JP6218073B2 - A highly sensitive near-infrared phosphorescent iridium complex for measuring oxygen concentration in cells and tissues - Google Patents
A highly sensitive near-infrared phosphorescent iridium complex for measuring oxygen concentration in cells and tissues Download PDFInfo
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- 229910052760 oxygen Inorganic materials 0.000 title claims description 45
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 title claims description 43
- 239000001301 oxygen Substances 0.000 title claims description 43
- 229910052741 iridium Inorganic materials 0.000 title description 14
- GKOZUEZYRPOHIO-UHFFFAOYSA-N iridium atom Chemical compound [Ir] GKOZUEZYRPOHIO-UHFFFAOYSA-N 0.000 title description 14
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- GETQZCLCWQTVFV-UHFFFAOYSA-N trimethylamine Chemical compound CN(C)C GETQZCLCWQTVFV-UHFFFAOYSA-N 0.000 description 4
- YZEUHQHUFTYLPH-UHFFFAOYSA-N 2-nitroimidazole Chemical compound [O-][N+](=O)C1=NC=CN1 YZEUHQHUFTYLPH-UHFFFAOYSA-N 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
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- LMSFUWKGADQXES-UHFFFAOYSA-N 2-[1h-pyrrol-2-yl-(2,4,6-trimethylphenyl)methyl]-1h-pyrrole Chemical compound CC1=CC(C)=CC(C)=C1C(C=1NC=CC=1)C1=CC=CN1 LMSFUWKGADQXES-UHFFFAOYSA-N 0.000 description 2
- ZNQVEEAIQZEUHB-UHFFFAOYSA-N 2-ethoxyethanol Chemical compound CCOCCO ZNQVEEAIQZEUHB-UHFFFAOYSA-N 0.000 description 2
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- -1 3-dimethylaminopropyl Chemical group 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- WLLGXSLBOPFWQV-UHFFFAOYSA-N MGK 264 Chemical compound C1=CC2CC1C1C2C(=O)N(CC(CC)CCCC)C1=O WLLGXSLBOPFWQV-UHFFFAOYSA-N 0.000 description 2
- 239000012327 Ruthenium complex Substances 0.000 description 2
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 description 2
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- LNJXVUXPFZKMNF-UHFFFAOYSA-K iridium(3+);trichloride;trihydrate Chemical compound O.O.O.Cl[Ir](Cl)Cl LNJXVUXPFZKMNF-UHFFFAOYSA-K 0.000 description 2
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- XHTYQFMRBQUCPX-UHFFFAOYSA-N 1,1,3,3-tetramethoxypropane Chemical compound COC(OC)CC(OC)OC XHTYQFMRBQUCPX-UHFFFAOYSA-N 0.000 description 1
- VBYDSMBICNUTKN-UHFFFAOYSA-N 3-hydrazinylbenzoic acid Chemical compound NNC1=CC=CC(C(O)=O)=C1 VBYDSMBICNUTKN-UHFFFAOYSA-N 0.000 description 1
- UQRONKZLYKUEMO-UHFFFAOYSA-N 4-methyl-1-(2,4,6-trimethylphenyl)pent-4-en-2-one Chemical group CC(=C)CC(=O)Cc1c(C)cc(C)cc1C UQRONKZLYKUEMO-UHFFFAOYSA-N 0.000 description 1
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- 125000002147 dimethylamino group Chemical group [H]C([H])([H])N(*)C([H])([H])[H] 0.000 description 1
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- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Pyrrole Compounds (AREA)
Description
本発明は、新規イリジウム錯体およびそれを用いた酸素濃度測定試薬、癌診断薬に関する。 The present invention relates to a novel iridium complex, an oxygen concentration measuring reagent using the same, and a cancer diagnostic agent.
生体内における低酸素環境は、我国の3大死亡原因であるがん、脳卒中、心筋梗塞などで共通して観測される。そのため、細胞および組織中の酸素濃度を非侵襲的にリアルタイムで測定する方法の開発は、細胞生物学や医療の分野において重要な課題である。
生体組織中における酸素濃度の定量方法としてこれまで(1)微小電極を組織に挿入して測定する方法、(2)常磁性プローブ分子のESR信号を用いる方法、(3)ニトロイミダゾール系プローブ分子の還元反応を用いる方法、(4)水溶性ポルフィリン、ルテニウム錯体の発光を用いる方法が知られている。(1)の微小電極を用いる方法は、電極近傍の一点における酸素分圧しか測定できず、また、侵襲性であるという欠点を持つ。(2)のESR信号に基づく方法ではリアルタイムでの酸素濃度計測はできない、(3)のニトロイミダゾール系薬剤を用いる方法は低酸素細胞内でニトロイミダゾールが還元されて細胞内タンパク質に結合しトラップされることを利用するが、この方法では、薬剤の代謝に時間を要するため、薬剤投与後数時間経過しないとデータが得られない、という欠点がある。一方、(4)の方法は、水溶性ポルフィリン誘導体やルテニウム錯体のりん光寿命が血中酸素濃度に依存して変化する(消光を受ける)ことを利用して酸素濃度を定量する方法である。この方法は、非侵襲で組織における酸素分圧を可視化できるという大きな利点を有するが、試薬が水溶性であるため、得られるデータは血中酸素濃度に限られる(非特許文献1)。
The hypoxic environment in the living body is commonly observed in cancer, stroke, myocardial infarction, etc., which are the three major causes of death in Japan. Therefore, the development of a method for measuring the oxygen concentration in cells and tissues non-invasively in real time is an important issue in the fields of cell biology and medicine.
