TWI573589B - Novel lipophilic n-substituted norcantharimide derivatives and uses thereo - Google Patents
Novel lipophilic n-substituted norcantharimide derivatives and uses thereo Download PDFInfo
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Description
本揭示內容是有關於一種新穎N-取代去甲基斑蝥素衍生物,及其用來治療癌症,特別是白血病,的用途。 The present disclosure is directed to a novel N-substituted desmethylcantharidin derivative and its use for the treatment of cancer, particularly leukemia.
斑蝥素(cantharidin,CA)是一種自斑蝥(Mylabris)中分離出來的活性化合物,已知其為絲胺酸/纈胺酸蛋白酶1(PP1)和蛋白酶2A(PP2A)的抑制劑,且已被證實具有毒殺癌細胞的能力,但不幸的是,它同時也具有腎臟毒性和肝毒性。因此,過去的研究都專注於找出一種新的斑蝥素衍生物或類似物,其仍保有毒殺癌細胞的能力但卻不會對正常組織造成傷害。據此,研究人員也找出兩種斑蝥素的衍生物,分別是去甲基斑蝥素(norcantharidin)和去甲基斑蝥亞胺(norcantharimide)。去甲基斑蝥素是斑蝥素去甲基化後的形式,具有較低的腎臟毒性,但去甲基化也相對地降低了斑蝥素的生物活性。至於去甲基斑蝥亞胺,研究發現當其N-上所帶烷基鏈愈長,由於烷基本身的親油性質,故可幫助細胞吸收去甲基斑蝥亞胺。 Cantharidin (CA) is an active compound isolated from Mylabris , known as an inhibitor of serine/proline protease 1 (PP1) and protease 2A (PP2A), and has been It has been shown to have the ability to kill cancer cells, but unfortunately it also has nephrotoxicity and hepatotoxicity. Therefore, past research has focused on finding a new cantharidin derivative or analog that still retains the ability to kill cancer cells without causing damage to normal tissues. Accordingly, the researchers also identified two cantharidin derivatives, norcantharidin and norcantharimide. Demethylcantharidin is a form of demethylation of cantharidin with low renal toxicity, but demethylation also relatively reduces the biological activity of cantharidin. As for the methyl spot quinone imine, it has been found that the longer the alkyl chain carried on the N-, due to the lipophilic nature of the alkyl group, helps the cells to absorb the demethylated smectin.
因此,相關領域需要一種斑蝥素的衍生物和 或類似物,其可對多種癌細胞表現出極佳的選擇性毒殺效果,但卻不會影響正常細胞。 Therefore, the related art requires a derivative of cantharidin and Or an analog that exhibits excellent selective toxicity to a variety of cancer cells without affecting normal cells.
本揭示內容至少部分係基於發現N-取代之去甲基斑蝥素衍生物其不具細胞毒性並可抑制癌細胞生長的特性而來。上述發現暗示本揭示內容中的N-取代之去甲基斑蝥素衍生物可作為能治療癌症之治療性藥劑的先導化合物。 The present disclosure is based, at least in part, on the discovery that N-substituted demethylcantharidin derivatives are not cytotoxic and can inhibit the growth of cancer cells. The above findings suggest that the N-substituted demethylcantharidin derivatives of the present disclosure are useful as lead compounds for the therapeutic agents for the treatment of cancer.
因此,本揭示內容之第一目的是關於一種式(I)化合物,
在一實例中,式(I)化合物是N-法尼氧基-7-氧二環[2,2,1]庚烷-2,3-二甲醯亞胺(NOC15)。在另一實例中,式(I)化合物是N-法尼基-7-氧二環[2,2,1]庚烷-2,3-二甲醯亞胺(NC15)。 In one example, the compound of formula (I) is N-farneoxy-7-oxabicyclo[2,2,1]heptane-2,3-dimethylimine (NOC15). In another example, the compound of formula (I) is N-farnesyl-7-oxabicyclo[2,2,1]heptane-2,3-dimethylimine (NC15).
因此,本揭示內容之第二目的是提供一種可用來治療癌症的醫藥品或藥學組合物。此醫藥品或藥學組合物包含一治療有效量之式(I)化合物,以及一藥學上可接受的佐劑。 Accordingly, a second object of the present disclosure is to provide a pharmaceutical or pharmaceutical composition that can be used to treat cancer. The pharmaceutical or pharmaceutical composition comprises a therapeutically effective amount of a compound of formula (I), and a pharmaceutically acceptable adjuvant.
可被本揭示內容之醫藥品或藥學組合物加以治療的癌症是選自以下群組中:白血病、肝癌、膀胱癌、直腸癌、腎癌、卵巢癌、子宮頸癌、子宮內膜癌、***癌、胰臟癌、肺癌、乳癌、黑色素細胞瘤和鱗狀細胞癌(squamous cell cancer,SCC)。在一實例中,該癌症是白血病;在另一實例中,該癌症是肝癌。 A cancer which can be treated by the pharmaceutical or pharmaceutical composition of the present disclosure is selected from the group consisting of leukemia, liver cancer, bladder cancer, rectal cancer, kidney cancer, ovarian cancer, cervical cancer, endometrial cancer, prostate Cancer, pancreatic cancer, lung cancer, breast cancer, melanoma, and squamous cell cancer (SCC). In one example, the cancer is leukemia; in another example, the cancer is liver cancer.
若以所述藥學組合物總重量為基準,本發明式(I)化合物約占該藥學組合物總重量的0.1%至99%(重量%)。在某些實施方式中,本發明式(I)化合物的量至少約為該藥學組合物總重量的1%。在特定實施方式中,本發明式(I)化合物的量至少約為該藥學組合物總重量的5%。在其他實施方式中,本發明式(I)化合物的量至少約為該藥學組合物總重量的10%。在另外實施方式中,本發明式(I)化合物的量至少約為該藥學組合物總重量的25%。 The compound of the formula (I) of the present invention comprises from about 0.1% to about 99% by weight based on the total weight of the pharmaceutical composition, based on the total weight of the pharmaceutical composition. In certain embodiments, the amount of the compound of formula (I) of the present invention is at least about 1% by weight based on the total weight of the pharmaceutical composition. In a particular embodiment, the amount of the compound of formula (I) of the present invention is at least about 5% by weight based on the total weight of the pharmaceutical composition. In other embodiments, the amount of the compound of formula (I) of the present invention is at least about 10% by weight based on the total weight of the pharmaceutical composition. In a further embodiment, the amount of the compound of formula (I) of the present invention is at least about 25% by weight based on the total weight of the pharmaceutical composition.
在某些實施方式中,本發明藥學組合物更包含另一種已知可改善癌症治療效果的治療藥劑,用以幫助延緩癌細胞的生長。較佳是,所揭示的醫藥品或藥學組合物更包含一種化療藥劑。 In certain embodiments, the pharmaceutical compositions of the present invention further comprise another therapeutic agent known to improve the therapeutic effect of cancer to help delay the growth of cancer cells. Preferably, the disclosed pharmaceutical or pharmaceutical composition further comprises a chemotherapeutic agent.
因此,本揭示內容之第三目的是關於所揭示的式(I)化合物或藥學組合物鈉來治療一患有癌症之個體的方法。所述方法包含對該亟需治療的個體施用一有效量之式(I)化合物或本發明之藥學組合物,以抑制該個體內癌細胞的生長。可以本揭示方法加以治療的癌症是選自以下群組中:白血病、肝癌、膀胱癌、直腸癌、腎癌、 卵巢癌、子宮頸癌、子宮內膜癌、***癌、胰臟癌、肺癌、乳癌、黑色素細胞瘤和鱗狀細胞癌(squamous cell cancer,SCC)。在一實例中,該個體患有白血病;在另一實例中,該個體患有肝癌。該個體較佳是哺乳動物,最佳是人類。 Accordingly, a third object of the present disclosure is directed to a method of treating a subject having cancer with respect to the disclosed compound of formula (I) or sodium of a pharmaceutical composition. The method comprises administering to the individual in need of treatment an effective amount of a compound of formula (I) or a pharmaceutical composition of the invention to inhibit growth of cancer cells in the subject. Cancers that can be treated by the disclosed methods are selected from the group consisting of leukemia, liver cancer, bladder cancer, rectal cancer, kidney cancer, Ovarian cancer, cervical cancer, endometrial cancer, prostate cancer, pancreatic cancer, lung cancer, breast cancer, melanoma, and squamous cell cancer (SCC). In one example, the individual has leukemia; in another example, the individual has liver cancer. The individual is preferably a mammal, preferably a human.
在某些實施方式中,本發明方法物更包含在施用本發明藥學組合物之前、同時和/或之後,對該個體施用另一種已知可改善癌症治療效果的治療藥劑,用以幫助抑制該個體體內癌細胞的生長或轉移。較佳是,本發明的醫藥品或藥學組合物是與另一種化療藥劑一同施用。 In certain embodiments, the method of the present invention further comprises administering to the individual another therapeutic agent known to improve the therapeutic effect of the cancer prior to, concurrently with, and/or after administration of the pharmaceutical composition of the present invention to help inhibit the treatment. Growth or metastasis of cancer cells in an individual. Preferably, the pharmaceutical or pharmaceutical composition of the invention is administered with another chemotherapeutic agent.
透過以下的詳細說明與附隨之申請專利範圍將可更了解本揭示內容的這些及其他特徵。需知以上的概述及以下的詳細說明僅為例示,用來闡述本揭示內容,而非用以限制本揭示內容之範疇。 These and other features of the present disclosure will be more apparent from the following detailed description and appended claims. The above summary and the following detailed description are merely illustrative, and are not intended to limit the scope of the disclosure.
