200822935 九、發明說明： 【發明所屬之技術領域】 本發明是有關於一種缺氧造影劑HL91-NI結構衍 生物，尤指製備一種可提高藥品的專一性，降低藥品 的極性，藉此提高藥物進入細胞内之藥量，進而提高 藥品的靈敏度之HL91結構衍生物者。 【先前技術】 目前臨床上能夠選擇性地積聚於缺氧細胞的核醫 藥物，乃以2-确基味吐（2-nitro_imidazole)類為主， 其反應機制主要是利用其硝基（nhro )官能基在進入 缺氧細胞後，被體内之黃嘌吟氧化酶（xanthine oxidase, X0)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内，而在具正 常含氧量之細胞中，氧氣可將藥物再氧化回到硝基， 而使其可以離開細胞，利用此特性可進行缺氧細胞之 核醫造影診斷。 以錯-99m-過錄酸鈉（99mTc-pertechnetate )標諸 2-nitroimidazole 之化合物主要分為 ΒΑΤΟ (boronic acid adduct of technetium dioxine) 、PnAO (propylene amine oxine)、Schiff-base 與 BnAO(butylenes amine oxine)等四類。HL91為1995年由Nicolini等人利用鉻 -99m-過鉻酸鈉所標誌的核醫藥物，其結構屬BnAO 類，此藥物水溶性極高（partition coefficient=0.1)，血 200822935 液清除率較快，背景活度低，進入細胞之藥量相對也 較低，琴此敏感度較差。故，上述習用並無法符合實 際使用時之所需。 【發明内容】 本發明之主要目的係在於，提供HL91結構衍生 物，以本發明之方法所製備之HL91結構衍生物，可 提高藥品的專一性，降低藥品的極性，藉此提高藥物 進入細胞内之藥量，進而提高藥品的靈敏度。 為達上述之目的，本發明係一種缺氧造影劑 HL91-NI結構衍生物，該HL91結構衍生物係為 HL91-NI，係由一 2-nitroimidazole 官能基結合 HL91 之結構製備而成；另一類HL91結構衍生物係為 HL91-ET，係修改該HL91化學結構並延長側邊碳鏈製 備而成。 【實施方式】 請參閱『第1及2圖』所示，係本發明之第一 HL91 結構衍生物結構示意圖及本發明之第二HL91結構衍 生物結構示意圖。如圖所示：本發明係一種缺氧造影 劑HL91-NI結構衍生物，利用一 2-硝基咪唑 (2-methyl-2-butene )為最初原料，利用有機合成的 方式（請參第3圖），即可製備出第一 HL91結構衍生 物1，該第一 HL91結構衍生物1係為HL91-NI，其 化學結構係如第1圖所示，上述之第一 HL91結構衍 200822935 生物 1 利用 2_nitroimidazole (N02+NH2)與 HL91 (+4 ++2)的雙重還原特性，因此可以提高藥品的專 一性；另因加入2-nitroimidazole後，降低藥品的極性， 藉此提高藥品進入細胞内之藥量，進而提高藥品的靈 敏度。 當修改該HL91化學結構並延長側邊碳鏈時，即 可製備出第二類HL91結構衍生物2，該第二種HL91 結構衍生物2係為HL91-ET，其化學結構係如第2圖 所示，上述之第二HL91結構衍生物2之側鏈長度越 長，極性越低，故可增加第二HL91結構衍生物2的 脂溶性，並可提高藥品的靈敏度，且並不至於增加太 高的背景值。 請參閱『第4、5及6圖』所示，係分別為本發 明進行放射化學純度分析之流程示意圖、本發明之第 一層析圖及本發明之第二層析圖。如圖所示：上述本 發明所製備之第一 HL91結構衍生物係可經鉻-99m過 鉻酸鈉進行新缺氧造影劑之標誌測試，然而進行放射 化學純度分析，其步驟包括： 步驟3 1 :製備一 HL91結構衍生物試劑，該HL91 結構衍生物試劑係由氣化亞錫，HL91_NI配位子、曱 基雙磷酸（MDP)及碳酸氫鈉所製備而成，若欲對第 二HL91結構衍生物進行分析，其試劑之HL91-NI配 位子係由HL91-ET配位子替代。 7 200822935 步驟3 2 :將該上述HL91結構衍生物試劑經鉻 -99m 過錯酸納（99mTc_pertechnetate)標諸。 步驟3 3 :選用兩種不同極性之溶劑作為展開劑 分別加入上述已標誌之HL91結構衍生物試劑，且分 別於SG-ITLC與Whatman# 1層析紙為固定相進行層析 分離，其中，上述兩種不同極性之溶劑分別為生理食 鹽液（normal saline)與 50%的乙睛（acetonitrile)。 如第5圖所示，係為已標誌之第一 HL91結構衍 生物試劑加入生理食鹽液為展開劑，於SG-ITLC層析 紙為固定相進行層析分離後，所得到之層析圖（内含 一第一層析曲線4 )可知，該展開劑前沿係並無波峰 出現，即表示無free-Tc出現。 如第6圖所示，係為已標誌之第一 HL91結構衍 生物試劑加入50%的乙睛為展開劑，於Whatman# 1層 析紙為固定相進行層析分離後，所得到之層析圖（内 含一第二層析曲線5 )可知，該滴樣品原點（original) 出現少量波峰，表示僅有少量（4.32% ) reduced_Tc 出現。由上述兩圖可知，第一 HL91結構衍生物的放 射化學純度可以>95%，效果相當良好。 請參閱『第7圖』所示，係本發明高壓液相層析 圖。如圖所示：本發明所製備之第一 HL91結構衍生 物以逆相的PRP-1 column，流動相（mobile phase )溶 劑選擇強極性的注射用水（含50mM醋酸納（sodium 200822935 acetate )) 與弱極性的四氫夫味 (tetrahydrofur§n，THF)，以梯度（gradient)的方式進 行液相層析。 請參閱『第8圖』所示，係本發明各藥物之細胞 吸收比較圖。如圖所示：藉由圖中可知（包含一液相 層析曲線6)，在11.51分鐘處有一主要波峰61存 在，於3.02分鐘處則有一小波峰6 2，顯示第一 HL91 結構衍生物的標誌效率>98%，此結果驗證上述放射 化學純度分析結果之正確性；並且鉻-99m-HL91_NI與 鉻-99m_HL91-ET於缺氧的環境下細胞的吸收效果於 不同的時間點皆大於傳統的鉻-99m-HL91，此即表示 HL91結構衍生物確實可以提升藥物進入細胞之藥 量，降低藥物的極性，提高藥物的敏感度 (sensitivity ) 〇 綜上所述，本發明係一種缺氧造影劑HLSU-NI結 構衍生物，可有效改善習用之種種缺點，以HL91結 構衍生物與上述配方進行混合，並使用鉻_99m過鉻酸 鈉進行標誌，利用層析法與高壓液相層析法方式進行 放射化學純度分析，以驗證鉻-99m標誌HL91結構衍 生物的放射化學純度皆> 95%，其標誌效率非常良 好。故可提高藥品的專一性，降低藥品的極性，藉此 提高藥物進入細胞内之藥量，進而提高藥品的靈敏 度，進而使本發明之産生能更進步、更實用、更符合 200822935 使用者之所須，確已符合發明專利申請之要件，爰依 法提出專利申請。 _ 惟以上所述者，僅為本發明之較佳實施例而已， 當不能以此限定本發明實施之範圍；故，凡依本發明 申請專利範圍及發明說明書内容所作之簡單的等效變 化與修飾，皆應仍屬本發明專利涵蓋之範圍内。 200822935 【圖式簡單說明】 _第1圖，係本發明之第一 HL91結構衍生物結構示意 圖。 第2圖，係本發明之第二HL91結構衍生物結構示意 圖。 第3圖，係本發明有機合成HL91-NI的化學合成步 驟。 第4圖，係本發明進行放射化學純度分析之流程示意 圖。 第5圖，係本發明之第一層析圖。 第6圖，係本發明之第二層析圖。 第7圖，係本發明高壓液相層析圖。 第8圖，係本發明各藥物之細胞吸收比較圖。 