JP2007031412A - Antitumor agent - Google Patents
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- JP2007031412A JP2007031412A JP2005221360A JP2005221360A JP2007031412A JP 2007031412 A JP2007031412 A JP 2007031412A JP 2005221360 A JP2005221360 A JP 2005221360A JP 2005221360 A JP2005221360 A JP 2005221360A JP 2007031412 A JP2007031412 A JP 2007031412A
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Abstract
Description
本発明は、新規化合物及び該化合物を有効成分とする抗腫瘍剤に関する。 The present invention relates to a novel compound and an antitumor agent comprising the compound as an active ingredient.
従来の抗腫瘍剤（抗癌剤）の多くは、ＤＮＡに直接作用するか、分裂時に作用する化合物が多く、生体の中でも骨髄細胞や毛根細胞など、増殖の盛んな正常細胞に対する副作用が問題であった。しかしながら最近では、細胞増殖のメカニズムが分子レベルで解明されてきたので、癌遺伝子の発現や癌遺伝子産物の機能を阻害する選択的な抗腫瘍剤の開発が可能になりつつある。従って、抗腫瘍剤の分野では、新しい作用機序を有し、特異性及び有効性に優れた抗腫瘍剤の開発が望まれている。 Many of the conventional antitumor agents (anticancer agents) act directly on DNA or have many compounds that act at the time of division, and there are problems with side effects on normal cells that are proliferating, such as bone marrow cells and hair root cells. . However, recently, since the mechanism of cell proliferation has been elucidated at the molecular level, it is becoming possible to develop selective antitumor agents that inhibit the expression of oncogenes and the function of oncogene products. Therefore, in the field of antitumor agents, development of antitumor agents having a new mechanism of action and excellent in specificity and effectiveness is desired.
このような状況の中、本発明者らは、新規な作用メカニズムによりがん細胞を選択的に攻撃する化合物をスクリーニングする方法を開発している（特開２００２−０６５２９８号公報）。 Under such circumstances, the present inventors have developed a method for screening a compound that selectively attacks cancer cells by a novel action mechanism (Japanese Patent Laid-Open No. 2002-065298).
本発明は、がん細胞を選択的に攻撃する新規化合物及びその製造方法、並びに該化合物を有効成分として含有する抗腫瘍剤を提供することを目的とする。 An object of this invention is to provide the novel compound which selectively attacks a cancer cell, its manufacturing method, and the antitumor agent which contains this compound as an active ingredient.
本発明者らは、上記課題を解決するために鋭意検討した結果、シシウドから採取される新規な化合物が優れた抗癌作用を有することを見出し、本発明を完成させるに至った。 As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that a novel compound collected from shisiudo has an excellent anticancer activity, and have completed the present invention.
即ち、本発明は以下の発明を包含する。
（１）下記式：
(1) The following formula:
（２）前記（１）記載の化合物を有効成分として含有する医薬。
（３）前記（１）記載の化合物を有効成分として含有する抗腫瘍剤。
（４）シシウド（Angelica pubescens）から抽出することを特徴とする、下記式：
(2) A medicament comprising the compound according to (1) as an active ingredient.
(3) An antitumor agent comprising the compound according to (1) as an active ingredient.
(4) The following formula, characterized in that it is extracted from Angelica pubescens:
本発明の化合物はがん細胞に対して選択的に細胞毒性を示す。また、本発明の化合物は、シシウドから抽出することにより容易に製造することができる。 The compounds of the present invention are selectively cytotoxic to cancer cells. Moreover, the compound of this invention can be easily manufactured by extracting from shishido.
以下、本発明について詳細に説明する。
本発明の化合物（以下、「Ａ２Ｍ７」とも言う）は以下の構造を有する：
The compounds of the present invention (hereinafter also referred to as “A2M7”) have the following structure:
本発明の化合物としては、上記化合物のみならず、その薬学的に許容される塩も包含される。 The compounds of the present invention include not only the above compounds but also pharmaceutically acceptable salts thereof.
薬学的に許容される塩としては、例えば、ギ酸、酢酸、プロピオン酸、トリフルオロ酢酸、酒石酸、リンゴ酸、マレイン酸、フマル酸、コハク酸、シュウ酸、メタンスルホン酸、ｐ−トルエンスルホン酸等の有機酸、塩酸、臭化水素酸、硫酸、リン酸等の無機酸等との塩が挙げられる。 Examples of the pharmaceutically acceptable salt include formic acid, acetic acid, propionic acid, trifluoroacetic acid, tartaric acid, malic acid, maleic acid, fumaric acid, succinic acid, oxalic acid, methanesulfonic acid, p-toluenesulfonic acid and the like. And salts with inorganic acids such as organic acids, hydrochloric acid, hydrobromic acid, sulfuric acid and phosphoric acid.