為讓本發明的上述與其他目的、特徵、優點與實施例能更明顯易懂,所附圖式之說明如下:第1圖繪示出依據本發明一實施方式,以化合物2、9或18分別處理HepG2肝癌細胞後,再以赫斯特染色法染色後所呈現的細胞核變化的照片;第2圖繪示出依據本發明一實施方式,以化合物2、9或18分別處理HepG2肝癌細胞後,細胞週期分析的結果; 第3圖繪示出依據本發明一實施方式,以化合物2、9或18分別處理HepG2肝癌細胞後,細胞凋亡的分析結果;第4圖繪示出依據本發明一實施方式,在巴豆醇-12-十四烷酸酯-13-乙酸酯(PMA)加離子黴素(ionomycin)活化的Jurkat T細胞模式中,(A)化合物2和(B)NOC15分別處理人類白血病細胞(Jurkat T細胞)及人類正常淋巴細胞後,其細胞存活的分析結果;第5A-5C圖分別繪示出依據本發明一實施方式,以NOC15分別處理人類白血病細胞(Jurkat T細胞)(A)0,(B)24或(C)48小時後,其細胞週期分析結果;第5D圖為第5A-5C圖之結果的量化分析;第6圖的柱狀圖繪示出依據本發明一實施方式,以NC15分別處理人類白血病細胞(Jurkat T細胞)及人類正常淋巴細胞約(A)24或(B)48小時後,其細胞存活的分析結果;第7A-7C圖分別繪示出依據本發明一實施方式,以NC15分別處理人類白血病細胞(Jurkat T細胞)(A)0,(B)24或(C)48小時後,其細胞週期分析結果;第7D圖為第7A-7C圖之結果的量化分析;第8A圖繪示出依據本發明一實施方式,經皮下注射L1210細胞之實驗動物以NOC15或NC15治療後,該實驗動物的存活曲線;第8B圖繪示出依據本發明一實施方式,經腹膜注射L1210細胞之實驗動物以NOC15或NC15治療後, 該實驗動物的存活曲線;第9圖繪示出依據本發明一實施方式,分別經皮下或腹膜注射L1210細胞之實驗動物,其體內(A)腫瘤,(B)肝臟及(C)脾臟之重量隨時間變化曲線;第10A圖繪示出依據本發明一實施方式,經皮下注射L1210細胞之實驗動物以NOC15或NC15治療後,該實驗動物的白血球細胞數目變化曲線;第10B圖繪示出依據本發明一實施方式,經腹膜注射L1210細胞之實驗動物以NOC15或NC15治療後,該實驗動物的白血球細胞數目變化曲線;第11A圖繪示出依據本發明一實施方式,以NOC15治療之實驗動物其身體體重重隨時間變化曲線;及第11B圖繪示出依據本發明一實施方式,以NC15治療之實驗動物其身體體重重隨時間變化曲線;根據慣常的作業方式,圖中各種特徵與元件並未依比例繪製,其繪製方式是為了以最佳的方式呈現與本發明相關的具體特徵與元件。此外,在不同圖式間,以相同或相似的元件符號來指稱相似的元件/部件。 The above and other objects, features, advantages and embodiments of the present invention will become more <RTIgt; <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; Photographs of nuclear changes exhibited by HepG2 liver cancer cells after staining with Hearst staining, respectively; Figure 2 depicts images of HepG2 liver cancer cells treated with Compounds 2, 9 or 18, respectively, according to an embodiment of the present invention. , the results of cell cycle analysis; 3 is a view showing the results of analysis of apoptosis after treatment of HepG2 liver cancer cells with Compounds 2, 9 or 18, respectively, according to an embodiment of the present invention; and FIG. 4 is a view showing crotyl alcohol according to an embodiment of the present invention. In the Jurkat T cell mode activated by -12-tetradecanoate-13-acetate (PMA) plus ionomycin, (A) Compound 2 and (B) NOC15 treated human leukemia cells (Jurkat T, respectively) Analysis of cell survival after cells and human normal lymphocytes; Figures 5A-5C respectively depict treatment of human leukemia cells (Jurkat T cells) (A)0 with NOC15, respectively, according to an embodiment of the present invention, B) the results of cell cycle analysis after 48 hours of 24 or (C); the 5D plot is a quantitative analysis of the results of 5A-5C; the histogram of Fig. 6 depicts an embodiment according to the present invention, NC15 treats the results of cell survival analysis of human leukemia cells (Jurkat T cells) and human normal lymphocytes after about 48 hours (A) 24 or (B), respectively; and Figures 7A-7C respectively illustrate an embodiment according to the present invention. By way of NC15, human leukemia cells (Jurkat T cells) (A) 0, (B) 24 or (C) 4 were treated separately. After 8 hours, the results of cell cycle analysis; Figure 7D is a quantitative analysis of the results of Figures 7A-7C; Figure 8A depicts experimental animals subcutaneously injected with L1210 cells with NOC15 or NC15 according to an embodiment of the present invention. Survival curve of the experimental animal after treatment; FIG. 8B depicts the experimental animal after intraperitoneal injection of L1210 cells treated with NOC15 or NC15 according to an embodiment of the present invention, Survival curve of the experimental animal; FIG. 9 depicts experimental animals in which L1210 cells were injected subcutaneously or intraperitoneally, respectively, in vivo (A) tumor, (B) liver and (C) spleen weight according to an embodiment of the present invention. Curves with time; FIG. 10A is a graph showing changes in the number of white blood cells in the experimental animals after subcutaneous injection of L1210 cells in the experimental animals treated with NOC15 or NC15 according to an embodiment of the present invention; FIG. 10B is a graph showing the basis In one embodiment of the present invention, the number of white blood cell cells in the experimental animal after the intraperitoneal injection of the L1210 cell is treated with NOC15 or NC15; FIG. 11A depicts the experimental animal treated with NOC15 according to an embodiment of the present invention. The body weight is plotted against time; and FIG. 11B is a graph showing the body weight of the experimental animals treated with NC15 according to an embodiment of the present invention; the various features and components in the drawing according to the conventional operation mode The drawings are not to scale, the manner In addition, similar elements/components are referred to by the same or similar element symbols throughout the different drawings.
為了使本揭示內容的敘述更加詳盡與完備,下文針對了本發明的實施態樣與具體實施例提出了說明性的描述;但這並非實施或運用本發明具體實施例的唯一形式。實施方式中涵蓋了多個具體實施例的特徵以及用以建構與操作這些具體實施例的方法步驟與其順序。 然而,亦可利用其他具體實施例來達成相同或均等的功能與步驟順序。 The description of the embodiments of the present invention is intended to be illustrative and not restrictive. The features of various specific embodiments, as well as the method steps and sequences thereof, are constructed and manipulated in the embodiments. However, other specific embodiments may be utilized to achieve the same or equivalent function and sequence of steps.
雖然用以界定本發明較廣範圍的數值範圍與參數均是約略的數值,此處已盡可能精確地呈現具體實施例中的相關數值。然而,任何數值本質上不可避免地含有因個別測試方法所致的標準偏差。在此處,「約」通常係指實際數值在一特定數值或範圍的正負10%、5%、1%或0.5%之內。或者是,「約」一詞代表實際數值落在平均值的可接受標準誤差之內,視本發明所屬技術領域中具有通常知識者的考量而定。除了實驗例之外,或除非另有明確的說明,當可理解此處所用的所有範圍、數量、數值與百分比(例如用以描述材料用量、時間長短、溫度、操作條件、數量比例及其他相似者)均經過「約」的修飾。因此,除非另有相反的說明,本說明書與附隨申請專利範圍所揭示的數值參數皆為約略的數值,且可視需求而更動。至少應將這些數值參數理解為所指出的有效位數與套用一般進位法所得到的數值。 Although numerical ranges and parameters are used to define a broad range of values for the present invention, the relevant values in the specific embodiments have been presented as precisely as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. As used herein, "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range. Alternatively, the term "about" means that the actual value falls within the acceptable standard error of the average, depending on the considerations of those of ordinary skill in the art to which the invention pertains. Except for the experimental examples, or unless otherwise explicitly stated, all ranges, quantities, values, and percentages used herein are understood (eg, to describe the amount of material used, the length of time, the temperature, the operating conditions, the quantity ratio, and the like. Are all modified by "about". Therefore, unless otherwise indicated to the contrary, the numerical parameters disclosed in the specification and the appended claims are intended to be At a minimum, these numerical parameters should be understood as the number of significant digits indicated and the values obtained by applying the general carry method.
除非本說明書另有定義,此處所用的科學與技術詞彙之含義與本發明所屬技術領域中具有通常知識者所理解與慣用的意義相同。此外,在不與上下文意衝突的情形下,本說明書所用的單數名詞涵蓋該名詞的複數型;而所用的複數名詞時亦涵蓋該名詞的單數型。 The scientific and technical terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention pertains, unless otherwise defined herein. In addition, the singular noun used in the specification encompasses the plural of the noun, and the plural noun of the noun is also included in the plural noun.
以下為本說明書中所用特定名詞的說明:在本文中,「抑制(inhibits,inhibiting,suppresses,suppressing)」一詞意指對一個體施用所述式(I) 化合物,以使至少45%、50%、55%、60%、65%、70%、75%、80%、85%、90%、95%或99%的癌細胞生長停滯,不再複製,因而能達到使癌細胞數目減少或腫瘤體積縮小,因此,「抑制」一詞在此亦表示能殺死癌細胞或誘發癌細胞凋亡。 In the following, the term "inhibits, inhibiting, suppresses, suppressing" means applying the formula (I) to a body. a compound such that at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% of the cancer cells are stagnant and no longer replicated, Therefore, the number of cancer cells can be reduced or the tumor volume can be reduced. Therefore, the term "inhibition" is also used herein to mean killing cancer cells or inducing apoptosis of cancer cells.
在本文中,「治療(treating or treatment)」一詞意指能部分或完全防治、減緩、減輕和/或管理與腫瘤相關之二次疾病或狀況的病徵。「治療」一詞意指對一表現出與腫瘤相關二次疾病或狀況之病徵的個體施加或投予本發明式(I)化合物,以部分或完全防治、減緩、減輕、延遲開始、抑制進展、降低嚴重度和/或減少與該腫瘤相關二次疾病或特徵之一或多個病徵的出現。與該腫瘤相關之二次疾病或特徵之病徵包括,但不限於,不明原因的體重減輕、發燒、疲累、疼痛和身體功能改變。可對表現出上述病徵、疾病或狀況之早期徵兆的個體施用治療,以便能降低發展出與腫瘤相關之二次疾病或狀況之病徵的風險。如果該一或多個病徵或臨床標記被減少,即稱該治療為有效的。或者,如果該一或多個病徵或臨床標記的進展被減少或停止,即稱該治療為有效的。 As used herein, the term "treating or treating" means a condition that partially or completely prevents, slows, reduces, and/or manages a secondary disease or condition associated with a tumor. The term "treatment" means the administration or administration of a compound of formula (I) of the present invention to an individual exhibiting a condition associated with a secondary disease or condition associated with a tumor, to partially or completely prevent, slow, alleviate, delay the onset, inhibit progression. The severity is reduced and/or the occurrence of one or more symptoms of a secondary disease or characteristic associated with the tumor is reduced. Symptoms of secondary diseases or characteristics associated with the tumor include, but are not limited to, unexplained weight loss, fever, fatigue, pain, and changes in body function. Treatment may be administered to an individual who exhibits early signs of the above mentioned signs, diseases or conditions in order to reduce the risk of developing a second disease or condition associated with the tumor. If the one or more signs or clinical markers are reduced, the treatment is said to be effective. Alternatively, the treatment is said to be effective if the progression of the one or more signs or clinical markers is reduced or stopped.
「一有效量(an effective amount)」一詞意此一用量在經過適當的給藥期間後,能夠達到對癌症治療而言,欲求的治療效果。換言之,該有效量可達到所亟求之抑制癌細胞的效果。 The term "an effective amount" means that the dosage can achieve the desired therapeutic effect for cancer treatment after an appropriate administration period. In other words, the effective amount can achieve the desired effect of inhibiting cancer cells.
「化合物(compound)」、「組合物 (composition)」、「藥劑(agent)」或「醫藥品(medicine or medicament)」等詞在此可互相替換,且都是指當施用.於一個體(人類或動物)時,能夠透過局部和/或全身性作用而誘發所亟求的藥學和/或生理反應的一種化合物或組合物。 "compound", "composition The words "composition", "agent" or "medicine or medicament" are used interchangeably herein, and are meant to be applied locally when applied to a body (human or animal). / or a compound or composition that induces a desired pharmaceutical and/or physiological response systemically.
「施用(administered、administering或、administration)」一詞在此係指直接施用所述的化合物或組合物,或施用活性化合物的前驅藥(prodrug)、衍生物(derivative)、或類似物(analog)等,而可於施用個體體內形成該活性化合物之一相當用量者。 The term "administered, administered" or "administration" as used herein refers to the direct administration of the compound or composition, or the administration of a prodrug, derivative, or analog of the active compound. Alternatively, a substantial amount of the active compound can be formed in the subject to be administered.
本文中交替使用「個體(subject)」或「患者(patient)」等詞,其係指可接受所述化合物和/或方法治療的動物(包括人類)。「個體」或「患者」在此涵蓋了雄性與雌性兩種性別,除非另有具體說明。因此「個體」或「患者」包含任何哺乳類動物,較佳為人類,其可因利用所述化合物進行治療而獲益。 The words "subject" or "patient" are used interchangeably herein to mean an animal (including a human) that is treatable by the compounds and/or methods. "Individual" or "patient" is used herein to encompass both male and female genders unless otherwise specified. Thus "individual" or "patient" encompasses any mammal, preferably a human, which may benefit from treatment with the compound.
本揭示內容至少有一部分是肇因於意外發現兩個親脂性N-取代之斑蝥素衍生物,其本身不具細胞毒性、且可延緩癌細胞,特別是白血球細胞,的生長特性而來。上述發現暗示本揭示內容中兩種N-取代之去甲基斑蝥素可作為能治療癌症(包括白血病)之治療性藥劑的先導化合物。 At least a portion of the disclosure is due to the accidental discovery of two lipophilic N-substituted cantharidin derivatives which are not cytotoxic in themselves and which delay the growth characteristics of cancer cells, particularly white blood cells. The above findings suggest that the two N-substituted desmethylcantharidins in the present disclosure are useful as lead compounds for the therapeutic agents of cancer, including leukemia.