As a method for quantifying oxygen concentration in living tissue, (1) a method of measuring by inserting a microelectrode into a tissue, (2) a method using an ESR signal of a paramagnetic probe molecule, and (3) of a nitroimidazole probe molecule A method using a reduction reaction and (4) a method using light emission of a water-soluble porphyrin or ruthenium complex are known. The method (1) using a microelectrode can measure only the oxygen partial pressure at one point in the vicinity of the electrode, and has a drawback of being invasive. The method based on the ESR signal of (2) cannot measure oxygen concentration in real time. The method of (3) using a nitroimidazole drug reduces nitroimidazole in hypoxic cells and binds to intracellular proteins and is trapped. However, since this method requires time for the metabolism of the drug, there is a drawback in that data cannot be obtained until several hours have passed after the drug administration. On the other hand, the method (4) is a method for quantifying the oxygen concentration by utilizing the fact that the phosphorescence lifetime of the water-soluble porphyrin derivative or ruthenium complex changes (subjects to quenching) depending on the blood oxygen concentration. This method has a great advantage that the oxygen partial pressure in the tissue can be visualized non-invasively, but since the reagent is water-soluble, the obtained data is limited to the blood oxygen concentration (Non-patent Document 1).
そこで、本発明者らのグループは、イリジウム錯体(BTP)の室温りん光(強度、寿命)を用いた生体組織中における酸素濃度計測方法を開発した(特許文献1)。BTPのりん光強度、寿命の測定から、リポソーム膜中の酸素濃度の定量、がん細胞を用いたりん光イメージング、担がんマウス中の腫瘍の可視化に成功した(非特許文献2)。さらに、近赤外光領域にりん光を示すイリジウム錯体BTPHSAを開発し、皮膚から約6-7mmにある腫瘍の可視化にも成功した(非特許文献2、特許文献2)。
しかしながら、BTPHSAは、りん光寿命が2.0μsと短く酸素応答性が低いため、正常組織と低酸素組織を区別することが難しい。一般に、近赤外光領域に発光(蛍光、りん光)を示す化合物の発光寿命は、エネルギーギャップ則に従い短寿命化する傾向にあり、室温でμs以上の発光寿命を与えるイリジウム錯体はほとんどない(非特許文献3)。
However, since BTPHSA has a short phosphorescence lifetime of 2.0 μs and low oxygen responsiveness, it is difficult to distinguish normal tissue from hypoxic tissue. In general, the emission lifetime of a compound that emits light (fluorescence, phosphorescence) in the near-infrared region tends to be shortened according to the energy gap law, and there is almost no iridium complex that gives an emission lifetime of μs or more at room temperature ( Non-patent document 3).
本発明は低酸素細胞・組織をイメージングあるいはそれらの酸素濃度定量のための長いりん光寿命(5μs以上)を有する近赤外りん光イリジウム錯体を提供することを課題とする。 An object of the present invention is to provide a near-infrared phosphorescent iridium complex having a long phosphorescence lifetime (5 μs or more) for imaging hypoxic cells / tissues or determining their oxygen concentration.
本発明者らは、上記の課題を解決すべく鋭意検討を重ねた結果、一般式(I)で表される化合物(以下、本発明の化合物又は本発明のイリジウム錯体と呼ぶことがある)が近赤外光領域でも長いりん光寿命を有し、低酸素細胞・組織のイメージングに有効であることを見出し、本発明を完成させた。 As a result of intensive studies to solve the above problems, the present inventors have found that the compound represented by the general formula (I) (hereinafter sometimes referred to as the compound of the present invention or the iridium complex of the present invention). It has been found that it has a long phosphorescence lifetime even in the near-infrared light region and is effective for imaging of hypoxic cells and tissues, and has completed the present invention.
即ち、本発明は以下の通りである。
[1]下記一般式(I)で表される化合物。
[2]下記いずれかの化合物である、[1]に記載の化合物。
[4][1]または[2]の化合物を含む癌診断薬。
That is, the present invention is as follows.
[1] A compound represented by the following general formula (I).
[2] The compound according to [1], which is any of the following compounds.
[4] A cancer diagnostic agent comprising the compound of [1] or [2].
本発明の化合物は、近赤外光領域に5μs以上のりん光寿命を有するため、低酸素細胞・組織のイメージングや酸素濃度の定量を効率よく行うことが可能である。本発明の化合物は、低酸素組織イメージング試薬、細胞および組織内酸素濃度測定試薬、酸素センサー、バイオセンサー、癌診断薬などとして有用である。 Since the compound of the present invention has a phosphorescence lifetime of 5 μs or more in the near-infrared light region, it is possible to efficiently perform imaging of hypoxic cells / tissues and quantification of oxygen concentration. The compound of the present invention is useful as a hypoxic tissue imaging reagent, a cell and tissue oxygen concentration measuring reagent, an oxygen sensor, a biosensor, a cancer diagnostic agent and the like.
