1377939 六、發明說明： 【發明所屬之技術領域】 本發明係祕-種含式I化合物.安卓幸且可胳抑獅細胞生長之藥 學組成物。 >1377939 VI. Description of the Invention: [Technical Field to Which the Invention Is Applicable] The present invention relates to a compound containing a compound of the formula I. Andrews is a pharmaceutical composition for growth of lion cells. >
【先前技術J 癌症-般可峨為惡題瘤，是―種赫。其特徵為雜組織的不正 常團塊’細於過度地細胞分裂。癌症細胞不具有正常_生長之限制， 會又與佔,貞正常留給其他細胞的範圍。癌症治療的種類包括化學治療、 手術放射線以及這些治療的結合。化學治療通常包括使用一個或多個抑 制癌細胞生·化合物。軸目前6經發展了許錢症化療_，但仍然 需要更有效的化學治療。 【發明内容】 化合物·安卓核其醫藥上可接受鹽，其可用於 抑制癌細胞生長，甚至治療或獅癌症，侧是乳癌。特定言之，本發明 ^提供-種胁抑制麻齡長，甚至治療或獅齡之轉組成物，包 有量之式I化„物_安卓幸或其醫藥上可接受鹽，與醫藥上可接受載體。 本發⑽、提供—细於抑制癌細胞生長之藥學組成物駐，經實驗證 ^ 物安卓幸對*同癌症細絲（包括：大腸直腸癌細胞·ΗΤ_29或 II6 ’肝癌細胞株·Huh7;肺腺癌細胞株韻9及乳癌細胞株捕於或 具高度抑制性，但對正常細胞（包括：纖維母細胞·及正 1377939 常乳腺上皮細胞视舰)職乎不具細鱗性；再者，經触症臨床化學 治療藥物(艾黴素Doxorubicin及賴Cisplatin)進行對照實驗，評估幻化 合物-安卓幸、艾歸、賴縣性漏_ (__他_231)之相對^制效 果，實驗證實在相狀舰τ 1 χ化合物·安轉蝴紐乳癌細胞 _Α-ΜΒ·2取效果優於艾黴素、賴’故其甚至可治療或預防癌症。[Prior Art J Cancer - can be regarded as a malignant tumor, is a species. It is characterized by an abnormal mass of the heterogeneous tissue that is finer than excessive cell division. Cancer cells do not have a normal-growth limit, which in turn is shared with other cells that are normally left to other cells. The types of cancer treatment include chemotherapy, surgical radiation, and a combination of these treatments. Chemotherapy typically involves the use of one or more compounds that inhibit cancer cells. Axis is currently developing 6 years of chemotherapy, but still needs more effective chemotherapy. SUMMARY OF THE INVENTION Compound·Android Nucleus is a pharmaceutically acceptable salt thereof, which is useful for inhibiting the growth of cancer cells, even for treating or lion cancer, and for breast cancer. In particular, the present invention provides a composition which inhibits the age of the numbness, even the treatment or the lion age, and the amount of the formula I _ _ _ android or its pharmaceutically acceptable salt, and medically The carrier (10), provides - a pharmaceutical composition that is finer than the growth of cancer cells, and is verified by the fact that the drug is for the same cancer filament (including: colorectal cancer cell, ΗΤ _29 or II6 ' liver cancer cell line· Huh7; lung adenocarcinoma cell line 9 and breast cancer cell lines are highly arrested or highly inhibitory, but do not have fine scales on normal cells (including: fibroblasts and positive 1377939 normal breast epithelial cells); Controlled clinical chemotherapeutic drugs (Dytomycin Doxorubicin and Lai Cisplatin) were used to evaluate the relative effects of phantom compounds - Andrews, Aigui, Lai County Sex _ (__He_231). Experiments have confirmed that in the phase ship τ 1 χ compound · An 蝴 纽 纽 纽 ΜΒ ΜΒ ΜΒ 取 取 取 取 取 取 取 取 取 取 取 取 取 优于 优于 优于 优于 优于 优于 优于 优于 优于 优于 优于 优于
式I化合物-安卓幸可擁有-或多鑛掌中心，因此具有各種立體異構 物形式。本發明中提及之式Ϊ化合物包括所有此等異構物。式t化合物具有 選擇性抑繼症_生㈣功效。由於其分子量極小，因此，可使用較低 劑量的式Ϊ化合物及其醫藥上可接受鹽，與醫藥上可接受載體，即可得到渴 望的治療效果。本發日縣-抑儲峨生長，甚至治療或獅癌症的藥學 組成物，係將—有效量之式！化合物及其醫藥上可接受鹽，用於抑制癌細 胞、或投予所需之病患（此病患具有癌症、癌症的症狀或傾向於癌症的體 質）以治癒、恢復、減輕、緩和、改變、治療、改善、改進或影響疾病、 疾病的症狀或傾向於疾病的體質為目的。此處使用的“有效量(⑽eftoive amount)”指有效量之式ί化合物_安卓幸及其醫藥上可接受鹽，具有抑制或 治療功效的量。纽量的改變是根據給藥的雜、讎侧(exdpientusage) 以及與其他共同使用(co-usage)的活性藥劑。 此處之“癌症，，意指細胞腫瘤。癌症細胞具有自主生長(aut〇n〇m〇us growth)的能力，即在不正常的狀態或條件下迅速增殖細胞生長。此處所指 之癌症係包含所有種類之細胞不當增生(CanCer〇US 或致癌過程 (oncogenicprocesses)、轉移性的組織或惡性轉換之細胞、組織或器官（與組 織病理學型態無關）或侵入階段。癌症的例子包括，但不限定於：癌症 (carcinoma)與惡性肉瘤(SarC〇ma)，例如乳癌(breast CanCer)、血癌(leukemia)、 惡性肉瘤(sarcoma)、淋巴瘤(lymphomas)、惡性骨肉瘤(osteosarcoma)、神經 膠質瘤(glioma)、嗜鉻細胞瘤(pheochromocytoma)、肝惡性禮瘤(hepatoma)、The compound of formula I - Andrew has the possession - or multi-mineral center and therefore has various stereoisomeric forms. The hydrazine compounds of the formula referred to in the present invention include all such isomers. The compound of formula t has a selective inhibitory effect (four) efficacy. Because of its extremely small molecular weight, a lower dose of a guanidine compound and a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier can be used to obtain a desired therapeutic effect. The Pharmacy Composition of this county, which inhibits the growth of sputum, and even treats or lion cancer, will be an effective amount! a compound and a pharmaceutically acceptable salt thereof for use in inhibiting cancer cells, or administering to a patient in need thereof (the patient has cancer, a symptom of cancer, or a constitution that is prone to cancer) to heal, restore, alleviate, alleviate, change To treat, improve, improve, or affect the disease, the symptoms of the disease, or the constitution that favors the disease. As used herein, "(10) eftoive amount" means an effective amount of a compound of the formula _ Andrew, and its pharmaceutically acceptable salt, having an inhibitory or therapeutic effect. The amount of change is based on the dosing, exdpientusage, and other co-usage active agents. Here, "cancer," means a cell tumor. The cancer cell has the ability to grow aut〇n〇m〇us, that is, rapidly proliferate cell growth under abnormal conditions or conditions. Contains all types of cellular inappropriate proliferation (CanCer〇US or oncogenic processes, metastatic tissue or malignant transformation of cells, tissues or organs (unrelated to histopathological type) or invasive stages. Examples of cancer include, but Not limited to: cancer and Sarc〇ma, such as breast cancer (breast cancer), leukemia, sarcoma, lymphomas, osteosarcoma, glial Gliaoma, pheochromocytoma, hepatoma,
