1260320 玫、發明說明： 【發明所屬之技術領域】 本發明有關一種化合物；特別是有關一種昨唾化合物 (carbazole compound)，及利用其使 DNA 四股結構體 (quardruplex)穩定之方法。 【先前技術】 端粒（telomere)，即染色體之端部，為真核細胞染色體穩定 性與複製不可或缺者。請參閱例如Williamson J.R.所著之4㈣1/. 1 994，23:703。：¾¾ 粒序歹丨J 在、名田月包分 裂期間變短，此係因為DNA之合成無法完全複製染色體之極 端部。端粒長度減少至臨界程度時，會導致基因不穩定、異常 之染色體融合、及細胞衰老。請參閱例如Hadey等人所著之 Cwrr. Dev.，1995，5:249。反之，腫瘤細胞之端粒在 細胞複製期間由於端粒酶（telomerase)之存在而不會變短，端粒 酶可使核苷酸加至染色體端部之端粒DNA上。請參閱Feng等 人所著之Sc/wce，1995, 269:1236。端粒酶在85%以上之腫瘤細 胞中會表現，但在大部分身體細胞中不會。請參閱Harley等人 所著之〜re，1990，345:458。因此，端粒酶逐漸成為癌症診 斷與化療之最佳標的。請參閱Blackburn E.H.等人所著之 1991， 350:569 。 端粒一般由富含鳥°票σ令之區域（G-rich motifs)(例如人類端 粒中之T2AG3)之許多重複片段（tandem repeats)所組成。請參閱 Morin G.B.所著之 Ce//，1989，59:521。體外（/" 試驗顯示 3 端富含鳥嘌呤之單股採取分子内之G四股結構 (G-quadruplex structure)。四股結構藉由環狀G -四重體 !26〇32〇 (quartet)之兀-兀相互作用而穩定，經由H〇〇糾een氫鍵所形 成“閱Gellert等人所著之p騰漏厂」—％·以队1962 ·2013因為在體外試驗已顯示將端粒dna摺疊成G_四股結 構體可抑制端粒酶活性，所以亦將G_四股結構體考慮為抗腫瘤 J之/曰在軚的。凊麥閱Zahler等人所著之愚加祀，1%1，。 【發明内容】 山本發明係基於意想不到之發現特定之昨唾化合物能使人 類端粒之G_四股結構體熱穩定。 在一方面，本發明之特徵在於下列化學式之咔唑化合物：BACKGROUND OF THE INVENTION 1. Field of the Invention This invention relates to a compound; particularly to a carbazole compound, and a method for stabilizing a DNA quadruplex thereof. [Prior Art] Telomere, the end of a chromosome, is indispensable for chromosomal stability and replication of eukaryotic cells. See, for example, 4 (4) 1/. 1 994, 23: 703 by Williamson J.R. :3⁄43⁄4 The granule 歹丨J is shortened during the splitting of the name, and this is because the synthesis of DNA cannot completely replicate the extreme ends of the chromosome. Decreased telomere length leads to genetic instability, abnormal chromosome fusion, and cellular senescence. See, for example, Hadey et al., Cwrr. Dev., 1995, 5:249. Conversely, telomeres of tumor cells do not become shorter during cell replication due to the presence of telomerase, which allows nucleotides to be added to the telomere DNA at the ends of the chromosome. See Sc/wce, 1995, 269:1236 by Feng et al. Telomerase is expressed in more than 85% of tumor cells, but not in most body cells. See Harley et al., re, 1990, 345: 458. Therefore, telomerase has gradually become the best target for cancer diagnosis and chemotherapy. See Blackburn E.H. et al. 1991, 350:569. Telomeres generally consist of a number of repeats that are rich in G-rich motifs (e.g., T2AG3 in human telomeres). See Ce//, 1989, 59: 521 by Morin G.B. In vitro (/" experiments show that the single-stranded guanine-rich single-strand adopts the intramolecular G-quadruplex structure. The four-strand structure is made of ring-shaped G-quadruplex! 26〇32〇 (quartet)兀-兀 interaction is stable, formed by H〇〇 een 氢 hydrogen bond “reading Gellert et al.'s p-tipping factory”—%· team 1962·2013 because it has been shown to fold telomere DNA in vitro The G_four-strand structure can inhibit telomerase activity, so the G_quad structure is also considered to be anti-tumor J. The buckwheat is written by Zahler et al., 1%1. SUMMARY OF THE INVENTION The present invention is based on the unexpected discovery that a particular sputum compound can thermally stabilize the G_quadruplex of human telomeres. In one aspect, the invention features a carbazole compound of the formula:
弋中B環各獨立為含有至少一個氮原子之雜芳基；） 與Y各獨立為CH或川心至化各獨立為H、C「C8烷基、C2_c 烯基C^C8炔基、CVC8環烷基、雜環烷基、芳基、_ ^基、〇H、Cl_c6烷氧基、芳氧基、雜芳氧基、NH2、Ci_C6^ 胺基、C^c!2二烷胺基、芳胺基、二芳胺基、或鹵基；心為H C」C8院基、C2_C8稀基、CrC8炔基、C3_C8環烧基、C3_C8縣 %烷基、芳基、或雜芳基；111與11各獨立為1、2、或3。 一上述咔唑化合物之一子集特徵在於八與8環各為含有一或 二個氮原子之雜芳基。在此等化合物中，n各為i R7各為Η;及X與γ各為CH或N。 「烷基」一詞指飽和之直鏈或支鏈烴基團（m〇iety)，例 1260320 CH3、-CH2-、或支鏈 c3h7。「 之直鏈或支鏈非芳香性之烴“」m具有至少-個雙鍵 -CH=CH_。「炔基」—詞指土團⑽吻）’例如-ch=ch2或 香性之烴基團，例如或如：鏈士=方 之環狀烴基團，例如環己基。「 、70土」一^日飽和 個環雜原子之飽和環^團4㈣基」―詞指具有至少— 衣狀基團，例如4-四氫哌σ南基。「芳基 詞指具有一或二個芳環之烴基 方基」 估踅1 ^ ^ ^ _ 团方基基團之貫例包括苯基、 伸本基、奈基、伸奈基、祐基、蒽基、 詞指具有一或多個含有入雜方基」 ^ ^ 1U雊原子之方裱之基團。雜芳基 t團之貫例包括呋喃基、伸呋喃基、第基、吡咯基、噻吩基、 %唑基、咪唑基、.坐基、吡啶基、嘧啶基、喹唑啉基、喹啉 基、異喹啉基、及_基。4氧基」—詞指含有氧基之直鍵 或支鏈、飽和或不飽和、非芳香性烴基團，例如_oc札或 -〇™=c2h5。「彡氧基」一詞指具有至少一個芳環及鍵結至芳環 之氧基之基團。「雜芳氧基」一詞指具有至少一個含有至少一 個環雜原子之芳環及鍵結至芳環之氧基之基團，例如4_吡啶 基。 本文中所述及之烷基、烯基、炔基、環烷基、雜環烷基、 芳基、雜芳基、烷氧基、芳氧基、雜芳氧基、烷胺基、二烷胺 基、芳胺基、及二芳胺基包括經取代或未經取代之基團。在環 燒基、雜環烧基、芳基、雜芳基、芳氧基、雜芳氧基、芳胺基、 及一芳胺基上之可能的取代基包括烧基、c2- c1()稀基、 C2-C1()炔基、C3-C8環烧基、〔5-(38環稀基、Cj-CjQ烧氧基、芳 基、芳氧基、雜芳基、雜芳氧基、胺基、Ci-CjQ烧胺基、Ci-Czo 二烧胺基、芳胺基、二芳胺基、經基、鹵基、硫基'c i - c i 〇烧 硫基、芳硫基、Cl-CiQ院續基、芳績基、氰基、硝基、酿基、 氧基、緩基、及竣Sa。另一方面，在烧基、稀基、炔基、燒 8 1260320 氧基、炫胺基、及二院胺基上可能之取代基包括除了 基、c2-C|0#s、C2_C|0快基之外之全部上述取代基。環貌基 與雜環烷基亦可與芳基或雜芳基稠合。 土 β上述之咔唾化合物包括其化合物本身以及其鹽及其前驅 ，物（prodrug)(若適用的話）。例如可在陰離子與咔唑化合物上 帶正電荷基（例如銨離子）之間形成鹽。適合之陰離子包括氣離 子、漠離^、蛾離子、硫酸根離子、亞硫酸根離子、過氣酸根 離子、六氟磷酸根離子、硝酸根離子、磷酸根離子、檸檬酸根 離子、甲糾酸根離子、三i乙酸根離子、及乙酸根離子。同乂 理，可在陽離子與咔唑化合物上帶負電荷基（例如羧基）之間形 成鹽。適合的陽離子包括鈉離子、鉀離子、鎂離子、鈣離子、 及銨陽離子例如四曱銨離子。前驅藥物之實例包括在投藥㈣ 者時能夠提供活性咔唑化合物之酉旨及其他醫藥丨可接受之;： 生物。 在另方面，本發明特徵在於一種使人類端粒或其他哺乳 動物端粒之G.四股結構體穩定之方法。該方法包括使端粒血上 迷相同化學式之昨唾化合物接觸。此方法可被用以治療任何需 抑制端粒酶活性之端粒酶相關疾病。此外，此方法可被用於體 ^忒驗（例如，鑑別端粒之G四股結構體）或用於體内動物模型 :驗或篩檢上述咔唑化合物做為治療端粒酶相關疾病（例如， :§症）之樂物之效力。本文中所述「治療」一詞指以賦與療效 目的例如治癒、減輕、改變、影響、改善、或防止端粒酶 j疾病、其症狀、或傾向於其之體質，對具有端粒酶相關疾 二旦此種疾病之症狀、或傾向於此等疾病之體質之患者予以有 之一或多種咔唑化合物之投藥。本文中所述之「有效量」， ^述—或多卿錢合_接受治療之患者具有療效所需 置 〇 1260320 此=本發明涵括-種含有有效量之至少—種上述昨唾化 合物及备樂上可接受之載體之醫藥組合物。 -種含有-或多㈣於治療端粒齡關疾病之上述味唾 化合物之組合物，及此種組合物於製造上述之治療用醫藥之 途’亦在本發明之範轉。 ’、 下列σ羊述本發明之一或多個具體實施例。從下列敘述及申 請專利範圍中將更了解本發明之其他特徵、目的、及優點。 【實施方式】 下列所示為舉例之本發明之咔唑化合物，化合物丨至The B ring in the oxime is independently a heteroaryl group containing at least one nitrogen atom;) and each of Y is independently CH or Chuan Xinzhi is independently H, C "C8 alkyl, C2_c alkenyl C^C8 alkynyl, CVC8 Cycloalkyl, heterocycloalkyl, aryl, _^yl, hydrazine H, Cl_c6 alkoxy, aryloxy, heteroaryloxy, NH2, Ci_C6^ amine, C^c!2 dialkylamino, An arylamino group, a diarylamino group, or a halogen group; the core is HC"C8, a C2_C8 dilute group, a CrC8 alkynyl group, a C3_C8 cycloalkyl group, a C3_C8 county % alkyl group, an aryl group, or a heteroaryl group; 11 each is 1, 2, or 3. A subset of the above carbazole compounds is characterized in that each of the eight and eight rings is a heteroaryl group containing one or two nitrogen atoms. In these compounds, n is each i R7 is deuterium; and X and γ are each CH or N. The term "alkyl" refers to a saturated straight or branched hydrocarbon group (m〇iety), such as 1260320 CH3, -CH2-, or a branched chain c3h7. "A straight or branched non-aromatic hydrocarbon ""m has at least one double bond -CH=CH_. "Alkynyl" - the term refers to a soil mass (10) kiss), such as -ch=ch2 or a fragrant hydrocarbon group, such as, for example, a cyclic hydrocarbon group such as a chain = square, such as a cyclohexyl group. ", 70 soil" is saturated with a ring of hetero atoms. The group of 4 (tetra)yl" means having at least a singular group such as 4-tetrahydropiperidinyl. "The aryl term refers to a hydrocarbon group having one or two aromatic rings." Estimated ^ 1 ^ ^ ^ _ The group of a group of a group includes a phenyl group, a stretching group, a naphthyl group, a naphthyl group, a ketone group, and a fluorene group. The term "base" refers to a group having one or more squares containing an atom of a ^^ 1U atom. Examples of heteroaryl t groups include furyl, furfuryl, thiol, pyrrolyl, thienyl, oxazolyl, imidazolyl, succinyl, pyridyl, pyrimidinyl, quinazolinyl, quinolyl , isoquinolinyl, and _ group. The term "oxy" refers to a straight or branched, saturated or unsaturated, non-aromatic hydrocarbon group containing an oxy group such as _ocza or -〇TM = c2h5. The term "decyloxy" refers to a group having at least one aromatic ring and an oxy group bonded to the aromatic ring. The term "heteroaryloxy" refers to a group having at least one aromatic ring containing at least one ring hetero atom and an oxy group bonded to the aromatic ring, such as a 4-pyridine group. Alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxy, aryloxy, heteroaryloxy, alkylamino, dioxane as described herein The amine group, the arylamine group, and the diarylamine group include a substituted or unsubstituted group. Possible substituents on cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aryloxy, heteroaryloxy, arylamino, and monoarylamine groups include alkyl, c2-c1() Dilute, C2-C1()alkynyl, C3-C8 cycloalkyl, [5-(38 ring dilute, Cj-CjQ alkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, Amine, Ci-CjQ acrylamine, Ci-Czo diamined amine, arylamino, diarylamine, thiol, thio, thio-ci-ci thiol, arylthio, Cl- CiQ's sequel, aryl, cyano, nitro, aryl, oxy, sulphonyl, and hydrazine. On the other hand, in the base, dilute, alkynyl, burnt 8 1260320 oxy, decylamine And the possible substituents on the amine group of the base and the second compound include all the above substituents except the group, c2-C|0#s, C2_C|0. The ring group and the heterocycloalkyl group may also be an aryl group. Or a heteroaryl fused. The above-mentioned sulfonium compound of the soil β includes the compound itself as well as a salt thereof and a prodrug thereof (if applicable), for example, a positively charged group on the anion and the carbazole compound (for example). Forming a salt between ammonium ions). Suitable anion pack Gas ion, desert ion, moth ion, sulfate ion, sulfite ion, peroxyacid ion, hexafluorophosphate ion, nitrate ion, phosphate ion, citrate ion, formazin ion, tri-acetic acid Root ion and acetate ion. The same cation can form a salt between the cation and a negatively charged group (such as a carboxyl group) on the carbazole compound. Suitable cations include sodium ion, potassium ion, magnesium ion, calcium ion, and An ammonium cation such as a tetra-ammonium ion. Examples of the prodrug include those which are capable of providing an active carbazole compound when administered (4) and other pharmaceuticals acceptable;: In another aspect, the invention features a human end A method for stabilizing the G. quadruplex structure of granules or other mammalian telomeres. The method comprises contacting the telomere blood with the same chemical formula of the same salivary compound. This method can be used to treat any end of the telomerase activity. Granzyme-associated diseases. In addition, this method can be used for physical testing (for example, to identify G-tetramers of telomeres) or for in vivo animal models: screening or screening The above carbazole compound serves as a therapeutic agent for the