本発明の化合物を医薬として用いる場合、予防又は治療目的に応じて各種の投与形態を採用することができ、該形態としては、例えば、経口剤、注射剤、坐剤、軟膏剤、貼付剤等のいずれでも良く、これらの投与形態は、各々当業者に公知慣用の製剤方法により製造できる。 When the compound of the present invention is used as a medicine, various administration forms can be adopted depending on the purpose of prevention or treatment. Examples of such forms include oral preparations, injections, suppositories, ointments, patches and the like. Each of these dosage forms can be produced by a conventional formulation method known to those skilled in the art.
経口用固形製剤を調製する場合は、本発明化合物に賦形剤、必要に応じて結合剤、崩壊剤、滑沢剤、着色剤、矯味・矯臭剤等を加えた後、常法により錠剤、被覆錠剤、顆粒剤、散剤、カプセル剤等を製造することができる。そのような添加剤としては、当該分野で一般的に使用されるものでよく、例えば、賦形剤としては、乳糖、白糖、塩化ナトリウム、ブドウ糖、デンプン、炭酸カルシウム、カオリン、微結晶セルロース、珪酸等を、結合剤としては、水、エタノール、プロパノール、単シロップ、ブドウ糖液、デンプン液、ゼラチン液、カルボキシメチルセルロース、ヒドロキシプロピルセルロース、ヒドロキシプロピルスターチ、メチルセルロース、エチルセルロース、シェラック、リン酸カルシウム、ポリビニルピロリドン等を、崩壊剤としては乾燥デンプン、アルギン酸ナトリウム、カンテン末、炭酸水素ナトリウム、炭酸カルシウム、ラウリル硫酸ナトリウム、ステアリン酸モノグリセリド、乳糖等を、滑沢剤としては精製タルク、ステアリン酸塩、ホウ砂、ポリエチレングリコール等を、着色剤としては、酸化チタン、酸化鉄等を、矯味・矯臭剤としては白糖、橙皮、クエン酸、酒石酸等を例示できる。 When preparing an oral solid preparation, after adding an excipient, a binder, a disintegrant, a lubricant, a coloring agent, a flavoring / flavoring agent, etc. to the compound of the present invention, if necessary, a tablet by a conventional method, Coated tablets, granules, powders, capsules and the like can be produced. Such additives may be those commonly used in the art. For example, excipients include lactose, sucrose, sodium chloride, glucose, starch, calcium carbonate, kaolin, microcrystalline cellulose, silicic acid As a binder, water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropyl starch, methylcellulose, ethylcellulose, shellac, calcium phosphate, polyvinylpyrrolidone, etc. Disintegrants include dry starch, sodium alginate, agar powder, sodium bicarbonate, calcium carbonate, sodium lauryl sulfate, stearic acid monoglyceride, lactose, etc., and lubricants include purified talc, stearate, C sand, polyethylene glycol, etc., as the coloring agent, titanium oxide, iron oxide, white as the flavoring agent sugar, orange peel, citric acid, can be exemplified tartaric acid.
経口用液体製剤を調製する場合は、本発明化合物に矯味剤、緩衡剤、安定化剤、矯臭剤等を加えて常法により内服液剤、シロップ剤、エリキシル剤等を製造することができる。この場合矯味・矯臭剤としては上記に挙げられたもので良く、緩衡剤としてはクエン酸ナトリウム等が、安定化剤としてはトラガント、アラビアゴム、ゼラチン等が挙げられる。 When preparing an oral liquid preparation, a liquid preparation, a syrup, an elixir or the like can be produced by a conventional method by adding a corrigent, a buffer, a stabilizer, a corrigent and the like to the compound of the present invention. In this case, the flavoring and flavoring agents may be those listed above, examples of the buffering agent include sodium citrate, and examples of the stabilizer include tragacanth, gum arabic, and gelatin.
注射剤を調製する場合は、本発明化合物にｐＨ調節剤、緩衡剤、安定化剤、等張化剤、局所麻酔剤等を添加し、常法により皮下、筋肉内及び静脈内用注射剤を製造することができる。この場合のｐＨ調節剤及び緩衡剤としてはクエン酸ナトリウム、酢酸ナトリウム、リン酸ナトリウム等が挙げられる。安定化剤としてはピロ亜硫酸ナトリウム、ＥＤＴＡ、チオグリコール酸、チオ乳酸等が挙げられる。局所麻酔剤としては塩酸プロカイン、塩酸リドカイン等が挙げられる。等張化剤としては、塩化ナトリウム、ブドウ糖等が例示できる。 When preparing an injection, a pH adjuster, a buffering agent, a stabilizer, an isotonic agent, a local anesthetic, etc. are added to the compound of the present invention, and injections for subcutaneous, intramuscular and intravenous injections are prepared by conventional methods. Can be manufactured. In this case, examples of the pH adjusting agent and the buffering agent include sodium citrate, sodium acetate, sodium phosphate and the like. Examples of the stabilizer include sodium pyrosulfite, EDTA, thioglycolic acid, thiolactic acid and the like. Examples of local anesthetics include procaine hydrochloride and lidocaine hydrochloride. Examples of isotonic agents include sodium chloride and glucose.
