TWI539965B - Hypoxia tissue contrast agent precursor BANI, its contrast agent and its preparation method - Google Patents
Hypoxia tissue contrast agent precursor BANI, its contrast agent and its preparation method Download PDFInfo
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本發明係關於一種造影劑前驅物，尤指一種包含有機配位子6-(2-硝基咪唑)己基-DL-2,3-雙[((三苯甲基)硫基)乙醯胺基]丙醯胺(6-(2-nitroimidazole)hexyl-DL-2,3-Bis-[((triphenylmethyl)thio)acetamido]propanamide，下稱為BANI)之造影劑前驅物、其造影劑及其製備方法。 The present invention relates to a contrast agent precursor, especially one comprising an organic ligand 6-(2-nitroimidazolyl)hexyl-DL-2,3-bis[((tritylmethyl)thio)acetamide Precursor precursor of 6-(2-nitroimidazole)hexyl-DL-2,3-Bis-[((triphenylmethyl)thio)acetamido]propanamide, hereinafter referred to as BANI), its contrast agent and its contrast agent Preparation.
很多疾病會引起缺血症狀，以致於氧的供應量不足以滿足組織器官的需求而造成缺氧現象。以腦中風、心肌梗塞及惡性腫瘤等重大疾病而言，其組織均會有缺氧現象發生，而此時缺氧可能是因為如心肌細胞等局部的血液循環急性或短暫性地功能下降，或者是像惡性腫瘤細胞慢性地沒有充分的氧氣供應。而因為缺氧是無法預知的，因此發展用以偵測心臟、腦部以及惡性腫瘤部位的等非侵入性技術就逐漸蓬勃。 Many diseases cause ischemic symptoms, so that the supply of oxygen is insufficient to meet the needs of tissues and organs, resulting in hypoxia. In the case of major diseases such as stroke, myocardial infarction, and malignant tumors, hypoxia may occur in tissues, and hypoxia may be due to acute or transient decline in local blood circulation such as cardiomyocytes, or It is like a malignant tumor cell that does not have a sufficient oxygen supply chronically. And because hypoxia is unpredictable, the development of non-invasive techniques to detect the heart, brain, and malignant tumors is growing.
所有哺乳類生物體之組織皆需要氧氣的供應方能滿足新陳代謝的需求。一般而言，經由微血管擴散至細胞之周圍的低濃度氧(約3~5torr)，即可進行細胞內新陳代謝的反應機制。但惡性腫瘤會改變組織內氧氣之供需平衡關係，因為其所增加的細胞團需有額外氧氣供應以維持細胞的活力。故為了滿足需求，惡性腫瘤會自行調整以促進新血管在病變組織的成長。然而其對氧需求的 增加量常常大於所能供應的量，當氧氣從微血管壁擴散出來即迅速被外層細胞代謝，無法擴散至腫瘤內層。較大之固形腫瘤其內部因氧氣及血液之供應不及而呈現缺氧狀況，長期缺氧之細胞則漸漸壞死，因此腫瘤依其體積及生物特性，常含有相當比例之缺氧或壞死細胞。 All mammalian organisms require a supply of oxygen to meet their metabolic needs. In general, a low-concentration oxygen (about 3 to 5 torr) that spreads around the cells through the microvasculature can perform a reaction mechanism of intracellular metabolism. However, malignant tumors alter the balance of supply and demand of oxygen in the tissue because the added cell mass requires additional oxygen supply to maintain cell viability. Therefore, in order to meet the demand, the malignant tumor will adjust itself to promote the growth of new blood vessels in the diseased tissue. However, its demand for oxygen The amount of increase is often greater than the amount that can be supplied. When oxygen diffuses out of the microvascular wall, it is rapidly metabolized by the outer cells and cannot spread to the inner layer of the tumor. Larger solid tumors have hypoxic conditions due to insufficient supply of oxygen and blood, and long-term hypoxic cells gradually become necrotic. Therefore, tumors often contain a considerable proportion of hypoxic or necrotic cells depending on their volume and biological characteristics.
因此在做核醫造影之技術上，若要觀察該些惡性腫瘤以做進一步診斷、追蹤，則透過缺氧組織做為觀察之標的或媒介即存在發展潛力。 Therefore, in the technique of nuclear angiography, if these malignant tumors are to be observed for further diagnosis and tracking, there is potential for development through hypoxic tissue as the target or medium of observation.
核醫造影，就是應用放射性同位素，而為病人做攝影檢查。其係將具有放射性同位素之造影劑經由靜脈注射，口服或吸入等方法而送入生物體內，待一段時間之後，這些造影劑會分佈到特定器官或組織，再利用核醫攝影儀器偵測特定器官或組織內藥物的分佈，例如是伽馬射線攝影機透過放射性同位素的伽馬射線撞擊碘化鈉晶體產生閃爍光來以產生影像；經顯像或電腦處理後，即可由醫師判讀診斷相關的生理變化。 Nuclear angiography is the application of radioisotopes to the photographic examination of patients. The contrast agent having a radioisotope is sent to a living body by intravenous injection, oral or inhalation, and after a period of time, the contrast agent is distributed to a specific organ or tissue, and the specific organ is detected by a nuclear medical photographic apparatus. Or the distribution of drugs in the tissue, such as a gamma ray camera that strikes sodium iodide crystals by radioactive isotope gamma rays to produce scintillation light to produce an image; after imaging or computer processing, the physician can interpret diagnostic-related physiological changes. .