本揭示內容一態樣是關於具有以下式(I)結構的化合物:
在一實施方式中,該式(I)化合物是N-法尼氧基-7-氧二環[2,2,1]庚烷-2,3-二甲醯亞胺(化合物9或NOC15)。在另一實施方式中,該式(I)化合物是N-法尼基-7-氧二環[2,2,1]庚烷-2,3-二甲醯亞胺(化合物18或NC15)。 In one embodiment, the compound of formula (I) is N-farneoxy-7-oxabicyclo[2,2,1]heptane-2,3-dimethylimine (Compound 9 or NOC 15) . In another embodiment, the compound of formula (I) is N-farnesyl-7-oxabicyclo[2,2,1]heptane-2,3-dimethylimine (Compound 18 or NC15) .
NOC15和NC15可分別透過本揭示內容實施例1所示的合成方法來製造。 NOC 15 and NC 15 can be produced by the synthesis method shown in Example 1 of the present disclosure, respectively.
依據一實施方式,依照流程1所述方式來製造NO-取代之去甲基斑蝥素衍生物,包括NOC15。詳言之,以呋喃(3)和順丁烯二酸酐(4)作為起始物,透過狄爾斯-阿德耳反應(Diels-Alder reaction)生成5,6-脫水去甲基斑蝥素(5)。接著,將化合物(5)氫化而獲得去甲基斑蝥素(2),其再於室溫下,於無水甲醇中,甲氧鈉存在下與羥胺氯化氫反應,而產生N-羥基去甲基斑蝥素(6)。接著,在碳酸鉀存在下,讓N-羥基去甲基斑蝥素(6)與溴化法尼烷(farnesyl bromide)反應生成N-法尼氧基-7-氧二環[2,2,1]庚烷-2,3-二甲醯亞胺(化合物9或NOC15)。 According to one embodiment, a NO-substituted demethylcantharidin derivative, including NOC15, is produced in the manner described in Scheme 1. In particular, using furan (3) and maleic anhydride (4) as starting materials, 5,6-anhydrodemethylcantharidin is produced by Diels-Alder reaction. 5). Next, the compound (5) is hydrogenated to obtain demethylcantharidin (2), which is further reacted with hydroxylamine hydrogen chloride in the presence of sodium methoxide in anhydrous methanol at room temperature to produce N-hydroxydemethylated quinone. Prime (6). Next, N-hydroxydesmethylcantharidin (6) is reacted with farnesyl bromide to form N-farnesyloxy-7-oxobicyclo[2,2,1 in the presence of potassium carbonate. Heptane-2,3-dimethylimine (Compound 9 or NOC 15).
流程1
流程1中的試劑與條件如下述:(a)***,室溫,48小時;(b)3大氣壓的氫氣,10% Pd/C,THF,室溫,8小時;(c)NaHCO3,NH2OH.HCl,MeOH,室溫,20小時;(d)K2CO3,溴化法尼烷,丙酮,迴流8-10小時。 The reagents and conditions in Scheme 1 are as follows: (a) diethyl ether, room temperature, 48 hours; (b) 3 atmospheres of hydrogen, 10% Pd/C, THF, room temperature, 8 hours; (c) NaHCO 3 , NH 2 OH. HCl, MeOH, room temperature, 20 hours; (d) K 2 CO 3 , hexanes bromide, acetone, reflux for 8-10 hours.
依據另一實施方式,依照流程2所述方式來製造N-取代之去甲基斑蝥素衍生物,包括NC15。詳言之,讓呋喃(3)和順丁烯二酸酐(4)在甲苯中反應生成二羥基去甲基斑蝥素(14)。接著,將化合物(14)氫化而獲得化合物(15),其再於室溫下,於碳酸鉀存在下,與溴化法尼烷(farnesyl bromide)反應生成N-法尼基-7-氧二環[2,2,1]庚烷-2,3-二甲醯亞胺(化合物18或NC15)。 According to another embodiment, the N-substituted demethylcantharidin derivative, including NC15, is made according to the procedure described in Scheme 2. In particular, furan (3) and maleic anhydride (4) are reacted in toluene to form dihydroxydesmethylcantharidin (14). Next, the compound (14) is hydrogenated to obtain the compound (15), which is further reacted with farnesyl bromide in the presence of potassium carbonate at room temperature to form N-farnesyl-7-oxygen. Ring [2,2,1]heptane-2,3-dimethylimine (Compound 18 or NC15).
流程2
流程2中的試劑與條件如下述:(a)甲苯,80℃,6小時;(b)3大氣壓的氫氣,10% Pd/C,THF,室溫,8-48小時;(c)K2CO3,溴化法尼烷,丙酮,迴流8-10小時。 The reagents and conditions in Scheme 2 are as follows: (a) toluene, 80 ° C, 6 hours; (b) 3 atmospheres of hydrogen, 10% Pd / C, THF, room temperature, 8-48 hours; (c) K 2 CO 3 , farnesene bromide, acetone, reflux for 8-10 hours.
諸如白血病、肝癌、膀胱癌、直腸癌、腎癌、卵巢癌、子宮頸癌、子宮內膜癌、***癌、胰臟癌、肺癌、乳癌、黑色素細胞瘤和鱗狀細胞癌(squamous cell cancer,SCC)之類的癌細胞的生長,可被濃度介於1-20μM的式(I)化合物抑制,例如1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20μM。依據一實施方式,50%肝癌細胞的體外生長可被8.2μM的NOC15(化合物9)所抑制;但此濃度卻不會傷害正常肝臟細胞。在另一實例中,50%肝癌細胞的體外生長可被10.7μM的NOC15(化合物9)所抑制;在又一實例中,8.3μM的NOC15(化合物9)可抑制50%之白血病細胞的體外生長。依據另一實施方式,50%白血病細胞(L1210) 的體外生長可被1.6μM的NC15(化合物18)所抑制。在另一實例中,50%白血病細胞(L1210)的體外生長可被2.6μM的NC15(化合物18)所抑制 Such as leukemia, liver cancer, bladder cancer, rectal cancer, kidney cancer, ovarian cancer, cervical cancer, endometrial cancer, prostate cancer, pancreatic cancer, lung cancer, breast cancer, melanoma and squamous cell cancer (squamous cell cancer, growth of cancer cells SCC) or the like, may be between 1-20 μ M concentration of compound to inhibit the formula (the I), for example 1,2,3,4,5,6,7,8,9,10,11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 μM . According to one embodiment, 50% of the in vitro growth of liver cancer cells can be NOC15 8.2 μ M (Compound 9) inhibited; however, this concentration but does not harm normal liver cells. In another example, 50% of in vitro growth of liver cancer cells can be NOC15 10.7 μ M (Compound 9) inhibited; In yet another example, NOC15 8.3 μ M (Compound 9) inhibit 50% of the leukemic cells Growth in vitro. According to another embodiment, 50% of leukemia cells in vitro (L1210) growth may be NC15 1.6 μ M (Compound 18) inhibited. In another example, 50% of leukemia cells in vitro (L1210) growth may be NC15 2.6 μ M (Compound 18) inhibited
因此,本發明也提供一種可用來治療癌症的醫藥品或藥學組合物。所述癌症包括,但不限於,白血病、肝癌、膀胱癌、直腸癌、腎癌、卵巢癌、子宮頸癌、子宮內膜癌、***癌、胰臟癌、肺癌、乳癌、黑色素細胞瘤和鱗狀細胞癌(squamous cell cancer,SCC)。在一較佳實施方式中,該癌症為白血病;在另一較佳實施方式中,該癌症為肝癌。 Accordingly, the present invention also provides a pharmaceutical or pharmaceutical composition useful for treating cancer. The cancer includes, but is not limited to, leukemia, liver cancer, bladder cancer, rectal cancer, kidney cancer, ovarian cancer, cervical cancer, endometrial cancer, prostate cancer, pancreatic cancer, lung cancer, breast cancer, melanoma, and scale Squamous cell cancer (SCC). In a preferred embodiment, the cancer is leukemia; in another preferred embodiment, the cancer is liver cancer.
本發明藥學組合物中包含一有效量之式(I)化合物以及一藥學上可接受的佐劑。一般來說,若以所述藥學組合物總重量為基準,則本發明式(I)化合物,約占該藥學組合物總重量的0.1%至99%(重量%)。在某些實施方式中,本發明式(I)化合物的量至少約為該藥學組合物總重量的1%。在特定實施方式中,本發明式(I)化合物物的量至少約為該藥學組合物總重量的5%。在其他實施方式中,本發明式(I)化合物的量至少約為該藥學組合物總重量的10%。在另外實施方式中,本發明式(I)化合物的量至少約為該藥學組合物總重量的25%。 The pharmaceutical compositions of the present invention comprise an effective amount of a compound of formula (I) and a pharmaceutically acceptable adjuvant. Generally, the compound of formula (I) of the present invention will comprise from about 0.1% to about 99% by weight based on the total weight of the pharmaceutical composition, based on the total weight of the pharmaceutical composition. In certain embodiments, the amount of the compound of formula (I) of the present invention is at least about 1% by weight based on the total weight of the pharmaceutical composition. In a particular embodiment, the amount of the compound of formula (I) of the present invention is at least about 5% by weight based on the total weight of the pharmaceutical composition. In other embodiments, the amount of the compound of formula (I) of the present invention is at least about 10% by weight based on the total weight of the pharmaceutical composition. In a further embodiment, the amount of the compound of formula (I) of the present invention is at least about 25% by weight based on the total weight of the pharmaceutical composition.
可依據藥學上常用的方法,例如Remington藥典(第17版,Alfonoso R.Gennaro編輯,Mack Publishing Company(Easton,Pa(1985))來製造本發明藥學組合物。藥學上可接受的佐劑是指那些可與配方中其他成分相容且可為生物系統接受的物質。 The pharmaceutical compositions of the present invention can be made according to methods commonly used in pharmacy, such as the Remington Pharmacopoeia (17th Ed., Alfonoso R. Gennaro, ed., Mack Publishing Company, Easton, Pa (1985). Pharmaceutically acceptable adjuvants refer to pharmaceutically acceptable adjuvants. Those that are compatible with the other ingredients in the formulation and are acceptable to biological systems.
可將本發明藥學組合物配方成固態劑型,其包括,但不限於藥錠,膠囊及藥袋(或藥包)。每一藥錠可包含各式佐劑(例如微晶纖維素、檸檬酸鈉、碳酸鈣、磷酸二鈣、和甘胺酸);以及各式崩散劑(如澱粉、藻酸和特定矽酸鹽)與顆粒黏結劑(聚乙烯吡咯烷酮、蔗糖、明膠和相思樹膠(acacia))。除此外,藥錠中還可包含諸如硬脂酸鎂、十二烷基硫酸鈉及滑石等的潤滑劑。與此相關的較佳材料包括乳糖或牛奶中的糖以及高分子量聚乙二醇。上述的固態劑型也可非必要地包括塗層或殼,例如腸衣塗層,以及用來改善任一藥物活性成分釋放速率的塗層。這類塗層的實例已為此領域中人士所熟知。在一實例中,所述藥學組合物是被配方成為藥錠。在另一實例中,所述藥學組合物是被配方成為充填在軟或硬明膠膠囊內或是封裝在生物可分解之藥包內的顆粒。當使用方式是口服用懸浮液和/或特效藥液(elixirs)時,可組合活性成分與各種甜味劑或風味劑、著色劑或染料一起配方,需要時還可加入乳化劑和/或懸浮劑,以及諸如水、酒精、丙二醇、甘油等稀釋劑。 The pharmaceutical compositions of the present invention can be formulated into solid dosage forms including, but not limited to, tablets, capsules and sachets (or sachets). Each tablet may contain various adjuvants (eg, microcrystalline cellulose, sodium citrate, calcium carbonate, dicalcium phosphate, and glycine); and various disintegrating agents (such as starch, alginic acid, and specific citrate). ) with particle binders (polyvinylpyrrolidone, sucrose, gelatin and acacia). In addition, a lubricant such as magnesium stearate, sodium lauryl sulfate, and talc may be contained in the tablet. Preferred materials associated therewith include sugars in lactose or milk as well as high molecular weight polyethylene glycols. The above solid dosage forms may also optionally include a coating or shell, such as a casing coating, and a coating to improve the rate of release of any of the pharmaceutically active ingredients. Examples of such coatings are well known to those skilled in the art. In one example, the pharmaceutical composition is formulated as a tablet. In another example, the pharmaceutical composition is a granule formulated to be filled in a soft or hard gelatin capsule or encapsulated in a biodegradable kit. When used in the form of oral suspensions and / or special effects (elixirs), the active ingredients may be combined with various sweeteners or flavors, colorants or dyes, and emulsifiers and/or suspensions may be added if desired. And diluents such as water, alcohol, propylene glycol, glycerin.