【主要元件符號說明】 第一 HL91結構衍生物1 第二HL91結構衍生物2 步驟：3 1〜3 3 第一層析曲線4 第二層析曲線5 液相層析曲線6 主要波峰6 1 200822935 小波峰6 2200822935 IX. Description of the invention: [Technical field of the invention] The present invention relates to a hypoxia contrast agent HL91-NI structural derivative, in particular to a method for improving the specificity of a drug, reducing the polarity of the drug, thereby improving the drug The HL91 structural derivative that enters the amount of drug in the cell, thereby improving the sensitivity of the drug. [Prior Art] At present, the nuclear medicines that can selectively accumulate in hypoxic cells in the clinic are mainly 2-nitro-imidazole, and the reaction mechanism is mainly to use the nitro group (nhro). After entering the hypoxic cells, the functional group is gradually reduced to the amine by the xanthine oxidase (X0) in the body. The reduced amine group cannot stay through the cell membrane due to the increased polarity of the molecule. Inside, in cells with normal oxygen content, oxygen can reoxidize the drug back to the nitro group, allowing it to leave the cell, and this feature can be used for nuclear diagnostic imaging of hypoxic cells. The compounds labeled 2-nitroimidazole with 99-Tm-pertechnetate are mainly classified into boronic acid adduct of technetium dioxine, PnAO (propylene amine oxine), Schiff-base and BnAO (butylenes amine oxine). ) and so on. HL91 is a nuclear medicine marked by chrome-99m-per sodium chromate by Nicolini et al. in 1995. Its structure belongs to BnAO class. The drug has high water solubility (partition coefficient=0.1), and blood 200822935 liquid clearance rate is faster. The background activity is low, and the amount of drug entering the cell is relatively low, and the sensitivity of the piano is poor. Therefore, the above-mentioned practices are not suitable for the actual use. SUMMARY OF THE INVENTION The main object of the present invention is to provide a HL91 structural derivative, the HL91 structural derivative prepared by the method of the invention can improve the specificity of the drug, reduce the polarity of the drug, thereby improving the entry of the drug into the cell. The amount of drug, which in turn increases the sensitivity of the drug. For the purpose of the above, the present invention is a hypoxia contrast agent HL91-NI structural derivative, which is HL91-NI, which is prepared by a structure in which a 2-nitroimidazole functional group is combined with HL91; The HL91 structural derivative is HL91-ET, which is prepared by modifying the chemical structure of the HL91 and extending the side carbon chain. [Embodiment] Please refer to the "Figures 1 and 2" for a schematic diagram of the structure of the first HL91 structural derivative of the present invention and a schematic diagram of the second HL91 structural derivative structure of the present invention. As shown in the figure: The present invention is a hypoxia contrast agent HL91-NI structural derivative, which utilizes 2-nitro-2-imene as the initial raw material and utilizes organic synthesis (see step 3). Fig.), the first HL91 structural derivative 1 can be prepared, and the first HL91 structural derivative 1 is HL91-NI, and its chemical structure is as shown in Fig. 1, and the first HL91 structural derivative 200822935 is described above. By using the double reduction property of 2_nitroimidazole (N02+NH2) and HL91 (+4 ++2), the specificity of the drug can be improved; and the addition of 2-nitroimidazole reduces the polarity of the drug, thereby increasing the entry of the drug into the cell. The amount of drug, which in turn increases the sensitivity of the drug. When the HL91 chemical structure is modified and the side carbon chain is extended, a second type of HL91 structural derivative 2 can be prepared. The second HL91 structural derivative 2 is HL91-ET, and its chemical structure is as shown in FIG. It is shown that the longer the side chain length of the second HL91 structural derivative 2 described above, the lower the polarity, so that the fat solubility of the second HL91 structural derivative 2 can be increased, and the sensitivity of the drug can be improved, and the increase is not too High background value. Please refer to the "Figures 4, 5 and 6" for the flow chart of the radiochemical purity analysis of the present invention, the first chromatogram of the present invention and the second chromatogram of the present invention. As shown in the figure: the first HL91 structural derivative prepared by the present invention can be tested by a new hypoxic contrast agent by using chromium-99m sodium perchromate, but the radiochemical purity analysis is carried out, and the steps thereof include: Step 3 1 : preparing a HL91 structural derivative reagent prepared by vaporized stannous, HL91_NI ligand, decyl diphosphate (MDP) and sodium hydrogencarbonate, if desired, for the second HL91 The structural derivative was analyzed and the HL91-NI ligand of the reagent was replaced by the HL91-ET ligand. 7 200822935 Step 3 2: The above HL91 structural derivative reagent was labeled with chromium-99m sodium peracid (99mTc_pertechnetate). Step 3 3: adding two solvents of different polarities as a developing agent to separately add the above-mentioned labeled HL91 structural derivative reagent, and separately separating and separating the SG-ITLC and Whatman # 1 chromatography paper as a stationary phase, wherein the above The two solvents of different polarities are normal saline and 50% acetonitrile. As shown in Fig. 5, the first HL91 structural derivative reagent which has been marked is added with physiological saline solution as a developing agent, and the chromatogram obtained after chromatographic separation on the SG-ITLC chromatography paper is carried out. It contains a first chromatogram 4). It can be seen that there is no peak in the front of the developer, which means that no free-Tc appears. As shown in Fig. 6, the obtained first HL91 structural derivative reagent is added with 50% acetonitrile as a developing solvent, and chromatographic separation is performed on Whatman# 1 chromatography paper as a stationary phase. The figure (containing a second tomogram 5) shows that a small number of peaks appear in the origin of the sample, indicating that only a small amount (4.32%) of reduced_Tc appears. As can be seen from the above two figures, the first HL91 structural derivative has a radiochemical purity of > 95%, and the effect is quite good. Please refer to Fig. 7 for the high pressure liquid chromatogram of the present invention. As shown in the figure: the first HL91 structural derivative prepared by the present invention uses a reverse phase PRP-1 column, a