4 1377939 黑色素瘤(melanoma)卵巢癌(ovarian cancer)、皮膚癌(Skin CanCer)、大腸癌 (colorectal cancer)、胃癌(gastric cancer)、胰臟癌②咖卿价 cancer)、腎臟癌 (renal cancer)、前列腺癌(prostate cancer)、睪丸癌(testicular cancer)、頭部與 頸部的癌症、腦癌(brain cancer)、食道癌(esophageal cancer)、膀胱癌(bladder cancer)、腎上腺皮質癌(adrenai eortical cancer)、肺癌(lung cancer)、支氣管 癌(bronchus cancer)、子宮内膜癌(endometrial cancer)、鼻咽癌(nas〇pharyngeal cancer)、子宮頸、肝癌(cervicai oriivercancer)或未知起始位置的癌症。 式I化合物-安卓幸是以有機溶劑萃取牛樟椴木培養成長中之牛樟芝 ^ (一真菌類），並經矽膠管柱分離純化製備而得；或另以化學合成方法製傷 而得。例如：由“牛樟椴木培養成長中之牛樟芝萃取”指自較適成長程度之牛 樟芝所萃取出的牛樟芝萃取物。為取得該牛樟芝萃取物，可使用本技術領 域中眾所周知的萃取技術。例如可將經乾燥與研磨之該牛樟芝懸浮在一溶 劑或者兩種或多種溶劑之混合液於一足夠長的時間。適合的溶劑的例子包 括，但不限定為：水、甲醇、乙醇、二氣曱烧(methyiene chi〇ride)、三氣甲 烷(chloroform)、丙酮(acetone)、醚類(ether)(例如乙醚(diethyl ether))與乙 酸乙酯酯類(ethyl acetate)與己烷（hexane)。之後移除固體殘餘物（例如藉 ^ 由過濾）得到該牛樟芝萃取物溶液，其可經矽膠管柱純化製備得式Ϊ化合物 -安卓幸。基本上，全世界近二十餘年在牛樟芝所含天然化合物的研究，除 多醣體等大分子外’總共發表了七十八個小分子化合物，其中包括三十一 個三萜類化合物且大都有相關藥理活性研究報告，尤其著重在該等之抗癌 活性，惟三結類化合物各別分子仍須在較高使用量’才能達到癌症臨床化 學治療藥物的效果【Geethangili M and Tzeng YM, Review 〇f pharmaeological effects oiAntrodia camphorata and its bioactive compounds. Evidence-based Complementary and Alternative Medicine, published online on Aug. 17, 2009* doi: 10.1〇93/ecam/nepl08】。但是，式I化合物-安卓幸自1995年首度報導在 牛樟芝中被發現後【Chiang HC，Wu DP，Chemg IW，Ueng CH. A SeSqUiterpene 5 1377939 lactone, phenyl and biphenyl compounds from Antrodia cinnamomea. 吵1995;39:613-616】，迄今十五年間，未再有任何相關報導， 其藥理活性報告亦付諸缺如。究其原因乃該式j化合物_安卓幸在一般之牛 樟之子實體含量頗低而不易分離取得，本發明在特定成長程度之牛樟芝中 可經萃取分離純化而製備取得。同時，本發明發現式工化合物_安卓幸對不 同癌症細胞株（包括：大腸直腸癌細胞-HT_29或HCT116 ;肝癌細胞株 -Huh7 ;肺腺癌細胞株-A549及乳癌細胞株-MCF7或MDAMB231)具高度 抑制性，但對正常細胞（包括：纖維母細胞_HS68及正常乳腺上皮細胞 -MCF10A)則幾乎不具細胞毒性；再者，經與癌症臨床化學治療藥物(艾黴素 Doxorubicin及順鉑Cisplatin)進行對照實驗，評估式I化合物_安卓幸、艾 黴素、順鉑對惡性乳癌細胞（MDA-MB-231)之相對抑制效果，實驗證實在 相同之使用劑量下，式I化合物-安卓幸抑制惡性乳癌細胞 之效果優於艾黴素Doxorubicin、順鉑Cisplatin，故其甚至可用於治療及/或 預防癌症。在此必須再強調的是：式I化合物_安卓幸乃是牛樟芝所含各種 天然化合物中，惟一經實驗證實抑制癌細胞效果較諸癌症臨床化學治療藥 物(艾黴素Doxorubicin及順鉑Cisplatin)為優之牛樟芝所含天然化合物。 在本發明之治療方法中，式I化合物-安卓幸或其醫藥上可接受鹽類可 同時給藥或分開給藥，以口服、非口服、經由吸入喷霧(inhalati〇n spray)或 — 藉由植入貯存器(implantedreservoir)的方式。此處所使用之“非口服”指皮下 (subcutaneous)、皮内（intracutaneous)靜脈内（intravenous)、肌肉内 (intramuscular)、關節内（intraarticular)動脈(intraarterial)、滑囊（腔）内 (intrasynovial)、胸骨内(intrastemal)蜘蛛膜下腔(intrathecal)、疾病部位内 (intraleaional)與頭顱内(intracranial)注射以及灌注技術。 本發明所使用式I化合物-安卓幸及/或其醫藥上可接受鹽類可與至少一 種固體、液體或半液體狀之賦形劑或輔助劑一同形成適當的藥劑形式。其 形式包括，但不限定於，藥錠、膠囊、乳劑(emulsions)、水性懸浮液(aqueous 6 1377939 suspensions)、分散液(dispersi〇ns)與溶液。雜一般所使用的載體(窗㈣包 •括⑽與玉該粉。-般也賴滑舰ubrieating agent)，例如硬脂酸鎮 (magnesiumstearate)加至藥錠巾。用於膠囊形摘稀釋劑㈣㈣包括乳糖 與經乾燥的玉米澱粉。當口服給藥為水性懸浮液或乳劑時，可懸浮或溶解 有效成分(active ingredient)於與乳化或懸浮劑結合的油相㈣灿㈣。如果 需要，可加入特定甜味、調味與著色劑。 本發明所制式I化合物安卓幸或其醫藥上可接受鶴亦可配製成無 菌注射成分（例如，水或油的懸浮液），例如利用本技術領域中已知的技術 ^使用適合的分散或增闕（例如τ·η 8〇)麟㈣。無敝賴劑也可 以將無g注射溶液絲浮液加人無雜非〃服之獅舰溶劑，例如丨,3 丁 二醇(U-Butanediol)中。可使用的載具(vehicles)與溶劑包括甘露醣醇 (mannitol)、水、林格氏液(R]nger’s s〇luti〇n)與等滲透壓氣化納溶液。此外， 無菌、固定油常作為溶劑或懸浮媒介（例如合成的單_或雙-甘油酯 (glycerides))。脂肪酸’例如油酸（〇ieic acid)與其甘油酯衍生物亦可用在 注射劑的調製，其為天_學上可接受的油，例如橄獅、顏^油㈣恤 oil) ’特別是於其聚氧乙基化的(polyoxyethylated)變化形式。這些油溶液或 ^ 懸浮液也可包含一長鏈醇類稀釋劑或分散劑，或者羧基甲基纖維素 (carboxymethyl cellulose )或類似的分散劑。 本發明所使用式I化合物-安卓幸或其醫藥上可接受鹽類亦可根據此技 術領域中所熟知的技術來配製成吸入成分。例如可製成鹽類溶液，利用苯 甲醇（benzyl alcohol )或其他適合的防腐劑、增強生物可利用性 (bioavailabi%)的吸附促進劑、碳氟化合物(fluorocarbon)或其他本技術領域 中熟知的助溶或分散劑來配製。 用於藥學組成物的載體必須是“可接受的”，其與配方的有效成分相容 (以及較佳為具有穩定有效成分之能力）以及不對病患有害。例如，助溶 劑（例如環狀糊精(cyclodextrins))(其與一個或多個萃取物的活性化合物形 成特定更可溶解的複合物），為了有效成分的傳送而作為藥理學上的輔藥。 其他載體的例子包括膠狀二氧化矽(colloidal silicon dioxide)、硬脂酸鎂、纖 維素與烧基琉酸鹽(sodium lauryl sulfate)。 另外，由於抗癌劑如以高劑量投予病患易產生毒性。是以，本發明醫 藥組合物為含有安全有效量之式j化合物_安卓幸，用於抑制癌細胞生長， 其中該安全有效量為Ο.ΟΙμΜ至1〇〇〇μΜ，較佳為0.5μΜ至50μΜ。施予個 別病人的特賴量是麵有可能存在因素而定，例如：職狀特定化合 物的活性、年齡、體重、—般健康狀況、性別、進食狀況、施用時間與路 徑、排泄率、醫熱質之組合、以及所欲絲之疾病的嚴重程度等。 1377939 【實施方式】 為了讓本發明之上述和其他目的、特徵、和優點能更明顯易懂’下文 特舉較佳實施例，作詳細說明如下： 實施例一、式I化合物_安卓幸之製備： 將選取之牛樟芝經過冷凍乾燥後粉碎，以曱酵熱迴流萃取後，將其過濾 刀別得到沈澱物以及甲醇萃取液兩部分。將甲醇萃取液經過減壓濃縮成黏 ^稠狀後’再利用氯仿和水萃取後，將氣仿層加入適量的石夕膠共同減壓濃縮 至粉末後，填入石夕膠管柱上端，進行矽膠管柱層析，純化製備式1化合物_ 安卓幸。 實施例二、生物活性分析方法： 1.冷凍細胞之活化 冷來細胞之活化原則為快速解凍，以避免冰晶重新結晶而對細胞 ^ 造成傷害，導致細胞之死亡。細胞活化後，約需數日，或繼代一至二 代，其細胞生長或特性表現才會恢復正常（例如產生單株抗體或是其 他蛋白質〕。冷柬的細胞快速解;東的方法為：將冷柬管由液氮或乾冰容 器中取出，立即放人37 °C水槽中快速解;東，輕搖冷束管使其在3分 鐘内全部融化，以观酒精擦拭保存管的外部，移入無_作台内。 