以下、本発明のイリジウム錯体を説明するに当たり、具体例を挙げて説明するが、本発明の趣旨を逸脱しない限り以下の内容に限定されるものではなく、適宜変更して実施することができる。 Hereinafter, the iridium complex of the present invention will be described with specific examples. However, the present invention is not limited to the following contents without departing from the gist of the present invention, and can be implemented with appropriate modifications.
本発明の化合物は下記一般式(I)で表される化合物である。
R1はそれぞれ独立に水素、ハロゲン、ヒドロキシル基、アミノ基、メルカプト基、又は炭素数1〜20の炭化水素基を示す。ここで、ハロゲンとしてはCl、BrまたはFが好ましい。アミノ基は、−NH2でもよいし、アルキルアミノ基でもよい。炭素数1〜20の炭化水素基は、直鎖アルキル基でもよいし、分岐鎖アルキル基でもよいし、環状アルキル基でもよい。また、不飽和結合を含んでいてもよいし、1以上の水素原子がハロゲン、ヒドロキシル基、アミノ基、メルカプト基などで置換されていてもよい。炭素数は好ましくは1〜10であり、より好ましくは1〜5であり、さらに好ましくは1〜3である。 R 1 independently represents hydrogen, halogen, hydroxyl group, amino group, mercapto group, or hydrocarbon group having 1 to 20 carbon atoms. Here, Cl, Br or F is preferable as the halogen. The amino group may be —NH 2 or an alkylamino group. The hydrocarbon group having 1 to 20 carbon atoms may be a straight chain alkyl group, a branched chain alkyl group, or a cyclic alkyl group. Further, it may contain an unsaturated bond, and one or more hydrogen atoms may be substituted with a halogen, a hydroxyl group, an amino group, a mercapto group, or the like. Preferably carbon number is 1-10, More preferably, it is 1-5, More preferably, it is 1-3.
R2はそれぞれ独立に水素原子又は炭素数1〜6の炭化水素基を示すが、直鎖アルキル基でもよいし、分岐鎖アルキル基でもよいし、環状アルキル基でもよい。また、不飽和結合を含んでいてもよいし、1以上の水素原子がハロゲン、ヒドロキシル基、アミノ基、メルカプト基などで置換されていてもよい。炭素数は好ましくは1〜3である。 Each R 2 independently represents a hydrogen atom or a hydrocarbon group having 1 to 6 carbon atoms, but may be a linear alkyl group, a branched alkyl group, or a cyclic alkyl group. Further, it may contain an unsaturated bond, and one or more hydrogen atoms may be substituted with a halogen, a hydroxyl group, an amino group, a mercapto group, or the like. Preferably carbon number is 1-3.
nは1〜5の整数を示すが好ましくは1〜3の整数であり、より好ましくは2である。 Although n shows the integer of 1-5, Preferably it is an integer of 1-3, More preferably, it is 2.
より具体的には、R1およびR2がいずれもメチル、nが2である以下の化合物が例示される。
本発明のイリジウム錯体は、後述の実施例に記載の方法に従って合成することができる。 The iridium complex of this invention is compoundable according to the method as described in the below-mentioned Example.
上記のようなイリジウム錯体は、細胞や組織などの環境下においたときに、該環境中の酸素濃度が低いときにより強いりん光を発する。したがって、りん光の強度に基づいて酸素濃度を測定することができる。すなわち、りん光が強いときに酸素濃度が低いというような判定ができる。また、あらかじめ酸素濃度とりん光強度の関係を求めておくことにより、酸素濃度を定量的に測定することも可能である。 When the iridium complex as described above is placed in an environment such as a cell or tissue, it emits stronger phosphorescence when the oxygen concentration in the environment is low. Therefore, the oxygen concentration can be measured based on the intensity of phosphorescence. That is, it can be determined that the oxygen concentration is low when phosphorescence is strong. It is also possible to quantitatively measure the oxygen concentration by obtaining the relationship between the oxygen concentration and the phosphorescence intensity in advance.
また、マウスやラットなどの実験動物あるいはヒトにイリジウム錯体を投与し、酸素濃度が低下している部位の検出などを行うこともできる。癌組織では酸素供給が不足しているので、酸素濃度が低下している部位の検出を行うことにより、癌組織を特異的に染色し、癌の診断薬として使用することもできる。 In addition, an iridium complex can be administered to a laboratory animal such as a mouse or rat or a human to detect a site where the oxygen concentration is lowered. Since the cancer tissue lacks oxygen supply, it can be used as a diagnostic agent for cancer by specifically staining the cancer tissue by detecting the site where the oxygen concentration is lowered.
