7.08-7.13 (2H, m), 7.16 (1H, td, J = 6.5, 1.5 Hz) , 7.24 (1H, dd, J = 5.0, 2.0 Hz), 7.39 (1H, td, J = 7.0, 1.0 Hz), 7.54 (1H, dd, J = 6.5, 1.5 Hz), 8.52 (1H, dd, J = 7.0, 1.0 Hz), 8.55 (1H, s), 9.13 (1H, s); ¹³ C-NMR (125 MHz, DMSO-d ₆ ) δ 119.8, 120.2, 120.4, 120.7, 122.6, 123.6, 124.6, 125.5 , 128.2, 129.9, 136.0, 142.5, 143.3, 144.5, 156.0, 160.8; IR (KBr): 3305, 1684, 1471, 1224 cm ^-1 ; MS (EI): m / z 314 (M ⁺ ); HRMS: Calcd for C ₁₆ H ₁₁ ClN ₂ O ₃ 314.0458, Found: 314.0459.

8-Hydroxy-2-imino-2H-chromene-3-carboxylic acid (3-chlorophenyl) amide (2f)
Yield: 65%; mp: 252-254 ° C; ¹ H-NMR (500 MHz, DMSO-d ₆ ) δ 7.07 (2H, m), 7.18 (1H, d, J = 8.0 Hz), 7.23 (1H, d, J = 8.0 Hz), 7.39 (1H, t, J = 8.0 Hz), 7.44 (1H, t, J = 8.0 Hz), 7.95 (1H, s), 8.50 (1H, s), 9.12 (1H, s); ¹³ C-NMR (125 MHz, DMSO-d ₆ ) δ 118.6, 119.6, 119.8, 120.1, 120.3, 120.6, 124.2, 124.6, 131.2, 133.9, 140.1, 142.5, 142.9, 144.5, 156.4, 160.7; IR (KBr): 3301, 1681, 1595, 1482 cm ^-1 ; MS (EI): m / z 314 (M ⁺ ); HRMS: Calcd for C ₁₆ H ₁₁ ClN ₂ O ₃ 314.0458, Found: 314.0458.

8-Hydroxy-2-imino-2H-chromene-3-carboxylic acid (4-chlorophenyl) amide (2g)
Yield: 91%; mp: 272-273 ° C; ¹ H-NMR (500 MHz, DMSO-d ₆ ) δ 7.07-7.11 (2H, m), 7.23 (1H, dd, J = 5.0, 2.5 Hz), 7.43 (1H, dd, J = 5.0, 2.0 Hz), 7.70 (1H, dd, J = 5.0, 2.0 Hz), 8.50 (1H, d, J = 1.5 Hz), 9.11 (1H, s); ¹³ C- NMR (125 MHz, DMSO-d ₆ ) δ 119.8, 120.2, 120.3, 120.6, 121.7, 124.6, 128.1, 129.5, 137.7, 142.5, 142.7, 144.5, 156.4, 160.5; IR (KBr): 3302, 1675, 1473, 1230 cm ^-1 ; MS (EI): m / z 314 (M ⁺ ); HRMS: Calcd for C ₁₆ H ₁₁ ClN ₂ O ₃ 314.0458, Found: 314.0459.

8-Hydroxy-2-imino-2H-chromene-3-carboxylic acid (2-methylphenyl) amide (2h)
Yield: 90%; mp: 263-264 ° C; ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 2.37 (3H, s), 7.08-7.16 (3H, m), 7.25-7.31 (3H, m ), 8.25 (1H, d, J = 7.6 Hz), 8.52 (1H, d, J = 1.2 Hz), 9.11 (1H, s), 12.62 (1H, s); ¹³ C-NMR (125 MHz, DMSO- d ₆ ) δ 18.4, 119.4, 119.6, 120.0, 120.2, 120.8, 124.0, 124.1, 126.3, 127.8, 130.3 137.0, 141.9, 142.2, 144.0, 156.0, 159.7; IR (KBr): 3299, 1682, 1593, 1470 cm ^-1 ; MS (EI): m / z 294 (M ⁺ ); HRMS: calcd for C ₁₇ H ₁₄ N ₂ O ₃ 294.1004, found 294.1008.

8-Hydroxy-2-imino-2H-chromene-3-carboxylic acid (3-methylphenyl) amide (2i)
Yield: 89%; mp: 241-242 ° C; ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 2.28 (3H, s), 6.93 (1H, d, J = 7.3 Hz), 7.08-7.11 ( 2H, m), 7.20 (1H, d, J = 7.3 Hz), 7.26 (1H, d, J = 7.3 Hz), 7.43 (1H, s), 7.52 (1H, d, J = 7.3 Hz), 8.49 ( 1H, d, J = 2.5 Hz), 9.09 (1H, s), 12.81 (1H, s); ¹³ C-NMR (125 MHz, DMSO-d ₆ ) δ 21.1, 116.7, 119.4, 119.7, 120.0, 124.1, 124.8, 128.9, 138.2, 138.4 142.0, 142.1, 144.0, 156.0, 159.7; IR (KBr): 3295, 1678, 1606, 1467 cm ^-1 ; MS (EI): m / z 294 (M ⁺ ); HRMS: calcd for C ₁₇ H ₁₄ N ₂ O ₃ 294.1004, found 294.1001.

