JP2015193584A - Novel heterocyclic compound and salt thereof, and luminescent substrate composition - Google Patents
Novel heterocyclic compound and salt thereof, and luminescent substrate composition Download PDFInfo
- Publication number
- JP2015193584A JP2015193584A JP2014189314A JP2014189314A JP2015193584A JP 2015193584 A JP2015193584 A JP 2015193584A JP 2014189314 A JP2014189314 A JP 2014189314A JP 2014189314 A JP2014189314 A JP 2014189314A JP 2015193584 A JP2015193584 A JP 2015193584A
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- Prior art keywords
- mmol
- luminescent substrate
- heterocyclic compound
- nmr
- salt
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- 150000002391 heterocyclic compounds Chemical class 0.000 title claims abstract description 57
- 150000003839 salts Chemical class 0.000 title claims description 37
- 239000000203 mixture Substances 0.000 title claims description 18
- 125000000217 alkyl group Chemical group 0.000 claims abstract description 4
- 125000004432 carbon atom Chemical group C* 0.000 claims abstract description 3
- 239000000126 substance Substances 0.000 claims description 20
- 230000007935 neutral effect Effects 0.000 abstract description 25
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- 239000007853 buffer solution Substances 0.000 abstract description 21
- 150000001875 compounds Chemical class 0.000 abstract description 15
- 238000005415 bioluminescence Methods 0.000 abstract description 14
- 230000029918 bioluminescence Effects 0.000 abstract description 14
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- 238000005481 NMR spectroscopy Methods 0.000 description 47
- 239000000243 solution Substances 0.000 description 41
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 38
- -1 dimethylamino compound Chemical class 0.000 description 36
- 238000004020 luminiscence type Methods 0.000 description 35
- 238000003786 synthesis reaction Methods 0.000 description 26
- 230000015572 biosynthetic process Effects 0.000 description 25
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical group C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 24
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- Plural Heterocyclic Compounds (AREA)
Abstract
【課題】pHが中性付近の緩衝液への溶解性に優れ、ホタル生物発光系における発光基質として利用可能な新規化合物の提供。【解決手段】下記一般式(I):[式中、R1は、−H、又は炭素数1〜3のアルキル基であり、R2は、−OH、−NH2、又は−N(CH3)2であり、R3は、−N=、−CH=、又は−CR4=で、ここで、R4は−CH2CH=CH2であり、但し、1つ以上のR3は−N=であり、nは0〜3の整数である]で表される複素環式化合物及びその塩。【選択図】図5The present invention provides a novel compound that is excellent in solubility in a buffer solution having a pH near neutral and can be used as a luminescent substrate in a firefly bioluminescence system. The following general formula (I): [wherein R 1 is —H or an alkyl group having 1 to 3 carbon atoms, and R 2 is —OH, —NH 2, or —N (CH 3) 2]. Yes, R3 is -N =, -CH =, or -CR4 =, where R4 is -CH2CH = CH2, where one or more R3 is -N = and n is 0-3. A heterocyclic compound represented by the following formula: [Selection] Figure 5
Description
本発明は、新規複素環式化合物及びその塩、並びに、発光基質組成物に関し、特には、pHが中性付近の緩衝液への溶解性に優れ、ホタル生物発光系における発光基質として利用可能な複素環式化合物に関するものである。 The present invention relates to a novel heterocyclic compound and a salt thereof, and a luminescent substrate composition, and in particular, has excellent solubility in a buffer solution having a pH near neutral and can be used as a luminescent substrate in a firefly bioluminescence system. The present invention relates to a heterocyclic compound.
生物発光系の中でも、ホタルの発光系は、発光効率に優れた系として知られている。該ホタルの発光系においては、発光基質であるホタルルシフェリンが、発光酵素のホタルルシフェラーゼと、アデノシン三リン酸(ATP)及びマグネシウムイオン(Mg2+)の存在下、励起状態のオキシルシフェリンに変換され、該オキシルシフェリンが基底状態へと失活する際に波長が約560nmの黄緑色の蛍光が発せられる。 Among the bioluminescent systems, the firefly luminescent system is known as a system excellent in luminous efficiency. In the firefly luminescence system, the luminescent substrate firefly luciferin is converted to excited oxyluciferin in the presence of the luminescent enzyme firefly luciferase, adenosine triphosphate (ATP) and magnesium ion (Mg 2+ ). When the oxyluciferin is deactivated to the ground state, yellow-green fluorescence having a wavelength of about 560 nm is emitted.