取出解凍之細胞懸浮液，緩緩加入有培養基之培養容器内（稀釋比例為 1:10-1:15)，混合均勻，放入c〇2培養箱培養。在解;東培養後隔日更換 培養基。 1377939 2.人類乳癌細胞之培養 此實驗選用三種不同的人類乳癌細胞株，分別為正常乳腺上皮細胞 (MCF10A cells)、人類乳癌前細胞（MCF-7 cells)及人類惡性乳癌細胞 (MDA-MB-231 cells)°MDA-MB-231細胞株培養於5%胎牛血清 (FBS)、2 mM 麩醯胺酸(glutamine) ’ lOOpg/ml 鍵徽素(streptomycin)和 100 U/ml 盤尼西林(penicillin)的 Dulbecco's Modified Eagle's Medium (DMEM)中；MCF-7細胞株培養於5%胎牛血清、2mM麩醯胺酸，100 pg/ml鍵徽素和100 U/ml盤尼西林、0.01 mg/ml騰島素(insulin)的 Dulbecco’s Modified Eagle's Medium (DMEM)中；MCF10A 細胞株培養 於5%馬血清(Horse Serum)、肌膚表皮生長因子(EGF)(最終濃度 20ng/ml)、氫羥腎上腺皮質素(Hydrocortisone)(最終濃度0.5 mg/ml)、霍 亂毒素(Cholera Toxin)(最終濃度100 ng/ml)、胰島素（最終濃度 l〇pg/ml)、4 mM麩酿胺酸、100 pg/ml鏈黴素和1〇〇 U/ml盤尼西林、 0.01 mg/ml 胰島素的 Dulbecco’s Modified Eagle's Medium (DMEM)中。 以上MDAMB231及MCF7兩種細胞以10 %胎牛血清作繼代培養。細 胞株放置於37 °C，5% C02，湮度95 %的培養箱中培養。4 1377939 melanoma ovarian cancer, skin canCer, colorectal cancer, gastric cancer, pancreatic cancer 2, renal cancer , prostate cancer, testicular cancer, head and neck cancer, brain cancer, esophageal cancer, bladder cancer, adrenal eortical Cancer), lung cancer, bronchus cancer, endometrial cancer, nas〇pharyngeal cancer, cervix, cervicai oriivercancer or cancer at an unknown starting point . The compound of the formula I-Android is extracted by extracting the growing Astragalus sinensis (a fungus) from the burdock wood by an organic solvent, and is obtained by separating and purifying the ruthenium tube column; or by chemically synthesizing the wound. For example, extracting from Antrodia camphorata, which is grown in the growth of burdock, refers to the extract of Antrodia camphorata extracted from the more suitable growth degree of Antrodia camphorata. To obtain the Antrodia camphorata extract, extraction techniques well known in the art can be used. For example, the dried and ground Astragalus lucidum can be suspended in a solvent or a mixture of two or more solvents for a sufficient period of time. Examples of suitable solvents include, but are not limited to, water, methanol, ethanol, methyiene chi 〇ride, chloroform, acetone, ether (eg diethyl ether) Diethyl ether)) with ethyl acetate and hexane. The solid residue is then removed (for example by filtration) to obtain the Antrodia camphorata extract solution, which can be purified by a gel column to prepare a compound of the formula - Andrew. Basically, the study of natural compounds contained in Antrodia camphorata in the world for more than 20 years, in addition to macromolecules such as polysaccharides, has published a total of 78 small molecule compounds, including 31 triterpenoids and most of them. Relevant pharmacological activity studies, especially focusing on these anticancer activities, but the individual molecules of the three nodal compounds still have to be used at higher doses to achieve the effect of cancer clinical chemotherapeutic drugs [Geethangili M and Tzeng YM, Review 〇 f pharmaological effects oiAntrodia camphorata and its bioactive compounds. Evidence-based Complementary and Alternative Medicine, published online on Aug. 17, 2009* doi: 10.1〇93/ecam/nepl08]. However, the compound of formula I - Android was first reported in 1995 after being discovered in Antrodia camphor [Chiang HC, Wu DP, Chemg IW, Ueng CH. A SeSqUiterpene 5 1377939 lactone, phenyl and biphenyl compounds from Antrodia cinnamomea. 39:613-616], there have been no reports in the past 15 years, and the pharmacological activity report has been invalidated. The reason is that the compound of the formula j_Android is well-known in the general bovine 樟 子 子 子 子 子 子 子 子 子 子 子 子 子 子 子 子 子 子 子 子 子 子 子 子 子 子 子 子 子 子 子 子 子 。 。 。 。 At the same time, the present invention finds that the compound of the formula_Android is for different cancer cell lines (including: colorectal cancer cell-HT_29 or HCT116; liver cancer cell line-Huh7; lung adenocarcinoma cell line-A549 and breast cancer cell line-MCF7 or MDAMB231) It is highly inhibitory, but it is almost non-cytotoxic to normal cells (including: fibroblasts _HS68 and normal mammary epithelial cells - MCF10A); in addition, it is clinically chemotherapeutic with cancer (Ixomycin Doxorubicin and Cisplatin Cisplatin) A comparative experiment was conducted to evaluate the relative inhibitory effect of the compound of formula I_Android, oxytetracycline and cisplatin on malignant breast cancer cells (MDA-MB-231). The experiment confirmed that the compound of formula I was used at the same dosage. The effect of inhibiting malignant breast cancer cells is superior to that of dioxin Doxorubicin and Cisplatin, so it can even be used for the treatment and/or prevention of cancer. It must be emphasized here that the compound of formula I_Android is the most natural compound contained in the burdock, and the only experimentally confirmed inhibition of cancer cells is superior