現有藥物之PnAO-NI與HL91均係含二胺二肟(diamine-dioxime)之有機配位子，其可鍵結鎝-99m(99mTc)等放射性同位素而有應用於缺氧組織造影劑之可能，但由於99mTc-PnAO-NI之脂溶性太高，而99mTc-HL91之脂溶性又太低，因此皆不適用為臨床上之缺氧組織造影劑。 The existing drugs PnAO-NI and HL91 are organic ligands containing diamine-dioxime, which can be bonded to radioactive isotopes such as 99-99m ( 99m Tc) and are used in hypoxic tissue contrast agents. Possibly, but because the fat solubility of 99m Tc-PnAO-NI is too high, and the fat solubility of 99m Tc-HL91 is too low, it is not suitable for clinical hypoxic tissue contrast agents.
因此，如何提出一種適用之缺氧組織造影劑，即係攸關對惡性腫瘤做核醫造影成敗與否之關鍵。 Therefore, how to propose a suitable hypoxic tissue contrast agent is the key to the success or failure of nuclear angiography of malignant tumors.
本發明之主要目的，係提供一種造影劑前驅物，其在結構上具有硝基咪唑(nitroimidazole)之官能基，能夠滯留於生物體之缺氧組織，同時亦具備雙官能基配位子之結構，能與放射性同位素錯合，使得放射性同位素能被帶到該些缺氧組織，因此具有作為標幟用途之潛力。 The main object of the present invention is to provide a contrast agent precursor which has a functional group of nitroimidazole in structure, can be retained in an anoxic tissue of a living body, and has a structure of a bifunctional ligand. It can be mismatched with radioisotopes so that radioisotopes can be carried to these anoxic tissues, thus having the potential to be used as a marker.
本發明之次要目的，係提供一種造影劑前驅物，其以三苯甲基為硫醇基之保護基，不但在化學性質穩定而有利於保存，且其在使用前可直接透過錯合反應而脫離，具有便利性。 A secondary object of the present invention is to provide a contrast agent precursor which has a triphenylmethyl group as a thiol group protecting group, which is not only chemically stable but also beneficial for preservation, and can directly pass a mismatch reaction before use. And it is convenient to leave.
本發明之再一目的，係提供一種造影劑前驅物之製備方法，其可有效製備出前述具有良好保存性、便利性以及針對生物體之缺氧組織具標幟用途之造影劑前驅物。 Still another object of the present invention is to provide a method for preparing a contrast agent precursor which can effectively prepare the aforementioned contrast agent precursor having good preservability, convenience, and use for anoxic tissue of a living body.
本發明之更一目的，係提供一種造影劑，其同時兼具了能滯留於生物體缺氧組織的有機官能基，而且也穩定的具有能被偵測之放射性同位素，因此能在相關儀器的檢測之下，透過觀察造影劑的分佈態樣而取得生物體缺氧組織、特別是惡性腫瘤之缺氧層的形態，提供醫生寶貴且充足的資訊以做診斷。 A further object of the present invention is to provide a contrast agent which simultaneously has an organic functional group which can be retained in an anoxic tissue of a living body, and which is stable and has a radioisotope which can be detected, and thus can be used in related instruments. Under the test, by observing the distribution of the contrast agent, the morphology of the hypoxic tissue of the organism, especially the malignant tumor, is obtained, and the doctor is provided with valuable and sufficient information for diagnosis.
故為了達到上述之目的，本發明揭示了一種缺氧組織造影劑前驅物、其造影劑及其製備方法，其在雙官能基配位子接上硝基咪唑或其衍生物，再與標幟用的放射性同位素相錯合，以發展成為一種缺氧組織造影劑；藉由使用此缺氧組織造影劑，不但可以輔助醫生做診斷，還可用來做為治療後之追蹤檢查，因此這在惡性腫瘤之診斷與治療領域上，得以成為不可或缺的有力工具。 Therefore, in order to achieve the above object, the present invention discloses an anoxic tissue contrast agent precursor, a contrast agent thereof and a preparation method thereof, which are provided with a nitroimidazole or a derivative thereof in a bifunctional ligand, and then The radioisotope used is misaligned to develop into an anoxic tissue contrast agent; by using this hypoxic tissue contrast agent, it can not only assist the doctor in making the diagnosis, but also can be used as a follow-up examination after treatment, so this is malignant. In the field of diagnosis and treatment of tumors, it has become an indispensable and powerful tool.