また、昨今、かかるホタルの発光系の発光基質の類似体として、多彩な発光波長を実現する化合物が合成されている。例えば、下記特許文献1には、ホタルルシフェリンのフェノール性水酸基を2級又は3級アミノ基で置換したルシフェリン誘導体が開示されている。また、下記特許文献2及び3には、ホタルルシフェリンと類似の分子構造を有するルシフェラーゼの発光基質が開示されている。
Recently, compounds that realize various emission wavelengths have been synthesized as analogs of the luminescent substrate of such a firefly luminescent system. For example,
これらのホタルルシフェリン類似体の中でも、長波長の光を発する発光基質は、長波長光は生体内での透過率が高いため、生体内深部の病巣を可視化するための標識材料として有望であり、例えば、和光純薬工業株式会社から商品名「アカルミネ」として、長波長光を発するホタルルシフェリン類似体が市販されている。 Among these firefly luciferin analogs, a luminescent substrate that emits light of long wavelength is promising as a labeling material for visualizing lesions in the deep part of the living body because long wavelength light has high transmittance in the living body, For example, a firefly luciferin analog that emits long-wavelength light is commercially available from Wako Pure Chemical Industries, Ltd. under the trade name “Akalumine”.
しかしながら、上記ホタル発光系の発光基質類似体は、多彩な発光波長を実現できるものの、水溶性が低く、特に、生体内深部の可視化に有用な長波長光を発する発光基質で顕著である。一般に、マウスやラット等の実験動物の生体内への投与においては、発光基質は1〜15mg/ml程度の溶解度を有することが必要であるが、上記の長波長光を発する発光基質は、水への溶解度が約0.1mg/mlであり、実用性に問題が有った。 However, although the above-mentioned firefly luminescent luminescent substrate analogs can realize various emission wavelengths, they have low water solubility, and are particularly prominent among luminescent substrates that emit long-wavelength light useful for visualization in the deep part of the living body. In general, in the administration to the living body of a laboratory animal such as a mouse or a rat, the luminescent substrate needs to have a solubility of about 1 to 15 mg / ml. Solubility in water was about 0.1 mg / ml, and there was a problem in practicality.
これに対して、本発明者らは、特定の分子構造を有し、ホタル生物発光系における発光基質として機能する水に難溶性の発光基質を、ハロゲン化水素で塩化することで、ホタル生物発光系における発光能を保持しつつ、水溶性が大幅に向上することを見出している。 On the other hand, the present inventors have developed a firefly bioluminescence by chlorinating a water-insoluble luminescent substrate having a specific molecular structure and functioning as a luminescent substrate in a firefly bioluminescent system with hydrogen halide. It has been found that the water solubility is greatly improved while maintaining the luminous ability in the system.
しかしながら、上記水に難溶性の発光基質のハロゲン化水素塩は、生体内への投与のために、pHが中性付近の緩衝液に添加すると、水に難溶性の発光基質が析出してしまうという問題があった。また、上記水に難溶性の発光基質のハロゲン化水素塩をpHが約2の酸性溶液として、実験動物の生体内へ投与すると、生体内の細胞が適切に活動するためにpHが7.4前後で調節されている血液(細胞外液)のバランスが崩れる等の問題があり、実験動物への投与は可能であるが好ましくない。 However, when the above-mentioned hydrogen halide salt of a luminescent substrate that is sparingly soluble in water is added to a buffer solution having a pH near neutral for administration into a living body, a luminescent substrate that is sparingly soluble in water will precipitate. There was a problem. In addition, when the above-mentioned hydrogen halide salt of a luminescent substrate that is sparingly soluble in water is administered as an acidic solution having a pH of about 2 to the living body of a laboratory animal, the pH is 7.4 in order for cells in the living body to act appropriately. There is a problem that the balance of blood (extracellular fluid) adjusted before and after is lost, and administration to experimental animals is possible but not preferred.
そこで、本発明の目的は、上記従来技術の問題を解決し、pHが中性付近の緩衝液への溶解性に優れ、ホタル生物発光系における発光基質として利用可能な新規化合物を提供することにある。 Accordingly, an object of the present invention is to provide a novel compound that solves the above-described problems of the prior art, has excellent solubility in a buffer solution having a pH near neutral, and can be used as a luminescent substrate in a firefly bioluminescence system. is there.