8-Methoxy-2-imino-2H-chromene-3-carboxylic acid (4-methylphenyl) amide (2j)
Yield: 89%; mp: 273-274 ° C; ¹ H-NMR (500 MHz, CDCl ₃ ) δ 2.36 (3H, s), 7.20 (2H, d, J = 8.1 Hz), 7.30-7.32 (3H, m), 7.63 (2H, d, J = 8.1 Hz), 9.04 (1H, s), 10.63 (1H, br); ¹³ C-NMR (125 MHz, DMSO-d ₆ ) δ 21.0, 119.9, 120.1, 120.2 , 120.5, 124.6, 130.0, 133.6, 136.3, 142.4, 142.5, 143.5, 144.5, 156.5, 160.0; IR (KBr): 3292, 1674, 1564, 1472 cm ^-1 ; MS (EI): m / z 294 (M ⁺ ); HRMS: calcd for C ₁₇ H ₁₄ N ₂ O ₃ 294.1004, found 294.1013.

8-Hydroxy-2-imino-2H-chromene-3-carboxylic acid (2-ethylphenyl) amide (2k)
Yield: 66%; mp: 209-210 ° C; ¹ H-NMR (500 MHz, DMSO-d ₆ ) δ 1.13 (3H, t, J = 7.7 Hz), 2.66 (2H, q, J = 7.7 Hz) , 7.04-7.07 (3H, m), 7.16-7.23 (3H, m), 8.16 (1H, d, J = 8.5 Hz), 8.47 (1H, d, J = 1.5 Hz), 9.06 (1H, s), 12.61 (1H, s); ¹³ C-NMR (125 MHz, DMSO-d ₆ ) δ 15.6, 28.2, 117.0, 118.9, 119.4, 119.6, 120.0, 120,1, 123.6, 124.1, 129.0, 138.3, 141.9, 142.0 , 144.0, 144.7, 155.9, 159.7; IR (KBr): 3297, 1669, 1569, 1472 cm ^-1 ; MS (EI): m / z 308 (M ⁺ ); HRMS: calcd for C ₁₈ H ₁₆ N ₂ O ₃ 308.1161, found 308.1153.

8-Hydroxy-2-imino-2H-chromene-3-carboxylic acid (3-ethylphenyl) amide (2l)
Yield: 78%; mp: 241-242 ° C; ¹ H-NMR (400 MHz, CDCl ₃ ) δ 1.14 (3H, t, J = 7.7 Hz), 2.65 (2H, q, J = 7.7 Hz), 6.99 (1H, d, J = 8.0 Hz), 7.11-7.12 (3H, m), 7.28 (1H, d, J = 8.0 Hz), 7.56 (1H, d, J = 8.0 Hz), 7.57 (1H, s) , 8.60 (1H, br), 12.36 (1H, br); ¹³ C-NMR (125 MHz, DMSO-d ₆ ) δ 14.5, 24.6, 119.4, 119.6, 120.0, 120.1, 121.5, 124.1, 124.3, 126.3, 128.8 , 134.0, 136.1, 141.9, 142.2, 144.0, 156.0, 159.8; IR (KBr): 3298, 1684, 1594, 1471 cm ^-1 ; MS (EI): m / z 308 (M ⁺ ); HRMS: calcd for C ₁₈ H ₁₆ N ₂ O ₃ 308.1161, found 308.1153.

8-Hydroxy-2-imino-2H-chromene-3-carboxylic acid (4-ethylphenyl) amide (2m)
Yield: 81%; mp: 259-260 ° C; ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 1.18 (3H, t, J = 7.9 Hz), 2.58 (2H, q, J = 7.9 Hz) , 7.08-7.10 (2H, m), 7.20-7.24 (3H, m), 7.59 (2H, d, J = 8.5 Hz), 8.49 (1H, d, J = 1.7 Hz), 9.08 (1H, s), 12.78 (1H, s); ¹³ C-NMR (125 MHz, DMSO-d ₆ ) δ 15.6, 27.6, 119.4, 119.6, 120.0, 120.8, 124.1, 124.1, 128.3, 136.0, 139.5, 141.9, 144.0, 146.1, 156.0 , 159.1; IR (KBr): 3296, 1669, 1599, 1473 cm ^-1 ; MS (EI): m / z 308 (M ⁺ ); HRMS: calcd for C ₁₈ H ₁₆ N ₂ O ₃ 308.1161, found 308.1153.

8-Hydroxy-2-imino-2H-chromene-3-carboxylic acid (2-hydroxyphenyl) amide (2n)
Yield: 48%; mp: 253-255 ° C; ¹ H-NMR (500 MHz, DMSO-d ₆ ) δ 6.80 (1H, td, J = 6.0, 2.0 Hz), 6.88-6.93 (2H, m), 7.21 (1H, dd, J = 4.5, 2.5 Hz), 8.37 (1H, d, J = 8.0 Hz), 8.48 (1H, s), 8.98 (1H, s), 9.96 (1H, br), 10.20 (1H , br); ¹³ C-NMR (125 MHz, DMSO-d ₆ ) δ 115.23, 119.5, 119.97, 120.05, 120.5, 121.0, 124.5, 124.6, 127.5, 142.0, 142.4, 142.5, 144.4, 147.7, 155.8, 160.1; IR (KBr): 3313, 1668, 1558, 1471 cm ^-1 ; MS (EI): m / z 296 (M ⁺ ); HRMS: Calcd for C ₁₆ H ₁₂ N ₂ O ₄ 296.0797, Found: 296.0798.

8-Hydroxy-2-imino-2H-chromene-3-carboxylic acid (3-hydroxyphenyl) amide (2o)
Yield: 40%; mp: 202-204 ° C; ¹ H-NMR (500 MHz, DMSO-d ₆ ) δ 6.52 (1H, dd, J = 4.5, 2.5 Hz), 6.97 (1H, dd, J = 7.0 , 1.0 Hz), 7.08-7.11 (2H, m), 7.14 (1H, t, J = 8.0 Hz), 7.22 (1H, dd, J = 4.5, 2.5 Hz), 7.28 (1H, t, J = 2.0 Hz ), 8.48 (1H, s), 9.08 (1H, s), 9.50 (1H, br), 10.20 (1H, br); ¹³ C-NMR (125 MHz, DMSO-d ₆ ) δ 107.1, 110.8, 111.7, 119.9, 120.2, 120.5, 120.6, 124.6, 130.3, 139.8, 142.5, 144.5, 156.5, 158.4, 160.1; IR (KBr): 3303, 1676, 1577, 1473 cm ^-1 ; MS (EI): m / z 296 ( M ⁺ ); HRMS: Calcd for C ₁₆ H ₁₂ N ₂ O ₃ 296.0797, Found: 296.0799.