to the cancer clinical chemotherapeutic drugs (Doxorubicin and Cisplatin). The natural compound contained in Antrodia camphorata. In the method of the present invention, the compound of the formula I - Andrews or a pharmaceutically acceptable salt thereof, may be administered simultaneously or separately, orally, parenterally, by inhalation spray or inhalation. By means of an implanted reservoir (implantedreservoir). As used herein, "non-oral" refers to subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intrasynovial, and intrasynovial. , intrastemal intrathecal, intraleacial and intracranial injection and perfusion techniques. The compound of formula I used in the present invention - Andrews &/or a pharmaceutically acceptable salt thereof, may be combined with at least one solid, liquid or semi-liquid excipient or adjuvant to form a suitable pharmaceutical form. Forms include, but are not limited to, tablets, capsules, emulsions, aqueous suspensions (aqueous 6 1377939 suspensions), dispersions (dispersi〇ns) and solutions. The carrier used in general (window (four) package includes (10) and jade powder. - also the ubrieating agent), such as magnesium stearate (magnesiumstearate) added to the drug towel. The capsule-shaped diluent (4) (iv) includes lactose and dried corn starch. When the oral administration is an aqueous suspension or emulsion, the active ingredient may be suspended or dissolved in an oil phase (4) which is combined with an emulsifying or suspending agent. Add specific sweetness, flavoring, and coloring agents if needed. The compound of the formula I of the present invention, Andrew or its pharmaceutically acceptable crane, may also be formulated as a sterile injectable component (for example, a suspension of water or oil), for example, using suitable techniques or techniques known in the art. Increase (for example, τ·η 8〇) Lin (4). The non-suppressant can also be used to add a non-g injection solution to the schooner solvent, such as bismuth, U-Butanediol. Vehicles and solvents that may be used include mannitol, water, Ringer's solution (R]nger's s〇luti〇n) and isotonic pressure gasification nanosolutions. In addition, sterile, fixed oils are often employed as a solvent or suspension medium (for example, synthetic mono- or glycerides). Fatty acids such as 油ieic acid and its glyceride derivatives can also be used in the preparation of injectables, which are _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ A polyoxyethylated variant. These oil solutions or suspensions may also contain a long chain alcohol diluent or dispersant, or carboxymethyl cellulose or a similar dispersing agent. The compound of formula I used in the present invention - Andrews or a pharmaceutically acceptable salt thereof, may also be formulated as an inhalation component according to techniques well known in the art. For example, a salt solution can be prepared, using benzyl alcohol or other suitable preservatives, bioavailabi% adsorption enhancers, fluorocarbons or other well known in the art. Formulated with a solubilizing or dispersing agent. The carrier for the pharmaceutical composition must be "acceptable" which is compatible with the active ingredients of the formulation (and preferably has the ability to have a stable active ingredient) and is not deleterious to the patient. For example, a solubilizing agent (e.g., cyclodextrins) which forms a specific more soluble complex with one or more active compounds of the extract, acts as a pharmacological adjuvant for the delivery of the active ingredient. Examples of other carriers include colloidal silicon dioxide, magnesium stearate, cellulose and sodium lauryl sulfate. In addition, since an anticancer agent is administered to a patient at a high dose, it is susceptible to toxicity. Therefore, the pharmaceutical composition of the present invention contains a safe and effective amount of a compound of formula j, which is used to inhibit the growth of cancer cells, wherein the safe and effective amount is Ο.ΟΙμΜ to 1〇〇〇μΜ, preferably 0.5μΜ. 50μΜ. The amount of special treatment given to individual patients may be determined by factors such as the activity of the specific compound in the occupation, age, weight, general health status, gender, eating status, administration time and route, excretion rate, medical fever. The combination of quality, and the severity of the disease. BRIEF DESCRIPTION OF THE DRAWINGS The above and other objects, features, and advantages of the present invention will become more <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; The selected Antrodia camphorata is lyophilized, pulverized, and refluxed with retort heat, and then filtered to obtain a precipitate and a methanol extract. The methanol