S1~S4‧‧‧步驟流程 S1~S4‧‧‧Step procedure
S11~S14‧‧‧步驟流程 S11~S14‧‧‧Step process
第一圖：其係為本發明之造影劑前驅物之化學結構圖；第二圖：其係為本發明之造影劑前驅物BANI之合成流程圖；第三圖：其係為本發明中，BANI前製之化合物之合成流程圖；第四圖：其係為本發明中，BANI前製之化合物之合成流程圖；以及第五圖：其係為本發明之造影劑之化學結構圖。 The first figure: it is the chemical structure diagram of the contrast agent precursor of the present invention; the second figure: it is the synthesis flow chart of the contrast agent precursor BANI of the present invention; the third figure: it is the invention, A synthetic scheme of a compound prepared by BANI; a fourth diagram: a synthesis scheme of a compound prepared by BANI in the present invention; and a fifth diagram: it is a chemical structure diagram of a contrast agent of the present invention.
為使本發明之特徵及所達成之功效有更進一步之瞭解與認識，謹佐以較佳之實施例及配合詳細之說明，說明如後：首先，請參考第一圖，其係為本發明之造影劑前驅物之化學結構圖；如圖所示，其係為前述之BANI，結構上具有長烷基及一個硝基咪唑(nitroimidazole)，可增加其脂溶性，且此硝基咪唑或其衍生物進入細胞後會進行一系列之還原氧化反應。 For a better understanding and understanding of the features and advantages of the present invention, the preferred embodiments and the detailed description are as follows: First, please refer to the first figure, which is the present invention. The chemical structure diagram of the contrast agent precursor; as shown in the figure, it is the aforementioned BANI, which has a long alkyl group and a nitroimidazole to increase its fat solubility, and the nitroimidazole or its derivative After the substance enters the cell, a series of reduction oxidation reactions are carried out.
此外，當硝基咪唑進入缺氧細胞後，其會被體內之黃嘌呤氧化酶(Xanthine oxidase)逐步還原成胺基(amine)，還原之胺基由於分子極性增大，無法通過細胞膜而停留在細胞內具有滯留性；反之在具正常含氧量之細胞中，氧氣可將期再氧化回到硝基，而使其可以離開細胞。 In addition, when the nitroimidazole enters the hypoxic cell, it is gradually reduced to an amine by Xanthine oxidase in the body, and the reduced amine group cannot stay through the cell membrane due to the increased polarity of the molecule. Retention in cells; conversely, in cells with normal oxygen levels, oxygen can be reoxidized back to the nitro phase, allowing it to leave the cell.
換言之，若細胞含有正常的氧量，則硝基咪唑或其衍生物將迅速逸出細胞而排出體外；而但若該細胞係處於缺氧狀態，則硝基咪唑或其衍生物將會滯留較長時間，因此其具有滯留於缺氧組織之特性，能在與放射性同位素接合後形成性能優越之缺氧組織造影劑。 In other words, if the cell contains normal oxygen, the nitroimidazole or its derivative will quickly escape from the cell and be excreted; but if the cell line is in anoxic state, the nitroimidazole or its derivative will remain. For a long time, it has the property of being retained in an anoxic tissue, and can form a hypoxic tissue contrast agent excellent in performance after being bonded to a radioisotope.
而在具有硝基咪唑的同時，此造影劑前驅物也具有雙官能基配位子，其可以透過五配位之方式與放射性同位素進行鍵結，形成穩定的金字塔構形，進而應用為一種造影劑。 While having nitroimidazole, the contrast agent precursor also has a bifunctional ligand which can be bonded to the radioisotope through a five-coordinated manner to form a stable pyramid configuration, which is then applied as an angiogram. Agent.
如前所述，本發明之造影劑前驅物具有滯留於缺氧組織之特性之硝基咪唑，而配合惡性腫瘤之癌細胞增長迅速，因此大部分都有缺氧層之特性，在經與放射性同位素鍵結後，所形成之造影劑就能停滯留惡性腫瘤所在之處，發揮造影劑之功能。 As described above, the contrast agent precursor of the present invention has nitroimidazole which is retained in the characteristics of anoxic tissue, and the cancer cells which cooperate with the malignant tumor grow rapidly, and therefore most of them have the characteristics of an oxygen-deficient layer, and undergo radioactivity. After isotope bonding, the formed contrast agent can stop where the malignant tumor is located and function as a contrast agent.
另外，本發明的化學結構中，BANI的硫醇基受有保護，化學性質穩定，也可延長其儲存時間。於此，係考量到硫醇基在中性或鹼性溶液中易於氧化，而其氧化後即不能與放射性同位素形成鍵結，故必須先行予以保護。硫醇基之保護方法很多，例如使用4-甲氧基苯(4-methoxybenzyl)或是三苯甲基(triphenylmethyl,CPh3)。由於三苯甲基與硫原子之間的鍵能較弱，當有重金屬存在時，此鍵結非常容易斷裂而形成重金屬與硫原子之鍵結，因此使用三苯甲基來保護硫醇基是較佳的的硫醇保護法，其反應條件較為簡單，只要加入氟化硼***複合物(boron trifluoride ethyl ether complex)，硫醇即轉化為三苯甲硫醚而達到保護的目的。 Further, in the chemical structure of the present invention, the thiol group of BANI is protected, chemically stable, and the storage time thereof is also prolonged. Here, it is considered that the thiol group is easily oxidized in a neutral or alkaline solution, and after it is oxidized, it cannot form a bond with a radioisotope, and therefore must be protected first. There are many methods for protecting a thiol group, for example, 4-methoxybenzyl or triphenylmethyl (CPh 3 ). Since the bond energy between the trityl group and the sulfur atom is weak, when a heavy metal is present, the bond is easily broken to form a bond between the heavy metal and the sulfur atom, so the use of the trityl group to protect the thiol group is The preferred thiol protection method has a simple reaction condition. As long as a boron trifluoride ethyl ether complex is added, the mercaptan is converted into triphenyl sulfide to achieve the purpose of protection.