坐剤を調製する場合は、本発明化合物に当業界において公知の製剤用担体、例えば、ポリエチレングリコール、ラノリン、カカオ脂、脂肪酸トリグリセライド等を、さらに必要に応じてツイーン（登録商標）のような界面活性剤等を加えた後、常法により製造することができる。 When preparing a suppository, a formulation carrier known in the art, such as polyethylene glycol, lanolin, cocoa butter, fatty acid triglyceride and the like, and an interface such as Tween (registered trademark) as necessary are added to the compound of the present invention. After adding an activator etc., it can manufacture by a conventional method.
軟膏剤を調製する場合は、本発明化合物に通常使用される基剤、安定剤、湿潤剤、保存剤等が必要に応じて配合され、常法により混合、製剤化される。基剤としては、流動パラフィン、白色ワセリン、サラシミツロウ、オクチルドデシルアルコール、パラフィン等が挙げられる。保存剤としては、パラオキシ安息香酸メチル、パラオキシ安息香酸エチル、パラオキシ安息香酸プロピル等が挙げられる。 When preparing an ointment, bases, stabilizers, wetting agents, preservatives and the like that are usually used for the compound of the present invention are blended as necessary, and mixed and formulated by a conventional method. Examples of the base include liquid paraffin, white petrolatum, white beeswax, octyldodecyl alcohol, paraffin and the like. Examples of the preservative include methyl paraoxybenzoate, ethyl paraoxybenzoate, and propyl paraoxybenzoate.
貼付剤を製造する場合は、通常の支持体に前記軟膏、クリーム、ゲル、ペースト等を常法により塗布すれば良い。支持体としては、綿、スフ、化学繊維からなる織布、不織布や軟質塩化ビニル、ポリエチレン、ポリウレタン等のフィルムあるいは発泡体シートが適当である。 When producing a patch, the ointment, cream, gel, paste or the like may be applied to a normal support by a conventional method. As the support, a woven fabric, nonwoven fabric, soft vinyl chloride, polyethylene, polyurethane, or a film or foam sheet made of cotton, suf, or chemical fiber is suitable.
上記の各投与単位形態中に配合されるべき本発明化合物の量は、これを適用すべき患者の症状によりあるいはその剤型等により一定ではないが、一般に投与単位形態あたり経口剤では約０．０１〜１０００ｍｇ、注射剤では約０．０１〜５００ｍｇ、坐剤では約０．０１〜１０００ｍｇとするのが望ましい。また、上記投与形態を有する薬剤の１日あたりの投与量は、患者の症状、体重、年齢、性別等によって異なり一概には決定できないが、通常成人１日あたり約０．０１〜５０００ｍｇ、好ましくは約０．０１〜１０００ｍｇとすれば良く、これを１日１回又は２〜４回程度に分けて投与するのが好ましい。 The amount of the compound of the present invention to be formulated in each of the above dosage unit forms is not constant depending on the symptoms of the patient to which the compound is to be applied or the dosage form thereof, but generally about 0. It is desirable that the dosage is 01 to 1000 mg, about 0.01 to 500 mg for injections, and about 0.01 to 1000 mg for suppositories. Further, the daily dose of the drug having the above dosage form varies depending on the patient's symptoms, body weight, age, sex, etc., and cannot be determined unconditionally, but is usually about 0.01 to 5000 mg per day for an adult, preferably The dose may be about 0.01 to 1000 mg, which is preferably administered once a day or divided into about 2 to 4 times a day.
本発明の化合物は、がん細胞が栄養状態や、酸素の供給状態が悪い条件下で生き延びるという性質を利用して、特開２００２−０６５２９８号公報に記載のスクリーニング方法により見出されたものである。本発明の化合物は、従来の抗腫瘍剤とは異なる新規な作用メカニズムによりがん細胞を選択的に攻撃する。 The compound of the present invention has been found by the screening method described in JP-A-2002-065298, taking advantage of the fact that cancer cells survive under nutrient conditions and poor oxygen supply conditions. is there. The compound of the present invention selectively attacks cancer cells by a novel mechanism of action different from conventional antitumor agents.