細胞内の酸素濃度を検出する場合は、イリジウム錯体を生体に添加してインキュベートした後、イリジウム錯体を励起してりん光を観察できるような蛍光顕微鏡、蛍光測定装置、蛍光イメージング装置などを用いてりん光を観察することができる。 When detecting intracellular oxygen concentration, add the iridium complex to the living body, incubate, and then use a fluorescence microscope, fluorescence measurement device, fluorescence imaging device, etc. that excites the iridium complex to observe phosphorescence. Phosphorescence can be observed.
以下に実施例及び比較例を挙げて本発明をさらに具体的に説明するが、本発明の趣旨を逸脱しない限り適宜変更することができる。従って、本発明の範囲は以下に示す具体例により限定的に解釈されるべきものではない。 Hereinafter, the present invention will be described more specifically with reference to examples and comparative examples, but can be appropriately changed without departing from the gist of the present invention. Therefore, the scope of the present invention should not be construed as being limited by the specific examples shown below.
メシチルジピリナート(MD)とフェニルピラゾール(PPZ)を配位子に有するイリジウム錯体PPZMDを合成した。さらにPPZMDの細胞親和性を高めるためジメチルアミノ基をPPZに導入したPPZ4DMMD、PPZ3DMMDを設計、合成した。 An iridium complex PPZMD with mesityl dipyrinate (MD) and phenylpyrazole (PPZ) as ligands was synthesized. Furthermore, PPZ4DMMD and PPZ3DMMD with dimethylamino group introduced into PPZ were designed and synthesized to increase the cell affinity of PPZMD.
合成手順を以下に示す。 The synthesis procedure is shown below.
PPZ4DMMDの合成
PPZ4COOH(1054mg,5.61mmol) (和光純薬より入手)をEtOH(60ml)に溶かし0.5MH2SO4を1ml加え100℃で一晩還流しPPZ4COOEt(956.8mg,4.42mmol)を得た。PPZ4COOEt(956.8mg,4.42mmol)と塩化イリジウム(III)・三水和物(IrCl33H2O)(790mg,2.24mmol)を2-エトキシエタノール(2EtOEtOH,60ml)、脱イオン水(20ml)に加え、140℃で一晩還流後ろ過し、PPZ4COOEt塩素架橋二核錯体(1.4g,1.06mmol)を得た。5-メシチルジピロメタン(290mg,1.10mmol)、2,3-ジクロロ-5,6-ジシアノ-1,4-ベンゾキノリン(DDQ)(250mg,1.10mmol)テトラヒドロフランに加え室温で1時間攪拌後、炭酸カリウム(K2CO3)(1g,7.23mmol)を加え再び15分攪拌し、さらにPPZ4COOEt塩素架橋二核錯体(730mg、0.055mmol))を加えて窒素ガス雰囲気下、100℃で一晩還流し、得られた生成物をクロロホルムで洗浄し、ろ過してカラムクロマトグラフィー(展開溶媒:クロロホルム:酢酸エチル=9:1)で原料を除去した後、リサイクル分取HPLC(日本分析工業、LC9225NEXT)で精製しPPZ4COOEtMDを得た。その後THF:H2O:EtOH=3:1:3溶液に水酸化リチウム一水和物(LiOH・H2O)を大過剰量用いて85℃で一晩還流し、その後pHが1程度になるまで0.1MHClを用いて、その後クロロホルムで分液を行いPPZ4COOHMD(173mg,0.21mmol)を得た。PPZ4COOHMD(83mg,0.10mmol)にN-ヒドロキシコハク酸イミド(NHS:34.5mg,0.30mmol)、1-エチル-3-(3-ジメチルアミノプロピル)カルボキシイミド塩酸塩(EDCHCl:57.5mg,0.30mmol)、N,N-ジメチルホルムアミド(DMF)を加え窒素雰囲気下で一晩攪拌した。その後トリメチルアミンを大過剰量加え一晩攪拌し得られた生成物をカラムクロマトグラフィー(展開溶媒:クロロホルム:メタノール=97:3、充填剤:アルミナ)を用いて精製し、目的物PPZ4DMMD(60mg,0.062mmol)を得た。
1H NMR (400MHz CDCl3) σ:8.026(d,2H), 7.39-7.37(s,1H),7.21-7.20(d,1H),7.052(s,2H),6.89-6.75(d,4H),6.78(s,1H),6.58-6.49(m,3H),6.38-6.37(d,2H),6.15-6.14(d,2H),3.40(d,2H),3.23-3.16(m,2H),2.88(s,1H),2.44-2.40(m,4H),2.34(s,2H),2.251-2.192(m,12H),2.207(s,6H),1.063(q,1H),1.24(s,1H),1.20(t,2H)
ESI-MS(positive):calcd.for C46H51IrN10O2:968.38,found:m/z=969.5([M]+)