treatment of telomerase-related diseases (for example, § ). The term "treatment" as used herein refers to the purpose of conferring therapeutic effects such as healing, alleviating, changing, influencing, and improving. Or preventing telomerase j disease, its symptoms, or its physique, one or more patients with telomerase-related diseases, symptoms of the disease, or physiques that favor these diseases. The administration of the carbazole compound. The "effective amount" described in the article, _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ a pharmaceutical composition comprising the above-mentioned sacrificial compound and a carrier acceptable for the above-mentioned. - a composition comprising the above-mentioned saliva compound containing - or more (d) for treating a telomere-aged disease, and the composition for the manufacture of the above-mentioned treatment The way of medicine is also used in the invention. The following sigma describes one or more specific embodiments of the invention. Other features, objects, and advantages of the invention will be apparent from the description and appended claims. [Embodiment] The following shows an oxazole compound of the present invention as exemplified,
10 126032010 1260320
揭厂、上述咔唑化合物可藉由技藝中習知之方法以及本文中所 '不之合成路徑製備。例如，可使3,6_二溴咔唑與含有具有至 個氣原子之雜芳基之烯烴在鈀觸媒之存在下反應，以製得 1物3,6-武（雜芳基-乙稀基）_昨嗤。然後以甲基蛾處理該中間 勿乂製得對應之峨鹽。另一實例中，使3，6-二胺口卡峻與含有具 有至少一個氮原子之雜芳基之甲醛反應，以製得中間物3,6-貳 (τ隹芳基-亞甲基_亞胺基）_昨嗤。類似的，亦可以甲基峨處理此 中間物而轉化成對應之碘鹽。 下列所示為上述化合物丨至8之合成示意圖。The above-mentioned carbazole compound can be prepared by a method known in the art and a synthetic route as described herein. For example, 3,6-dibromocarbazole can be reacted with an olefin having a heteroaryl group having up to a gas atom in the presence of a palladium catalyst to produce a compound 3,6-wu (heteroaryl-B Thin base) _ Yesterday. The intermediate bismuth salt is then prepared by treating the intermediate with a methyl moth. In another example, a 3,6-diamine group is reacted with formaldehyde containing a heteroaryl group having at least one nitrogen atom to produce an intermediate 3,6-fluorene (τ隹 aryl-methylene group). Imino)) Yesterday. Similarly, the intermediate can be treated with methyl hydrazine to convert to the corresponding iodide salt. The following is a schematic diagram showing the synthesis of the above compounds 丨 to 8.
RR
R = Br 或 NH；> VR•或、R·R = Br or NH;> VR• or R·
化合物1, 3, 5,及7: R·=吡啶2-基，_定4-基，或 吡哄-2-基； X = CH 或 N;及 Y = CH 或 N. 化合物2, 4, 6,及8: R" = 1-甲基雌-2-基， 1-甲基批錠冬基，或 4-甲基u比啡正離予：2-基； X = CH 或 N;及 Y = CH 或 N. 化合物1至8之合成分別詳述於實施例1至8。製備其他 D卡4 ^合物時，可將上述示意圖中所示之吡啶基或吡畊基以另 11 1260320 一含有至少一 原子之雜芳基（例如D比略基、味σ坐基、喊σ定基、 或吲哚基）置換。 上述合成路徑中所使用之化學藥品可包括例如溶劑、試 劑、觸媒、保護基劑、及去保護基劑。上述之方法可在本文中 所特定敘述之步驟之前或者之後包括額外之步驟，以附加或移 除適合之保護基，以便最終能合成得到咔唑化合物。此外，可 以另外之次序或順序進行各種合成步驟以獲得所欲之咔唑化 合物。可使用於合成可應用之咔唑化合物之合成化學之變化及 保護基之方法（保護及去保護）係技藝中已知，及包括例如彼等 在 R. Larock 戶斤著之 Comprehensive Organic Transformations， VCH Publishers 出版（西元 1989 年）；T.W. Greene 與 P.G.M. Wuts 所著之 Protective Groups in Organic Synthesis ^ % 2 ^ JohnCompounds 1, 3, 5, and 7: R·=pyridine 2-yl, _4-yl, or pyridin-2-yl; X = CH or N; and Y = CH or N. Compound 2, 4, 6, and 8: R" = 1-methylest-2-yl, 1-methylindolyl winter base, or 4-methylu than morphine is given: 2-yl; X = CH or N; Y = CH or N. The synthesis of compounds 1 to 8 is detailed in Examples 1 to 8, respectively. When preparing other D-card 4 conjugates, the pyridyl group or pyridinyl group shown in the above schematic diagram may be further substituted with at least one atom of a heteroaryl group (for example, D is slightly base, taste σ sits on the base, shouting σ定基,或吲哚基) substitution. The chemicals used in the above synthetic route may include, for example, a solvent, a reagent, a catalyst, a protective base, and a deprotecting base. The above methods may include additional steps before or after the steps specifically recited herein to add or remove suitable protecting groups to ultimately synthesize the carbazole compound. In addition, various synthetic steps can be carried out in another order or order to obtain the desired carbazole compound. Methods (protection and deprotection) that can be used to synthesize synthetic chemical changes and protecting groups of applicable oxazole compounds are known in the art, and include, for example, those of R. Larock, Comprehensive Organic Transformations, VCH Publishers Publishing (AD 1989); TW Greene and PGM Wuts by Protective Groups in Organic Synthesis ^ % 2 ^ John