本発明の化合物を含有する抗腫瘍剤を投与することにより治療できる癌・悪性腫瘍としては、特に制限はなく、例えば頭頸部癌、食道癌、胃癌、結腸癌、直腸癌、肝臓癌、胆のう・胆管癌、膵臓癌、肺癌、乳癌、卵巣癌、膀胱癌、前立腺癌、睾丸腫瘍、骨・軟部肉腫、悪性リンパ腫、白血病、子宮頸癌、皮膚癌、脳腫瘍等が挙げられる。 The cancer / malignant tumor that can be treated by administering the antitumor agent containing the compound of the present invention is not particularly limited. For example, head and neck cancer, esophageal cancer, stomach cancer, colon cancer, rectal cancer, liver cancer, gallbladder Examples include bile duct cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, testicular tumor, bone / soft tissue sarcoma, malignant lymphoma, leukemia, cervical cancer, skin cancer, brain tumor and the like.
次に本発明の化合物の製造方法について説明する。
本発明の化合物は化学合成により製造することもできるが、例えば、以下のようにしてシシウド（Angelica pubescens）（生薬名、唐独活）から抽出することにより得ることもできる。
Next, the manufacturing method of the compound of this invention is demonstrated.
Although the compound of this invention can also be manufactured by chemical synthesis, it can also be obtained, for example, by extracting from Shishido (Angelica pubescens) (namely, crude drug name, Tangkoku active) as follows.
シシウド（Angelica pubescens）の根をアルコール（例えば、メタノール、エタノール、プロパノール又はそれらと水との混合物）に浸し、これを超音波処理した後、１時間〜数日間放置する。次いで、抽出液に塩化メチレンを加えて超音波処理する。塩化メチレンに不溶性の物質を除去し、塩化メチレン溶液を濃縮して、残渣をシリカゲルカラムクロマトグラフィにかける。溶離液としては、例えば、クロロホルム-メタノール、ヘキサン−酢酸エチル、酢酸エチル-ベンゼン等の系が挙げられる。本発明の化合物を含む画分を集め、溶媒を留去して本発明の化合物を得る。各成分の分離が不十分な場合は、異なる溶離液を組み合わせてクロマトグラフィを複数回行ってもよいし、或いは、他の分離精製手段、例えば濃縮、溶媒抽出、濾過、再結晶、各種クロマトグラフィ等を用いることにより精製するとよい。 The root of Angelica pubescens is immersed in alcohol (for example, methanol, ethanol, propanol or a mixture thereof with water), sonicated, and then left for 1 hour to several days. Next, methylene chloride is added to the extract and sonicated. Material insoluble in methylene chloride is removed, the methylene chloride solution is concentrated, and the residue is subjected to silica gel column chromatography. Examples of the eluent include chloroform-methanol, hexane-ethyl acetate, ethyl acetate-benzene, and the like. The fractions containing the compound of the present invention are collected and the solvent is distilled off to obtain the compound of the present invention. If the separation of each component is insufficient, the chromatography may be performed multiple times by combining different eluents, or other separation and purification means such as concentration, solvent extraction, filtration, recrystallization, various chromatography, etc. It is good to refine | purify by using.
以下、実施例により本発明を更に具体的に説明するが、本発明の範囲はこれらの実施例に限定されるものではない。 EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the scope of the present invention is not limited to these examples.
本実施例で使用した主な装置、分析機器等は以下のとおりである。
旋光度：
日本分光DIP-140デジタル旋光計
赤外吸収スペクトル：
島津IR-408赤外分光光度計（CHCl3溶液中）
核磁気共鳴（ＮＭＲ）スペクトル：
日本電子JNM-LA400分光計（内部標準としてテトラメチルシラン(TMS）を使用し、化学シフトをδ値として表示)
円二色性（ＣＤ）スペクトル：
日本分光J-805分光旋光計
高分解能高速原子衝突質量スペクトル（ＨＲ−ＦＡＢ−ＭＳ）：
日本電子JMS-700T分析計（グリセロールをマトリックスとして使用）
カラムクロマトグラフィ：
BW-820MHシリカゲル（富士シリシア化学，春日井）
分取および分析薄層クロマトグラフィ（ＴＬＣ）：
シリカゲル60F254（厚さ0.25 mm若しくは0.5 mm，ドイツ・メルク）、又はRP-18F254S（厚さ0.25 mm，メルク）のプレコートプレート
The main devices and analytical instruments used in this example are as follows.