PPZ4COOH (1054 mg, 5.61 mmol) (obtained from Wako Pure Chemical Industries, Ltd.) was dissolved in EtOH (60 ml), 1 ml of 0.5MH 2 SO 4 was added and refluxed at 100 ° C. overnight to obtain PPZ4COOEt (956.8 mg, 4.42 mmol). PPZ4COOEt (956.8 mg, 4.42 mmol) and iridium (III) chloride trihydrate (IrCl 3 3H 2 O) (790 mg, 2.24 mmol) in 2-ethoxyethanol (2 EtOEtOH, 60 ml) and deionized water (20 ml) In addition, the mixture was refluxed at 140 ° C. overnight and filtered to obtain PPZ4COOEt chlorine-bridged binuclear complex (1.4 g, 1.06 mmol). 5-mesityldipyrromethane (290 mg, 1.10 mmol), 2,3-dichloro-5,6-dicyano-1,4-benzoquinoline (DDQ) (250 mg, 1.10 mmol) added to tetrahydrofuran and stirred at room temperature for 1 hour Potassium carbonate (K 2 CO 3 ) (1 g, 7.23 mmol) was added and stirred again for 15 minutes, and further PPZ4COOEt chlorine-bridged binuclear complex (730 mg, 0.055 mmol)) was added at 100 ° C. overnight under a nitrogen gas atmosphere. After refluxing, the resulting product was washed with chloroform, filtered, and the raw material was removed by column chromatography (developing solvent: chloroform: ethyl acetate = 9: 1), followed by recycle preparative HPLC (Nippon Analytical Industry, LC9225NEXT To obtain PPZ4COOEtMD. After that, the THF: H 2 O: EtOH = 3: 1: 3 solution was refluxed overnight at 85 ° C. with a large excess of lithium hydroxide monohydrate (LiOH · H 2 O). 0.1M HCl was used until it was, and then liquid separation was performed with chloroform to obtain PPZ4COOHMD (173 mg, 0.21 mmol). PPZ4COOHMD (83 mg, 0.10 mmol) to N-hydroxysuccinimide (NHS: 34.5 mg, 0.30 mmol), 1-ethyl-3- (3-dimethylaminopropyl) carboximide hydrochloride (EDCHCl: 57.5 mg, 0.30 mmol) N, N-dimethylformamide (DMF) was added, and the mixture was stirred overnight under a nitrogen atmosphere. Thereafter, the product obtained by adding a large excess of trimethylamine and stirring overnight was purified using column chromatography (developing solvent: chloroform: methanol = 97: 3, filler: alumina), and the target product PPZ4DMMD (60 mg, 0.062). mmol).
1 H NMR (400 MHz CDCl 3 ) σ: 8.026 (d, 2H), 7.39-7.37 (s, 1H), 7.21-7.20 (d, 1H), 7.052 (s, 2H), 6.89-6.75 (d, 4H) , 6.78 (s, 1H), 6.58-6.49 (m, 3H), 6.38-6.37 (d, 2H), 6.15-6.14 (d, 2H), 3.40 (d, 2H), 3.23-3.16 (m, 2H) , 2.88 (s, 1H), 2.44-2.40 (m, 4H), 2.34 (s, 2H), 2.251-2.192 (m, 12H), 2.207 (s, 6H), 1.063 (q, 1H), 1.24 (s , 1H), 1.20 (t, 2H)
ESI-MS (positive): calcd.for C 46 H 51 IrN 10 O 2 : 968.38, found: m / z = 969.5 ([M] + )
PPZ3DMMDの合成
3-ヒドラジノ安息香酸(1g,6.57mmol)と1,1,3,3-テトラメトキシプロパン(0.6ml,3.66mg)を酢酸(3.0ml)イオン水(1.0ml)混合溶液に加え、マイクロウェーブ合成装置を用いて、150℃で5分加熱しPPZ3COOH(850mg,4.52mmol)を得た。PPZ3COOH(850mg,4.52mmol)をEtOH(60ml)に溶かし、0.5MH2SO4を1ml加え100℃で一晩還流しPPZ3COOEt(750mg,3.47mmol)を得た。PPZ3COOEt(750mg,3.47mmol)と塩化イリジウム(III)・三水和物(IrCl3・3H2O)(630mg,1.79mmol)を2-エトキシエタノール(2EtOEtOH,60ml)、脱イオン水(20ml)に加え、140℃で一晩還流後、ろ過しPPZCOOEt塩素架橋二核錯体を得た。
5-メシチルジピロメタン(423mg,1.60mmol)、2,3-ジクロロ-5,6-ジシアノ-1,4-ベンゾキノリン(DDQ,)(363mg,1.86mmol)テトラヒドロフランに加え室温で1時間攪拌後、炭酸カリウム(K2CO3)(1g,7.23mmol)を加え再び15分攪拌、PPZ3COOEt塩素架橋二核錯体を加えて窒素ガス雰囲気下、100℃で一晩還流し、得られた生成物をクロロホルムで洗浄し、ろ過してカラムクロマトグラフィー(展開溶媒:クロロホルム:メタノール=9:1)で原料を除去した後、リサイクル分取HPLC(日本分析工業、LC9225NEXT)で精製しPPZ3COOEtMD(484mg、0.55mg)を得た。その後PPZ3COOEtMDにTHF:H2O:EtOH=3:1:3溶液と水酸化リチウム一水和物(LiOH・H2O)大過剰量用いて85℃で一晩還流し、その後pHが1程度になるまで0.1MHClを用い、その後クロロホルムで分液を行いPPZ3COOHMD(369mg,0.446mg)を得た。PPZ3CCOHMD(110mg,0.133mmol)にN-ヒドロキシコハク酸イミド(NHS)(46mg,0.400mol)、1-エチル-3-(3-ジメチルアミノプロピル)カルボキシイミド塩酸塩(EDC・HCl)(77mg,0.402mmol)、N,N-ジメチルホルムアミド(DMF)を加え窒素雰囲気下で一晩攪拌した。その後トリメチルアミンを大過剰量加え得られた生成物をカラムクロマトグラフィー(展開溶媒:クロロホルム:メタノール=97:3、充填剤:アルミナ)を用いて精製し、目的物PPZ3DMMD(80mg,0.083mmol)を得た。