extract is concentrated under reduced pressure into a thickened shape, and then extracted with chloroform and water. The gas-like layer is added to an appropriate amount of Shiqijiao and concentrated under reduced pressure to a powder, and then filled into the upper end of the Shixi rubber tube column. Purification of the compound of formula 1 by gel column chromatography, _ Andrew. Example 2: Biological activity analysis method: 1. Activation of frozen cells The principle of activation of cold cells is rapid thawing to avoid recrystallization of ice crystals and cause damage to cells, leading to cell death. After activation of the cells, it takes about a few days, or one to two generations of subculture, and its cell growth or characteristic performance will return to normal (for example, producing monoclonal antibodies or other proteins). The cells of cold Cambodia are quickly solved; the eastern method is: Remove the cold tube from the liquid nitrogen or dry ice container, and immediately put it into the 37 °C water tank for quick solution; East, gently shake the cold tube to melt it in 3 minutes, wipe the outside of the storage tube with alcohol, and move in. No _ in the table. Take out the thawed cell suspension, slowly add the culture medium in the culture medium (dilution ratio: 1:10-1:15), mix well, and put it into the c〇2 incubator for cultivation. After the culture in East, the medium was changed every other day. 1377939 2. Culture of human breast cancer cells Three different human breast cancer cell lines were selected for this experiment: normal breast epithelial cells (MCF10A cells), human breast precancerous cells (MCF-7 cells) and human malignant. Breast cancer cells (MDA-MB-231 cells)°MDA-MB-231 cells were cultured in 5% fetal bovine serum (FBS), 2 mM glutamine 'lOOpg/ml streptomycin and 100 U/ml Dulbecco's M of penicillin In odified Eagle's Medium (DMEM); MCF-7 cell line is cultured in 5% fetal bovine serum, 2 mM glutamic acid, 100 pg/ml bondin and 100 U/ml penicillin, 0.01 mg/ml temsin (insulin) In Dulbecco's Modified Eagle's Medium (DMEM); MCF10A cells were cultured in 5% horse serum (Horse Serum), skin epidermal growth factor (EGF) (final concentration 20 ng/ml), Hydrocorretisone (final) Concentration 0.5 mg/ml), Cholera Toxin (final concentration 100 ng/ml), insulin (final concentration l〇pg/ml), 4 mM bran tyrosine, 100 pg/ml streptomycin and 1〇 〇U/ml penicillin, 0.01 mg/ml insulin in Dulbecco's Modified Eagle's Medium (DMEM). The above MDAMB231 and MCF7 cells were subcultured with 10% fetal bovine serum. The cell line was placed at 37 °C, 5% C02. Incubate in a 95% incubator.
3.細胞藥物處理： 所有實驗的細胞皆培養於含10°/〇胎牛血清的培養液中，待細胞長到 約八成滿時，將舊培養液抽乾並以PBS緩衝液(碟酸鹽緩衝液)溶液清洗 細胞後，加入10毫升不含血清之培養液。依實驗目的不同加入不同的 藥物，於37。(：恆溫培養箱中進行反應。 10 ③ 4、細胞毒性實驗(cytotoxicity): 將惡性乳癌細胞（MDA-MB-231 cells)、人類乳癌前細胞（mCF-7 cells)及正常乳房上皮細胞(MCF10A cells)放置於96孔培養盤(culture plate)(2000細胞/孑匕），以及將其隔夜培養於100 μΐ的完全DMEM中。 將50 μΐ包含式I化合物-安卓幸(0.5-10 μΜ)之完全DMEM等量樣品加 入培養盤的不同孔中。另外，控制組則只加入100 μΐ的完全DMEM。 於培養2天之後，以績跌羅丹明B (sulforhodamine Β)(—蛋白質結合 染劑）分析測定每孔中的細胞數目。簡單地說，固定細胞於10%三氣 醋酸（trichloroacetic acid)中，與以0.4%磺酰羅丹明B將其染色。染色 20分鐘後再以1%乙酸清洗，之後，將與細胞結合的磺酰羅丹明β溶 解於10 mM的Tris base中。以微量滴定盤檢測器(microtiter plate reader) 在562 nm下測定吸光值(optical density)。上述之方法亦用來測試Huh7 肝癌細胞、A549肺腫瘤細胞與HT29及HCT116大腸癌細胞株等細胞 對式I化合物-安卓幸的敏感性。 5.細胞型態變化的分析 細胞凋亡(apoptosis)與細胞壞死(necrosis)在型態學上有很大的差 異’特徵有細胞萎縮、脫落，染色質濃縮等現象。將惡性乳癌細胞 (MDA-MB-231)放置於petri dish培養盤(皮氏培養孤），待細胞貼底後， 給予適當包含式I化合物-安卓幸(0.5-10 μΜ)之藥物濃度，至於培養箱 中一段時間後，以倒立顯微鏡觀察細胞型態，並拍照紀錄之。 1377939 6.細胞週期與細胞凋亡檢測： 惡性乳癌細胞(MDAMB231)經式I化合物-安卓幸處理後’以腺蛋 白-乙二胺四乙酸(trypsin-EDTA)處理細胞，與培養液一起收集，離心後 去掉上清液，並以4°C磷酸鹽緩衝液清洗後，加入1毫升冰冷的75% 酒精，放入4。<：冰箱過夜，以固定細胞。離心後，續以1毫升PBS懸 浮，加入適量的核糖核酸A(RNaseA)，於37°C作用30分鐘，最後加 入 40mg/ml 之碘化丙鍵(propidium iodide) (PI，Sigma Chemical Co.，cat.3. Cell drug treatment: All the cells in the experiment were cultured in a culture medium containing 10 °/fetal fetal bovine serum. When the cells were grown to about 80% full, the old culture solution was drained and used in PBS buffer (disc acid salt). After washing the cells with the buffer) solution, 10 ml of serum-free medium was added. Add different drugs according to the purpose of the experiment, at 37. (: Reaction in a constant temperature incubator. 10 3 4. Cytotoxicity: MDA-MB-231 cells, human breast cancer cells (mCF-7 cells), and normal breast epithelial cells (MCF10A) Cells were placed in a 96-well culture plate (2000 cells/孑匕) and cultured overnight in 100 μΐ of complete DMEM. 50 μΐ containing the compound of formula I - Andrew (0.5-10 μΜ) A full DMEM sample was added to the different wells of the plate. In addition, the control group was only 100 μΐ of complete DMEM. After 2 days of culture, the sulforhodamine ( (protein binding agent) was analyzed. The number of cells in each well was determined. Briefly, the cells were fixed in 10% trichloroacetic acid and stained with 0.4% sulforhodamine B. After 20 minutes of staining, it was washed with 1% acetic acid. Thereafter, the cell-bound sulforhodamine β was dissolved in 10 mM Tris base, and the optical density was measured at 562 nm using a microtiter plate reader. Test Huh7 liver cancer The sensitivity of A549 lung tumor cells and HT29 and HCT116 colorectal cancer cell lines to the compound of formula I-Android. 