8-Hydroxy-2-imino-2H-chromene-3-carboxylic acid (4-hydroxyphenyl) amide (2p)
Yield: 46%; mp: 231-233 ° C; ¹ H-NMR (500 MHz, DMSO-d ₆ ) δ 6.76 (2H, d, J = 7.8 Hz), 7.06-7.10 (2H, m), 7.19- 7.23 (1H, m), 7.47 (2H, d, J = 7.8 Hz), 8.46 (1H, s), 9.03 (1H, s), 9.34 (1H, br), 12.58 (1H, br); ¹³ C- NMR (125 MHz, DMSO-d ₆ ) δ 116.0, 119.96, 120.03, 120.5, 120.7, 121.7, 124.6, 130.5, 142.1, 142.4, 144.5, 154.5, 156.5, 159.6; IR (KBr): 3287, 1669, 1513, 1473 cm ^-1 ; MS (EI): m / z Calcd for C ₁₆ H ₁₂ N ₂ O ₄ 296.0797, Found: 296.0796.

8-Hydroxy-2-imino-2H-chromene-3-carboxylic acid (3,4-difluorophenyl) amide (2q)
Yield: 83%; mp: 279-281 ° C; ¹ H-NMR (500 MHz, DMSO-d ₆ ) δ 7.07-7.12 (2H, m), 7.24 (1H, dd, J = 7.0, 2.5 Hz), 7.32-7.34 (1H, m), 7.44 (1H, q, J = 9.0 Hz), 7.95 (1H, ddd, J = 10.1, 7.5, 2.3 Hz), 8.50 (1H, d, J = 1.0 Hz), 9.11 (1H, s); ¹³ C-NMR (125 MHz, DMSO-d ₆ ) δ 109.3 (d, J = 22.0 Hz), 116.1, 117.2, 117.8 (d, J = 18.3 Hz), 118.6 (d, J = 15.9 Hz), 119.9 (dd, J = 33.0, 7.3 Hz), 121.2, 124.1, 125.3, 142.2, 142.5, 142.7, 143.5, 144.5, 156.4, 160.7; IR (KBr): 3301, 1679, 1517, 1473 cm - ¹ ; MS (EI): m / z 316 (M ⁺ ); HRMS: calcd for C ₁₆ H ₁₀ F ₂ N ₂ O ₃ 316.0659, found 316.0654.

8-Hydroxy-2-imino-2H-chromene-3-carboxylic acid (3,4,5-trifluorophenyl) amide (2r)
Yield: 83%; mp: 277-279 ° C; ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 7.08-7.12 (2H, m), 7.23 (1H, dd, J = 6.7, 2.4 Hz), 7.65 (2H, dd, J = 10.1, 6.5 Hz), 8.50 (1H, s), 9.13 (1H, s), 10.24 (1H, s), 13.12 (1H, s); ¹³ C-NMR (125 MHz, DMSO-d ₆ ) δ 104.7, 104.9, 119.7, 120.5, 120.7, 124.7, 134.8, 142.5, 143.2, 144.5, 149.8, 156.2, 161.1; IR (KBr): 3306, 1680, 1531, 1473 cm ^-1 ; MS ( (EI): m / z 334 (M ⁺ ); HRMS: calcd for C ₁₆ H ₉ F ₃ N ₂ O ₃ 334.0565, found 334.0560.

8-Hydroxy-2-imino-2H-chromene-3-carboxylic acid (2-trifluoromethylphenyl) amide (2s)
Yield: 64%; mp: 244-245 ° C; ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 7.07-7.14 (2H, m), 7.25 (1H, dd, J = 6.7, 2.4 Hz), 7.38 (1H, t, J = 8.0 Hz), 7.72 (1H, t, J = 8.0 Hz), 7.76 (1H, d, J = 8.0 Hz), 8.21 (1H, d, J = 8.0 Hz), 8.55 ( 1H, d, J = 1.2 Hz), 9.16 (1H, s), 10.25 (1H, br), 13.10 (1H, s); ¹³ C-NMR (125 MHz, DMSO-d ₆ ) δ 119.5, 119.6, 120.1 , 120.5, 120.8, 124.4, 125.2, 125.7, 126.3, 133.4, 135.5, 142.3, 143.2, 144.2, 155.7, 160.9; IR (KBr): 3294, 1677, 1589, 1469 cm ^-1 ; MS (EI): m / z 348 (M ⁺ ); HRMS: calcd for C ₁₇ H ₁₁ F ₃ N ₂ O ₃ 348.0722, found 348.0732.

8-Hydroxy-2-imino-2H-chromene-3-carboxylic acid (3-trifluoromethylphenyl) amide (2t)
Yield: 71%; mp: 256-257 ° C; ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 7.06-7.15 (2H, m), 7.24 (1H, dd, J = 6.1, 2.5 Hz), 7.49 (1H, d, J = 8.0 Hz), 7.62 (1H, t, J = 8.0 Hz), 7.79 (1H, d, J = 8.0 Hz), 8.22 (1H, s), 8.52 (1H, s), 9.18 (1H, s), 13.20 (1H, s); ¹³ C-NMR (125 MHz, DMSO-d ₆ ) δ 115.7, 119.3, 119.6, 119.9, 120.2, 122.7, 123.3, 124.2, 129.6, 130.0, 130.3, 139.0, 142.0, 142.5, 144.0, 155.9, 160.4; IR (KBr): 3282, 1668, 1575, 1470 cm ^-1 ; MS (EI): m / z 348 (M ⁺ ); HRMS: calcd for C ₁₇ H ₁₁ F ₃ N ₂ O ₃ 348.0722, found 348.0732.