Wiley and Sons 出版（西元 1991 年）；L. Fieser and M. Fieser 所 著之 Fieser and Fieserfs Reagents for Organic Synthesis，John Wiley and Sons 出版（西元 1994 年）；及 L. Paquette 編輯之 Encyclopedia of Reagents for Organic Synthesis，John Wiley and Sons出版（西元1995年）及其後續版本中所述者。 如此合成所得之昨σ坐化合物可進一步以例如管柱層析 法、南壓液相層析法、或再結晶之方法純化。 注意到咔唑化合物含有至少二個雙鍵，可進一步含有一或 多個不對稱中心。因此，其可產生消旋物及消旋混合物、單一 鏡像體、個別非鏡像異構物、非鏡像異構物混合物、及順-或 反-或Ε-或Ζ-雙鍵異構物形式。全部之此種異構物形式均為本 發明所涵括者。 一種含有有效量之至少一種上述咔唑化合物及醫藥上可 接受之載體之醫藥組合物亦在本發明之範疇。再者，本發明涵 蓋一種使人類端粒或其他哺乳動物端粒之G-四股結構體穩定 12 1260320 之方法。此方法可藉由斟g ^ 一 糟甶對患者予以有效量之一或多種咔唑化合 物“才又藥而用以/台療具有端粒酉每相關疾病之患者。此種 可错^建康醫護專家基於任何適當診斷方法之結果鑑別出;。 §使用本發明之方、、土、Λ + 方法>〇療具有端粒酶相關疾病之电 日寺，可藉由技藝中孰知夕士、、n 〜考 …、 方法決定有效劑量。例如，動物盘人 類之劑量（以毫克/平方 / 於F⑽eiCh等人所著之（西^身/表面積為準）相互關係見述 219。身體表面浐可士凡9队年、CancerChemotherRep5Q 表面積可由病患之身高及體重大 Scientific Tables Gpi^ dl / 阅例如 ， 奶卿⑽价A，Ardley出版，紐約， 西兀1970年，第537百 吐， 、'Ί η , / 、。卡唑化合物之有效量範圍可在約 0.1mg/Kg 至約 i00m /Kg 夕 ^ ,,... g g之間。有效劑量則有變化，如孰習此 …社 例如要治療之疾病型態、投藥路徑、賦带 劑之使用、及與其他治療❹之可純而定。 料 可以非經腸、口服、鼻腔、直 有一或多鍤卜、儿人， 皮饜或頰之方式將具 rd?义 物之組合物予以投藥以治療端粒酶相關 疾病。本文中所使用之「非經腸」一詞指皮 =關 肌肉内、關節内、動脈内、滑液 =内、 技術。 》頭-月内注射’以及任何適合之注入 滅囷可庄射之組合物可為纟無毒性 | 釋劑或溶劑中之溶液或懸浮液，例如在 可#用夕可to 5 ’丁 一醇中之溶液。 …广 文之載劑及溶射舉例為甘露糖醇、水、R咖， 浴液、及等張氣化納溶液。此外，固定子尺Rln⑽s 溶劑或懸浮介質（例如合成之甘油單或二酸‘ =習用之 油酸及其甘油酸酯衍生物 曰肪齩，例如 醫華上可接為夕、山 衣備血官庄射劑，為天铁 -条7接又之油，例如撖慢油或薇麻油 天…、 化之形式。此等油溶液或懸浮 匕…^乙基 月匕3有長鏈醇稀釋劑或分散 13 1260320 劑，或缓甲基纖維素或類似之分散劑。亦可使用其他 :界面活性劑例如Tweens或Spans或其他類似乳化 力，，其-般使用於製造醫藥上可接受 之口體液肚、或其他劑量形式，供配方目的之用。 口服投樂之組合物可為任何口服上可接受之劑量，勺 括膠囊、錠、乳劑、及水性懸浮劑、分散劑、及溶液= 劑，-般使用之載體包括乳糖及玉米興粉。潤滑劑例如硬脂酸 鎂亦為典型上所添加者。對於膠囊形式之口服投藥，可使用之 =包燥玉米殿粉。當以水性懸浮劑或乳劑 又'才α ^生成伤懸序或溶解於與乳化劑或懸浮劑組合一 二之油相中。若需要，可添加特定之增甜劑、風味劑、或著色 可«醫藥配方技#中熟知之技術製備鼻腔氣溶膠或吸 亡組成物。例如，可將此種組合物製備為鹽水中之溶液，使用 本甲％或其他適合之保存劑、增強生物可用性之吸附促進劑、 氟碳化物’及/或其他技藝中已知之溶解或分散劑。亦可以检 劑或直腸投藥之形式將具有一或多種上述活性化合物之έ且合 物予以投藥。 ^ 慣常將醫藥上可接受之載體與一或多種上述活性化合物 -起使用。醫藥組合物中之載體必須為「可接受」，意即，其 與組合物之活性成份可相容（及較佳能夠使活性成份穩定）及不 曰對接文治療之患者有害。可使用一或多種溶解劑做為醫藥賦 形劑以傳遞上述化合物。其他載體之實例包括膠態二氧化石夕、 硬脂酸鎮、纖維素、硫酸月桂㈣、及D&C黃色第Κ)號（D&C Yellow #1〇) 〇 可藉由體外試驗（參閱下述實施例9)預先篩選本發明之咔 唑化合物於穩定G-四股結構體之效力。熟習此技藝者亦知其他 14 1260320 方法。 下列特定實施例僅做為說明，並不以任何方式限制本發明 其他揭示之部分。相信熟習此技藝者基於本文之敘述能夠使用 本發明至最完全之程度。本文中所引述之出版物均完整併入本 文以做參考。 【實施例】 實施例1 :化合物1之製備· 3,6-貳-(2-吡啶-4-基-乙烯基）_9H_ 昨嗤 依照下述步驟製備化合物1 : 將3,6-二溴咔唑（1.63克，5毫莫耳，Aidnch公司製造）添 加至含有乙酸鈀（11)(15毫克，Strem公司製造）及三_鄰曱苯膦 (td-o-tolyl phosphme) (150 毫克，Aldrich 公司製造）之混合物 之高壓燒瓶中。在此燒瓶中添加混合溶液（三乙胺5mL/乙腈 15mL)及4-乙烯基吡啶（2克，20亳莫耳，Merck公司製造）。將 燒瓶吹氮氣ίο分鐘後密封。保持在約1〇5〇c下反應二天，收 集沉澱，及以H2〇/CH2C12洗滌二次。過濾所得之不ϋ之固體 及溶解於THF，然後以無水MgS〇4乾燥。自thf淚液再结晶 後過濾，獲得化合物1為黃色粉末（雇旦· ⑺木（產里· 62 %，熔點 (mp)>300°C) 〇 lHNMR(CD3〇D):58_42(d，h5.7Hz，4H)，8.25(s,2H)， 7.65 (d，J = 8.4 Ηζ，2Η), 7·56 (d，h 16 2 Hz, 2h)，7 45 (d / = 5.7 Hz, 4H), 7.42 (d, J = 8.4 Hz, 2H), 7 〇7 rn r h /.υ/ (d，j 二 ΐ6·2 Hz，2H)。 元素分析（ΕΑ)(373 + 1·5Η2〇):計算佶❻目兹付〇/、 |斤值（靦祭值％) C : 83.64 (78_20)，Η : 5.09 (5·14)，Ν ·· 11.26 (ίο.38)。 實施例2:化合物2之製備：3,6|(2_(1_甲基吼錠_4_基)_乙稀 基）-9Η-咔唑二碘化物 依照下述步驟製備化合物2 : 15 1260320 在將實施例1所獲得之^ ' ，-一（4-乙稀基D比咬）昨σ坐斑 CH3I於丙酮中迴流後，自甲 二 ^與過里 2，為橘紅色粉末（產率：92。/， 後侍化a物 Z /〇，mp>300oC) 〇 !H NMR (DMSO-d6)： 5 8 77 ^ 〇 S.77 (d，j = 6 9 H 4h 2H), 8.19 (d5J= 6.9 HZ?4H) 8 20 ^ ⑴，&20 (d，15 9 Hz，2H)，7 J = 8.7 Hz? 2H)? 7.64 (d5 J = g 7 R ’ 2H)。 8·7Ηζ, 2H), 7.53(d,J^15.9Hz, EA (65 7+1·0Η2Ο):計瞀佶 r驻曰 _ 4 值（蜆察值 ％) c : 51.14 (49 87)， Η : 3·81 (4·03)，N : 6_39 (6·32)。 實施例3 :化合物3之製備：λ 6十 口卡唾 衣彳有3,6遣-(2-吼啶-2-基-乙烯基）_9Η_ 一以類似於實施例1所述之步驟製備化合物3。 貫施例4 :化合物4之製備：3 才μ 〇u ^ ，6•'貳-(2-(1-曱基吡錠_2_基）_乙稀 基）-9Η-咔唑二碘化物 〜以類似於實施例2所述之步驟製備化合物4。 貫施例5 :化合物5之製備· 口卡。坐 · 3,6-武-(2-吡畊-2-基-乙烯基）-9Η· 以類似於實施例i所述之步驟製備化合物5。 只2』6 .化合物6之製備：3,6·武-(2-(4-甲基Π比哄-4-正離子 _2_暴）-乙烯基）-9H-咔唑二碘化物 以類似於實施例2所述之步驟製備化合物6。 昨嗤^ 7 ·化合物7之衣備· N，NK吼咬-4-基亞甲基）-9H- 二胺 以類似於實施例i所述之步驟製備化合物7。 某）8 ·化合物8之製備：NH((1-甲基口比錠-4-基）亞甲 土 ^ $咔唑-3,6-二胺二碘化物 以类員似於實施例2所述之步驟製備化合物8。 16 1260320 實施例9:昨嗤化合物與DNA二股結構體（duplexes)及四股結 構體之間之結合 自Applied Biosystems公司購得小牛胸腺（ct-DNA)及寡核 符酸（oligonucleotides) AT、LD、GC、G10、LQ1、LQ2、LQ4、Published by Wiley and Sons (Ever. 1991); Fieser and Fieserfs Reagents for Organic Synthesis by L. Fieser and M. Fieser, published by John Wiley and Sons (1994); and Encyclopedia of Reagents for Organic by L. Paquette Synthesis, published by John Wiley and Sons (Eng. 1995) and subsequent editions. The sigma compound thus synthesized can be further purified by, for example, column chromatography, Southern pressure liquid chromatography, or recrystallization. It is noted that the carbazole compound contains at least two double bonds and may further contain one or more asymmetric centers. Thus, it can produce racemates and racemic mixtures, single mirror images, individual non-image isomers, non-imagewise isomer mixtures, and cis- or trans- or Ε- or Ζ-double bond isomer forms. All such isomeric forms are encompassed by the present invention. A pharmaceutical composition comprising an effective amount of at least one of the above carbazole compounds and a pharmaceutically acceptable carrier is also within the scope of the invention. Furthermore, the present invention encompasses a method of stabilizing the G-quadruplex of human telomeres or other mammalian telomeres 12 1260320. This method can be used to administer one or more carbazole compounds in an effective amount by 斟g ^ 甶 “ 才 才 才 才 才 台 台 台 台 台 台 台 台 台 具有 具有 具有 具有 建 建 建 建 建 建 建 建The health care professional identifies it based on the results of any appropriate diagnostic method; § using the formula, soil, sputum + method of the present invention> treatment of a telomerase-related disease of the electric eclipse, which can be learned by the skill , n ~ test ..., the method determines the effective dose. For example, the human dish dose (in mg / square / F (10) eiCh et al. (West body / surface area) relationship 219. Body surface 浐Ke Shifan 9 team year, CancerChemotherRep5Q surface area can be the height and body size of the patient Scientific Tables Gpi ^ dl / read