在某些實施方式中,本發明藥學組合物是被配方成為適合口服的液體劑型。此類液體配方可更包括用來維持pH值的緩衝液。也可將此液體配方充填在軟膠囊內。此液體劑型可為一種溶液,懸浮液,乳化液,微乳化液,沉澱或是可攜帶本發明式(I)化合物、其之藥學上可接受的衍生物、異構物、代謝物、鹽類或溶合物之任何液體介質。此液體可被設計成能改善本發明式(I) 化合物或其藥學上可接受鹽類之溶解度,以便形成一種含有藥物的乳化液或分散液。當使用這種劑型時,是將活性化合物與至少一種藥學上可接受的佐劑(包括,但不限於,上述佐劑)一起混合後而形成。 In certain embodiments, the pharmaceutical compositions of the invention are formulated as liquid dosage forms suitable for oral administration. Such liquid formulations may further include a buffer to maintain the pH. This liquid formulation can also be filled in a soft capsule. The liquid dosage form can be a solution, suspension, emulsion, microemulsion, precipitate or can carry the compound of the formula (I) of the present invention, its pharmaceutically acceptable derivatives, isomers, metabolites, salts Or any liquid medium of the solvate. This liquid can be designed to improve the formula (I) of the present invention The solubility of the compound or a pharmaceutically acceptable salt thereof to form a drug-containing emulsion or dispersion. When such a dosage form is employed, it is formed by mixing the active compound with at least one pharmaceutically acceptable adjuvant, including, but not limited to, the above adjuvants.
若以腸胃外(parenterally)方式施用,可將本發明化合物配方成為液態的藥學組合物,其可為能以靜脈內注射、肌肉內注射、皮下注射或腹膜內注射等方式施用的無菌溶液或懸浮液。可用來製造上述無菌注射溶液或懸浮液的稀釋劑包括,但不限於,1,3-丁二醇、甘露醇、水、林格氏溶液、等張性氯化鈉溶液。也可使用脂肪酸(如,油酸)及其之甘油酯衍生物,或是天然藥學可接受的油(如,橄欖油或菜籽油)來製造可供注射用的溶液或懸浮液。這類油性溶液或懸浮液中也可包含用來稀釋的醇類或羧甲基纖維素或類似的分散劑。也可使用其他常用的界面活性劑(如,Tweens或Spans系列)或乳化劑,或藥學領域製造配方時常用來增強生物可利用性的藥劑。 If administered parenterally, the compound of the invention may be formulated as a liquid pharmaceutical composition which may be sterile solution or suspension which can be administered by intravenous, intramuscular, subcutaneous or intraperitoneal injection. liquid. Diluents which can be used in the manufacture of such sterile injectable solutions or suspensions include, but are not limited to, 1,3-butanediol, mannitol, water, Ringer's solution, isotonic sodium chloride solution. Fatty acids (e.g., oleic acid) and their glyceride derivatives, or natural pharmaceutically acceptable oils (e.g., olive oil or rapeseed oil) can also be used to make solutions or suspensions for injectable use. Alcohols or carboxymethylcellulose or similar dispersing agents for dilution may also be included in such oily solutions or suspensions. Other commonly used surfactants (e.g., Tweens or Spans series) or emulsifiers, or agents commonly used in the pharmaceutical arts to enhance bioavailability can be used.
在某些其他實施方式中,亦可將本發明上述醫藥品或藥學組合物製成多種適用於黏膜給藥(mucosal application)的劑型,如經頰(buccal)和/或舌下(sublingual)藥物劑型單元,以遞送藥物穿過口腔黏膜。可使用多種生物可降解且藥學可接受的高分子佐劑,此種佐劑可使得藥學組合物具有可接受的吸附效果以及所欲的藥物釋放模式,且可和經頰和/或舌下藥物劑型單元中所含的欲施用活性成分或其他成分相容。一般來說,上述的高分 子佐劑包含親水性聚合物,其可黏附至口腔黏膜的濕潤表面。高分子佐劑的實施例包括但不限於丙烯酸聚合物與共聚物(acrylic acid polymers and copolymers);水解聚乙烯醇(hydrolyzed polyvinylalcohol);聚乙烯氧化物(polyethylene oxides);聚丙烯酸酯(polyacrylates);乙烯聚合物與共聚物(vinyl polymers and copolymers);聚乙烯吡咯烷酮;葡萄糖(dextran);瓜膠(guar gum);果膠(pectins);澱粉;及纖維素聚合物(cellulosic polymers)。 In certain other embodiments, the above-described pharmaceutical or pharmaceutical compositions of the present invention may also be formulated into a variety of dosage forms suitable for mucosal applications, such as buccal and/or sublingual drugs. A dosage unit to deliver a drug through the oral mucosa. A wide variety of biodegradable and pharmaceutically acceptable polymeric adjuvants can be used which provide the pharmaceutical compositions with acceptable adsorption and desired drug release patterns, as well as buccal and/or sublingual drugs. The active ingredient or other ingredients to be administered in the dosage unit are compatible. Generally speaking, the above high scores The sub-adjuvant comprises a hydrophilic polymer that adheres to the wet surface of the oral mucosa. Examples of polymeric adjuvants include, but are not limited to, acrylic acid polymers and copolymers; hydrolyzed polyvinyl alcohol; polyethylene oxides; polyacrylates; Vinyl polymers and copolymers; polyvinylpyrrolidone; dextran; guar gum; pectins; starch; and cellulosic polymers.
此外,還可將本發明藥學組合物配方成適合局部塗抹的劑型,其可利用此領域中孰知的各種皮膚可接受的惰性佐劑來製造。適合經皮膚、局部和黏膜施用的劑型包含,但不限於溶液、乳霜、乳液、軟膏、凝膠、噴霧劑、氣霧劑、皮膚貼片、等其他所屬領域具有通常知識者已知的劑型。典型的惰性佐劑可以是水、酒精、聚乙烯吡咯烷酮、聚乙二醇、礦物油、硬脂酸醇和凝膠物質。上述所有劑型和佐劑都是藥學領域中孰知的,對本發明藥學組合物來說,劑型的選擇並非關鍵。 In addition, the pharmaceutical compositions of the present invention may also be formulated in a form suitable for topical application, which may be made using various dermatologically acceptable inert adjuvants known in the art. Dosage forms suitable for transdermal, topical, and mucosal administration include, but are not limited to, solutions, creams, lotions, ointments, gels, sprays, aerosols, dermal patches, and the like, which are known to those of ordinary skill in the art. . Typical inert adjuvants can be water, alcohol, polyvinylpyrrolidone, polyethylene glycol, mineral oil, stearic alcohol, and gel materials. All of the above dosage forms and adjuvants are well known in the pharmaceutical arts, and the choice of dosage form is not critical to the pharmaceutical compositions of the present invention.
因此,本揭示內容之第三目的是提供一種治療一罹患或可能罹患癌症之個體的方法。此方法包括對該個體施用上述本發明之藥學組合物(其包含一有效量之式(I)化合物),以便抑制該個體體內癌細胞的生長或轉移。適合以本發明方法進行治療的癌症包括,但不限於白血病、肝癌、膀胱癌、直腸癌、腎癌、卵巢癌、子宮頸癌、子宮內膜癌、***癌、胰臟癌、肺癌、乳癌、黑色素 細胞瘤和鱗狀細胞癌。在一實例中,該個體患有白血病;在另一實例中,該個體患有肝癌。 Accordingly, a third object of the present disclosure is to provide a method of treating an individual suffering from or likely to have cancer. The method comprises administering to the individual a pharmaceutical composition of the invention described above (which comprises an effective amount of a compound of formula (I)) for inhibiting the growth or metastasis of cancer cells in the subject. Cancers suitable for treatment by the methods of the invention include, but are not limited to, leukemia, liver cancer, bladder cancer, rectal cancer, kidney cancer, ovarian cancer, cervical cancer, endometrial cancer, prostate cancer, pancreatic cancer, lung cancer, breast cancer, melanin Cell tumors and squamous cell carcinoma. In one example, the individual has leukemia; in another example, the individual has liver cancer.
在某些實施方式中,述本發明之藥學組合物(其包含一有效量之式(I)化合物)可經由口服、靜脈注射或肌肉注射方式施用至一個體身上,所施用的有效用量約為1-100毫克/公斤個體體重間。每天可施用至該個體身上的有效用量約為10、20、30、40、50、60、70、80、90或100毫克/公斤體重;較佳是約30-70毫克/公斤體重,例如約30、40、50、60、70毫克/公斤體重;更佳是約50毫克/公斤體重。這些劑量可以單次施用或是分成多次在一天內施用。 In certain embodiments, the pharmaceutical compositions of the present invention comprising an effective amount of a compound of formula (I) can be administered to a subject via oral, intravenous or intramuscular injection at an effective level of about 1-100 mg / kg body weight. An effective amount to be administered to the individual per day is about 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 mg/kg body weight; preferably about 30-70 mg/kg body weight, for example about 30, 40, 50, 60, 70 mg / kg body weight; more preferably about 50 mg / kg body weight. These doses can be administered in a single administration or divided into multiple administrations within one day.
在某些實施方式中,所述方法更包括在施用本發明式(I)化合物的同時、之前或之後,對該患者施用另一種已知可改善治療效果的藥物。該可與本發明式(I)化合物一同施用的藥物可以是一種化療藥物,例如烷化劑、拓樸酶抑制劑、細胞毒性劑、抗微管劑及其他。 In certain embodiments, the method further comprises administering to the patient another drug known to improve the therapeutic effect, simultaneously with, before or after administration of the compound of formula (I) of the invention. The drug which can be administered together with the compound of the formula (I) of the present invention may be a chemotherapeutic drug such as an alkylating agent, a tocopherylase inhibitor, a cytotoxic agent, an anti-microtubule agent and the like.
一般來說,本發明藥學組合物(或式(I)化合物)可經由任何適當的途徑施用,例如口服(如,口服用膠囊、藥包(sachets)、懸浮液或藥錠)、或腸胃外(parenterally)等適當方法施用。腸胃外(parenterally)施用方式包括例如肌肉內注射、靜脈注射、皮下注射或腹膜內注射等系統性方式施用。或是,也可透過穿皮膜方式施用,如局部皮膚塗抹或是吸入性(如支氣管內、鼻腔內、口腔內或鼻滴劑等);或是直腸內方式施用。施用時可單獨給藥或併同習知藥學可接受佐劑一起給藥。在 較佳實施方式中,可經由口服方式(如,透過食物)將本發明藥學組合物(包含式(I)化合物)投予個體。 In general, the pharmaceutical compositions of the invention (or compounds of formula (I)) can be administered by any suitable route, for example, orally (e.g., orally, in capsules, sachets, suspensions or elixirs), or parenterally. (parenterally) and the like are applied by appropriate methods. Parenterally administered means include systemic administration such as intramuscular injection, intravenous injection, subcutaneous injection or intraperitoneal injection. Alternatively, it may be applied by means of a film, such as topical skin application or inhalation (such as intrabronchial, intranasal, intraoral or nasal drops); or intrarectal administration. Administration may be carried out alone or in combination with conventional pharmaceutically acceptable adjuvants. in In a preferred embodiment, a pharmaceutical composition of the invention (comprising a compound of formula (I)) can be administered to an individual via oral means (e.g., through food).