而本發明所揭示之造影劑前驅物之製備方法，則請參考第二圖，其係包含步驟：步驟S1：水解甲基-DL-2,3-雙[((三苯甲基)硫基)乙醯基]丙酸酯methyl(DL-2,3-bis[((triphenylmethyl)-thio)acetamido]propionate)，生成DL-2,3-雙[((三苯甲基)硫基)乙醯基]丙酸(DL-2,3-Bis- [((triphenylmethyl)thio)acetamido]propionic acid)；步驟S2：使用6-胺基己醇(6-aminohexanol)與DL-2,3-雙[((三苯甲基)硫基)乙醯基]丙酸進行醯胺化反應，生成6-羥基己基-DL-2,3-雙[((三苯甲基)硫基)乙醯胺基]丙醯胺(6-hydroxyhexyl DL-2,3-Bis[((triphenylmethyl)thio)acetamido]-propanamide)；步驟S3：使用P-甲苯磺醯氯(P-toluenesulfornyl chloride)與6-羥基己基-DL-2,3-雙[((三苯甲基)硫基)乙醯胺基]丙醯胺進行取代反應，生成6-甲苯磺醯己基-DL-2,3-雙[((三苯甲基)硫基) For the preparation method of the contrast agent precursor disclosed in the present invention, please refer to the second figure, which comprises the steps of: Step S1: Hydrolysis of methyl-DL-2,3-bis[((trityl)thio) ) propyl-2,3-bis[((triphenylmethyl)-thio)acetamido]propionate), DL-2,3-bis[((trityl)thio)) Mercapto]propionic acid (DL-2,3-Bis- [((triphenylmethyl)thio)acetamido]propionic acid); Step S2: using 6-aminohexanol and DL-2,3-bis[((tritylmethyl)thio)ethenyl ]Proline acid undergoes amidoximation to form 6-hydroxyhexyl-DL-2,3-bis[((trityl)thio)ethinyl]propanamine (6-hydroxyhexyl DL-2,3 -Bis[((triphenylmethyl)thio)acetamido]-propanamide); Step S3: using P-toluenesulfornyl chloride with 6-hydroxyhexyl-DL-2,3-bis[((triphenyl) Substituting a reaction with a sulfhydryl)acetamido]propanamine to form 6-toluenesulfonyl-DL-2,3-bis[((trityl)thio)
乙醯胺基]丙醯胺(6-toluenesulfonylhexyl-DL-2,3-Bis[((triphenyl-methyl)-thio)acetamido]propanamide)；步驟S4：使用2-硝基咪唑(2-nitroimidazole)與6-甲苯磺醯己基-DL-2,3-雙[((三苯甲基)硫基)乙醯胺基]丙醯胺進行取代反應，生成BANI。 6-toluenesulfonylhexyl-DL-2,3-Bis[((triphenyl-methyl)-thio)acetamido]propanamide); Step S4: using 2-nitroimidazole 6-Toluenesulfonyl-DL-2,3-bis[((trityl)thio)ethinyl]propanamine was subjected to a substitution reaction to produce BANI.
於步驟S1中，其水解反應係以氫氧化鉀(KOH)或甲氧化鈉(CH3NaO)為催化劑，並在甲醇(CH3OH)溶液中進行反應，同時使用鹽酸(HCl)中和之。 In the step S1, the hydrolysis reaction is carried out by using potassium hydroxide (KOH) or sodium methoxide (CH 3 NaO) as a catalyst, and reacting in a methanol (CH 3 OH) solution while neutralizing with hydrochloric acid (HCl). .
步驟S2在進行醯胺化反應之前，會先將DL-2,3-雙[((三苯甲基)硫基)乙醯基]丙酸之羧酸結構活化，然後再以1,3-二環己基碳二亞胺(1,3-dicyclohexylcarbodiimide,DCC)以及N-羥基丁二醯亞胺(N-hydroxysuccinimide,NHS)為反應劑，並於氯仿(chloroform,trichloromethane,ChCl3)溶液中進行醯胺化反 應，反應溫度為50℃，反應時間則為24小時為佳。 Step S2 activates the carboxylic acid structure of DL-2,3-bis[((trityl)thio)ethinyl]propionic acid prior to the guanidation reaction, and then 1,3- Dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) are used as reactants in chloroform (trichloromethane, ChCl 3 ) solution. The hydrazide reaction has a reaction temperature of 50 ° C and a reaction time of 24 hours.