1H NMR (400MHz CDCl3) σ:8.12(d,2H), 7.80(s,2H),7.07-6.98(m,4H),6.88-6.79(m,5H),6.48-6.39(m,6H),6.14-6.13(d,2H),2.48(m,3H),2.34(s,3H),2.25-2.23(s,12H),2.03(s,6H),1.68(m,3H),1.24(s,2H),0.86(s,1H)
ESI-MS(positive):calcd.for C46H51IrN10O2
ESI-MS(positive):calcd.for C46H51IrN10O2:968.38,found:m/z=969.4([M]+)
Microwave synthesis by adding 3-hydrazinobenzoic acid (1 g, 6.57 mmol) and 1,1,3,3-tetramethoxypropane (0.6 ml, 3.66 mg) to a mixed solution of acetic acid (3.0 ml) and ionic water (1.0 ml) Using the apparatus, it was heated at 150 ° C. for 5 minutes to obtain PPZ3COOH (850 mg, 4.52 mmol). PPZ3COOH (850 mg, 4.52 mmol) was dissolved in EtOH (60 ml), 1 ml of 0.5MH 2 SO 4 was added, and the mixture was refluxed at 100 ° C. overnight to obtain PPZ3COOEt (750 mg, 3.47 mmol). PPZ3COOEt (750 mg, 3.47 mmol) and iridium (III) chloride trihydrate (IrCl 3 3H 2 O) (630 mg, 1.79 mmol) in 2-ethoxyethanol (2 EtOEtOH, 60 ml) and deionized water (20 ml) In addition, after refluxing at 140 ° C. overnight, filtration was performed to obtain a PPZCOOEt chlorine-bridged binuclear complex.
Add 5-mesityldipyrromethane (423mg, 1.60mmol), 2,3-dichloro-5,6-dicyano-1,4-benzoquinoline (DDQ,) (363mg, 1.86mmol) tetrahydrofuran and stir at room temperature for 1 hour After that, potassium carbonate (K 2 CO 3 ) (1 g, 7.23 mmol) was added and stirred again for 15 minutes, and PPZ3COOEt chlorine-bridged binuclear complex was added and refluxed at 100 ° C. overnight under a nitrogen gas atmosphere. After washing with chloroform, filtering and removing the raw material by column chromatography (developing solvent: chloroform: methanol = 9: 1), it was purified by recycle preparative HPLC (Nippon Analytical Industry, LC9225NEXT) and PPZ3COOEtMD (484 mg, 0.55 mg). Then reflux to PPZ3COOEtMD at 85 ° C overnight using THF: H 2 O: EtOH = 3: 1: 3 solution and a large excess of lithium hydroxide monohydrate (LiOH · H 2 O), then pH is about 1 Then, 0.1M HCl was used until separation was performed, followed by liquid separation with chloroform to obtain PPZ3COOHMD (369 mg, 0.446 mg). PPZ3CCOHMD (110 mg, 0.133 mmol) and N-hydroxysuccinimide (NHS) (46 mg, 0.400 mol), 1-ethyl-3- (3-dimethylaminopropyl) carboximide hydrochloride (EDCHCl) (77 mg, 0.402) mmol), N, N-dimethylformamide (DMF) was added, and the mixture was stirred overnight under a nitrogen atmosphere. Thereafter, the product obtained by adding a large excess of trimethylamine was purified using column chromatography (developing solvent: chloroform: methanol = 97: 3, filler: alumina) to obtain the target product PPZ3DMMD (80 mg, 0.083 mmol). It was.