for example, Dai Qing (10) price A, published by Ardley, New York, West 1970, the 537th spit,, 'Ί The effective amount of the η, /, and the carbazole compound may range from about 0.1 mg/Kg to about i00 m /Kg ^^, ... gg. The effective dose may vary, such as 孰 此 ...... Disease type, administration route, use of the agent And other treatments may be pure. It may be administered parenterally, orally, nasally, directly or in one or more babies, children, skin or buccal to treat the composition with rd? Telomerase-related diseases. The term "parenteral" as used herein refers to skin = intramuscular, intra-articular, intra-arterial, synovial fluid = internal, technique. "head-intra-month injection" and any suitable injection The composition of the cockroach can be a solution or suspension in a non-toxic | release or solvent, for example, a solution which can be used in 5 'butanol. Examples are mannitol, water, R coffee, bath, and isotonic sodium solution. In addition, the immobilizer Rln (10)s solvent or suspension medium (such as synthetic glycerol mono or diacids = = conventional oleic acid and its glycerol The acid ester derivative is a fat sputum. For example, the medicinal medicinal product can be used as a stagnation, and the mountain smear is prepared by the blood-supplied squid, which is the oil of the Tiantie-Article 7 and the oil, such as the simmering oil or the sesame oil. These oil solutions or suspensions ^...^ Ethyl hydrazine 3 has a long-chain alcohol diluent or dispersion 13 1260 320 agents, or slow methylcellulose or similar dispersing agents. Others: surfactants such as Tweens or Spans or other similar emulsifying powers, which are generally used in the manufacture of pharmaceutically acceptable oral fluids, or Other dosage forms for formulation purposes. The oral dosage composition can be any orally acceptable dosage, including capsules, ingots, emulsions, and aqueous suspensions, dispersing agents, and solutions. The carrier includes lactose and corn powder. Lubricants such as magnesium stearate are also typically added. For oral administration in capsule form, it can be used as a dry corn powder. When an aqueous suspension or emulsion is used, it can be in the form of a suspension or dissolved in an oil phase in combination with an emulsifier or suspending agent. If desired, a specific aerosol or flavor composition can be prepared by adding a specific sweetener, flavor, or coloring technique well known in the art. For example, such compositions can be prepared as solutions in saline, using % or other suitable preservatives, adsorption enhancers that enhance bioavailability, fluorocarbons' and/or other dissolution or dispersing agents known in the art. . It is also possible to administer a pharmaceutically acceptable compound having one or more of the above active compounds in the form of a test or a rectal administration. ^ It is customary to use a pharmaceutically acceptable carrier with one or more of the above active compounds. The carrier in the pharmaceutical composition must be "acceptable", i.e., it is compatible with the active ingredients of the composition (and preferably is capable of stabilizing the active ingredient) and is not deleterious to the patient treated by the docking. One or more solubilizing agents may be used as a pharmaceutical excipient to deliver the above compounds. Examples of other carriers include colloidal silica dioxide, stearic acid town, cellulose, lauric acid sulfate (IV), and D&C yellow Κ) (D&C Yellow #1〇) 〇 can be tested by in vitro (see The following Example 9) pre-screens the effectiveness of the carbazole compound of the present invention in stabilizing the G-quad structure. Those skilled in the art are also aware of other 14 1260320 methods. The following specific examples are merely illustrative and are not intended to limit the scope of the invention in any way. It is believed that those skilled in the art will be able to use the invention to the fullest extent. The publications cited herein are hereby incorporated by reference in their entirety. EXAMPLES Example 1: Preparation of Compound 1 · 3,6-fluorenyl-(2-pyridin-4-yl-vinyl)_9H_ Previously, Compound 1 was prepared according to the following procedure: 3,6-dibromoindole Oxazole (1.63 g, 5 mmol, manufactured by Aidnch) was added to contain palladium acetate (11) (15 mg, manufactured by Strem) and td-o-tolyl phosphme (150 mg, Aldrich) In a high pressure flask of a mixture made by the company. A mixed solution (triethylamine 5 mL / acetonitrile 15 mL) and 4-vinylpyridine (2 g, 20 Torr, manufactured by Merck) were added to the flask. The flask was purged with nitrogen for ί. The reaction was maintained at about 1 〇 5 〇 c for two days, and the precipitate was collected and washed twice with H 2 〇/CH 2 C 12 . The resulting solid was filtered and dissolved in THF, then dried over anhydrous MgS. After recrystallization from thf tears, it was filtered to obtain Compound 1 as a yellow powder (Employed (7) wood (Cory 62%, melting point (mp) > 300 ° C) 〇lHNMR (CD3〇D): 58_42 (d, h5) .7Hz, 4H), 8.25(s, 2H), 7.65 (d, J = 8.4 Ηζ, 2Η), 7·56 (d, h 16 2 Hz, 2h), 7 45 (d / = 5.7 Hz, 4H) , 7.42 (d, J = 8.4 Hz, 2H), 7 〇7 rn rh /.