步驟S3的取代反應係於吡啶(Pyridine)溶液中進行，並以二氯甲烷為溶劑，反應溫度為室溫，反應時間3小時。 The substitution reaction in the step S3 is carried out in a solution of pyridine (pyridine), and the reaction temperature is room temperature and the reaction time is 3 hours.
步驟S4的取代反應係於無水二甲基甲醯胺(Dimethylformamide,DMF)溶液中進行，並以碳酸銫(Cs2CO3)為反應劑，反應溫度為70℃，反應時間24小時。 The substitution reaction of the step S4 is carried out in a solution of anhydrous dimethylformamide (DMF), and cesium carbonate (Cs 2 CO 3 ) is used as a reactant, and the reaction temperature is 70 ° C, and the reaction time is 24 hours.
另外，本發明對於步驟S1當中所使用的DL-2,3-雙[((三苯甲基)硫基)乙醯基]丙酸酯亦有揭示其製備方法，請參考第三圖和第四圖，其係包含步驟：步驟S11：將乙硫醇酸甲基酯(thioglycolic acid methy ester)之硫醇基以三苯甲基(triphenylmethyl)保護，生成三苯甲基乙硫醇酸甲基酯(triphenylmethyl thioglycolic acid methyl ester)；步驟S12：水解三苯甲基乙硫醇酸甲基酯，生成三苯甲基乙硫醇酸(triphenylmethyl thioglycolic acid)；步驟S13：將DL-2,3-二胺基丙酸(DL-2,3-diaminopropionic acid)於甲醇中進行酯化反應，生成DL-2,3-二胺基丙酸甲酯(methyl DL-2,3-diamino-propionate)；以及步驟S14：將DL-2,3-二胺基丙酸甲酯與三苯甲基乙硫醇酸進行醯胺化反應，生成DL-2,3-雙[((三苯甲基)硫基)乙醯基]丙酸酯。 In addition, the present invention also discloses a preparation method for the DL-2,3-bis[((trityl))thio)ethyl]propionate used in the step S1, please refer to the third figure and the first Four figures, comprising the steps of: Step S11: protecting a thiol group of thioglycolic acid methy ester with triphenylmethyl to form a trityl ethanethiol methyl group Triphenylmethyl thioglycolic acid methyl ester; Step S12: Hydrolysis of trityl ethane thioglycolic acid methyl ester to form triphenylmethyl thioglycolic acid; Step S13: DL-2,3- Diaminopropionic acid (DL-2,3-diaminopropionic acid) is esterified in methanol to form methyl DL-2,3-diamino-propionate; And step S14: subjecting methyl DL-2,3-diaminopropionate and tritylethanethiol acid to amidoxime reaction to form DL-2,3-bis[((tritylmethyl)sulfide) Base) ethyl phthalate] propionate.
於步驟S11中，其於將硫醇基以三苯甲基保護時，係以三氟化硼***複合物(boron trifluoride ethyl ether complex)為 催化劑，以三氯甲烷為溶劑，反應溫度室溫，反應時間5小時。 In step S11, when the thiol group is protected with trityl group, the boron trifluoride ethyl ether complex is used as the boron trifluoride ethyl ether complex. The catalyst was treated with chloroform as a solvent, the reaction temperature was room temperature, and the reaction time was 5 hours.
此些硫醇保護基在日後要進行去除時，係將BANI溶於三氟醋酸中，加入過量之三乙矽烷，即可使三苯甲基從硫醇上脫離而形成不溶於三氟醋酸之固體，可用過濾法去除或用正己烷清洗，方法簡便。 When these thiol protecting groups are to be removed in the future, BANI is dissolved in trifluoroacetic acid, and an excess of triethyl decane is added to remove the trityl group from the thiol to form insoluble in trifluoroacetic acid. The solid can be removed by filtration or washed with n-hexane, and the method is simple.
而在步驟S12之水解反應，則如同步驟S1，其係以氫氧化鉀或甲氧化鈉為催化劑，並在甲醇溶液中進行。 On the other hand, in the hydrolysis reaction in the step S12, as in the step S1, potassium hydroxide or sodium methoxide is used as a catalyst, and it is carried out in a methanol solution.
而至於步驟S14之醯胺化反應，係以DCC以及NHS為反應劑，並於無水四氫呋喃溶液中進行，反應溫度為室溫，反應時間1小時。另外，此醯胺化反應中所使用的DL-2,3-二胺基丙酸甲酯之莫耳數，係為三苯甲基乙硫醇酸的兩倍。 The amidation reaction in the step S14 is carried out by using DCC and NHS as a reactant in an anhydrous tetrahydrofuran solution, and the reaction temperature is room temperature, and the reaction time is 1 hour. Further, the molar number of DL-2,3-diaminopropionate methyl ester used in the guanidation reaction was twice that of trityl ethanethiol acid.
透過本發明於上述所揭示之步驟，即可製備有機配位子BANI，其在結構上所具有的2-硝基咪唑得以在應用時滯留於缺氧組織，例如惡性腫瘤因癌細胞增長迅速而產生的缺氧層，因此具有應用為造影劑之優勢；而其雙官能基配位子也可錯合放射性同位素，使之藉由放射性同位素而可被相關儀器所偵測。 By the steps of the present invention disclosed above, an organic ligand BANI can be prepared, and the structurally possessed 2-nitroimidazole can be retained in hypoxic tissue during application, for example, malignant tumors grow rapidly due to cancer cells. The resulting oxygen-deficient layer has the advantage of being used as a contrast agent; its bifunctional ligand can also be mismatched with a radioisotope, which can be detected by the relevant instrument by means of a radioisotope.