Optical rotation:
Infrared absorption spectrum of JASCO DIP-140 digital polarimeter:
Shimadzu IR-408 infrared spectrophotometer (in CHCl 3 solution)
Nuclear magnetic resonance (NMR) spectrum:
JEOL JNM-LA400 spectrometer (uses tetramethylsilane (TMS) as internal standard and displays chemical shift as δ value)
Circular dichroism (CD) spectrum:
JASCO J-805 Spectrophotometer High Resolution Fast Atom Collision Mass Spectrum (HR-FAB-MS):
JEOL JMS-700T analyzer (glycerol is used as matrix)
Column chromatography:
BW-820MH silica gel (Fuji Silysia Chemical, Kasugai)
Preparative and analytical thin layer chromatography (TLC):
Silica gel 60F 254 (thickness 0.25 mm or 0.5 mm, Merck, Germany) or RP-18F 254S (thickness 0.25 mm, Merck) pre-coated plate
実施例１：シシウドからのＡ２Ｍ７の抽出
図１に示す工程に従って、シシウドの根から本発明の化合物「Ａ２Ｍ７」を抽出した。
シシウド（Angelica pubescens）の根（生薬名、唐独活；乾燥重量900 g）を70％エタノール-水（3L）で2時間２回超音波処理し、さらに22時間放置して抽出し、70％エタノール-水エキス（142 g）を得た。このエキス（69 g）を塩化メチレン（700 mL）で1時間超音波処理（2回）して塩化メチレン可溶部（9.1 g）を得た。塩化メチレン可溶部（6.0 g）をシリカゲルカラムクロマトグラフィ（内径3.5 cm×53 cm）に付し、クロロホルム（以下、CHCl3）-メタノール（以下、MeOH）系で溶出して以下の画分（fr.1〜10）を得た。
fr. 1：CHCl3-MeOH（99:1） 溶出部，1.3 g
fr. 2：CHCl3-MeOH（98:2） 溶出部，1.74 mg
fr. 3：CHCl3-MeOH（97:3） 溶出部，1.7 g
fr. 4：CHCl3-MeOH（96:4） 溶出部，1.3 g
fr. 5：CHCl3-MeOH（95:5） 溶出部，247 mg
fr. 6：CHCl3-MeOH（92:8） 溶出部，83 mg
fr. 7：CHCl3-MeOH（90:10）溶出部，91 mg
fr. 8：CHCl3-MeOH（85:15）溶出部，290 mg
fr. 9：CHCl3-MeOH（80:20）溶出部，159 mg
fr.10：CHCl3-MeOH（70:30）溶出部，42 mg
Example 1 Extraction of A2M7 from Sisius The compound "A2M7" of the present invention was extracted from the roots of Sisiod according to the process shown in FIG.
The roots of herb (Angelica pubescens) (namely herbal medicine, Tang Duo Active; dry weight 900 g) were sonicated twice in 70% ethanol-water (3 L) for 2 hours, left to extract for another 22 hours, and extracted with 70% ethanol. -A water extract (142 g) was obtained. This extract (69 g) was sonicated with methylene chloride (700 mL) for 1 hour (twice) to obtain a methylene chloride soluble part (9.1 g). The methylene chloride soluble part (6.0 g) was subjected to silica gel column chromatography (inner diameter 3.5 cm × 53 cm) and eluted with chloroform (hereinafter referred to as CHCl 3 ) -methanol (hereinafter referred to as MeOH) system. .1-10) were obtained.
fr 1:. CHCl 3 -MeOH ( 99: 1) eluate, 1.3 g
fr. 2: CHCl 3 -MeOH (98: 2) elution part, 1.74 mg
fr. 3: CHCl 3 -MeOH (97: 3) elution part, 1.7 g
fr. 4: CHCl 3 -MeOH (96: 4) elution part, 1.3 g
fr. 5: CHCl 3 -MeOH (95: 5) elution part, 247 mg
fr. 6: CHCl 3 -MeOH (92: 8) elution part, 83 mg
fr. 7: CHCl 3 -MeOH (90:10) elution part, 91 mg
fr. 8: CHCl 3 -MeOH (85:15) elution part, 290 mg
fr. 9: CHCl 3 -MeOH (80:20) elution part, 159 mg
fr.10: CHCl 3 -MeOH (70:30) elution part, 42 mg
Fr. 3（1.0 g）を再度シリカゲルカラムクロマトグラフィ（内径3.5 cm×25 cm）に付し、ヘキサン-酢酸エチル(0-100％)系で溶出して以下の画分（fr.3-1〜3-5）を得た。
fr. 3-1，266 mg
fr. 3-2， 36 mg
fr. 3-3， 42 mg
fr. 3-4， 11 mg
fr. 3-5，250 mg
Fr. 3 (1.0 g) was again subjected to silica gel column chromatography (inner diameter 3.5 cm × 25 cm) and eluted with hexane-ethyl acetate (0-100%) system to obtain the following fractions (fr.3-1 to 3-5) was obtained.
fr. 3-1, 266 mg
fr. 3-2, 36 mg
fr. 3-3, 42 mg
fr. 3-4, 11 mg
fr. 3-5, 250 mg
次いで、Fr. 3-5をさらに順相分取TLC（酢酸エチル/ベンゼン＝25/75）により分離して本発明の化合物Ａ２Ｍ７（25 mg）を無色アモルファス固体として得た。 Next, Fr. 3-5 was further separated by normal phase preparative TLC (ethyl acetate / benzene = 25/75) to obtain Compound A2M7 (25 mg) of the present invention as a colorless amorphous solid.