υ/ (d,j ΐ6·2 Hz, 2H) Elemental analysis (ΕΑ)(373 + 1·5Η2〇): Calculation佶❻目兹付〇/, | 斤值(腼腼值%) C : 83.64 (78_20), Η : 5.09 (5·14), Ν ·· 11.26 (ίο.38). Example 2: Compound 2 Preparation: 3,6|(2_(1_methyloxime_4_yl)-ethlyl)-9Η-carbazole diiodide Compound 2 was prepared according to the following procedure: 15 1260320 obtained in Example 1 ^ ', -1 (4-Ethyl D to bite) Yesterday σ sitting spot CH3I in acetone reflux, from A 2 ^ and over 2, for orange-red powder (yield: 92. /, Hou Shi A substance Z / 〇, mp > 300oC) 〇!H NMR (DMSO-d6): 5 8 77 ^ 〇S.77 (d,j = 6 9 H 4h 2H), 8.19 (d5J= 6.9 HZ?4H) 8 20 ^ (1), & 20 (d, 15 9 Hz, 2H), 7 J = 8.7 Hz? 2H )? 7.64 (d5 J = g 7 R ' 2H). 8·7Ηζ, 2H), 7.53(d, J^15.9Hz, EA (65 7+1·0Η2Ο): Calculate r 曰 曰 4 value (visual value %) c : 51.14 (49 87), Η : 3·81 (4·03), N: 6_39 (6·32). Example 3: Preparation of compound 3: λ 6 ten-port card sputum has 3,6--- 2-acridin-2- -vinyl)_9Η_ A compound 3 was prepared in a similar manner to that described in Example 1. Example 4: Preparation of Compound 4: 3 μ μ 〇 u ^ , 6•'贰-(2-(1-曱) Pyridinium-2-yl)-ethidyl)-9-oxazole diiodide~ Compound 4 was prepared in a similar manner to that described in Example 2. Example 5: Preparation of Compound 5 · Port. · 3,6-Wu-(2-pyroxy-2-yl-vinyl)-9Η· Preparation of compound 5 in a similar manner to that described in Example i. Only 2:6. Preparation of compound 6: 3,6 Wu-(2-(4-methylindole-4-indol-2-d)-vinyl)-9H-carbazole diiodide Compound 6 was prepared in a similar manner to that described in Example 2. Yesterday 77 7 · Preparation of Compound 7 · N, NK 吼 4-ylmethylene)-9H-diamine Compound 7 was prepared in a similar manner to that described in Example i. a) 8 · Preparation of compound 8: NH ((1-methylpyranium-4-yl) methylene terephthalate ^ carbazole-3,6-diamine diiodide as a member of Example 2 The procedure described is to prepare compound 8. 16 1260320 Example 9: Binding between compound and DNA duplexes and tetraplexes from the Biosystems Corporation. The calf thymus (ct-DNA) and oligos were purchased from Applied Biosystems. Acid (oligonucleotides) AT, LD, GC, G10, LQ1, LQ2, LQ4,
Tetl2、Apt、〇xyl2、〇xy28、Huml2 苻酸之序列如下： 、及Hum24。 此等 AT: 5，-(AT)6-3’ (SEQ ID NO:l) LD: 5，-GCGCA2T2GCGC-3’ (SEQ ID NO:2) GC: 5，-(GC)6-3’ (SEQ ID NO:3) G10: 5、d(G)i〇-3’ (SEQ ID NO:4) LQ1: 5，-TG4T-3’ (SEQ ID NO:5) LQ2: 5，-T2G4T2-3’ (SEQ ID NO:6) LQ4: 5，-T4G4-3， (SEQ ID NO:7) Tetl2: 5，-(T2G4)2-3’ (SEQ ID NO:8) Apt: 5，-G2T2G2TGTG2T2G2-3’ (SEQ ID NO:9) Oxy 12: 5，-G4T4G4-3’ (SEQ ID NO:10) Oxy28: 5，-G4(T4G4)3-3， (SEQ ID NO:ll) Huml 2: 5，-(T2AG3)2-3’ (SEQ ID NO:12) Hum24: 5,-(T2AG3)4-3’ (SEQ ID NO:13) 其中 ，ct-DNA及寡核苷酸AT、 LD、及GC可形成 股 結構體（duplexes)，而寡核苷酸 G10、LQ卜 LQ2、LQ4、Tetl2、 Apt、Oxy 12、Oxy28、Huml 2、及 Hum24 可形成四股結構體。 例如，Apt能夠形成極安定之单分子四股結構體’具有二個G-四重體，由一端之一個側邊TGT環（lateral TGT loop)與另一端 之二個平行TT環（parallel TT loop)所連接。Hum24能夠形成單 分子四股結構體，具有在G-四重體一端之一個對角T2A環 (diagonal T2A-loop)及在G-四重體另一端之二個平行T2A環 17 1260320 (parallel T2A loop)。Tetl2 及 Huml2 能夠形成具有側邊環（lateral loops)之二聚體（dimeric)之髮夹四股結構體（hairpin quadruplex)。Oxy 1 2能夠形成二聚體髮夾四股結構體，其在四 重體各端具有對角環（diagonal loop)。 咔唑化合物及其與DNA二股結構體及四股結構體之錯合物之 吸收、螢光、及圓形二色性光譜（circu丨ardichroism，CD)分 析 吸收光譜分析 將上述各寡核苻酸與10 mM Tris-HCl (pH 7.5)及150 mM NaCl之溶液混合，及在90QC下去活性（denature)達2分鐘。然 後使混合物緩慢冷卻至室溫，及使用前貯存於4QC下超過2 天。然後將受測化合物添加至此溶液中以形成化合物/DNA錯 合物。使用Hitachi U3200 UV-可見光光譜儀對所得之錯合物進 行吸收分析。 記錄化合物2及其與各核苷酸之錯合物之吸收光譜圖。結 果顯示，在DNA二股結構體之存在下，化合物2之吸收峰自 約435nm紅位移至約450nm，及在DNA四股結構體之存在下， 更紅位移至約460nm。此外，莫耳吸收係數在DNA二股結構 體之存在下減少約15%，在DNA四股結構體之存在下，減少 約35%。此等光譜之改變顯示化合物2與二股結構體及四股結 構體結合。 螢先分析 將化合物2與十一個DNA混合，及在λ£Χ与430nm下，在 Ι-cm樣品測試槽中進行螢光分析，使用Hitachi F40 10螢光光 譜儀，2nm之頻寬。亦對化合物4或6在Hum24存在下進行螢 光分析。結果顯示各化合物2、4及6之螢光微弱。然而，螢 光強度意想不到的，在DN A二股結構體存在時，增加一個數 18 1260320 量級（order of magnitude)，及在DNA四股結構體存在時，增加 兩個數量級。進一步，化合物2與DNA二股結構體之錯合物 及化合物2與DNA四股結構體之錯合物之螢光波峰分別在約 5 50nm 及約 5 75nm 〇 此等結果使人聯想到可使用化合物2、4、及6以區別DN A 二股結構體與DNA四股結構體◦此外，此等化合物在與DNA 四股結構體結合時擁有增強之螢光，及因此，尚可做為電泳時 對於DNA四股結構體之生物標記。 CD分析 對8個DNA四股結構體及其與化合物2之錯合物進行CD 分析。在具有 2nm 頻寬之 Jasco J-715 偏光儀（spectropolarimeter) 上掃描平均10次，獲得CD光譜。掃描速度為50nm/分鐘及步 進解析度（step resolution)為 0.2nm。 在化合物2與DNA四股結構體結合之前後，所偵測到之 DNA四股結構體CD光譜並無可察覺到之改變，使人聯想到 DNA四股結構體並不會因與化合物2結合而扭曲。CD光譜亦 確認 Hum 12、Hum24、〇xyl2、Oxy28、Tetl2、及 Apt 中絕大 多數為逆向（antiparallel)四股結構體。 進一步，對於Hum24及其與化合物2、4、6、及8之錯合 物進行溫度相依性之CD分析。結果顯示Hum24四股結構體之 熔化溫度在四個受測化合物之各化合物存在時均會增加，顯示 改良之熱穩定性。 聚丙烯醯胺凝膠電泳（PAGE) 進一步，使用 PAGE 在 10 mM Tris-HCl 及 150 mM NaCl 之20%天然凝膠（pH 7.5)中分析上述化合物/DNA錯合物。在100 V/cm之電場下，在4QC下進行凝膠電泳達15小時。以260nm 處之吸收度決定DNA濃度，並將DNA濃度調整至每單位結構 1260320 約10μΜ。在以UV投射陰影（shadowing)照相後，將凝膠於一 含有10 μΜ之受測化合物、10 mM Tris-HCl、及150 mM NaCl 之溶液（pH 7.5)中於室溫下進行後染色（post-stained) 10分鐘。 然後以蒸餾水沖洗凝膠及在254nm UV光下使用數位相機照 相。 在完成 AT、LD、GC、G10、LQ1、LQ2、LQ4、Tetl2、 〇xy!2、Huml2、Apt、〇xy28、及Hum24之電泳後，以化合物 2將凝膠後染色。結果顯示大部分DNA在254nm UV光下呈現 營光帶（bands)。特言之，大部分DNA在凝膠中以單一帶之形 式遷移，但是Tetl2及LQ4以二條帶的形式遷移。 赢 使用預染色之凝膠供選擇性試驗。將0.1 μΜ受測化合物溶 液一開始以10 μΜ之不同DNA溶液培養10分鐘。然後在100 V/cm之電場中在4°C下進行凝膠電泳達6小時。使用Bio-Rad 衫像價測器（imaging detector)在254 nm UV光下將全部預染色 之凝膠照相。 在對 AT、LD、GC、G10、LQ卜 LQ2、LQ4、Tetl2、0xyl2、 Hum!2、Apt、〇xy28、及Hum24進行電泳之前使用以化合物2 預染色之凝膠。