在某些實施方式中,相較於控制組來說,接受本發明藥學組合物治療的個體其壽命至少可延長10-40%,且其腫瘤體積和白血球數目均較控制組(亦即,未接受本發明化合物治療的個體)來得小。 In certain embodiments, an individual treated with a pharmaceutical composition of the invention has a lifespan of at least 10-40% longer than the control group, and both the tumor volume and the number of white blood cells are greater than the control group (ie, no Individuals treated with the compounds of the invention are small.
當可理解,本發明化合物的劑量會因個體而異,這不僅是因為所用的特定化合物或組合物、給藥途徑、化合物(單獨或連同一或多種藥物)於患者體內所引發之所欲反應等因素之不同,還可能受到其他因素影響,例如:欲治療症狀的疾病狀態或嚴重程度;患者的年齡、性別或體重、患者的健康狀況;以及欲治療的病理狀態的嚴重程度、患者於同時進行的其他醫療或特殊飲食內容;以及本領域通常知識者可想到的其他因素;而負責照料的醫療人員最終可基於這些因素而判斷出適當的劑量。可調整給藥劑量與形式以提供較佳的治療反應。治療有效量同時也是指化合物或組合物所致的毒性或有害的效果不及於其所帶來的治療利益。在較佳的情形中,本發明之化合物或組合物於投藥時,應採用適當的劑量並持續一段時間,以減少出現症狀的次數和/或嚴重程度。 It will be understood that the dosage of the compound of the invention will vary from individual to individual, not only because of the particular compound or composition employed, the route of administration, the compound (either alone or in combination with one or more drugs) Other factors may also be affected by other factors, such as the state or severity of the disease to be treated; the age, sex or weight of the patient, the health of the patient; the severity of the pathological condition to be treated, and the patient at the same time Other medical or special dietary content to be performed; and other factors conceivable by those of ordinary skill in the art; and the medical personnel responsible for care may ultimately determine the appropriate dosage based on these factors. The dosage and form of administration can be adjusted to provide a preferred therapeutic response. A therapeutically effective amount also refers to a toxic or detrimental effect of a compound or composition that is less than the therapeutic benefit it brings. In preferred instances, the compounds or compositions of the invention, when administered, should be administered in an appropriate dosage for a period of time to reduce the number and/or severity of symptoms.
以下實施例是用來闡明本揭示內容特定態樣,並幫助習知技藝者了解並實施本揭示內容。但本揭示內容範疇並不限於這些實施例中。 The following examples are presented to illustrate certain aspects of the present disclosure and to assist those skilled in the art to understand and practice the present disclosure. However, the scope of the disclosure is not limited to these embodiments.
實施例Example
材料與方法Materials and Methods
細胞培育及動物Cell culture and animals
用於本發明的細胞株包括人類肝癌細胞株HepG2、人類膀胱癌細胞株BFTC905、人類直腸癌細胞株HT29和SW480、和人類白血球細胞株HL60和Jurkat T;以及小鼠淋巴白血病細胞株L1210。每一細胞株都培養並維持在杜必可氏改良伊果培養基(Dulbecco's modified Eagle media(DMEM))或RPMI 1640培養基,其中添加了10%熱失活胎牛血清、1%盤尼西林/鏈黴素,並於5%CO2/95%空氣、37℃環境下培育。 Cell lines used in the present invention include human hepatoma cell line HepG2, human bladder cancer cell line BFTC905, human rectal cancer cell lines HT29 and SW480, and human leukocyte cell lines HL60 and Jurkat T; and mouse lymphocytic leukemia cell line L1210. Each cell lines were cultured and maintained in a DU will be Igor's modified medium (Dulbecco 's modified Eagle media ( DMEM)) or RPMI 1640 medium, added with 10% heat-inactivated fetal calf serum, 1% penicillin / streptavidin And cultured in 5% CO 2 /95% air at 37 °C.
DBA/2小鼠(約7週大,18-22公克)係購自樂斯科生物科技公司,並眷養在沒有病原菌的環境中,可自由食用水及飼料。透過皮下或腹腔注射將L1210細胞植入動物體內,以建立起同源移植腫瘤。所有實驗動物的管理與使用都符合臺北榮民總醫院動物管理委員會所頒定的規則。 DBA/2 mice (approximately 7 weeks old, 18-22 grams) were purchased from Lesco Biotech and were maintained in an environment free of pathogens and were free to consume water and feed. L1210 cells are implanted into the animal by subcutaneous or intraperitoneal injection to establish a homologous transplant tumor. The management and use of all experimental animals are in accordance with the rules set by the Animal Management Committee of Taipei Veterans General Hospital.
MTT分析MTT analysis
MTT分析是一種比色分析,其可量測酵素(即,還原酶)在活細胞內將黃色的四唑化合物-溴化3-(4,5-二甲噻唑)-2,5-二苯四銼(3-[4,5-dimethylthiazol]-2,5-diphenyltetrazolium bromide,簡稱MTT)還原成甲(formazane)的活性。只有在細胞存活時,才能進行這種還原作用;因此通常會利用MTT分析來評估細胞存活與增殖的能力。簡言之,使細胞和不同濃度的試驗化合物(如,式(1)化合物)接觸 48小時;之後加入MTT染料(500μg/ml),並讓還原反應進行1小時;而後加入500μL的異丙醇以終止還原反應。利用分光光度計測量樣本在540nm之波長下的吸光度。當以未接受藥物處理的細胞存活率為100%時,則可抑制50%細胞生長的藥物濃度即為IC50值。所有數值都以3次獨立實驗的平均值±標準偏差來表示。 MTT assay is a colorimetric assay that quantifies the enzyme (ie, reductase) in a living cell with a yellow tetrazolium compound - 3-(4,5-dimethylthiazole)-2,5-diphenyl bromide. Reduction of 3-[4,5-dimethylthiazol]-2,5-diphenyltetrazolium bromide (MTT) into a (formazane) activity. This reduction can only be carried out when the cells are alive; therefore, MTT assays are often used to assess the ability of cells to survive and proliferate. Briefly, the cells and test compounds at various concentrations (e.g., of Formula (1) compound) 48 hours; after addition of MTT dye (500 μ g / ml), and so that the reduction reaction for 1 hour; followed by addition of 500 μ L Isopropyl alcohol to terminate the reduction reaction. The absorbance of the sample at a wavelength of 540 nm was measured using a spectrophotometer. When the cell survival rate of the untreated drug is 100%, the drug concentration which can inhibit 50% of cell growth is the IC 50 value. All values are expressed as the mean ± standard deviation of 3 independent experiments.
巴豆醇-12-十四烷酸酯-13-乙酸酯(PMA)加離子黴素(ionomycin)活化的Jurkat T細胞模式Jurkat T cell pattern activated by crotonol-12-tetradecanoate-13-acetate (PMA) plus ionomycin
細胞存活率實驗利用96孔盤進行。將100μl細胞懸浮液(NHL或Jurkat T細胞,5×103個細胞/孔,無血清培養基)接種於孔中,再將96孔盤於培養箱中培養24小時(先預培養22小時,再用PMA 5ng/ml加離子黴素250ng/ml培養2小時)。然後將不同濃度的去甲基斑蝥素(0,2,4,15,30和60μM)或NOC15(0、0.25、0.5、1、2和4μM)加入到孔中的培養基。藥物作用24小時後,NHL和Jurkat T細胞的細胞從活率以細胞計數試劑盒-8(CCK-8,Sigma公司)進行測定,並計算出去甲基斑蝥素和NOC15對NHL細胞和Jurkat T細胞的半數抑制濃度(IC50)。 Cell viability experiments were performed using 96-well plates. 100 μl of cell suspension (NHL or Jurkat T cells, 5×10 3 cells/well, serum-free medium) was inoculated into the wells, and the 96-well plates were cultured in an incubator for 24 hours (pre-culture for 22 hours, then Incubation with PMA 5 ng/ml plus ionomycin 250 ng/ml for 2 hours). Different concentrations of demethylcantharidin (0, 2, 4, 15, 30 and 60 μM) or NOC 15 (0, 0.25, 0.5, 1, 2 and 4 μM) were then added to the medium in the wells. After 24 hours of drug action, the cell viability of NHL and Jurkat T cells was measured by Cell Counting Kit-8 (CCK-8, Sigma), and methylcantharidin and NOC15 were extracted to NHL cells and Jurkat T cells. The half-inhibitory concentration (IC 50 ).
赫斯特33528染色(Hoechst 33528 staining)Hearst 33528 staining (Hoechst 33528 staining)
依據已知方法(Huang et al.,Cytotechnology 2009,59,201-208)來實施赫斯特33528染色。簡言之,先以40μM去甲基斑蝥素、NOC15或NC15處理細胞48小時,接著以PBS沖洗後再以赫斯特33528染色(最終濃度為10μg/mL),然後在螢光顯微鏡下檢視玻片。呈現小細 胞核的細胞或是高螢光強度或細胞核呈現斷片的細胞都認定屬於凋亡的細胞。 The Hurst 33528 staining was carried out according to a known method (Huang et al., Cytotechnology 2009, 59, 201-208). Briefly, 40 μ M to demethyl-to cantharidin, NOC15 or NC15 cells were treated for 48 hours and then rinsed with PBS and then stained in Hoechst 33528 (final concentration 10 μ g / mL), and then fluorescence View the slide under the microscope. Cells that exhibit small nuclei or cells that exhibit high fluorescence intensity or nucleus fragments are considered to belong to apoptotic cells.
細胞週期分析 自培養基中取得經培育之細胞(不論是否經過本發明之化合物(即,經10-60μM之去甲基斑蝥素、NOC15或NC15處理約48小時)預先處理),接著,在4℃下以75%冰冷的酒精處理該些細胞至少經過一夜,使細胞固定。之後,以PBS沖洗後在37℃下以RNA酶(DNAase,200μg/mL,500μL)處理細胞約1.5小時,離心後將細胞沉澱物重新懸浮在適量的碘化丙啶(propidium iodide,PI)(40μg/ml)中,然後在黑暗中於20-25℃下培育約20分鐘後,再以流式細胞儀測定出不同週期的細胞並計數其數目。 Cell cycle analysis was acquired from the culture medium of cells incubated (compound of the present invention, whether or not through (i.e., after 10-60 μ M of demethylated cantharidin, NOC15 or NC15 for about 48 hours) pre-treatment), then, in The cells were treated with 75% ice-cold alcohol at 4 ° C for at least one night to immobilize the cells. Thereafter, rinsed with PBS and RNA enzyme (DNAase, 200μg / mL, 500 μ L) at 37 [deg.] C cells were treated for about 1.5 hours, after centrifugation the cell pellet was resuspended in an appropriate amount of propidium iodide (propidium iodide, PI (40 μg/ml), and then incubated at 20-25 ° C for about 20 minutes in the dark, and then cells of different cycles were measured by flow cytometry and counted.
凋亡細胞百分比是依照製造商提供的流程以帶有綠螢光蛋白的第五膜粘連蛋白試劑套組(annexin-V/FITC kit/propidium iodine(PI)))搭配流式細胞儀來檢測。為幫助偵測凋亡的細胞,先將處理過的細胞以1,000g的速度離心5分鐘,接著重新懸浮並以PBS清洗,再以第五膜粘連蛋白/碘化丙啶(annexin-V/PI)進行染色。 The percentage of apoptotic cells was determined by flow cytometry with a fifth membrane-fixing protein kit (annexin-V/FITC kit/propidium iodine (PI)) with green fluorescent protein according to the manufacturer's protocol. To help detect apoptotic cells, the treated cells were first centrifuged at 1,000 g for 5 minutes, then resuspended and washed with PBS, followed by a fifth membrane-adhesin/propidium iodide (annexin-V/PI). ) to perform dyeing.