mobile phase solvent to select a highly polar water for injection (containing 50 mM sodium acetate (sodium 200822935 acetate)) and The weakly polar tetrahydrofurt (THF) was subjected to liquid chromatography in a gradient manner. Please refer to Fig. 8 for a comparison of cell uptake of each drug of the present invention. As shown in the figure: As can be seen from the figure (including a liquid chromatography curve 6), a major peak 61 exists at 11.51 minutes, and a small peak 62 at 3.02 minutes, showing the first HL91 structural derivative. Marking efficiency > 98%, this result verifies the correctness of the above radiochemical purity analysis results; and the absorption effect of chromium-99m-HL91_NI and chromium-99m_HL91-ET in anoxic environment is greater than that at the different time points. Chromium-99m-HL91, which means that the HL91 structural derivative can really increase the amount of drug into the cell, reduce the polarity of the drug, and improve the sensitivity of the drug. In summary, the present invention is an anoxic angiography. The HLSU-NI structural derivative can effectively improve the disadvantages of the conventional use, and the HL91 structural derivative is mixed with the above formula, and marked with chromium_99m sodium perchromate, using chromatography and high pressure liquid chromatography. The radiochemical purity analysis was carried out to verify that the radiochemical purity of the chromium-99m-labeled HL91 structural derivative was > 95%, and its labeling efficiency was very good. Therefore, the specificity of the medicine can be improved, the polarity of the medicine can be lowered, thereby increasing the amount of the medicine into the cell, thereby improving the sensitivity of the medicine, thereby making the invention more progressive, practical, and more suitable for the user of 200822935. It must have met the requirements of the invention patent application and filed a patent application in accordance with the law. The above is only the preferred embodiment of the present invention, and the scope of the present invention is not limited thereto; therefore, the simple equivalent changes made according to the scope of the invention and the contents of the description of the invention are Modifications are still within the scope of the invention. 200822935 [Simplified description of the drawings] Fig. 1 is a schematic view showing the structure of the first HL91 structural derivative of the present invention. Fig. 2 is a schematic view showing the structure of a second HL91 structural derivative of the present invention. Figure 3 is a chemical synthesis step of the organic synthesis HL91-NI of the present invention. Fig. 4 is a schematic flow chart showing the radiochemical purity analysis of the present invention. Figure 5 is a first chromatogram of the present invention. Figure 6 is a second chromatogram of the present invention. Figure 7 is a high pressure liquid chromatogram of the present invention. Fig. 8 is a comparison chart of cell absorption of each of the drugs of the present invention. [Main component symbol description] First HL91 structural derivative 1 Second HL91 structural derivative 2 Step: 3 1~3 3 First tomographic curve 4 Second tomographic curve 5 Liquid chromatography curve 6 Main peak 6 1 200822935 Wavelet Peak 6 2