実施例２：Ａ２Ｍ７の同定
実施例１で得られた化合物について解析を行った。
1H NMR(400 MHz, CDCl3)：δ 1.61 (3H, s, H-13), 1.65 (3H, s, H-12), 3.38 (2H, ddd, J = 16.3, 9.5, 8.0 Hz, H-9), 5.21 (1H, dd, J = 9.5, 8.0 Hz, H-10), 6.14 (1H, d, J = 15.9, H-8'), 6.24 (1H, d, J = 9.5 Hz, H-2), 6.79 (1H, d, J = 8.3 Hz, H-6), 6.83 (2H, d, J = 8.5 Hz, H-3', 5'), 7.28 (2H, d, J = 8.5 Hz, H-2', 6'), 7.31 (1H, d, J = 8.3 Hz, H-5), 7.35 (1H, d, J = 15.9, H-7'), 7.68 (1H, d, J = 9.5 Hz, H-3).
13C NMR(100 MHz, CDCl3)：δ 21.1 (C-13), 22.1 (C-12), 27.5 (C-9), 82.2 (C-11), 89.1 (C-10), 106.9 (C-6), 111.98 (C-3), 113.0 (C-4a), 113.6 (C-8), 114.5 (C-7'), 115.9 (C-3',5'), 116.1 (C-8'), 126.6 (C-1'), 128.9 (C-5), 129.8 (C-2',6'), 144.3 (C-4), 151.1 (C-8a), 158.3 (C-4'), 161.5, (C-2), 164.1 (C-7), 166.5 (C-9').
IR (CHCl3) cm-1： 3280, 1910, 1705, 1610, 1585, 1506, 1490, 1460, 1405, 1388, 1370, 1325, 1260, 1168, 1140, 1065, 980, 835.
CD λmax (EtOH, 0.255 mM) nm： 338 ([θ] +34314), 298 ([θ] -16932).
HR-FAB-MS： 393.1295 ［Calcd for C23H21O6 (M+H)+ 393.1338］.
［α］D 25： + 218.7 (c = 0.025, CHCl3)。
Example 2: Identification of A2M7 The compound obtained in Example 1 was analyzed.
1 H NMR (400 MHz, CDCl 3 ): δ 1.61 (3H, s, H-13), 1.65 (3H, s, H-12), 3.38 (2H, ddd, J = 16.3, 9.5, 8.0 Hz, H -9), 5.21 (1H, dd, J = 9.5, 8.0 Hz, H-10), 6.14 (1H, d, J = 15.9, H-8 '), 6.24 (1H, d, J = 9.5 Hz, H -2), 6.79 (1H, d, J = 8.3 Hz, H-6), 6.83 (2H, d, J = 8.5 Hz, H-3 ', 5'), 7.28 (2H, d, J = 8.5 Hz , H-2 ', 6'), 7.31 (1H, d, J = 8.3 Hz, H-5), 7.35 (1H, d, J = 15.9, H-7 '), 7.68 (1H, d, J = (9.5 Hz, H-3).
13 C NMR (100 MHz, CDCl 3 ): δ 21.1 (C-13), 22.1 (C-12), 27.5 (C-9), 82.2 (C-11), 89.1 (C-10), 106.9 (C -6), 111.98 (C-3), 113.0 (C-4a), 113.6 (C-8), 114.5 (C-7 '), 115.9 (C-3', 5 '), 116.1 (C-8' ), 126.6 (C-1 '), 128.9 (C-5), 129.8 (C-2', 6 '), 144.3 (C-4), 151.1 (C-8a), 158.3 (C-4'), 161.5, (C-2), 164.1 (C-7), 166.5 (C-9 ').
IR (CHCl 3) cm -1: 3280, 1910, 1705, 1610, 1585, 1506, 1490, 1460, 1405, 1388, 1370, 1325, 1260, 1168, 1140, 1065, 980, 835.
CD λ max (EtOH, 0.255 mM) nm: 338 ([θ] +34314), 298 ([θ] -16932).
HR-FAB-MS: 393.1295 [Calcd for C 23 H 21 O 6 (M + H) + 393.1338].
[Α] D 25 : + 218.7 (c = 0.025, CHCl 3 ).
上記の分析結果より、本発明の化合物は以下の化学構造を有する8,9-ジヒドロ-8(S)-[1-[[3-(4-ヒドロキシフェニル)-1-オキソ-2(E)-プロペニル]オキシ]-1-メチルエチル]-2H-フロ[2,3-h]-1-ベンゾピラン-2-オンであることが示された。 From the above analysis results, the compound of the present invention has 8,9-dihydro-8 (S)-[1-[[3- (4-hydroxyphenyl) -1-oxo-2 (E) having the following chemical structure: -Propenyl] oxy] -1-methylethyl] -2H-furo [2,3-h] -1-benzopyran-2-one.