產生同源腫瘤及活體治療Produce homologous tumors and in vivo therapy
實驗開始第1天,以皮下注射或腹膜腔內注射方式將L1210細胞(每次注射1x106細胞)注入實驗動物體內。7天過後,以腹腔注射的方式給予實驗組動物試驗化合物(即,NOC15或NC15),連續7天。試驗過程中每天測量並紀錄實驗動物的體重。 Start of the experiment on day 1, intraperitoneally or subcutaneously injecting the L1210 cells (1x10 6 cells per injection) into the experimental animals. After 7 days, the test group animals (i.e., NOC15 or NC15) were administered by intraperitoneal injection for 7 consecutive days. The body weight of the experimental animals was measured and recorded daily during the test.
完成試驗後,將實驗動物犧牲,並取出腫瘤、肝臟及脾臟,並秤重,同時測量白血球細胞數目。 After the test was completed, the experimental animals were sacrificed, and the tumor, liver, and spleen were taken out and weighed, and the number of white blood cells was measured.
實施例1 合成本發明化合物Example 1 Synthesis of a Compound of the Invention
1.1 合成N-法尼氧基-7-氧二環[2,2,1]庚烷-2,3-二甲醯亞胺(化合物9或NOC15)1.1 Synthesis of N-farnesyloxy-7-oxabicyclo[2,2,1]heptane-2,3-dimethylimine (Compound 9 or NOC15)
依以下流程1所述方式,合成NOC15化合物。 The NOC15 compound was synthesized in the manner described in Scheme 1 below.
7-氧二環[2,2,1]庚烯-2,3-二羧酸酐(化合物5)7-oxobicyclo[2,2,1]heptene-2,3-dicarboxylic anhydride (compound 5)
在***中混合呋喃(furan)(40毫升,550毫莫耳)及順丁烯二酐(10克,102毫莫耳),並於室溫下反應約48小時,接著收集沉澱,乾燥後可得化合物5之無色 固體(產率:93.3%),其熔點約為121-122℃,並以1H-NMR及13C-NMR鑑定其結構,光譜數據如下:1H NMR(CDCl3)δ 6.58(s,2H,H-5,6),5.47(s,2H,H-1,4),3.19(s,2H,H-2,3);13C NMR(CDCl3)δ 170.1(C=O),137.2(C-5,C-6),82.4(C-1,C-4),48.9(C-2,C-3)。 Furan (40 ml, 550 mmol) and maleic anhydride (10 g, 102 mmol) were mixed in diethyl ether and reacted at room temperature for about 48 hours, then the precipitate was collected and dried. The colorless solid of Compound 5 (yield: 93.3%) having a melting point of about 121-122 ° C and its structure was identified by 1 H-NMR and 13 C-NMR. The spectral data are as follows: 1 H NMR (CDCl 3 ) δ 6.58 (s, 2H, H-5, 6), 5.47 (s, 2H, H-1, 4), 3.19 (s, 2H, H-2, 3); 13 C NMR (CDCl 3 ) δ 170.1 (C =O), 137.2 (C-5, C-6), 82.4 (C-1, C-4), 48.9 (C-2, C-3).
LC-MS(ESI-,m/z)C8H6O4:理論值為166.03,實際量測值為164.90[M-H]-。 LC-MS (ESI -, m / z) C 8 H 6 O 4: theoretical value of 166.03, the actual measurement value of 164.90 [MH] -.
7-氧二環[2,2,1]庚烷-2,3-二羧酸酐(化合物2)7-oxobicyclo[2,2,1]heptane-2,3-dicarboxylic anhydride (compound 2)
在前述化合物5(4.7克,28.3毫莫耳)的THF(200毫升)溶液中加入10%之Pd/C(470毫克),在3大氣壓的氫氣、及室溫下攪拌反應混合物約8-12小時。以Celite 545®過濾反應混合物,真空乾燥後可得化合物2之無色固體(4.3克,產率:91.4%),其熔點約為116-118℃,並以1H-NMR及13C-NMR鑑定其結構,光譜數據如下:1H NMR(CDCl3)δ 5.05(dd,J=2.3,2.2Hz,2H,H-1,4),3.18(s,2H,H-2,3),1.91-1.89(m,2H,H-5,6),1.65-1.63(m,2H,H-5,6);13C NMR(CDCl3)δ 171.1(C=O),80.2(C-1,C-4),50.6(C-2,C-3),28.1(C-5,C-6);LC-MS(ESI-,m/z)C8H8O4:理論值為168.04,實際量測值為166.86[M-H]-。 10% Pd/C (470 mg) was added to a solution of the above compound 5 (4.7 g, 28.3 mmol) in THF (200 ml), and the mixture was stirred at room temperature under atmospheric pressure of 3 atm. hour. Celite 545® reaction mixture was filtered, and dried in vacuo to give compound 2 may be of a colorless solid (4.3 g, yield: 91.4%) having a melting point of about 116-118 deg.] C, and in 1 H-NMR and 13 C-NMR identified Its structure and spectral data are as follows: 1 H NMR (CDCl 3 ) δ 5.05 (dd, J = 2.3, 2.2 Hz, 2H, H-1, 4), 3.18 (s, 2H, H-2, 3), 1.91 1.89 (m, 2H, H-5, 6), 1.65-1.63 (m, 2H, H-5, 6); 13 C NMR (CDCl 3 ) δ 171.1 (C=O), 80.2 (C-1, C -4), 50.6 (C-2, C-3), 28.1 (C-5, C-6); LC-MS (ESI - , m/z) C 8 H 8 O 4 : theoretical value 168.04, actual The measured value is 166.86 [MH] - .
N-羥基-7-氧二環[2,2,1]-5-庚烷-2,3-二甲醯亞胺(化合物2)N-hydroxy-7-oxobicyclo[2,2,1]-5-heptane-2,3-dimethylimine (Compound 2)
在前述化合物2(5.04克,30毫莫耳)的無水 甲醇(200毫升)溶液中,加入甲基氧化鈉(1.62克,30毫莫耳)和羥基胺氯化氫(2.08克,30毫莫耳)。在室溫下攪拌反應混合物約20小時,過濾反應混合物,真空濃縮乾燥後以CHCl3再結晶可得化合物6的無色結晶(產率:63%)。其熔點約為168-169℃,分離出來的化合物利用1H-NMR及13C-NMR進行結構解析與鑑定。光譜數據如下:1H NMR(D2O)δ 4.93(s,2H,H-1,4),3.21(s,2H,H-2,3),1.89-1.86(m,2H,H-5,6),1.78-1.74(m,2H,H-5,6);13C NMR(D2O)δ 177.0,78.8,46.9,28.0;LC-MS(ESI-,m/z)C8H9NO4:理論值為183.05,實際量測值為206.00[M-H]-。 To a solution of the aforementioned compound 2 (5.04 g, 30 mmol) in anhydrous methanol (200 ml), sodium methoxide (1.62 g, 30 mM) and hydroxylamine hydrogen chloride (2.08 g, 30 mM) . The reaction mixture was stirred at room temperature for about 20 hours, the reaction mixture was filtered, dried and concentrated in vacuo to CHCl 3 Recrystallization available compound 6 as colorless crystals (yield: 63%). Its melting point is about 168-169 ° C, and the isolated compound is analyzed and identified by 1 H-NMR and 13 C-NMR. The spectral data are as follows: 1 H NMR (D 2 O) δ 4.93 (s, 2H, H-1, 4), 3.21 (s, 2H, H-2, 3), 1.89-1.86 (m, 2H, H-5) , 6), 1.78-1.74 (m, 2H, H-5, 6); 13 C NMR (D 2 O) δ 177.0, 78.8, 46.9, 28.0; LC-MS (ESI - , m/z) C 8 H 9 NO 4 : The theoretical value is 183.05, and the actual measured value is 206.00 [MH] - .
N-法尼氧基-7-氧二環[2,2,1]庚烷-2,3-二甲醯亞胺(化合物9或NOC15)N-farnesyloxy-7-oxabicyclo[2,2,1]heptane-2,3-dimethylimine (Compound 9 or NOC15)
在前述化合物6(1毫莫耳)的無水丙酮溶液中,加入溴化法尼烷(1毫莫耳)和K2CO3(3毫莫耳),讓混合物迴流反應8-10小時。過濾反應混合物,真空濃縮後以矽膠管柱純化(乙酸乙酯/正己烷為洗液),可得化合物9的無色液體(產率:28%)。分離出來的化合物利用1H-NMR及13C-NMR進行結構解析與鑑定。光譜數據如下:1H NMR(D2O)δ 5.42(td,J=0.9,7.7Hz,1H,H-2’),5.11-5.07(m,2H,H-6’,10’),4.85(q,J=2.4Hz,2H,H-1,4),4.62(d,J=7.7Hz,2H,H-1’),2.83(s,2H,H-2,3),2.09-2.04(m,6H,H-5,6,H-4’,8’),1.99-1.96(m,2H,H-5’),1.89-1.86(m,2H,H-6’),1.72(d,J-0.7Hz, 3H,CH3),1.68(s,3H,CH3),1.63-1.62(m,2H,H-5,6),1.60(s,3H,CH3),1.59(s,3H,CH3);13C-NMR(CDCl3):δ 171.6,147.3,135.3,131.3,124.3,123.5,116.1,78.5,73.0,47.4,39.65,39.63,28.7,26.7,26.1,25.7,17.7,16.6,16.0;LC-MS(ESI-,m/z)C23H33NO4:理論值為387.24,實際量測值為410.21[M+Na]-。 To a solution of the aforementioned compound 6 (1 mmol) in dry acetone, bromofurane (1 mmol) and K 2 CO 3 (3 mmol) were added, and the mixture was refluxed for 8 to 10 hours. The reaction mixture was filtered, concentrated in vacuo purified by silica gel column (ethyl acetate / hexane as a lotion), a Compound 9 as a colorless liquid (yield: 28%). The isolated compound was subjected to structural analysis and identification by 1 H-NMR and 13 C-NMR. The spectral data are as follows: 1 H NMR (D 2 O) δ 5.42 (td, J = 0.9, 7.7 Hz, 1H, H-2'), 5.11-5.07 (m, 2H, H-6', 10'), 4.85 (q, J = 2.4 Hz, 2H, H-1, 4), 4.62 (d, J = 7.7 Hz, 2H, H-1 '), 2.83 (s, 2H, H-2, 3), 2.09-2.04 (m, 6H, H-5, 6, H-4', 8'), 1.99-1.96 (m, 2H, H-5'), 1.89-1.86 (m, 2H, H-6'), 1.72 ( d, J-0.7Hz, 3H, CH 3 ), 1.68 (s, 3H, CH 3 ), 1.63-1.62 (m, 2H, H-5, 6), 1.60 (s, 3H, CH 3 ), 1.59 ( s, 3H, CH 3 ); 13 C-NMR (CDCl 3 ): δ 171.6, 147.3, 135.3, 131.3, 124.3, 123.5, 116.1, 78.5, 73.0, 47.4, 39.65, 39.63, 28.7, 26.7, 26.1, 25.7, 17.7,16.6,16.0; LC-MS (ESI - , m / z) C 23 H 33 NO 4: theoretical value of 387.24, the actual measurement value of 410.21 [m + Na] -.
1.2 合成N-法尼基-7-氧二環[2,2,1]庚烷-2,3-二甲醯亞胺(化合物18或NC15)1.2 Synthesis of N-farnesyl-7-oxabicyclo[2,2,1]heptane-2,3-dimethylimine (Compound 18 or NC15)
依以下流程2所述方式,合成NC15化合物。 The NC15 compound was synthesized as described in Scheme 2 below.