8-Hydroxy-2-imino-2H-chromene-3-carboxylic acid (4-trifluoromethylphenyl) amide (2u)
Yield: 78%; mp: 271-272 ° C; ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 7.07-7.13 (2H, m), 7.25 (1H, dd, J = 6.7, 2.4 Hz), 7.74 (2H, d, J = 8.6 Hz), 7.88 (2H, d, J = 8.6 Hz), 8.53 (1H, d, J = 1.8 Hz), 9.17 (1H, s), 13.24 (1H, s); ¹³ C-NMR (125 MHz, DMSO-d ₆ ) δ 119.3, 119.5, 119.6, 119.9, 120.2, 124.1, 126.3, 141.7, 142.0, 144.0, 155.9, 160.4; IR (KBr): 3335, 1687, 1579, 1474 cm ^-1 ; MS (EI): m / z 348 (M ⁺ ); HRMS: calcd for C ₁₇ H ₁₁ F ₃ N ₂ O ₃ 348.0722, found 348.0732.

8-Hydroxy-2-imino-2H-chromene-3-carboxylic acid (2,4-ditrifluoromethylphenyl) amide (2v)
Yield: 60%; mp: 236-237 ° C; ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 7.10-7.15 (2H, m), 7.27 (1H, dd, J = 7.0, 2.1 Hz), 8.04 (1H, s), 8.12 (1H, d, J = 8.6 Hz), 8.59 (1H, s), 8.62 (1H, d, J = 8.6 Hz), 9.23 (1H, s), 13.61 (1H, s ); ¹³ C-NMR (125 MHz, DMSO-d ₆ ) δ 118.9, 119.2, 119.6, 120.0, 120.1, 120.3, 122.0, 123.3, 124.2, 124.6, 125.0, 130.3, 139.2, 142.1, 143.5, 144.0, 155.5, 160.0; IR (KBr): 3333, 1680, 1561, 1470 cm ^-1 ; MS (EI): m / z 416 (M ⁺ ); HRMS: calcd for C ₁₈ H ₁₀ F ₆ N ₂ O ₃ 416.0596, found 416.0600 .

8-Hydroxy-2-imino-2H-chromene-3-carboxylic acid (2-isopropylphenyl) amide (2w)
Yield: 76%; mp: 235-236 ° C; ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 1.38 (6H, d, J = 6.7 Hz), 3.39 (1H, sept, J = 6.7 Hz) , 7.25-7.49 (4H, m), 7.51 (1H, dd, J = 8.0, 1.5 Hz), 8.24 (1H, dd, J = 8.0, 1.5 Hz), 8.69 (1H, d, J = 1.5 Hz), 9.26 (1H, s), 12.78 (1H, s); ¹³ C-NMR (125 MHz, DMSO-d ₆ ) δ 22.9, 27.5, 119.4, 119.6, 120.0, 120.2, 122.6, 124.1, 124.8, 125.3, 126.0, 135.2, 139.0, 142.0, 142.2, 144.0, 156.0, 160.0; IR (KBr): 3305, 1676, 1564, 1474 cm ^-1 ; MS (EI): m / z 322 (M ⁺ ); HRMS: calcd for C ₁₉ H ₁₈ N ₂ O ₃ 322.1317, found 322.1321.

8-Hydroxy-2-imino-2H-chromene-3-carboxylic acid (3-isopropylphenyl) amide (2x)
Yield: 80%; mp: 235-236 ° C; ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 1.21 (6H, d, J = 6.7 Hz), 2.90 (1H, sept, J = 6.7 Hz) , 7.02 (1H, d, J = 8.0 Hz), 7.08-7.10 (2H, m), 7.22 (1H, dd, J = 6.1, 3.1 Hz), 7.29 (1H, t, J = 8.0 Hz), 7.47 ( 1H, s), 7.55 (1H, d, J = 8.0 Hz), 8.49 (1H, d, J = 1.2 Hz), 9.08 (1H, s), 12.81 (1H, s); ¹³ C-NMR (125 MHz , DMSO-d ₆ ) δ 23.8, 33.4, 117.2, 117.5, 119.4, 119.6, 120.0, 122.1, 124.1, 129.0, 138.3, 141.9, 142.0, 144.0, 149.4, 155.9, 159.7; IR (KBr): 3296, 1667, 1569, 1470 cm ^-1 ; MS (EI): m / z 322 (M ⁺ ); HRMS: calcd for C ₁₉ H ₁₈ N ₂ O ₃ 322.1317, found 322.1321.

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. After inoculating the E. coli in 1 L of LB culture medium and culturing at 37 ° C until the turbidity at 600 nm becomes 0.4-0.6, isopropyl-β-D-galactopyranoside (IPTG) is brought 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 min, 4 ° C), and 10 mM Tris-HCl (pH) containing 0.5 mM EDTA and 5 mM 2-mercaptoethanol (2-ME). 8.0). This 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 the coomassie brilliant blue (CBB) R250 staining after analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

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 Shirashi et al. [Shiraishi et al., Biochem. J., 334, 399. -405].

4. Measurement of AKR1C3 inhibitory activity
The dehydrogenase activity of AKR1C isoforms (AKR1C1, AKR1C2, AKR1C3) was measured spectrophotometrically (Ex. 340 nm, Em. 455 nm) for the production rate of NADPH in the following reaction system. Standard reaction system contains 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 values of the inhibitor are 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 In the 2.0 mL reaction system, it calculated from the inhibition rate when the inhibitor of 5 different concentrations was added. These inhibition constants were expressed as the mean value ± standard deviation of at least 3 measurements.