結果顯示LQ2、LQ4、〇xyl2、〇xy28、及Hum24 之四股結構體在254 nm UV光下呈現螢光帶。進一步，Tetl2_ 在預染色凝膠電泳中僅呈現第三螢光帶，與後染色凝膠電泳中 觀察到之二條帶形式不同。此第三螢光帶係因為Tetl2為線形 四聚合體。另一方面，在化合物2與LD、AT、GC、G10、LQ1、 Huml2、及Apt之四股結構體之錯合物上檢測不到螢光帶。 而 Apt、Tetl2、及 Huml2 不呈 如上述，Hum24、〇xyl2、及Oxy28在其逆向四股結構體 中含有至少一個對角環，及Apt、Tetl2、及Huml2在其逆向四 股結構體中不含對角環。在與化合物2結合實合時，Hum24、 〇xyl2、及〇xy28呈現螢光帶， 20 1260320 見因哽向四股結構體所 物可區別呈有斜自广 螢先f。此觀察使人聯想到化合 ，、有對角％之逆向四股結構體 向四股結構體。 ”个，、有對角％之迚 此外山’勞光係因化合物2與LQ2(含有Τ2尾端）及lq4(含 有τ尾端）及叫(不含τ尾端^線形四㈣ :二物而呈現。此等結果使人聯想到化合物2亦能夠區別具 有不同長度τ尾端之線形四聚物。 為研究DNA檢測之靈敏度，將含有〇1μΜ之化合物2之 之與含有2.5至〇·〇〇5μΜ之Hum24之溶液一起培養。然後 、、字混合物進行電泳分析。靈敏度試驗顯示Q l_之化合物：溶私 液：檢測Ο.ΟΙμΜ之Hum24四股結構體之存在，顯示化合物2 為-種檢測HUm24之存在之靈敏生物標記。再者，結果亦顯示 0·1μΜ之化合物2溶液能夠檢測〇1μΜ之lq4四股結構體、 〇.1μΜ之0xy28四股結構體、及〇 25_之^12四股結構體 中少量線形四聚物之存在。 其他具體實施例 本說明書中所揭示之全部特徵可以任何組合方式組合。本 說明書中揭示之各特徵可被另一具有相同、均等、或類似目的 之特徵取代。因此，除非另有陳述’否則所揭示之特徵僅為均_ 等或類似特徵之一般系列之實例。 由上述，熟習此技藝者能夠輕易確定本發明之必要特性， 及能夠對本發明予以種種改變與修飾，以合於種種使用及狀 况，而不背離其精神及範疇。因此，其他具體實施例亦在下列 申請專利範圍之範®壽内。 21 1260320 【圖式簡單說明】 【符號說明】 無。The sequences of Tetl2, Apt, 〇xyl2, 〇xy28, Huml2 citric acid are as follows: , and Hum24. These AT: 5,-(AT)6-3' (SEQ ID NO:l) LD: 5,-GCGCA2T2GCGC-3' (SEQ ID NO: 2) GC: 5,-(GC)6-3' ( SEQ ID NO: 3) G10: 5, d(G)i〇-3' (SEQ ID NO: 4) LQ1: 5, -TG4T-3' (SEQ ID NO: 5) LQ2: 5, -T2G4T2-3 '(SEQ ID NO:6) LQ4: 5,-T4G4-3, (SEQ ID NO:7) Tetl2: 5,-(T2G4)2-3' (SEQ ID NO:8) Apt: 5,-G2T2G2TGTG2T2G2- 3' (SEQ ID NO: 9) Oxy 12: 5, -G4T4G4-3' (SEQ ID NO: 10) Oxy28: 5, -G4(T4G4)3-3, (SEQ ID NO: ll) Huml 2: 5 ,-(T2AG3)2-3' (SEQ ID NO: 12) Hum24: 5,-(T2AG3)4-3' (SEQ ID NO: 13) wherein ct-DNA and oligonucleotides AT, LD, and GC can form duplexes, while oligonucleotides G10, LQ, LQ2, LQ4, Tetl2, Apt, Oxy 12, Oxy28, Huml 2, and Hum24 can form a four-strand structure. For example, Apt is capable of forming a very stable single-molecule four-strand structure with two G-quadruplexes, one lateral TGT loop at one end and two parallel TT loops at the other end (parallel TT loop) Connected. Hum24 is capable of forming a single-molecule four-strand structure with a diagonal T2A-loop at one end of the G-quadruplex and two parallel T2A loops at the other end of the G-quadruplex 17 1260320 (parallel T2A loop ). Tetl2 and Huml2 are capable of forming a hairpin quadruplex having a dimer of lateral loops. Oxy 1 2 is capable of forming a dimeric hairpin four-strand structure having a diagonal loop at each end of the quadruplex. Absorption, fluorescence, and circular dichroism (CD) analysis of oxazole compounds and their complexes with DNA two-stranded structures and four-stranded structures are analyzed by absorption spectroscopy. A solution of 10 mM Tris-HCl (pH 7.5) and 150 mM NaCl was mixed and deactivated at 90QC for 2 minutes. The mixture was then slowly cooled to room temperature and stored at 4QC for more than 2 days prior to use. The test compound is then added to this solution to form a compound/DNA complex. The resulting complex was subjected to absorption analysis using a Hitachi U3200 UV-Vis spectrometer. The absorption spectrum of Compound 2 and its complex with each nucleotide was recorded. The results show that in the presence of the DNA strand structure, the absorption peak of Compound 2 is shifted from about 435 nm to about 450 nm, and in the presence of the DNA quadruplex, it is more red shifted to about 460 nm. In addition, the molar absorption coefficient is reduced by about 15% in the presence of the DNA strand structure and by about 35% in the presence of the DNA strand structure. These spectral changes show that Compound 2 binds to the two-strand structure and the four-strand structure. Fluorescence analysis Compound 2 was mixed with eleven DNAs and subjected to fluorescence analysis in a Ι-cm sample test cell at λ£Χ and 430 nm using a Hitachi F40 10 fluorescence spectrometer with a bandwidth of 2 nm. Fluorescence analysis was also performed on Compound 4 or 6 in the presence of Hum24. The results showed that the fluorescence of each of Compounds 2, 4 and 6 was weak. However, the fluorescence intensity is unexpectedly increased by a number of 18 1260320 orders of magnitude in the presence of the DN A two-strand structure, and by two orders of magnitude in the presence of the DNA four-strand structure. Further, the complex peak of the compound 2 and the DNA double-stranded structure and the complex peak of the compound 2 and the DNA quadruplex are respectively at about 50 nm and about 5 75 nm, and the results are reminiscent of the use of the compound 2 , 4, and 6 to distinguish the DN A two-strand structure from the DNA four-strand structure. In addition, these compounds have enhanced fluorescence when combined with the DNA four-strand structure, and thus, can be used as a DNA quadruplex structure when electrophoresed. Biomarker of the body. CD analysis CD analysis was performed on eight DNA four-stranded structures and their complexes with Compound 2. An average of 10 scans were performed on a Jasco J-715 polarizer with a 2 nm bandwidth to obtain a CD spectrum. The scanning speed was 50 nm/min and the step resolution was 0.2 nm. After the