7-氧二環[2,2,1]-5-庚烯-2,3-二甲醯胺(化合物14)7-oxobicyclo[2,2,1]-5-heptene-2,3-dimethylguanamine (compound 14)
將順丁烯二醯亞胺(化合物13)(1.5克,15毫莫耳)的甲苯(30毫升)溶液加熱至80℃,之後加入呋喃(5.4毫升,75毫莫耳),並在80℃下攪拌6小時,冷卻至室溫後收集沉澱,再以矽膠色層管柱純化(乙酸乙酯/正己烷為洗液),可得化合物14之無色晶體(2.1克,產率:87%),其熔點約為162-163℃,並以1H-NMR及13C-NMR鑑定其結構,光譜數據如下:1H-NMIR(CDCl3):δ 8.13(s,1H,NH),6.50(s,2H,H-5,6),5.29(s,2H,H-1.4),2.87(s,2H,H-2,3);13C-NMR(CDCl3):δ 176.2,136.8,81.2,48.9;LC-MS(ESI-,m/z)C8H7NO3理論值為165.04,實際量測值為164.02[M-H]-。 A solution of maleimide (Compound 13 ) (1.5 g, 15 mmol) in toluene (30 mL) was heated to 80 ° C then added furan (5.4 mL, 75 mmol) and at 80 ° C After stirring for 6 hours, the mixture was cooled to room temperature, and then the precipitate was collected, and then purified on a silica gel column (ethyl acetate / n-hexane as a washing liquid) to obtain a colorless crystal of compound 14 (2.1 g, yield: 87%) Its melting point is about 162-163 ° C, and its structure is identified by 1 H-NMR and 13 C-NMR. The spectral data are as follows: 1 H-NMIR (CDCl 3 ): δ 8.13 (s, 1H, NH), 6.50 ( s, 2H, H-5, 6), 5.29 (s, 2H, H-1.4), 2.87 (s, 2H, H-2, 3); 13 C-NMR (CDCl 3 ): δ 176.2, 136.8, 81.2 , 48.9; LC-MS (ESI - , m / z) C 8 H 7 NO 3 The theoretical value is 165.04, the actual measured value is 164.02 [MH] - .
7-氧二環[2,2,1]庚烷-2,3-二甲醯胺(化合物15)7-oxobicyclo[2,2,1]heptane-2,3-dimethylguanamine (compound 15)
在前述化合物14(0.5克,2.8毫莫耳)的THF(20毫升)溶液中加入10%之Pd/C(50毫克),在室溫下、氫氣存在的環境下,攪拌反應混合物約4小時。以Celite545®過濾反應混合物,真空乾燥後可得化合物15(即,NC15)之無色固體(424毫克,產率:84%),其熔點約為188-190℃,並以1H-NMR及13C-NMR鑑定其結構,光譜數據如下:1H-NMR(CDCl3):δ 8.60(s,1H,NH),4.91(dd,J=2.4,3.3Hz,2H,H-1,4),2.92(s,2H,H-2,3),1.89-1.85(m,2H,H-5,6),1.61-1.57(m,2H,H-5,6); 13C-NMR(CDCl3):δ 177.2,79.1,51.3,28.5;LC-MS(ESI-,m/z),C8H9NO3理論值為167.06,實際量測值為166.03[M-H]-。 10% Pd/C (50 mg) was added to a solution of the above compound 14 (0.5 g, 2.8 mmol) in THF (20 ml), and the mixture was stirred for about 4 hours at room temperature in the presence of hydrogen. . Celite545® reaction mixture was filtered, and dried in vacuo to afford compound may be 15 (i.e., NC15) of a colorless solid (424 mg, yield: 84%) having a melting point of about 188-190 deg.] C, and at 13 and 1 H-NMR The structure was identified by C-NMR, and the spectral data were as follows: 1 H-NMR (CDCl 3 ): δ 8.60 (s, 1H, NH), 4.91 (dd, J = 2.4, 3.3 Hz, 2H, H-1, 4), 2.92 (s, 2H, H-2, 3), 1.89-1.85 (m, 2H, H-5, 6), 1.61-1.57 (m, 2H, H-5, 6); 13 C-NMR (CDCl 3 ): δ 177.2, 79.1, 51.3, 28.5; LC-MS (ESI - , m/z), C 8 H 9 NO 3 The theoretical value is 167.06, the actual measured value is 166.03 [MH] - .
N-法尼基-7-氧二環[2,2,1]庚烷-2,3-二甲醯亞胺(化合物18或NC15)N-farnesyl-7-oxabicyclo[2,2,1]heptane-2,3-dimethylimine (Compound 18 or NC15)
在前述化合物15(1毫莫耳)的無水丙酮溶液(25毫升)中,加入溴化法尼烷(1毫莫耳)和K2CO3(3毫莫耳),讓混合物迴流反應8-10小時。過濾反應混合物,真空濃縮後以矽膠管柱純化(乙酸乙酯/正己烷為洗液),可得化合物18的無色液體(產率:28%)。分離出來的化合物利用1H-NMR及13C-NMR進行結構解析與鑑定。光譜數據如下:1H-NMR(CDCl3):δ 5.10-5.02(m,3H,H-2’,6’,10’),4.84(dd,J=2.2,2.3Hz,2H,H-1,4),4.03(d,J=7.0Hz,2H,H-1’),2.82(s,2H,H-2,3),2.06-2.01(m,4H,H-4’,8’),1.97-1.92(m,4H,H-5’,9’),1.84-1.81(m,2H,H-5,6),1.75(s,3H,CH3),1.67(s,3H,CH3),1.60(s,3H,CH3),1.59(s,3H,CH3),1.58-1.55(m,2H,H-5,6);13C-NMR(CDCl3):δ 176.8,140.9,135.3,131.2,124.3,123.7,117.2,78.9,50.0,39.7,39.5,37.0,28.6,26.8,26.3,25.7,17.7,16.4,16.0;LC-MS(ESl-,m/z),C23H33NO3理論值為371.25,實際量測值為394.2[M-H]-。 In a solution of the aforementioned compound 15 (1 mmol) in dry acetone (25 ml), br.nibrane (1 mmol) and K 2 CO 3 (3 mmol) were added and the mixture was refluxed. 10 hours. The reaction mixture was filtered, and then purified, mjjjjjjj The isolated compound was subjected to structural analysis and identification by 1 H-NMR and 13 C-NMR. The spectral data are as follows: 1 H-NMR (CDCl 3 ): δ 5.10-5.02 (m, 3H, H-2', 6', 10'), 4.84 (dd, J = 2.2, 2.3 Hz, 2H, H-1 , 4), 4.03 (d, J = 7.0 Hz, 2H, H-1 '), 2.82 (s, 2H, H-2, 3), 2.06-2.01 (m, 4H, H-4', 8') , 1.97.1.22 (m, 4H, H-5', 9'), 1.84-1.81 (m, 2H, H-5, 6), 1.75 (s, 3H, CH 3 ), 1.67 (s, 3H, CH 3 ), 1.60 (s, 3H, CH 3 ), 1.59 (s, 3H, CH 3 ), 1.58-1.55 (m, 2H, H-5, 6); 13 C-NMR (CDCl 3 ): δ 176.8, 140.9, 135.3, 131.2, 124.3, 123.7, 117.2, 78.9, 50.0, 39.7, 39.5, 37.0, 28.6, 26.8, 26.3, 25.7, 17.7, 16.4, 16.0; LC-MS (ESl - , m/z), C 23 The theoretical value of H 33 NO 3 is 371.25, and the actual measured value is 394.2 [MH] - .
實施例2 本發明化合物的抗增生活性分析Example 2 Analysis of anti-proliferative activity of the compounds of the present invention
以細胞存活試驗來分析本發明式(I)化合物抑制各式癌細胞,包括肝癌HepG2細胞、膀胱癌BFTC905細胞、直腸癌HT29細胞和SW480細胞及白血病HL60細胞生長的效果,並以化合物2、及其他習知抗癌藥物(包括5-FU、順鉑和阿黴素)作為對照組。結果示於表1。 The cell survival assay was used to analyze the effects of the compound of the formula (I) of the present invention on the growth of various cancer cells including liver cancer HepG2 cells, bladder cancer BFTC905 cells, rectal cancer HT29 cells, SW480 cells and leukemia HL60 cells, and the compounds 2 and Other conventional anticancer drugs (including 5-FU, cisplatin and doxorubicin) were used as a control group. The results are shown in Table 1.
表1 本發明式(I)化合物對各式癌細胞生長的影響
如表1所示,NOC15和NC15兩化合物對所測試的5種細胞都具有毒性,且其細胞毒性至少與對照組化合物(如,化合物2、5-FU、順鉑和阿黴素)不相上下,或更佳。此外,還觀察到5-FU和順鉑無法抑制白血病細胞株HL60的生長,但卻會被NOC15和NC15兩化合物抑制。 As shown in Table 1, both NOC15 and NC15 compounds were toxic to the five cells tested, and their cytotoxicity was at least as low as that of the control compounds (eg, compound 2, 5-FU, cisplatin, and doxorubicin). Up and down, or better. In addition, 5-FU and cisplatin were also observed to inhibit the growth of leukemia cell line HL60, but were inhibited by both NOC15 and NC15 compounds.
實施例3 NOC15和NC15抑制人類肝癌細胞增生Example 3 NOC15 and NC15 inhibit human liver cancer cell proliferation
3.1 細胞存活試驗3.1 Cell survival test
在此實施方式中,依據材料和方法中所述細胞存活試驗,來測試NOC15和NC15兩化合物抑制人類肝癌細胞HepG2生長的效果,結果示於表2。 In this embodiment, the effects of NOC15 and NC15 compounds inhibiting the growth of human hepatoma cells HepG2 were tested according to the cell survival assay described in Materials and Methods. The results are shown in Table 2.
表2 NOC15和NC抑制肝癌細胞生長
由表2數據可知,NOC15和NC15的活性至少是化合物2的3-4倍,亦即,只需要約1/3或1/4的化合物2的量,NOC15或NC15即可達成與化合物2相同程度的細胞抑制效果。 As can be seen from the data in Table 2, the activity of NOC15 and NC15 is at least 3-4 times that of Compound 2, that is, only about 1/3 or 1/4 of Compound 2 is required, and NOC15 or NC15 can be the same as Compound 2. Degree of cytostatic effect.
3.2 細胞核型態變化3.2 Cell nuclear type changes
為了進一步探索NOC15和NC15造成細胞凋亡的原因,分別以化合物2、NOC15和NC15處理HepG2細胞約48小時,然後以赫斯特33528染色並以螢光顯微鏡檢視細胞型態。 To further explore the causes of apoptosis in NOC15 and NC15, HepG2 cells were treated with Compound 2, NOC15 and NC15 for about 48 hours, respectively, and then stained with Hearst 33528 and examined for cell morphology by fluorescence microscopy.
第1圖為經過化合物2、NOC15和NC15處理後的細胞核照片,相對於控制組細胞核,可觀察到經化合物2、NOC15或NC15處理後的細胞,明顯萎縮,出現染色質沉積和DNA斷片。 Fig. 1 is a photograph of the nucleus after treatment with Compound 2, NOC15 and NC15. Compared with the control group, the cells treated with Compound 2, NOC15 or NC15 were observed to be significantly atrophied, and chromatin deposition and DNA fragmentation occurred.
3.3 細胞週期和細胞凋亡分析3.3 Cell cycle and apoptosis analysis
以10-60μM之化合物2、NOC15或NC15處理HepG2細胞約48小時,接著以流式細胞儀分析細胞週期和細胞凋亡的情況,並以PI對G0/G1周期細胞染色。結果分別示於第2、3圖。 Compound of 10-60 μ M 2, NOC15 or NC15 treated HepG2 cells for about 48 hours, followed by flow cytometry analysis of cell cycle and apoptosis of cells, and PI staining to the cells of the G0 / G1 period. The results are shown in Figures 2 and 3, respectively.