請參考第五圖，其係為本發明使用BANI有機配位子而架構為缺氧組織造影劑之化學結構。與造影劑前驅物不同的是，此時BANI已與放射性同位素M相結合，而保護用的三苯甲基則在錯合反應時能自動脫離，不必事先移除。於此，M係核醫造影領域常使用的放射性同位素，例如(99mTc)或錸-188(188Re)，而本發明中較佳的選擇係為99mTc。99mTc之半衰期為6小時，長短適中，其加馬射線能量為140.6KeV，不易被人體組織減弱，而且係產自核 反應器，價格低廉，取用上方便也不需另做純化。 Please refer to the fifth figure, which is a chemical structure of the present invention using a BANI organic ligand to construct an anoxic tissue contrast agent. Unlike the contrast agent precursor, BANI has been combined with the radioisotope M at this time, and the protective trityl group is automatically detached in the case of a mismatch reaction without prior removal. Here, M is a radioisotope commonly used in the field of nuclear medicine, for example, ( 99m Tc) or 铼-188 ( 188 Re), and a preferred choice in the present invention is 99m Tc. The half-life of 99m Tc is 6 hours, the length is moderate, the energy of the added horse is 140.6KeV, which is not easy to be weakened by human tissue, and it is produced from the nuclear reactor, and the price is low. It is convenient to use and does not need to be purified.
當此種造影劑在進入生物體體內後，將會透過BANI有機配位子結構所提供的2-硝基咪唑官能基而滯留於生物體內的缺氧組織，例如其惡性腫瘤，因此只要透過正子射出造影術或單光子射出造影術，偵測99mTc或188Re等放射性同位素的分佈態樣，就可清楚地觀察到惡性腫瘤的狀態，這對於正確診斷疾病而提升醫療品質有相當大的助益。 When the contrast agent enters the living body, it will retain the hypoxic tissue in the living body through the 2-nitroimidazole functional group provided by the BANI organic ligand structure, such as a malignant tumor, so Ejection angiography or single photon emission angiography, detection of the distribution of radioisotopes such as 99m Tc or 188 Re, can clearly observe the state of malignant tumors, which is quite helpful for the correct diagnosis of diseases and improve medical quality. beneficial.
以下為本發明於製備BANI之完整操作實施例： The following is a complete operational example of the invention for preparing BANI:
【合成Triphenylmethyl thioglycolic acid methyl ester】取methyl thioglycolate(5.0mL，55.0mmol)及triphenylmethanol(14.3g，55.0mmol)共溶於三氯甲烷(80mL)，慢慢滴入boron trifluoride ethyl ether complex(6.9mL，55.0mmol)在室溫攪拌，應用TLC(chloroform：hexane=1：1)追蹤反應，待起始物完全消失後，用水(2×100mL)清洗反應液。有機相經無水硫酸鈉乾燥後減壓蒸乾，獲得產物Triphenylmethyl thioglycolic acid methyl ester(下稱為化合物1)(18.6g，97.5%)。 [Synthesis of Triphenylmethyl thioglycolic acid methyl ester] methyl thioglycolate (5.0 mL, 55.0 mmol) and triphenylmethanol (14.3 g, 55.0 mmol) were dissolved in chloroform (80 mL), and slowly dropped into boron trifluoride ethyl ether complex (6.9 mL, After stirring at room temperature, the reaction was followed by TLC (chloroform:hexane = 1:1). After the starting material disappeared completely, the mixture was washed with water (2×100 mL). The organic phase was dried over anhydrous sodium sulfate and evaporated to dryness to dryness to give the product triphenylmethyl thioglycolic acid methyl ester (hereinafter referred to as compound 1) (18.6 g, 97.5%).
【合成Triphenylmethyl thioglycolic acid】取化合物1(18.6g，53.5mmol)置於10%氫氧化鉀之甲醇溶液(300mL)中，在室溫攪拌直到完全溶解。減壓濃縮後，用50%甲醇水溶液(100mL)將殘留物溶解，滴入濃鹽酸直到pH值為6，用三氯甲烷(3×100mL)淬取，有機相經無水硫酸鈉乾燥後減壓蒸乾溶劑，得Triphenylmethyl thioglycolic acid(下稱為化合物2)(17.9g，~100%)。 [Synthesis of Triphenylmethyl thioglycolic acid] Compound 1 (18.6 g, 53.5 mmol) was taken in 10% potassium hydroxide in methanol (300 mL) and stirred at room temperature until completely dissolved. The organic layer was dried over anhydrous sodium The solvent was evaporated to give triphenylmethyl thioglycolic acid (hereinafter referred to as Compound 2) (17.9 g, -100%).