binding of Compound 2 to the DNA four-strand structure, there was no appreciable change in the detected CD spectrum of the four-strand structure of DNA, reminiscent of the fact that the DNA four-strand structure was not distorted by the binding to Compound 2. The CD spectrum also confirmed that most of Hum 12, Hum24, 〇xyl2, Oxy28, Tetl2, and Apt are antiparallel four-strand structures. Further, a CD analysis of temperature dependence of Hum24 and its mismatches with Compounds 2, 4, 6, and 8 was performed. The results show that the melting temperature of the four-membered structure of Hum24 increases in the presence of each compound of the four test compounds, indicating improved thermal stability. Polyacrylamide gel electrophoresis (PAGE) Further, the above compound/DNA complex was analyzed by PAGE in a 20% natural gel (pH 7.5) of 10 mM Tris-HCl and 150 mM NaCl. Gel electrophoresis was carried out at 4QC for 15 hours under an electric field of 100 V/cm. The DNA concentration was determined by the absorbance at 260 nm, and the DNA concentration was adjusted to about 10 μM per unit structure of 1260320. After photographing with UV shadowing, the gel was post-stained at room temperature in a solution containing 10 μM of test compound, 10 mM Tris-HCl, and 150 mM NaCl (pH 7.5). -stained) 10 minutes. The gel was then rinsed with distilled water and photographed with a digital camera under 254 nm UV light. After completion of electrophoresis of AT, LD, GC, G10, LQ1, LQ2, LQ4, Tetl2, 〇xy!2, Huml2, Apt, 〇xy28, and Hum24, the gel was post-stained with Compound 2. The results showed that most of the DNA exhibited camping bands at 254 nm UV light. In particular, most of the DNA migrates in the form of a single band in the gel, but Tetl2 and LQ4 migrate in the form of two bands. Win Use a pre-stained gel for selective testing. A 0.1 μΜ test compound solution was initially incubated with 10 μΜ of the different DNA solution for 10 minutes. Gel electrophoresis was then carried out at 4 ° C for 6 hours in an electric field of 100 V/cm. All pre-stained gels were photographed under 254 nm UV light using a Bio-Rad shirt imaging detector. A gel pre-stained with Compound 2 was used before electrophoresis of AT, LD, GC, G10, LQ, LQ2, LQ4, Tetl2, 0xyl2, Hum!2, Apt, 〇xy28, and Hum24. The results show that the four structures of LQ2, LQ4, 〇xyl2, 〇xy28, and Hum24 exhibit a fluorescent band at 254 nm UV light. Further, Tetl2_ exhibited only a third fluorescent band in the prestaining gel electrophoresis, which was different from the two bands observed in post-staining gel electrophoresis. This third fluorescent band is because Tetl2 is a linear tetramer. On the other hand, no fluorescent band was detected on the complex of the compound 2 and the four structures of LD, AT, GC, G10, LQ1, Huml2, and Apt. While Apt, Tetl2, and Huml2 are not as described above, Hum24, 〇xyl2, and Oxy28 contain at least one diagonal ring in their reverse four-strand structure, and Apt, Tetl2, and Huml2 do not contain a pair in their reverse four-strand structure. Corner ring. When combined with compound 2, Hum24, 〇xyl2, and 〇xy28 exhibit a fluorescent band, and 20 1260320 can be distinguished from the four-strand structure by oblique self-growth. This observation is reminiscent of a compound, with a diagonal of four percent reverse structure to a four-strand structure. ",, there is a diagonal of 迚 迚 迚 ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' These results are reminiscent of the fact that Compound 2 can also distinguish linear tetramers with different lengths of τ. For the purpose of studying the sensitivity of DNA detection, it will contain 〇1μΜ of compound 2 and contain 2.5 to 〇·〇. 〇5μΜ of Hum24 solution was cultured together. Then, the word mixture was subjected to electrophoresis analysis. Sensitivity test showed that Q l_ compound: lysate: detection of the presence of um.ΟΙμΜ Hum24 four-strand structure, showing that compound 2 was detected The sensitive biomarker of the presence of HUm24. Furthermore, the results also show that the compound 1 solution of 0·1μΜ can detect the lq4 four-strand structure of 〇1μΜ, the 0xy28 four-strand structure of 〇.1μΜ, and the four-strand structure of 〇25_之^12 The presence of a small number of linear tetramers. Other embodiments All of the features disclosed in this specification can be combined in any combination. The features disclosed in this specification can be otherwise identical, equal, or similar. The features are superseded. Therefore, unless otherwise stated, the features disclosed are merely examples of the general series of the same or similar features. From the foregoing, those skilled in the art can readily determine the essential characteristics of the present invention and can Various changes and modifications are made to suit various uses and conditions without departing from the spirit and scope thereof. Therefore, other specific embodiments are also within the scope of the following patent application scope. 21 1260320 [Simplified illustration] Explanation of symbols] None.