5. Cell culture
CWR22Rv1, PC3 and SC3 cells were cultured in a carbon dioxide incubator under conditions of 37 ° C. and 5% CO ₂ . As growth medium, CWR22Rv1 cells consist of 10% (v / v) FBS, 100 U / ml penicillin-G potassium, 100 μg / ml streptomycin sulfate and 10 mM [4- (2-hydroxyethyl) -1-piperazineethane sulfonic acid] HEPES RPMI1640 containing a buffer solution (pH 7.0) was used. As a growth medium for PC3 and SC3 cells, DMEM (pH 7.4) containing 10% (v / v) FBS, 100 U / ml penicillin-G potassium and 100 μg / ml streptomycin sulfate was used. For cell detachment, Dulbecco's phosphate buffered saline (DPBS) (pH 7.4) containing 0.25% trypsin and 0.02% ethylenediaminetetraacetic acid (EDTA) was used.

6. Measurement of cell growth ability and evaluation of agonist activity and antagonist activity for androgen receptor Seed cells suspended in growth medium in 3 × 10 ⁴ cells / 300 µL each in a 48-well multiplate and place in a CO ₂ incubator. Cultured overnight. CWR22Rv1 cells contain antibiotics and 2% charcoal stripped fetal bovine serum (CS-FBS) and are replaced with phenol red-free RPMI medium. PC3 cells have antibiotics and 2% FBS The culture medium was replaced with a medium containing 2 hours and cultured for 2 hours. After adding the sample to the medium and further culturing for 24 hours, the number of viable cells after 24 and 72 hours was measured according to the measurement of cell viability. The absorbance of MTT after each treatment time was standardized using human lymphoma-derived U937 cells, and the number of viable cells was calculated. SC-3 cells expressing wild-type androgen receptor were used for evaluation of agonist activity and antagonist activity against androgen receptor. SC-3 cells suspended in a growth medium were seeded at 4 × 10 ⁴ cells / 2 mL each in a 3.5 mm dish and cultured overnight in a CO ₂ incubator. Replace with RPMI medium containing antibiotics and 2% CS-FBS, and without phenol red. After 2 hours of culture, add samples of various concentrations to the medium and further culture for 24 hours. It was measured according to the measurement of cell viability.

7. Measurement of PSA mRNA expression level Total RNA was isolated from cells using TRIzol reagent and RNAlater reagent. Single-stranded complementary DNA (cDNA) was prepared using ReverTra Ace qPCR RT Master Mix (Toyobo). Using the prepared cDNA (5μg) as a template and using PSA specific primers (Forward primer: 5'-CCTCCTGAAGAATCGATTCCT-3 '(SEQ ID NO: 1) and reverse primer: 5'-GAGGTCCACACACTGAAGTT-3' (SEQ ID NO: 2)) Quantitative PCR (qPCR) was performed using THUNDERBIRD SYBR qPCR Mix (Toyobo). As an internal standard, human β-actin cDNA was amplified, and the Takara Thermal Cycler Dice Real-Time PCR System was used for qPCR.

8. Measurement of the amount of testosterone Cells suspended in a growth medium were seeded at 3 × 10 ⁴ cells / 200 μL in a 96-well multiplate and cultured overnight in a CO ₂ incubator. The medium was replaced with a phenol red-free medium containing antibiotics and 2% CS-FBS, and cultured for 2 hours. The sample was added to the medium, and further cultured for 24 hours, and the medium was collected. The amount of testosterone in the medium was measured using Testosterone EIA kit (Cayman).

9. Measurement of the amount of AR in the nucleus The treated cells were washed twice with DPBS, and then detached using a cell scraper. The collected cells were centrifuged (1,500 × g, 3 minutes), the supernatant was removed, and buffer A [1 M HEPES (pH 7.9), 1 M KCl, 0.5 M EDTA, 1 M dithiothreitol (DTT), 100 The suspension was suspended in 300 μl of mM phenylmethylsulfonyl fluoride (PMSF) and allowed to stand for 15 minutes under ice cooling. Next, 10% NP-40 was added, and centrifugation (500 × g, 3 minutes) was performed. After removing the supernatant, the suspension was suspended in 600 μl of buffer A and centrifuged (500 × g, 1 minute). After removing the supernatant, the suspension was suspended in 80 μl of buffer B [1 M HEPES (pH 7.9), 5 M NaCl, 0.5 M EDTA, 1 M DTT, 100 mM PMSF], and allowed to stand for 15 minutes under ice cooling. Next, the suspension is centrifuged (15,000 xg, 15 minutes), the supernatant is removed, and a Urea buffer containing 8 M Urea, 10 mM Tris (hydroxymethyl) aminomethane, 50 mM Na ₂ H ₂ PO ₄ is added. The suspension was used as a nuclear extract. The amount of AR in the nucleus was measured by Western blot using an antiandrogen receptor (AR) antibody.

10. DNA fragmentation Cells were washed 3 times with DPBS, and then detached using a cell scraper. The collected cells were dissolved in 100 μL of cell lysis buffer [1 M Tris-HCl (pH 7.4), 0.5 M EDTA (pH 8.0), 10% Triton X-100], and the DNA fragment was extracted. After standing at 4 ° C for 10 minutes, 2 μL of RNase A solution dissolved in 10 mg / ml TE buffer [1M Tris-HCl (pH 7.4), 0.5 M EDTA (pH 8.0)] is added to the supernatant obtained by centrifugation. In addition, it was incubated at 37 ° C. for 60 minutes. Next, 2 μL of 10 mg / ml proteinase K solution was added and incubated at 50 ° C. for 30 minutes, then 5 μL and 120 μL of 5 M NaCl and isopropanol were added and left at −30 ° C. overnight. After centrifugation, the isolated DNA was dissolved in 20 μL of TE buffer, electrophoresed on a 2% agarose gel, stained with ethidium bromide, and detected under UV irradiation.