【合成Methyl DL-2,3-diaminopropionate dihydrochloride】取DL-2,3-diaminopropionic acid monohydrochloride(5.0g，35.6mmol)溶於甲醇(500mL)，在冰浴冷卻下，慢慢滴入thionyl chloride(25mL)，滴完後移走冰浴，在室溫下攪拌隔夜，減壓濃縮，得無色固體之DL-2,3-diaminopropionic acid monohydrochloride(下稱為化合物3)(6.8g，100%)。 [Synthesis of Methyl DL-2,3-diaminopropionate dihydrochloride] DL-2,3-diaminopropionic acid monohydrochloride (5.0 g, 35.6 mmol) was dissolved in methanol (500 mL), and slowly poured into thionyl chloride (25 mL) under ice cooling. After the completion of the dropwise addition, the ice bath was removed, stirred at room temperature overnight, and concentrated under reduced pressure to give DL-2,3-diaminopropionic acid monohydrochloride (hereinafter referred to as compound 3) (6.8 g, 100%) as a colorless solid.
【合成Methyl DL-2,3-bis[((triphenylmethyl)thio)-acetamido]propionate】 [Synthesis of Methyl DL-2,3-bis[((triphenylmethyl)thio)-acetamido]propionate]
(1)取化合物3(0.57g，3.0mmol)溶於甲醇(20mL)中，加入氫氧化鉀(0.33g，6.0mmol)之甲醇(10mL)溶液，攪拌5分鐘後過濾，減壓蒸乾溶劑。 (1) The compound 3 (0.57 g, 3.0 mmol) was dissolved in methanol (20 mL), and then a solution of potassium hydroxide (0.33 g, 6.0 mmol) in methanol (10 mL). .
(2)取化合物2(2.0g，6.0mmol)，1,3-dicyclohexyl-carbodiimide(1.85g，9.0mmol)及N-hydroxysuccinimide(0.83g，7.2mmol)溶於無水四氫呋喃(60mL)，在室溫下攪拌1小時後有固體析出，減壓過濾，棄固體。 (2) Compound 2 (2.0 g, 6.0 mmol), 1,3-dicyclohexyl-carbodiimide (1.85 g, 9.0 mmol) and N-hydroxysuccinimide (0.83 g, 7.2 mmol) were dissolved in anhydrous tetrahydrofuran (60 mL) at room temperature After stirring for 1 hour, a solid precipitated, and the mixture was filtered under reduced pressure to give a solid.
(3)將步驟(2)所得之濾液倒入步驟(1)之殘留物中，攪拌隔夜，過濾後將濾液減壓蒸乾，用二氯甲烷(2×30mL)萃取殘留物，合併後之二氯甲烷溶液經水洗及用無水硫酸鈉除水後減壓濃縮，使用液相層析法(SiO2，CHCl3：EtOAc=4：1)得固體產物(下稱為化合物4)(1.3g，57%)。 (3) The filtrate obtained in the step (2) is poured into the residue of the step (1), stirred overnight, filtered, and the filtrate is evaporated to dryness under reduced pressure, and the residue is extracted with dichloromethane (2×30 mL). methylene chloride solution was washed with water and dried over anhydrous sodium sulfate and concentrated under reduced pressure after addition of water, the use of liquid chromatography (SiO 2, CHCl 3: EtOAc = 4: 1) to give a solid product (hereinafter referred to as compound 4) (1.3g , 57%).
【合成DL-2,3-Bis[((triphenylmethyl)thio)acetamido]-propionic acid】 [Synthesis DL-2,3-Bis[((triphenylmethyl)thio)acetamido]-propionic acid]
取化合物4(0.7g，0.9mmol)溶於10%氫氧化鉀之甲醇溶液(10mL)中，在室溫攪拌30分鐘後用冰浴冷卻，加入水(20mL)，並用6N鹽酸中和至pH為6.0，用三氯甲烷(3×80mL)萃取。合 併後之三氯甲烷溶液經無水硫酸鈉除水後，減壓蒸乾得產物(下稱為化合物5)(0.7g，100%)。 Compound 4 (0.7 g, 0.9 mmol) was dissolved in 10% EtOAc EtOAc (EtOAc) (EtOAc)EtOAc. It was 6.0 and extracted with chloroform (3 x 80 mL). Combined After the chloroform solution was removed by anhydrous sodium sulfate, the product was evaporated to dryness (yield: Compound 5) (0.7 g, 100%).
【合成6-hydroxyhexyl DL-2,3-Bis[((triphenylmethyl)thio)-acetamido]propanamide】 [Synthesis of 6-hydroxyhexyl DL-2,3-Bis[((triphenylmethyl)thio)-acetamido]propanamide]
取化合物5(1.06g，1.44mmol)、N-hydroxysuccinimide(0.20g，1.73mmol)、1,3-dicyclohexylcarbodiimide(0.45g，2.16mmol)、6-aminohexanol(0.17g，1.44mmol)加入氯仿(100mL)，並加熱(50℃)攪拌隔夜。真空濃縮後加入丙酮溶解，抽氣過濾取濾液濃縮，使用液相層析法(SiO2,CHCl3：CH3OH=95：5)分離純化，得產物(下稱為化合物6)(0.54g，45%)。 Compound 5 (1.06 g, 1.44 mmol), N-hydroxysuccinimide (0.20 g, 1.73 mmol), 1,3-dicyclohexylcarbodiimide (0.45 g, 2.16 mmol), 6-aminohexanol (0.17 g, 1.44 mmol) were added to chloroform (100 mL) And heated (50 ° C) to stir overnight. After concentration in vacuo, it was dissolved in acetone, and the filtrate was concentrated by suction filtration, and purified by liquid chromatography (SiO 2 , CHCl 3 : CH 3 OH = 95:5) to give the product (hereinafter referred to as compound 6) (0.54 g) , 45%).