1 H NMR (400 MHz CDCl 3 ) σ: 8.12 (d, 2H), 7.80 (s, 2H), 7.07-6.98 (m, 4H), 6.88-6.79 (m, 5H), 6.48-6.39 (m, 6H) , 6.14-6.13 (d, 2H), 2.48 (m, 3H), 2.34 (s, 3H), 2.25-2.23 (s, 12H), 2.03 (s, 6H), 1.68 (m, 3H), 1.24 (s , 2H), 0.86 (s, 1H)
ESI-MS (positive) : calcd.for C 46 H 51 IrN 10 O 2
ESI-MS (positive): calcd.for C 46 H 51 IrN 10 O 2 : 968.38, found: m / z = 969.4 ([M] + )
<吸収・りん光スペクトルの測定>
PPZMD、PPZ4DMMD、PPZ3DMMDをテトラヒドロフラン(THF)に溶解し、室温で吸収・りん光スペクトルを測定した。BTPHSAも同様に測定した。結果を図1に示す。また、これらのイリジウム錯体の光極大波長λabs、りん光極大波長λphos、空気中、脱気下のりん光量子収率(Φp、Φp 0)、りん光寿命(τp、τp 0)および酸素応答性τp0/τpを表1に示す。吸収スペクトルの波長は、BTPHSAに比べると短波長シフトしているが、りん光スペクトルは近赤外領域まで延びていることがわかる(図1)。
PPZMD, PPZ4DMMD, and PPZ3DMMD were dissolved in tetrahydrofuran (THF), and absorption / phosphorescence spectra were measured at room temperature. BTPHSA was measured similarly. The results are shown in FIG. In addition, these iridium complexes have a light maximum wavelength λabs, a phosphorescence maximum wavelength λ phos , a phosphorescence quantum yield (Φ p , Φ p 0 ), and a phosphorescence lifetime (τ p , τ p 0 ) in air and under deaeration. Table 1 shows the oxygen responsiveness τp0 / τp. Although the wavelength of the absorption spectrum is shifted by a shorter wavelength than BTPHSA, it can be seen that the phosphorescence spectrum extends to the near infrared region (FIG. 1).
また、PPZ,PPZ4DMMD,PPZ3DMMDは、483 nmでのモル吸光係数が3.59×104 M-1cm-1、3.60×104 M-1cm-1、3.45×104 M-1cm-1となり、BTPHSAに比べて吸収効率が増大した。さらに、PPZ4DMMD、PPZ3DMMDでは、りん光寿命τp 0が10-20 μsと大幅な増大が見られ、それに伴って酸素応答性も40以上まで増加させることができた(表1)。 PPZ, PPZ4DMMD and PPZ3DMMD have molar extinction coefficients at 483 nm of 3.59 × 10 4 M −1 cm −1 , 3.60 × 10 4 M −1 cm −1 and 3.45 × 10 4 M −1 cm −1 . The absorption efficiency increased compared with BTPHSA. Furthermore, in PPZ4DMMD and PPZ3DMMD, the phosphorescence lifetime τ p 0 was significantly increased to 10-20 μs, and the oxygen responsiveness could be increased to 40 or more (Table 1).
なお、表1において、りん光寿命は以下のようにして計算した。
酸素によるりん光消光は次のStern-Volmerの式に従うと考えられる。
Phosphorescence quenching by oxygen is thought to follow the following Stern-Volmer equation.
<細胞での評価>
HeLa細胞の培養液にPPZ3DMMD、PPZ4DMMDを2 mM添加し、2時間培養後に測定したりん光顕微画像を図2に示す。その結果、PPZMDは脂溶性が高いため細胞にほとんど取り込まれなかったが(図示せず)、カチオン性のアミノ基を導入したPPZ3DMMD、PPZ4DMMDは細胞に取り込まれ、PPZ3DMMDの方が高い取り込み効率を示した(図2)。
<Evaluation with cells>
FIG. 2 shows a phosphorescence microscopic image measured after adding 2 mM of PPZ3DMMD and PPZ4DMMD to the culture medium of HeLa cells and culturing for 2 hours. As a result, PPZMD was hardly taken into cells due to its high lipid solubility (not shown), but PPZ3DMMD and PPZ4DMMD introduced with cationic amino groups were taken into cells, and PPZ3DMMD showed higher uptake efficiency. (FIG. 2).
また、HeLa細胞の培養液にBTPHSA、PPZ4DMMD、PPZ3DMMを2 mM添加し、常酸素(20 %)、低酸素(2.5 %)培養下で2時間培養後に測定したりん光顕微画像を図3に示す。その
結果、PPZ3DMMD、PPZ4DMMDは、細胞内においてBTPHSAに比べてより高い酸素応答性を示した。
In addition, Fig. 3 shows a phosphorescence microscopic image measured after 2 hours of culturing in normal oxygen (20%) and hypoxia (2.5%) culture with 2 mM BTPHSA, PPZ4DMMD, PPZ3DMM added to the HeLa cell culture medium. . As a result, PPZ3DMMD and PPZ4DMMD showed higher oxygen responsiveness in cells than BTPHSA.
図4に培養細胞のりん光寿命測定装置を示す。励起光にはNd:YAGレーザーの第2高調波(532 nm、パルス幅:1 ns、繰り返し20 kHz)または半導体レーザー(488 nm、パルス幅:25 ns、繰り返し15 kHz)を使用し、時間相関単一光子計数法に基づいて発光減衰曲線を測定した。 FIG. 4 shows an apparatus for measuring the phosphorescence lifetime of cultured cells. The second harmonic of the Nd: YAG laser (532 nm, pulse width: 1 ns, repetition rate 20 kHz) or semiconductor laser (488 nm, pulse width: 25 ns, repetition rate 15 kHz) or time correlation is used as the excitation light. The emission decay curve was measured based on the single photon counting method.