11. Apoptosis-related factor western blot The treated cells were washed three times with DPBS, and then detached using a cell scraper. The collected cells were suspended in Urea buffer and the cell membrane was disrupted by sonication. The cell lysate was centrifuged (15,000 × g, 15 minutes, 4 ° C.), and the supernatant was used as a cell extract. After cleaved PARP and β-actin were separated by SDS-PAGE using 12.5% polyacrylamide gel and Bax and Bcl-2 were 15% polyacrylamide gel, proteins on the gel were electrically transferred to a PVDF membrane. The PVDF membrane was blocked with BSA or skim milk, and then reacted sequentially with a primary antibody against Bcl-2, Bax, Cleaved PARP and human β-actin and a secondary antibody labeled with horseradish peroxidase. Antibody-reactive protein was detected by chemiluminescence using ECL enhanced chemiluminescence detection kit (GE healthcare).

12. Fluorescent immunostaining Cells suspended in growth medium were seeded in 24-well multiplates at 7.5 × 10 ⁴ cells / 500 μL each and cultured overnight at 37 ° C. and 5% CO ₂ . After removing the medium, the cells were washed twice with DPBS. 300 μL of 4% paraformaldehyde phosphate buffer solution was added and fixed for 10 minutes, 300 μL of DPBS containing 0.1% Triton X-100 and 100 mM glycine was added, and the mixture was allowed to stand for 10 minutes. Add 300 μL of DPBS containing 0.1% Tween 20 and 1% BSA, block for 1 hour, wash twice with DPBS, and then dilute with DPBS 300: 1 in primary antibody (anti-Caspase-3 antibody) at 4 ° C. Incubated overnight. After washing twice with PBS, the mixture was incubated in Alexa Fluoro-488-labeled rabbit secondary antibody solution diluted with DPBS at 500: 1 for 1 hour at room temperature, protected from light. After washing twice with PBS, excess water was removed, and a cover glass was fixed on the slide glass using a mounting agent (DAPI fluoromount-G). The fluorescent immunostained cells were set on a confocal laser microscope LSM700 (Carl Zeiss) and observed for fluorescence. Images were captured using a 100 × oil immersion objective of an inverted fluorescence microscope.

13. In vivo antitumor activity assessment
A group of 5 × 10 ⁵ CWR22Rv1 cells transplanted subcutaneously into 6-week-old Balb / c nu / nu mice and randomly administered DMSO as a control (n = 15) and 100 mg / kg of compound 2l Groups (n = 10) were prepared and administered intraperitoneally twice a week. The body weight and tumor diameter (major axis, minor axis) were measured once a week, and the tumor volume was calculated as an approximate value (major axis) ² × minor axis / 2.

<Result>
1. Research on the creation of novel AKR1C3 inhibitors based on chromene derivatives The structure optimization studies were conducted using lead compounds that were found in advance and had a chromene skeleton with AKR1C3 inhibitory activity. Since it became clear that the marine group at position 8 was essential for AKR1C3 inhibitory activity, 24 8-hydroxychromene derivatives were synthesized and evaluated for AKR1C3 inhibitory activity (Table 1). In addition to 2a, which is unsubstituted as well as 2d having a fluoro group at the 4-position, all derivatives 2b-m in which a benzene ring is introduced with a fluoro group, a chloro group, a methyl group, or an ethyl group have strong AKR1C3 inhibitory activity Among them, 2j having a methyl group at the 4-position showed the strongest inhibitory activity with an IC ₅₀ value of 9.1 nM. The inhibitory activities of derivatives 2q and 2r with the number of fluoro groups increased to 2 or 3 were slightly lower than 2d. Substituting the methyl group of the benzene ring with a fluoromethyl group did not change the inhibitory effect of 2s-v. In addition, the inhibitory activities of derivatives 2w and 2x with one carbon chain extended from 2j decreased. On the other hand, 2o having a hydroxyl group at the 3 position showed strong inhibitory activity, whereas 2n and 2p having a hydroxyl group at the 2 and 4 positions hardly inhibited AKR1C3.

表2. ヒトAKR1Cサブファミリー酵素間での阻害選択性 Next, in order to examine whether the inhibitory activity of these 8-hydroxychromene derivatives is specific to AKR1C3, 2c-f, 2j and 2l against AKR1C subfamily enzymes AKR1C1, AKR1C2, and AKR1C4 showing high structural similarity to AKR1C3 The inhibitory activity was evaluated (Table 2). These enzymes were hardly inhibited by 8-hydroxychromene derivatives. Therefore, it was found that these 8-hydroxychromene derivatives not only show strong AKR1C3 inhibitory activity but also have high selectivity.

Table 2. Inhibition selectivity between human AKR1C subfamily enzymes

  The prostatic cancer cell proliferation effect of 2d, 2j and 2l, which showed potent and selective AKR1C3 inhibitory activity, was variously examined using the CWR22Rv1 cell line that highly expresses AKR1C3.