【合成6-toluenesulfonylhexyl DL-2,3-Bis[((triphenyl-methyl)thio)acetamido]propanamide】 [Synthesis of 6-toluenesulfonylhexyl DL-2,3-Bis[((triphenyl-methyl)thio)acetamido]propanamide]
取化合物6(0.55g，0.66mmol)溶於無水二氯甲烷(10mL)及無水Pyride(1mL)中、在冰浴狀態下緩慢加入P-toluenesulfonyl chloride(0.50g，2.63mmol)並在室溫下攪拌3小時，再加入水(10mL)並加入濃鹽酸調PH值為酸性。以二氯甲烷(80mL)萃取二次，取其有機層，再以1N鹽酸水溶液清洗，取其有機層加入經無水硫酸鈉除水後，減壓蒸乾，使用液相層析法(SiO2,CHCl3：CH3OH=95：5)分離純化，得產物(下稱為化合物7)(0.42g，65%)。 Compound 6 (0.55 g, 0.66 mmol) was dissolved in anhydrous dichloromethane (10 mL) and anhydrous Pyride (1 mL), and P-toluenesulfonyl chloride (0.50 g, 2.63 mmol) was slowly added in an ice bath at room temperature After stirring for 3 hours, water (10 mL) was added and concentrated hydrochloric acid was added to adjust the pH to be acidic. In (80 mL) and extracted twice with dichloromethane, whichever organic layer was then washed with 1N aqueous hydrochloric acid, the organic layer was added whichever dried over anhydrous sodium After addition of water, evaporated to dryness, using liquid chromatography (SiO 2 , CHCl 3 : CH 3 OH = 95: 5) was isolated and purified to give the product (hereinafter referred to as compound 7) (0.42 g, 65%).
【合成6-(2-nitroimidazole)hexyl-DL-2,3-Bis-[((triphenylmethyl)thio)acetamido]propanamide，下稱為BANI】 [Synthesis of 6-(2-nitroimidazole)hexyl-DL-2,3-Bis-[((triphenylmethyl)thio)acetamido]propanamide, hereinafter referred to as BANI]
取化合物7(0.44g，0.45mmol)、2-nitroimidazole(0.05g，0.45mmol)、Cs2CO3(0.15g，0.45mmol)加入無水DMF(10mL)， 並加熱(70℃)攪拌隔夜。上真空加熱(50℃)抽乾，再加入氯仿溶解，取溶者減壓濃縮，使用液相層析法(SiO2,CHCl3：CH3OH=95：5)分離純化，得產物BANI(0.18g，42%)。 Solution of compound 7 (0.44g, 0.45mmol), 2 -nitroimidazole (0.05g, 0.45mmol), Cs 2 CO 3 (0.15g, 0.45mmol) was added in anhydrous DMF (10mL), and heated (70 deg.] C) was stirred overnight. It was dried under vacuum (50 ° C), dissolved in chloroform, concentrated under reduced pressure, and purified by liquid chromatography (SiO 2 , CHCl 3 : CH 3 OH = 95:5) to give product BANI ( 0.18 g, 42%).
配合透過上述之步驟，所製備之BANI即具有良好的保存性質，待使用前與放射性同位素錯合後，所形成之造影劑即可透過BANI的官能基作用而滯留於生物體的缺氧組織，並同時致使放射性同位素能夠確實抵達該些缺氧組織於生物體內的位置，發揮造影劑之功效，故本發明無疑提供了一種具實用價值之缺氧組織造影劑前驅物、其造影劑及其製備方法。 In combination with the above steps, the prepared BANI has good preservation properties. After being mismatched with the radioisotope before use, the formed contrast agent can be retained in the anoxic tissue of the organism through the functional group of BANI. At the same time, the radioactive isotope can surely reach the position of the hypoxic tissue in the living body and exert the effect of the contrast agent, so the invention undoubtedly provides a practical hypoxic tissue contrast agent precursor, the contrast agent and the preparation thereof. method.
惟以上所述者，僅為本發明之較佳實施例而已，並非用來限定本發明實施之範圍，舉凡依本發明申請專利範圍所述之形狀、構造、特徵及精神所為之均等變化與修飾，均應包括於本發明之申請專利範圍內。 The above is only the preferred embodiment of the present invention, and is not intended to limit the scope of the present invention, and the variations, modifications, and modifications of the shapes, structures, features, and spirits described in the claims of the present invention. All should be included in the scope of the patent application of the present invention.
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