5. Analysis of cell type changes Apoptosis and necrosis in morphology There is a big difference in the characteristics of cell atrophy, shedding, chromatin condensation and other phenomena. Place the malignant breast cancer cells (MDA-MB-231) in a petri dish (Pearl culture), after the cells are bottomed, The appropriate concentration of the drug containing the compound of formula I - Android (0.5-10 μΜ) was given. After a period of time in the incubator, the cell type was observed with an inverted microscope and photographed. 1377939 6. Cell cycle and apoptosis detection : Malignant breast cancer cells (MDAMB231) were treated with the compound of formula I - Android, and treated with adenosine-EDTA, collected with the culture medium, centrifuged and the supernatant removed, and 4 After washing with °C phosphate buffer, add 1 ml of ice-cold 75% alcohol and put in 4. <: refrigerator overnight to fix the cells. After centrifugation, continue to suspend in 1 ml of PBS, add appropriate amount of ribonucleic acid A (RNaseA ), at 37 ° C Over 30 minutes, and finally added 40mg / ml of propidium iodide key (propidium iodide) (PI, Sigma Chemical Co., cat.
No P-4170)避光作用半小時，以35mm尼龍網（nylon mesh)過濾後， 以495nm波長激發後，於637nm波長彳貞測細胞螢光含量，以流式細 4 胞儀進行分析。 實施例三、生物活性測定之結果 1.式I化合物-安卓幸可抑制不同腫瘤細胞之生長。 挑選不同腫瘤細胞株包括：大腸直腸癌細胞-HT-29或HCT116 ; 肝癌細胞株-Huh7 ;肺腺癌細胞株-A549及乳癌細胞株_MCF7或 MDAMB231來探討式I化合物-安卓幸對不同癌細胞之毒殺作用。結果 β 發現式I化合物可顯著抑制不同遽瘤細胞之生長，對腫瘤細胞所造成之 半抑制濃度（IC5〇值)分別介於0.6〜4.5μΜ之間，其中又以對惡性乳癌細 胞（MDA-MB-231 cells)之毒殺效果最強（圖一）。值得注意的是，以纖 維母細胞(HS68)及正常乳腺上皮細胞(MCF10A cells)進一步分析式！化 合物對正常細胞生長之影響，則發現式I化合物並不會造成兩株正常細 胞之生長抑制（圖二）。由此得知，式I化合物-安卓幸的確具有選擇性 毒殺癌細胞之能力，其中又以乳癌的毒殺效果最為顯著。No P-4170) After being shielded from light for half an hour, it was filtered through a 35 mm nylon mesh, and after excitation at a wavelength of 495 nm, the fluorescence content of the cells was measured at a wavelength of 637 nm, and analyzed by a flow cytometer. Example 3 Results of Biological Activity Assay 1. The compound of Formula I - Andrew fortunes inhibits the growth of different tumor cells. Selection of different tumor cell lines including: colorectal cancer cell-HT-29 or HCT116; liver cancer cell line-Huh7; lung adenocarcinoma cell line-A549 and breast cancer cell line _MCF7 or MDAMB231 to explore the compound of formula I - Andrew fortunately for different cancers The poisoning effect of cells. Results β found that the compound of formula I can significantly inhibit the growth of different tumor cells, and the semi-inhibitory concentration (IC5 〇 value) caused by tumor cells is between 0.6 and 4.5 μΜ, respectively, which is in the pair of malignant breast cancer cells (MDA- MB-231 cells) have the strongest poisoning effect (Figure 1). It is worth noting that further analysis is performed with fibroblasts (HS68) and normal mammary epithelial cells (MCF10A cells)! The effect of the compound on normal cell growth revealed that the compound of formula I did not cause growth inhibition in both normal cells (Fig. 2). It is thus known that the compound of the formula I - Andrew has the ability to selectively kill cancer cells, and the most effective killing effect of breast cancer.
12 137.7939 2，式I化合物-安卓幸與化學治療藥物艾黴素(D〇x〇rubicin)及順鉑 (Ckplatin)的比較 利用SRB的方法來比較式工化合物_安卓幸、d〇x〇mbidn與 cisplatin對於惡性乳癌細胞ce㈣的效用DD〇x〇mbidn 與cisplatin為市面上常使用的抗乳癌藥物之一，d〇xorubicin主要是嵌 入DNA的含氮鹽基中與DNA形成複合物、抑制聚合晦的活性，或是 藉著產生具有毒性的超氧游離根攻擊DNA，都可影響癌細胞合$dna; cisplatin的作用機轉主要以共價鍵鍵結至癌細胞之DNA，造成雙股 ^ DNA間交互連結，進而抑制腫瘤細胞DNA合成。在投與藥物48小時 後，在比較發現doxorubicin在5〜ΙΟμΜ之間，對於惡性乳癌細胞 (MDA-MB-231 cells)維持了 60-70%的毒性，而式I化合物在濃度5μΜ 以上毒性卻比doxorubicin高’在ΙΟμΜ竟高於30%。Cisplatin對於惡 性乳癌細胞（MDA-MB-231cells)在5μΜ的濃度下細胞毒性與控制組 相差無幾’在ΙΟμΜ才有30°/❶的毒性（圖三）。 ^ 3.式1化合物-安卓幸對惡性乳癌細胞及正常乳腺上皮細胞生長之影 響。 由實驗結果得知式I化合物-安卓幸的確對乳癌細胞具有較佳之抑 制作用。因此進一步將惡性乳癌細胞（MDA-MB-231 cells)、人類乳癌 前細胞（MCF-7 cells)及正常乳房上皮細胞(MCF10A cells)利用 Sulforhodamine B (SRB assay)分析的方法測定式I化合物對於細胞生長 之景>響。SRB分析法的原理是利用染劑（SulforhodamineB，SRB)與細 胞質蛋白質的鹼性氨基酸鍵結而呈色，主要用於量測細胞的增生與存 活率。結果發現，投與藥物48小時後，惡性乳癌細胞株(MDAMB231) 及人類乳癌前細胞(MCF7)隨著式I化合物濃度的上升其細胞毒性顯著 13 增加’在式I化合物濃度1_處理下分別可抑制8〇%及6〇%細胞存 活率（表-及表-)。而正常乳腺上皮細胞⑽^·)之細胞存活率並未 隨著式I化合物濃度的增加而有所影響(表三)。故由此可知式“匕合物_ 安卓幸麟·«細胞(MDAMB231及人難麟_ (MCF7)有 很高的細胞毒性反而對於正常乳腺上皮細胞(MCF1GA)並無太高的細 胞毒性。表一為以SRB 分析評估式I化合物_安卓 幸對惡性乳癌細胞（MDAMB231)之細胞毒性。表二為以SRB分析評估 式I化合物-安卓幸對惡性乳癌細胞（MCF7)之細胞毒性。