2. Evaluation of the effectiveness of novel AKR1C3 inhibitors as prostate cancer therapeutics AKR1C3 is highly effective in suppressing prostate cancer cell growth of 2d, 2j and 2l, which showed strong AKR1C3 inhibitory activity in studies using purified enzymes. Various studies were performed using CWR22Rv1 cells. To limit the source of androgen to pregnenolone or androstenedione, RPMI1640 without phenol red containing 2% CS-FBS was used as the medium. First, the pregnenolone concentration that induces cell proliferation in CWR22Rv1 cells was optimized. Pregnenolone increased the number of living cells in a concentration-dependent manner, and the effect was highest at 20 nM (FIG. 1A). Therefore, 20 nM pregnenolone in which the most induction of cell proliferation was observed was used for the subsequent examination. Pregnenolone-induced cell proliferation was inhibited by the AKR1C3 inhibitors 2d, 2j and 2l, as well as the AR antagonists flutamide and enzalutamide, and the CYP17A1 inhibitor abiraterone (Figure 1B). . The growth inhibitory effects of these AKR1C3 inhibitors were not as good as abiraterone, but were comparable to flutamide and enzalutamide.

  In order to elucidate the androgen-dependent cell growth suppression mechanism by the novel AKR1C3 inhibitor, the amount of testosterone in the cells was measured. In order to clarify the effects of AKR1C3 inhibitor, 2d, 2j and 2l were added in the presence of finasteride, a 5α reductase (SRD5A1, 2) inhibitor that catalyzes the reductive metabolism of testosterone and androstenedione. Compared with the group added with finasteride alone, the amount of testosterone produced was significantly reduced (FIG. 2A). Testosterone, a reduced metabolite of AKR1C3, is reduced and metabolized intracellularly by SRD5A1 and 2 to 5α-DHT. AR is a ligand-binding domain that is present in the cytoplasm in a state of binding to heat shock protein (HSP) 90, and by binding to 5α-DHT, HSP90 is released, forms a dimer, and moves into the nucleus. AR then binds to androgen receptor element (ARE) present in the promoter region on the DNA, and promotes transcription of target genes such as PSA and TMPRSS2, thereby enhancing cell proliferation. Therefore, when the effects of 2d, 2j and 2l on AR translocation into the nucleus were examined using Western blotting, the amount of AR expression in the nucleus that was significantly increased by the addition of androstenedione decreased with the addition of 2d, 2j and 2l. (Fig. 2B).

  Since 2d and 2j inhibited the nuclear translocation of AR, the effects of 2d, 2j and 2l on the expression of PSA mRNA, the target gene of AR, were examined using real-time PCR. As a result, PSA mRNA expression increased by pregnenolone treatment was not as strong as abiraterone by the addition of 2d, 2j and 2l, but decreased to the same extent as flutamide and enzalutamide (FIG. 2C).

Using prostate cancer PC3 cells that do not express AR, various effects of 2d, 2j and 2l on androgen-independent cell proliferation were examined. In addition to androgen synthesis, AKR1C3 is responsible for the mitogen-activated protein kinase (MAPK) pathway and phosphatidyl via the biosynthesis of prostaglandin F (PGF) receptor ligands PGF _2α and 9α, 11β-PGF _2. It has been reported that phosphatidylinositol-3 kinase (PI3K) pathway is involved in the growth of cancer cells by activating the pathway. Therefore, we investigated the effects of 2d, 2j and 2l on cell proliferation using PC3 cells that endogenously express AKR1C3. As a result, although the effect at 24 hours after addition of 2d, 2j and 2l was small, after 72 hours, a significant decrease in the number of viable cells was confirmed compared to control cells to which DMSO was added (Fig. 3). ).

The main AR ligands testosterone and 5α-DHT are synthesized by reduction at position 17 with AKR1C3. Therefore, it is assumed that the ligand binding sites of AR and AKR1C3 are similar. To date, no compound that inhibits both AKR1C3 and AR has been reported, and it is epoch-making if the androgen synthesis pathway can be inhibited at multiple points of action. Thus, AR agonist activity and antagonist activity of various 8-hydroxychromene derivatives were evaluated using breast cancer SC-3 cells expressing wild type AR. SC-3 cells were treated with 10 μM of various 8-hydroxychromene derivatives or 1 nM 5α-DHT, and the agonist activity was evaluated by measuring the number of viable cells after 24 hours. As a result, the number of viable cells increased significantly when 5α-DHT was added compared to the control group added with DMSO, but slightly increased when 4 derivatives (2f, 2g, 2q, 2r) were added. Only a decrease was observed, and no effect on the number of viable cells was observed when 12 other derivatives (2a-e, 2j-p) were added. Therefore, it was revealed that the 8-hydroxychromene derivative developed this time does not show an AR agonist activity (FIG. 4A). Next, in the presence of 1 nM DHT, the number of viable cells after 24 hours of SC-3 cells pretreated with various concentrations of AR antagonist hydroxyflutamide (HF) or various 8-hydroxychromene derivatives was measured. Based on the number of living cells in the DHT single treatment group, the AR antagonist activity was evaluated by calculating the IC ₅₀ value. As a result, the IC ₅₀ values of eight derivative (2b-e, 2j-m ) as compared to an IC ₅₀ value of HF, although inhibitory activity was low about 10-fold showed AR antagonist effect (Figure 4B ).

  Using CWR22Rv1 cells, the combined effect of AKR1C3 inhibitors 2d, 2j and 2l and existing therapeutic agents was examined. As a result, pregnenolone-induced cell proliferation was significantly suppressed by treatment with 5 μM 2d, 2j and 2l, flutamide, abiraterone and enzalutamide. In addition, when using any of the therapeutic agents flutamide, abiraterone, and enzalutamide, the combined treatment of 2d, 2j, and 2l significantly suppressed cell growth compared to the single treatment, so existing therapeutic agents It was shown that anti-cancer activity can be enhanced by the combined use of AKR1C3 and AKR1C3 inhibitor (Fig. 5).