実施例３：Ａ２Ｍ７の抗腫瘍活性に対する栄養因子の影響
本発明の化合物Ａ２Ｍ７のヒト膵臓がん細胞PANC-1に対する細胞毒性を、以下に示す栄養成分が異なる培地ａ）〜ｅ）を使用して調べた。
ａ）ＮＤＭ ：栄養欠乏培地。ＤＭＥＭの電解質だけを含んでいる培地
ｂ）ＮＤＭ＋Ｓｅ ：１０％ウシ胎児血清を含むＮＤＭ培地
ｃ）ＮＤＭ＋Ｓｅ＋ＡＡ：１０％ウシ胎児血清及びアミノ酸混合物（ＤＭＥＭに含まれるアミノ酸を同濃度で添加）を含むＮＤＭ培地
ｄ）ＮＤＭ＋Ｇｌｃ ：グルコースを１mg/mlの濃度で含むＮＤＭ培地
ｅ）ＤＭＥＭ ：ＤＭＥＭ培地
Example 3: Effect of trophic factors on the antitumor activity of A2M7 The cytotoxicity of compound A2M7 of the present invention against human pancreatic cancer cells PANC-1 was determined using the following media a) to e) having different nutritional components. Examined.
a) NDM: nutrient-deficient medium. Medium containing only DMEM electrolyte b) NDM medium containing NDM + Se: 10% fetal calf serum c) NDM + Se + AA: 10% fetal calf serum and amino acid mixture (added amino acids contained in DMEM at the same concentration) d) NDM + Glc: NDM medium containing glucose at a concentration of 1 mg / ml e) DMEM: DMEM medium
上記各培地に細胞を播種し、これにＡ２Ｍ７を所定の濃度で添加し、２４時間培養した。培養終了後、ＷＳＴ−８法で生細胞数を測定した。その結果を図２に示す。図中、縦軸の「ＯＤ」は生細胞数の測定のために添加された発色試薬の吸光度であり、吸光度が高いほど生細胞数が多いことを示す。 Cells were seeded in each of the above media, and A2M7 was added thereto at a predetermined concentration and cultured for 24 hours. After completion of the culture, the number of viable cells was measured by the WST-8 method. The result is shown in FIG. In the figure, “OD” on the vertical axis represents the absorbance of the coloring reagent added for the measurement of the number of viable cells, and the higher the absorbance, the greater the number of viable cells.
この実験結果から、アミノ酸が欠乏している場合に、Ａ２Ｍ７はがん細胞に対して強い毒性を示すことが分かった。 From this experimental result, it was found that A2M7 shows strong toxicity to cancer cells when amino acids are deficient.
実施例４：栄養欠乏培地におけるＡ２Ｍ７の抗腫瘍活性
栄養欠乏培（ＮＤＭ）におけるＡ２Ｍ７のヒト膵臓がん細胞PANC-1に対する細胞毒性を、種々の濃度で通常培地（ＤＭＥＭ）と比較した。
Example 4: Antitumor activity of A2M7 in nutrient-deficient medium The cytotoxicity of A2M7 to human pancreatic cancer cells PANC-1 in nutrient-deficient medium (NDM) was compared with normal medium (DMEM) at various concentrations.
それぞれの培地を用いて実施例３と同様の方法により細胞を培養し、増殖阻害活性について試験した。その結果を図３に示す。 Cells were cultured in the same manner as in Example 3 using each medium and tested for growth inhibitory activity. The result is shown in FIG.
本実験の結果によれば、炭素源や窒素源のない栄養欠乏培（ＮＤＭ）ではＡ２Ｍ７濃度が１０ng/mlを超えるとがん細胞に対して強い細胞毒性を示し、それ以上の濃度ではがん細胞はすべて死滅した。しかし、通常の培地であるＤＭＥＭではＡ２Ｍ７濃度１０μg/mlまでほとんど毒性を示さなかった。この結果から、Ａ２Ｍ７のがん細胞に対する細胞毒性について栄養飢餓選択性が証明された。 According to the results of this experiment, nutrient-deficient culture (NDM) without a carbon source or nitrogen source shows strong cytotoxicity against cancer cells when the A2M7 concentration exceeds 10 ng / ml, and cancer at higher concentrations. All cells died. However, DMEM, which is a normal medium, showed almost no toxicity up to an A2M7 concentration of 10 μg / ml. This result demonstrated the nutrient starvation selectivity for cytotoxicity of A2M7 to cancer cells.