表三為以 SRB分析評估式I化合物_安卓幸對正常乳腺上皮細胞（MCF1〇A)之細 胞毒性。 表—、以SRB分析評估式丨化合物對惡性乳癌細胞(MDAMB231)之細胞毒性 式丨化合物 濃度（μΜ) 吸光度 相對細胞 存活率(％) 0 0.484 ± 0.009 100 0.5 0.301 ± 0.004 62.1 1 ——— 0_103± 0.001 21.2 3 0.082 ± 0.001 17.1 5 0.057 ± 0.004 11.8 10 0.032 ± 0.006 6.6 相對細胞存活率(％) =(式I化合物處理組之吸光值)/(控制組之吸光值)X 1〇〇 1377939 表二、以SRB分析評估式I化合物對人類乳癌前細胞（MCF7)之細胞毒性 式1化合物 濃度（"M) 吸光度 相對細胞 存活率(％) 0 0.192 ± 0_005 100 0.5 0.148 ± 0.002 77 1 0.114± 0_003 59.5 3 0.091 ± 0.006 47.7 5 0.068 ± 0.005 35.8 10 0.046 ± 0_001 24.1 ‘ 相對細胞存活率(％) = (式丨化合物處理組之吸光值)/(控制組之吸光值)X 1 〇〇 表三、以SRB分析評估式I化合物對正常乳腺上皮細胞(MCF10A)之細胞毒性 式丨化合物 吸光度 相對細胞 濃度（_ 一1 -— 存活率(％) 0 2.352 ± 0_002 100 0.5 2.265 ± 0.001 97.3 1 2.194 ± 0.004 93.2 3 2.143 ± 0.008 91.1 5 2.067 ± 0.003 87.8 10 1.935 ± 0.004 82.2 相對細胞存活率(％) =(式I化合物處理組之吸光值)/(控制組之吸光值)X 100 15 1377939 4.測定式I化合物-安卓幸對惡性乳癌細胞株（丨）細胞型 態的影響。 本發明進-步酬式I化合物_安卓幸引起惡性乳癌峨的細胞毒 殺現象是否為經由細胞凋亡所引起。首先就細胞型態而言，由於細胞 凋亡活化了許多内生性的蛋白酵素，會顯著導致細胞骨架的破壞 (cytoskeletal disruption)、細胞萎縮(ceu shrinkage)、細胞膜上會有泡狀 物(membmnce blebbing)的產生’並且有染色質濃縮(chf〇matin condensation)的現象。由實驗結果中’清楚觀察到將式〗化合物給予惡 性乳癌細胞MDA-MB-231後’細胞型態上有明顯萎縮的變化，並且可 明顯的觀察到凋亡小體(apoptoticbody)。此與未加式I化合物處理的控 制組細胞相比，外觀有顯著的不同。此外，也可觀察到許多細胞有脫 落現象(detachment)而懸浮於培養液中（圖四）。 5.式I化合物-安卓幸造成惡性乳癌細胞MDA-MB-231細胞凋亡的分 析以及其對細胞週期的影響 由於正常人體的DNA是雙套的(2N)，而凋亡細胞DNA斷裂成小 一 片斷，因此會比正常G0/G1期細胞有較低的染色密度(stainability)，經 piOpidiumiodide(PI)染色後，以流式細胞儀分析後，可將凋亡細胞顯現 出來’而形成sub G1 peak。因此，本發明進一步對惡性乳癌細胞 MDA-MB-231細胞進行PI染色，利用流式細胞儀分析式I化合物-安卓 幸對細胞週期的影響。由實驗結果發現，式I化合物（ΙμΜ和5 μΜ) 作用48小時後，會些微增加G0/G1的比例。相對的，隨著時間和濃 度的增加，經式I化合物處理的細胞’subGl的比例亦跟著明顯增加（圖 五）。 16 1377939 【圖式簡單說明】 圖一、以SRB分析評估式I化合物-安卓幸對不同癌症細跑株（包括.大 腸直腸癌細胞-HT-29或HCT116;肝癌細胞株-Huh7;肺腺癌細胞株 -A549及乳癌細胞株-MCF7或MDAMB231)之細胞毒性。 圖二、以SRB分析評估式I化合物-安卓幸對正常細胞（包括^纖維母細 胞-HS68及正常乳腺上皮細胞-MCF10A)之細胞毒性。 圖三、以SRB分析比較式I化合物安卓幸及癌症臨床化學治療藥物(艾 微素Doxorubicin及順始Cisplatin)評估對惡性乳癌細胞（MDA-MB-231) 之細胞毒性。 圖四、式I化合物-安卓幸造成惡性乳癌細胞株（MDA-MB-231)的細胞 型態改變。 圖五、式I化合物-安卓幸造成惡性乳癌細胞株（MDA-MB-231)的細胞 週期改變並造成subGJj的細胞增多。 【主要元件符號說明】 M. 1712 137.7939 2, a compound of formula I - Andrew fortunes with the chemotherapeutic drug dioxin (D〇x〇rubicin) and cisplatin (Ckplatin) using SRB method to compare formula compounds _ Andrew Xing, d〇x〇mbidn The effect of cisplatin on malignant breast cancer cell ce(4) DD〇x〇mbidn and cisplatin are one of the commonly used anti-breast cancer drugs on the market. d〇xorubicin mainly forms a complex with DNA and inhibits polymerization in the nitrogen-containing base of DNA. The activity, or by attacking DNA by producing toxic superoxide free roots, can affect cancer cells to bind to $dna; the action of cisplatin is mainly covalently bonded to the DNA of cancer cells, resulting in double strands of DNA Interacting with each other to inhibit tumor cell DNA synthesis. After 48 hours of administration of the drug, it was found that doxorubicin was between 5 and ΙΟμΜ, maintaining 60-70% toxicity to malignant breast cancer cells (MDA-MB-231 cells), while the compound of formula I was more than 5 μΜ. Higher than doxorubicin 'in ΙΟμΜ actually higher than 30%. The cytotoxicity of Cisplatin for malignant breast cancer cells (MDA-MB-231 cells) at a concentration of 5 μΜ was comparable to that of the control group, which was 30 °/❶ toxicity at ΙΟμΜ (Fig. 3). ^ 3. Compound of Formula 1 - Andrew's effect on the growth of malignant breast cancer cells and normal mammary epithelial cells. From the experimental results, it was found that the compound of the formula I - Andrew, did have a better inhibitory effect on breast cancer cells. Therefore, the MFA-MB-231 cells, human breast precancerous cells (MCF-7 cells) and normal breast epithelial cells (MCF10A cells) were further assayed by the method of Sulforhodamine B (SRB assay) for the cells of the formula I. The scene of growth > The principle of SRB analysis is to use coloring agent (Sulforhodamine B, SRB) to form a color with the basic amino acid bond of cytoplasmic protein, which is mainly used to measure the proliferation and survival rate of cells. The results showed that after administration of the drug for 48 hours, the malignant breast cancer cell line (MDAMB231) and human breast precancerous cells (MCF7) increased significantly with the increase of the concentration of the compound of formula I, and the cytotoxicity was significantly increased by 13% under the treatment of compound I of formula I. It can inhibit the cell survival rate of 8〇% and 6〇% (Table- and Table-). The cell viability of normal mammary epithelial cells (10)^·) did not affect the concentration of the compound of formula I (Table 3). Therefore, it can be known that the formula "Well _ Android Xing Lin · « cells (MDAMB231 and human yulin _ (MCF7) have high cytotoxicity but not too high cytotoxicity to normal mammary epithelial cells (MCF1GA). One was to evaluate the cytotoxicity of the compound of