  It is generally known that apoptotic cell death is involved in the antitumor effect of anticancer agents. Therefore, we examined the effect of enzalutamide, 2d and 2j on apoptosis (Fig. 6). The combined treatment of Enzalutamide, 2d, and 2j enhanced DNA fragmentation, a phenomenon characteristic of apoptosis, compared to single treatment (FIG. 6A). Moreover, PRP degradation, increased Bax expression, decreased Bcl-2 expression, and increased active caspase-3 were also observed by the combined treatment of 2d and 2j (FIGS. 6B and C). Furthermore, the survival rate of CWR22Rv1 cells was significantly reduced by the combined use of 2d and 2j, compared to the treatment with enzalutamide alone (FIG. 6D).

  A similar study was performed using abiraterone, a CRPC therapeutic agent with a different mechanism of action (Fig. 7). As in the case of Enzalutamide, the combined treatment of abiraterone, 2d, and 2j resulted in DNA fragmentation, PARP degradation, increased Bax expression, decreased Bcl-2 expression, and increased active caspase-3. The cell viability was significantly reduced and the cell viability was significantly reduced.

  Furthermore, the effect of enhancing both CRPC inhibitor-induced apoptosis by 2l was verified (FIG. 8). By the combined treatment, DNA fragmentation, PARP degradation, and active caspase-3 increase were all increased compared to the single treatment, and the cell viability was significantly reduced.

Of the three compounds examined this time, 2j is a compound not described in the literature, in which only the CAS number is registered. On the other hand, 2l was an unpublished new compound. Therefore, the in vivo antitumor effect of 2l, a novel compound, was examined (FIG. 9). Groups of 5x10 ⁵ CWR22Rv1 cells implanted subcutaneously in 6-week-old Balb / c nu / nu mice, and randomly administered DMSO as a control (n = 15) and 2 mg of 100 mg / kg (n = 10) was prepared and administered intraperitoneally twice a week. The body weight and tumor diameter (major axis, minor axis) were measured once a week, and the tumor volume was calculated by the formula (major axis) ² × minor axis / 2 as an approximate value. In the group administered with 2 l, a remarkable tumor size suppression effect was shown.

<Discussion>
A number of 8-hydroxychromene derivatives have been found that exhibit potent and selective AKR1C3 inhibitory activity. That is, the present inventors have succeeded in developing a novel AKR1C3 inhibitor with high utility. A novel AKR1C3 inhibitor suppressed the growth of prostate cancer cells. In addition, in vivo antitumor effects were confirmed by experiments using representative compounds. One important thing was that the novel AKR1C3 inhibitor also showed cytostatic activity against prostate cancer cells not expressing AR, and was expected to be applied to CRPC treatment.

  On the other hand, the novel AKR1C3 inhibitor also showed an AR antagonistic action (not showing an AR agonistic activity). There are no reports of compounds having such characteristics. A novel AKR1C3 inhibitor can inhibit the androgen synthesis pathway at multiple points of action (AKR1C3 and AR), and can be expected to have a strong PSA lowering effect.

  The combination of existing drugs and new AKR1C3 inhibitors showed enhanced anticancer activity. This fact confirms that a novel AKR1C3 inhibitor exerts a therapeutic effect based on a mechanism of action different from that of existing drugs, and also means that a therapeutic strategy with a high therapeutic effect can be achieved by using in combination with existing drugs.

  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 in treating 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. The AKR1C3 inhibitor of the present invention can be easily synthesized and is suitable for mass production. If the AKR1C3 inhibitor of the present invention is applied to cancer treatment, it can contribute to the improvement of treatment opportunities for patients and consequently to the reduction of national medical expenses. The AKR1C3 inhibitor of the present invention is particularly expected to be used and applied to CRPC therapeutics.

  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.

SEQ ID NO: 1: Description of artificial sequence: Forward primer SEQ ID NO: 2: Description of artificial sequence: Reverse primer

Claims (11)

AKR1C3 inhibitor represented by the following chemical formula 1:

However, R1 in the formula is a hydrogen atom, a hydroxy group, a halogen atom, or a hydrocarbon group which may have a substituent.   In the above formula, R1 is a hydrogen atom, a fluoro group, a chloro group, a methyl group, an ethyl group, a hydroxy group (except for the 2nd and 4th positions), a difluoro group, a trifluoro group, a trifluoromethyl group, and a ditrifluoromethyl. The AKR1C3 inhibitor according to claim 1, which is a group or an isopropyl group.   R1 in the above formula is a hydrogen atom, 2-fluoro, 3-fluoro, 4-fluoro, 2-chloro, 3-chloro, 4-chloro, 2-methyl, 3-methyl, 4-methyl, 2-ethyl, 3 -Ethyl, 4-ethyl, 3-hydroxy, 3,4-difluoro, 3,4,5-trifluoro, 2-trifluoromethyl, 3-trifluoromethyl, 4-trifluoromethyl, 2,4-ditrifluoro The AKR1C3 inhibitor according to claim 1, which is methyl, 2-isopropyl or 3-isopropyl. The AKR1C3 inhibitor according to claim 1, which is represented by any one of the following chemical formulas 2 to 4:

  The anticancer agent which contains the AKR1C3 inhibitor as described in any one of Claims 1-4, or its pharmacologically acceptable salt as an active ingredient.   The anticancer drug according to claim 5, which is used for the treatment or prevention of prostate cancer, breast cancer, hepatocellular carcinoma, non-small cell lung cancer or leukemia.   The anticancer drug according to claim 5, which is used for treatment or prevention of prostate cancer.   The anticancer drug according to claim 7, wherein the prostate cancer is castration resistant prostate cancer.   The anticancer drug according to claim 7 or 8, which is used in combination with an antiandrogen drug.   A method for treating or preventing cancer, comprising a step of administering a therapeutically effective amount of the anticancer drug according to claim 5 to a cancer patient.   A research reagent comprising the AKR1C3 inhibitor according to any one of claims 1 to 4.

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