本発明者らは、上記目的を達成するために鋭意検討した結果、特定の複素環を有する化合物が、ホタル生物発光系における発光基質として機能する上、pHが中性付近の緩衝液への溶解性に優れることを見出し、本発明を完成させるに至った。 As a result of intensive studies to achieve the above object, the present inventors have found that a compound having a specific heterocyclic ring functions as a luminescent substrate in a firefly bioluminescence system and dissolves in a buffer solution having a pH near neutral. As a result, the present invention was completed.
即ち、本発明によれば、下記一般式(I):
本発明の複素環式化合物の好適例においては、前記一般式(I)中のR1が−Hである。この場合、複素環式化合物の発光効率及びpHが中性付近の緩衝液への溶解性が更に良好である。 In a preferred example of the heterocyclic compound of the present invention, R 1 in the general formula (I) is —H. In this case, the luminous efficiency and solubility of the heterocyclic compound in a buffer solution having a neutral pH are even better.
本発明の複素環式化合物の他の好適例においては、前記一般式(I)中のR2が−N(CH3)2である。この場合、複素環式化合物の発光効率が更に優れる。 In another preferred embodiment of the heterocyclic compound of the present invention, R 2 in the general formula (I) is —N (CH 3 ) 2 . In this case, the luminous efficiency of the heterocyclic compound is further improved.
本発明の複素環式化合物の中でも、下記化学式(II)、(III)又は(IV):
また、本発明によれば、前記複素環式化合物の塩が提供され、該塩も、pHが中性付近の緩衝液への溶解性に優れ、ホタル生物発光系における発光基質として機能する。 In addition, according to the present invention, a salt of the heterocyclic compound is provided, and the salt is also excellent in solubility in a buffer solution having a pH near neutral, and functions as a luminescent substrate in a firefly bioluminescence system.
また、本発明によれば、前記複素環式化合物又は塩を含む発光基質組成物が提供され、該発光基質組成物は、pHが中性付近でも安定であり、発光酵素のホタルルシフェラーゼと共にホタル生物発光系を構成できる。 In addition, according to the present invention, there is provided a luminescent substrate composition comprising the heterocyclic compound or salt. The luminescent substrate composition is stable even when the pH is near neutral, and together with the firefly luciferase, a firefly organism. A light emitting system can be constructed.
本発明によれば、pHが中性付近の緩衝液への溶解性に優れ、ホタル生物発光系における発光基質として利用可能な新規化合物を提供することができる。 ADVANTAGE OF THE INVENTION According to this invention, the novel compound which is excellent in the solubility to the buffer solution near pH neutral, and can be utilized as a luminescent substrate in a firefly bioluminescence system can be provided.
以下に、本発明を詳細に説明する。本発明の複素環式化合物は、上記一般式(I)で表される。本発明の複素環式化合物は、ジヒドロチアゾール環に加えて、複素環をもう一つ有し、該複素環は、ベンゼン環よりも極性が高い。そのため、本発明の複素環式化合物は、水溶性が高く、また、pHが中性付近(好ましくはpHが4〜10、より好ましくはpHが6〜8)の緩衝液への溶解性に優れる。また、本発明の複素環式化合物は、分子構造がホタルルシフェリンと類似しているため、ホタル生物発光系における発光基質として利用できる。 The present invention is described in detail below. The heterocyclic compound of the present invention is represented by the above general formula (I). The heterocyclic compound of the present invention has another heterocyclic ring in addition to the dihydrothiazole ring, and the heterocyclic ring is more polar than the benzene ring. Therefore, the heterocyclic compound of the present invention has high water solubility, and is excellent in solubility in a buffer solution having a pH near neutral (preferably pH 4 to 10, more preferably pH 6 to 8). . Moreover, since the heterocyclic compound of the present invention has a molecular structure similar to firefly luciferin, it can be used as a luminescent substrate in a firefly bioluminescence system.
<一般式(I)で表される複素環式化合物>
上記一般式(I)中、R1は、−H(水素)又は炭素数1〜3のアルキル基である。ここで、アルキル基としては、メチル基、エチル基、n−プロピル基、i−プロピル基が挙げられる。なお、R1としては、発光効率及びpHが中性付近の緩衝液への溶解性の観点から、−Hが好ましい。
<Heterocyclic compound represented by general formula (I)>
In said general formula (I), R < 1 > is -H (hydrogen) or a C1-C3 alkyl group. Here, examples of the alkyl group include a methyl group, an ethyl group, an n-propyl group, and an i-propyl group. R 1 is preferably -H from the viewpoint of luminous efficiency and solubility in a buffer solution having a pH near neutral.