実施例５：Ａ２Ｍ７の細胞毒性発現のメカニズム
本発明の化合物のがん細胞に対する毒性発現のメカニズムを解析するため、ヒト膵臓がん細胞PANC-1細胞の栄養欠乏培地中での生存にとって必須であるPI3キナーゼ及びAKTに対する影響を調べた。
Example 5: Mechanism of cytotoxic expression of A2M7 In order to analyze the mechanism of cytotoxic expression of the compound of the present invention against cancer cells, it is essential for the survival of human pancreatic cancer cells PANC-1 cells in nutrient-deficient media The effects on PI3 kinase and AKT were investigated.
実験は、セリンスレオニンキナーゼAKTのリン酸化をリン酸化AKT特異的抗体を用いてウエスタンブロット法で検出することにより行った。具体的な実験手順は以下のとおりである。 The experiment was performed by detecting phosphorylation of serine threonine kinase AKT by Western blotting using a phosphorylated AKT specific antibody. The specific experimental procedure is as follows.
ヒト膵がん細胞PANC-1及び本発明の化合物（A2M7） 10 μg/mlをDMEM培地に加えた。所定の時間経過後に細胞をはがし、１０％ＳＤＳを含むサンプルバッファーで抽出してウエスタンブロット法でリン酸化AKT検出した。その結果を図４に示す。この結果から、本発明の化合物によりAKTのリン酸化が増強されることが示された。 Human pancreatic cancer cell PANC-1 and 10 μg / ml of the compound of the present invention (A2M7) were added to the DMEM medium. After a predetermined time, the cells were peeled off, extracted with a sample buffer containing 10% SDS, and phosphorylated AKT was detected by Western blotting. The result is shown in FIG. From this result, it was shown that the phosphorylation of AKT is enhanced by the compound of the present invention.
また、ヒト膵がん細胞PANC-1細胞を、本発明の化合物の存在下（10 μg/ml）又は非存在下で各種の培地（DMEM、NDM+Se+AA、NDM+Se+Glc、NDM+Se+AA+Glc、NDM）において２．５時間培養し、培養終了後に細胞をはがし、１０％ＳＤＳを含むサンプルバッファーで抽出してウエスタンブロット法でリン酸化AKT検出した。その結果を図５に示す。この結果から、本発明の化合物により、グルコース又はアミノ酸の欠乏によるAKTのリン酸化、血清による定常状態のAKTのリン酸化のいずれも増強されることが示された。
なお、AKTの総量（tAKT）は、リン酸化に関係なくAKTに反応する抗体で検出した。
In addition, human pancreatic cancer cell PANC-1 cells can be cultured in various media (DMEM, NDM + Se + AA, NDM + Se + Glc, NDM) in the presence (10 μg / ml) or absence of the compound of the present invention. + Se + AA + Glc, NDM) for 2.5 hours, and after completion of the culture, the cells were detached, extracted with a sample buffer containing 10% SDS, and phosphorylated AKT was detected by Western blotting. The result is shown in FIG. From these results, it was shown that the compound of the present invention enhances both phosphorylation of AKT due to glucose or amino acid deficiency and steady-state phosphorylation of AKT by serum.
The total amount of AKT (tAKT) was detected with an antibody that reacts with AKT regardless of phosphorylation.
これまで本発明者らが見出した抗腫瘍活性を有する化合物の多くはPI3キナーゼを阻害するものであったが、本発明の化合物によりAKTのリン酸化が増強されるという事実を考慮すると、本発明の化合物はこれまでの化合物とは異なった作用機序により抗腫瘍活性を示すものと考えられる。即ち、栄養状態が悪い場合、細胞は生存を維持するためにその適応反応としてAKTが活性化するが、本発明の化合物はAKTの活性化を阻害するのではなく、逆にAKTの活性化を促進しつつがん細胞を細胞死にいたらしめるという、従来の知見とは異なる機構で抗腫瘍活性が発現されることが示唆された。 Many of the compounds having antitumor activity that the present inventors have found so far inhibit PI3 kinase, but considering the fact that phosphorylation of AKT is enhanced by the compounds of the present invention, the present invention This compound is considered to exhibit antitumor activity by a mechanism of action different from that of the conventional compounds. That is, when the nutritional state is poor, AKT is activated as an adaptive response in order to maintain the survival of the cells, but the compound of the present invention does not inhibit the activation of AKT but conversely activates AKT. It was suggested that antitumor activity is expressed by a mechanism different from the conventional knowledge of causing cancer cells to die while promoting.
本発明の化合物はがん細胞に対して選択的に細胞毒性を示すとともに、従来の抗腫瘍剤とは異なる新規な抗癌活性メカニズムを有しており、副作用の少ない抗腫瘍剤として期待される。 The compound of the present invention is selectively cytotoxic to cancer cells, has a novel anticancer activity mechanism different from conventional antitumor agents, and is expected as an antitumor agent with few side effects. .
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