formula I by SRB analysis - Andrews for malignant breast cancer cells (MDAMB231). Table 2 shows the cytotoxicity of the compound of formula I - Andrews for malignant breast cancer cells (MCF7) by SRB analysis. SRB analysis was used to evaluate the cytotoxicity of the compound of formula I_Android for normal mammary epithelial cells (MCF1〇A). Table--Score analysis of the cytotoxic sputum compound concentration of malignant breast cancer cells (MDAMB231) by SRB analysis (μΜ Absorbance vs. cell viability (%) 0 0.484 ± 0.009 100 0.5 0.301 ± 0.004 62.1 1 ——— 0_103± 0.001 21.2 3 0.082 ± 0.001 17.1 5 0.057 ± 0.004 11.8 10 0.032 ± 0.006 6.6 Relative cell viability (%) = (absorbance of the compound treated group of formula I) / (absorbance of the control group) X 1〇〇1377939 Table 2. Evaluation of the cytotoxicity of the compound of formula I on human breast cancer pre-cells (MCF7) by SRB analysis 1 Compound concentration ("M) Absorbance versus cell viability (%) 0 0.192 ± 0_005 100 0.5 0.148 ± 0.002 77 1 0.114 ± 0_003 59.5 3 0.091 ± 0.006 47.7 5 0.068 ± 0.005 35.8 10 0.046 ± 0_001 24.1 ' Relative cell viability (%) = (absorbance of the compound treatment group) / (absorbance of the control group) X 1 〇〇 Table 3. Evaluation of the cytotoxic 丨 compound of the compound of formula I against normal mammary epithelial cells (MCF10A) by SRB analysis Absorbance vs. cell concentration (_1 - survival rate (%) 0 2.352 ± 0_002 100 0.5 2.265 ± 0.001 97.3 1 2.194 ± 0.004 93.2 3 2.143 ± 0.008 91.1 5 2.067 ± 0.003 87.8 10 1.935 ± 0.004 82.2 Relative cell viability ( %) = (absorbance of the compound treated group of formula I) / (absorbance of the control group) X 100 15 1377939 4. Determination of the effect of the compound of formula I - Andrews on the cell type of malignant breast cancer cell line (丨). The present invention is a compound of the formula I. The cytotoxicity of the cancer caused by malignant breast cancer is caused by apoptosis. First, in terms of cell type, many endogenous proteolytic enzymes are activated by apoptosis, which can cause cytoskeletal disruption, cue shrinkage, and bubble on the cell membrane (membmnce blebbing). 'produces' and has the phenomenon of chf〇matin condensation. From the experimental results, it was clearly observed that the compound of the formula was administered to the malignant breast cancer cell line MDA-MB-231, and there was a significant atrophy change in the cell type, and an apoptotic body was clearly observed. This is a significant difference in appearance compared to control group cells treated with no compound of formula I. In addition, many cells were observed to have detachment and were suspended in the culture medium (Fig. 4). 5. Compound of formula I - Andrews forcing apoptosis in malignant breast cancer cell line MDA-MB-231 and its effect on cell cycle Since normal human DNA is double-set (2N), apoptotic cell DNA breaks into small A fragment, therefore, has a lower staining stainability than normal G0/G1 phase cells. After staining with piOpidiumiodide (PI), the apoptotic cells can be visualized by flow cytometry to form sub G1. Peak. Therefore, the present invention further performs PI staining on the malignant breast cancer cell line MDA-MB-231, and analyzes the effect of the compound of the formula I-Android on the cell cycle by flow cytometry. From the experimental results, it was found that the compound of the formula I (ΙμΜ and 5 μΜ) slightly increased the ratio of G0/G1 after 48 hours of action. In contrast, as time and concentration increased, the proportion of cells treated with the compound of formula I was also significantly increased (Fig. 5). 16 1377939 [Simple description of the diagram] Figure 1. Evaluation of the compound of formula I by SRB analysis - Andrew fortunately for different cancer races (including colorectal cancer cells - HT-29 or HCT116; liver cancer cell line - Huh7; lung adenocarcinoma) Cytotoxicity of cell line-A549 and breast cancer cell line-MCF7 or MDAMB231. Figure 2. Evaluation of the cytotoxicity of the compound of formula I by the SRB assay - Andrews for normal cells, including fibroblasts-HS68 and normal mammary epithelial cells-MCF10A. Figure 3. Comparison of the cytotoxicity of the compound of formula I with the clinical chemotherapeutic drug of cancer (Axorubicin Doxorubicin and Cisplatin) by SRB analysis for malignant breast cancer cells (MDA-MB-231). Figure 4. Compound of Formula I - Andrew fortuned to cause cell type changes in a malignant breast cancer cell line (MDA-MB-231). Figure 5. Compound of Formula I - Andrews has caused a change in the cell cycle of the malignant breast cancer cell line (MDA-MB-231) and caused an increase in the cell count of subGJj. [Main component symbol description] M. 17