図5にこの装置を使って得られたBTPHSA、PPZ3DMMDを取り込んだHeLa細胞のりん光減衰を示す。図5のりん光減衰は、どれも2成分からなる指数関数減衰にフィットできる。BTPHSAの20 % O2条件下、2.5 % O2条件下の平均りん光寿命は、それぞれ0.797 μs、1.45 μsとなり、低酸素下では寿命の増加が見られた。PPZ3DMMDでは、それぞれ2.71 μs、9.62 μsとなり、著しい寿命の増加が見られた。すなわち、τp 0が増加したことにより酸素応答性が増大したことがわかる。 FIG. 5 shows phosphorescence decay of HeLa cells incorporating BTPHSA and PPZ3DMMD obtained using this apparatus. All of the phosphorescence decays in FIG. 5 can fit into an exponential decay consisting of two components. The average phosphorescence lifetimes of BTPHSA under 20% O 2 and 2.5% O 2 conditions were 0.797 μs and 1.45 μs, respectively. With PPZ3DMMD, the lifetime was increased to 2.71 μs and 9.62 μs, respectively. That is, it can be seen that the oxygen responsiveness increased as τ p 0 increased.
<インビボでの評価>
図6に、In vivo酸素計測用の寿命測定システムの概略図を示す。励起用のファイバー(1本)に対して受光側のファイバーを6本のバンドルとして検出系の感度を向上させた。図6のシステムを用いて測定した担がんマウスの正常組織、腫瘍組織のりん光減衰曲線をそれぞれ図7、図8に示す。プローブとしてPPZ4MDDMを用いた。PPZ4MDDM 50 nmolを担がんマウス(マウス扁平上皮がんSCC7細胞を移植後約2週間)の尾静脈から投与し、LD (488
nm)を励起光としてりん光減衰を観測した。正常組織4箇所(図7左図の1-4)のPPZ4MDDMのりん光減衰速度はほぼ等しいのに対し、腫瘍部のりん光減衰は位置依存性が見られ、正常組織との境界に近いところ(図8、下側の実線)では減衰速度の増加すなわち酸素濃度の増加が見られた(図8)。
<Evaluation in vivo>
FIG. 6 shows a schematic diagram of a lifetime measurement system for in vivo oxygen measurement. The sensitivity of the detection system has been improved by using six fibers on the light-receiving side for one fiber for excitation. The phosphorescence decay curves of normal tissues and tumor tissues of cancer-bearing mice measured using the system of FIG. 6 are shown in FIGS. 7 and 8, respectively. PPZ4MDDM was used as a probe. PPZ4MDDM 50 nmol was administered from the tail vein of tumor-bearing mice (approximately 2 weeks after transplantation of mouse squamous cell carcinoma SCC7 cells), and LD (488
Phosphorescence decay was observed using nm) as excitation light. Whereas the phosphorescence decay rate of PPZ4MDDM in four normal tissues (1-4 in the left figure in Fig. 7) is almost the same, the phosphorescence decay of the tumor is position-dependent and close to the boundary with the normal tissue In FIG. 8, the lower solid line shows an increase in attenuation rate, that is, an increase in oxygen concentration (FIG. 8).
図9に正常組織と腫瘍部のりん光減衰曲線を比較して示す。腫瘍組織では正常組織に比べてPPZ4MDDMのりん光寿命が大きく増加しているのがわかる。すなわち、腫瘍が低酸素状態に陥っていることを明確に示すことができた。 FIG. 9 shows a comparison of phosphorescence decay curves of normal tissue and tumor. It can be seen that the phosphorescent lifetime of PPZ4MDDM is greatly increased in tumor tissues compared to normal tissues. That is, it was possible to clearly show that the tumor was in a hypoxic state.
図10にゲート付ICCDカメラを用いて、図8と同一の担がんマウスの腫瘍部の寿命イメージングを行った結果を示す。担がんマウスの尾静脈からPPZ4DMMDを50 nmol投与し、腫瘍部(直径0.5-1.0 cm)にLD (488 nm、 25 ns)のパルス光を照射し、ゲート付ICCDカメラを使って励起後1.5 〜30 μsの時間分解画像を測定した。腫瘍の中で中心部は赤、周辺は黄色となり(白黒写真では中心部が黒、周辺は白となり)、中心部の方が長寿命すなわち低酸素に陥っていることがわかった。 FIG. 10 shows the results of lifetime imaging of the tumor part of the same tumor-bearing mouse as in FIG. 8 using a gated ICCD camera. 50 nmol of PPZ4DMMD was administered from the tail vein of tumor-bearing mice, the tumor (diameter 0.5-1.0 cm) was irradiated with LD (488 nm, 25 ns) pulsed light, and 1.5 g after excitation using a gated ICCD camera. A time-resolved image of ˜30 μs was measured. The tumor was red in the center and yellow in the periphery (black in the black-and-white photo and white in the periphery), and the center was found to have a longer life span, that is, hypoxia.
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