如第2圖所示,控制組(未經化合物處理)中僅有少數細胞出現凋亡,同樣情況也發生在以低濃度(10μM)化合物2處理的細胞群中。隨著化合物2的濃度從20μM增加到40μM,凋亡細胞比率以從1.6%提升到16.7%。類似的,當細胞以10-60μM的化合物18進行處理時,凋亡細胞比率以從3.7%提升到19.8%。此外,化合物9和18(即,NOC15和NC15)都可以誘導細胞進入G2/M週期。 As shown in Figure 2, only a few cells in the control group (without compound treatment) developed apoptosis, as did the cell population treated with low concentration (10 μM) of Compound 2. As the concentration of Compound 2 increased from 20 μM to 40 μM, the apoptotic cell ratio increased from 1.6% to 16.7%. Similarly, when cells were treated with 10-60 μM of Compound 18, the apoptotic cell ratio increased from 3.7% to 19.8%. In addition, compounds 9 and 18 (i.e., NOC 15 and NC 15) can induce cells to enter the G2/M cycle.
第3圖數據顯示,NOC15和NC15造成的細胞死亡與使用劑量相關,凋亡的細胞比率會隨著所用NOC15和NC15濃度增加而增加。至於化合物2,只有當濃度高達60μM時,才會出現明憲的細胞毒性。 The data in Figure 3 shows that the cell death caused by NOC15 and NC15 is related to the dose used, and the percentage of apoptotic cells increases with the concentration of NOC15 and NC15 used. As for Compound 2, the cytotoxicity of the Constitution was observed only when the concentration was as high as 60 μM.
實施例4 NOC15抑制人類白血病細胞增生Example 4 NOC15 inhibits proliferation of human leukemia cells
4.1 細胞存活試驗4.1 Cell survival test
在此實施方式中,以巴豆醇-12-十四烷酸酯-13-乙酸酯(PMA)加離子黴素(ionomycin)活化的Jurkat T細胞模式來評估NOC15對人類白血病細胞(Jurkat T細胞)和正常淋巴細胞(NHL)的影響。 In this embodiment, the Jurkat T cell pattern activated by crotyl alcohol-12-tetradecanoate-13-acetate (PMA) plus ionomycin is used to evaluate NOC15 on human leukemia cells (Jurkat T cells). ) and the effects of normal lymphocytes (NHL).
如第4A圖所示,化合物2對正常淋巴細胞(NHL)幾乎沒有毒性,但卻可殺死人類白血病細胞(Jurkat T細胞)。此外,如果Jurkat T細胞預先以巴豆醇-12-十四烷酸酯-13-乙酸酯(PMA)加離子黴素處理的話(已知巴豆醇-12-十四烷酸酯-13-乙酸酯(PMA)可保護細胞不致凋亡),則可稍微提高細胞存活率。類似的結果也可在NOC15處理過的細胞群中看到(第4B圖)。以上結果表示化合物2和NOC15同樣都是 經由致腫瘤活化蛋白酶路徑(mitogen-activated protein kinase(MAPK)pathwat)來抑制腫瘤細胞的生長。 As shown in Figure 4A, Compound 2 is almost non-toxic to normal lymphocytes (NHL) but kills human leukemia cells (Jurkat T cells). In addition, if Jurkat T cells are previously treated with crotyl alcohol-12-tetradecanoate-13-acetate (PMA) plus ionomycin (known crotyl-12-tetradecanoate-13-B The acid ester (PMA) protects cells from apoptosis and slightly increases cell viability. Similar results can also be seen in the NOC15 treated cell population (Fig. 4B). The above results indicate that both compound 2 and NOC15 are also Tumor cell growth is inhibited via a mitogen-activated protein kinase (MAPK) pathway.
4.2 細胞週期分析4.2 Cell cycle analysis
以1.4μM之NOC15處理白血病細胞約24或48小時,接著以流式細胞儀分析細胞週期的情況,並以PI對G0/G1周期細胞染色。結果分別示於第5A-5D圖。 Leukemia cells were treated with 1.4 μM NOC15 for about 24 or 48 hours, followed by analysis of cell cycle by flow cytometry and staining of G0/G1 cycle cells with PI. The results are shown in Figures 5A-5D, respectively.
可發現只有少部分的控制組細胞處於G0/G1周期(第5A圖),但如果經過NOC15處理24或48小時,則處於G0/G1周期的細胞數目將增加(第5B、5C圖);同時,以NOC15處理白血病細胞48小時後,可發現留在G2/M週期的細胞數目也會增加。第5A-5C圖數據經量化後繪示在第5D圖。 It can be found that only a small number of control group cells are in the G0/G1 cycle (Fig. 5A), but if treated with NOC15 for 24 or 48 hours, the number of cells in the G0/G1 cycle will increase (Fig. 5B, 5C); After 48 hours of treatment of leukemia cells with NOC15, the number of cells remaining in the G2/M cycle was also increased. The data of Fig. 5A-5C is quantized and shown in Fig. 5D.
實施例5 NC15抑制人類白血病細胞增生Example 5 NC15 inhibits proliferation of human leukemia cells
5.1 細胞存活試驗5.1 Cell survival test
NC15對正常淋巴細胞(NHL)和人類白血病細胞的毒殺效果顯示在第6圖。類似化合物2和NOC15的效果,NC15對正常淋巴細胞(NHL)同樣幾乎沒有毒性,但卻可殺死人類白血病細胞(Jurkat T細胞),且其毒殺效果與劑量呈現正相關。當以NC15處理細胞24小時後,存活細胞比率隨著NC15濃度增加而下降(第6A圖),以NC15處理細胞48小時,其結果也類似。 The toxicity of NC15 on normal lymphocytes (NHL) and human leukemia cells is shown in Figure 6. Similar to the effects of Compound 2 and NOC15, NC15 is almost non-toxic to normal lymphocytes (NHL), but kills human leukemia cells (Jurkat T cells), and its toxicity is positively correlated with dose. After 24 hours of treatment with NC15, the viable cell ratio decreased as the concentration of NC15 increased (Fig. 6A), and cells were treated with NC15 for 48 hours, and the results were similar.
5.2 細胞週期分析5.2 Cell cycle analysis
以2.513μM之NC15處理白血病細胞約24或48小時,接著以流式細胞儀分析細胞週期的情況,並以PI對G0/G1周期細胞染色。結果分別示於第7圖。 2.513 μ M to the NC15 treated leukemia cells of about 24 or 48 hours, followed by cell cycle analysis by flow cytometry and PI staining to the cells of G0 / G1 period. The results are shown in Figure 7, respectively.
大部分控制組細胞(未接受任何藥物處理)都處於G0/G1周期,當細胞經過NOC15處理24小時後,處於G0/G1周期以下(subG1周期)的細胞數目將開始增加(第7B圖),並在48小時後達到一明顯的數量(第7C圖);同時還可發現留在G0/G1周期的細胞數目相較於控制組而言,明顯減少(第7D圖)。第7A-7C圖數據經量化後繪示在第7D圖。 Most of the control group cells (not receiving any drug treatment) were in the G0/G1 cycle, and the number of cells below the G0/G1 cycle (subG1 cycle) began to increase after the cells were treated with NOC15 for 24 hours (Fig. 7B). A significant amount was reached after 48 hours (Fig. 7C); it was also found that the number of cells remaining in the G0/G1 cycle was significantly reduced compared to the control group (Fig. 7D). The data of Fig. 7A-7C is quantized and shown in Fig. 7D.
5.3 NOC15和/或NC15可縮減植入有人類白血病細胞L1210之動物身上的腫瘤5.3 NOC15 and / or NC15 can reduce tumors in animals implanted with human leukemia cells L1210
依據「材料與方法」段落中的操作步驟將L1210細胞接種在實驗動物體內。在活體治療試驗中,經由腹腔注射對實驗動物施用18毫克/公斤的NOC15或NC15,連續施用7天。然後,在指定時間分別量測腫瘤大小、以及實驗動物的肝臟、脾臟重量和白血球細胞數目。 L1210 cells were seeded in experimental animals according to the procedures in the "Materials and Methods" section. In the in vivo treatment test, experimental animals were administered 18 mg/kg of NOC15 or NC15 via intraperitoneal injection for 7 consecutive days. Then, the tumor size, and the liver, spleen weight, and white blood cell count of the experimental animals were measured at the designated time.
NOC15或NC15對皮下或腹腔接種L1210細胞的影響繪示於第8圖。可發現,NOC15和NC15都可有效地延長動物的生命,對接受皮下注射L1210細胞的動物來說,其生命週期延長了約40%;對腹腔接種L1210細胞的動物來說,其生命週期延長了約14-18%。此外,以NOC15或NC15治療的SC-腫瘤小鼠,其腫瘤重量明顯縮小許多(第9A圖),至於其肝臟與脾臟的重量,則明顯有改善(相較於控制組而言)(第9B、9C圖)。此外,相較於控制組而言,白血球數目在治療後第14天也有明顯下降(第10圖)。 The effect of NOC15 or NC15 on subcutaneous or intraperitoneal inoculation of L1210 cells is shown in Figure 8. It can be found that both NOC15 and NC15 can effectively prolong the life of the animal, and the life cycle of the animals receiving the subcutaneous injection of L1210 cells is extended by about 40%; for the animals inoculated with L1210 cells per abdomen, the life cycle is prolonged. About 14-18%. In addition, SC-tumor mice treated with NOC15 or NC15 showed a significant reduction in tumor weight (Fig. 9A), and their liver and spleen weights were significantly improved (compared to the control group) (9B) , 9C picture). In addition, the number of white blood cells decreased significantly on the 14th day after treatment compared to the control group (Fig. 10).
以傳統化療藥物治療癌症最大的缺點是會造成患者體重減輕、一旦患者無法維持正常體重,也將間接導 致治療效果不佳。在本實施方式中,發明人發現,相較於控制組,在癌症治療期間,給予患者18毫克/公斤體重的NOC15可逐步改善患者體重減少的情況(第11A圖);同樣情況下,NC15則可使動物體重恢復到與控制組動物相同的程度(第11B圖)。數據顯示NC15除了抑制腫瘤生長外,其也沒有一般傳統化療藥物會造成使用者體重減輕的缺點,因此NC15具有開發成新一代抗癌藥物(特別是抗白血病)的潛力。 The biggest disadvantage of treating cancer with traditional chemotherapy drugs is that it will cause the patient to lose weight. Once the patient is unable to maintain normal weight, it will also be indirectly guided. The treatment effect is not good. In the present embodiment, the inventors have found that administering 18 mg/kg of NOC15 to a patient during a cancer treatment can gradually improve the patient's weight loss during the cancer treatment (Fig. 11A); in the same case, NC15 The animal's body weight was restored to the same extent as the control group animals (Fig. 11B). The data show that in addition to inhibiting tumor growth, NC15 does not have the disadvantage that conventional chemotherapeutic drugs cause weight loss in users. Therefore, NC15 has the potential to develop into a new generation of anticancer drugs (especially anti-leukemia).
總合以上,本發明所合成的兩種新穎親脂性之N-取代去甲基斑蝥素衍生物除了具有可毒殺多種癌細胞的活性外,且不會對正常細胞造成傷害,因此具有開發成新一代抗癌藥物(特別是抗白血病)的潛力。 In summary, the two novel lipophilic N-substituted demethylcantharidin derivatives synthesized by the present invention have the activity of being toxic to kill a plurality of cancer cells, and do not cause damage to normal cells, so they are developed into new ones. The potential of a generation of anticancer drugs (especially anti-leukemia).
當可理解上述實施方式與實施例僅為例示,且熟習此技藝者可對齊進行各種修飾。上文提出之說明書、實施例與資料的目的在於使本說明書的結構完備,並作為實作本發明之例示。雖然本揭示內容已以實施方式揭露如上,然其並非用以限定本揭示內容,任何熟習此技藝者,在不脫離本揭示內容之精神和範圍內,當可作各種之更動與潤飾,因此本揭示內容之保護範圍當視後附之申請專利範圍所界定者為準。 It will be understood that the above-described embodiments and examples are merely illustrative, and that those skilled in the art can align various modifications. The description, examples, and materials set forth above are intended to be illustrative of the present invention and are illustrative of the invention. The present disclosure has been disclosed in the above embodiments, but it is not intended to limit the disclosure, and any person skilled in the art can make various changes and refinements without departing from the spirit and scope of the disclosure. The scope of protection of the disclosure is subject to the definition of the scope of the patent application.
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