上記一般式(I)中、R2は、−OH(ヒドロキシ基)、−NH2(アミノ基)、又は−N(CH3)2(ジメチルアミノ基)である。なお、R2としては、発光効率の観点から、−N(CH3)2が好ましい。 In the general formula (I), R 2 is —OH (hydroxy group), —NH 2 (amino group), or —N (CH 3 ) 2 (dimethylamino group). As the R 2, from the viewpoint of emission efficiency, -N (CH 3) 2 is preferred.
また、上記一般式(I)中、R3は、−N=、−CH=、又は−CR4=で、ここで、R4は−CH2CH=CH2(アリル基)であり、但し、1つ以上のR3は−N=である。なお、本発明においては、合成の容易性の観点から、4つのR3の内、1つが−N=で、3つが−CH=であることが好ましく、即ち、本発明の複素環式化合物は、ピリジン環を有することが好ましい。また、本発明においては、pHが中性付近の緩衝液への溶解性の観点から、4つのR3の内、2つが−N=で、2つが−CH=であることが好ましく、即ち、本発明の複素環式化合物は、ピラジン環、ピリミジン環、ピリダジン環を有することも好ましく、ピラジン環を有することが更に好ましい。 In the general formula (I), R 3 is —N═, —CH═, or —CR 4 ═, where R 4 is —CH 2 CH═CH 2 (allyl group), provided that One or more of R 3 is —N═. In the present invention, from the viewpoint of ease of synthesis, it is preferable that one of the four R 3 s is —N═ and three are —CH═, that is, the heterocyclic compound of the present invention is It preferably has a pyridine ring. Further, in the present invention, from the viewpoint of solubility in a buffer solution having a pH near neutral, it is preferable that two of the four R 3 are -N = and two are -CH =, The heterocyclic compound of the present invention preferably has a pyrazine ring, a pyrimidine ring and a pyridazine ring, and more preferably has a pyrazine ring.
上記一般式(I)中、nはビニレン単位(−CH=CH−)の繰り返し数を示し、0〜3の整数である。nの数が大きい程、発光波長が長くなるため、生体内深部の可視化の観点から、nは2又は3であることが好ましく、入手容易性の観点から、nは2であることが好ましい。 In said general formula (I), n shows the repeating number of vinylene units (-CH = CH-), and is an integer of 0-3. Since the emission wavelength becomes longer as the number of n is larger, n is preferably 2 or 3 from the viewpoint of visualization of the deep part in the living body, and n is preferably 2 from the viewpoint of availability.
上記一般式(I)で表される複素環式化合物は、特に限定されるものではないが、以下のようにして合成することができる。例えば、出発物質として2−アミノ−5−シアノピリジン、5−アミノ−2−シアノピリジン等のシアノピリジン誘導体を使用し、所望により、ヨードメタンと反応させて、ジメチルアミノ体を得、次いで、水素化ジイソブチルアミニウムと反応させて、アルデヒド体を生成させる。或いは、出発物質として2−アミノ−5−ブロモ−ピラジン等のブロモピラジン誘導体を使用し、所望により、ヨードメタンと反応させて、ジメチルアミノ体を得、次いで、ノルマルブチルリチウム等のアルキルリチウムと反応させた後、N,N−ジメチルホルムアミド等のアミド化合物と反応させて、アルデヒド体を生成させる。次いで、該アルデヒド体を4−ホスホノクロトン酸トリエチルと反応させてエチルエステル体を得、次いで、該エチルエステル体を水酸化ナトリウム水溶液中でカルボキシル体に変換する。一方、D−システイン−S−トリチル化合物を塩化水素、1,4−ジオキサン溶液存在下、メタノール溶液中で反応させて、メチルエステル体を準備し、次いで、該メチルエステル体と前記カルボキシル体とを反応させて、アミド体を生成させる。次いで、該アミド体をトリフルオロ酢酸無水物、トリフルオロメタンスルホン酸無水物等の酸無水物によって環化させて複素環を生成させ、チアゾリン体を得る。次いで、所望により、チアゾリン体のメチルエステル部分を加水分解して、チアゾリン環を有するカルボキシル体を得る。また、出発物質を適宜変更したり、種々の置換基を導入したりする等して、或いは、他の合成経路を利用して、所望の複素環式化合物を得ることができる。 The heterocyclic compound represented by the general formula (I) is not particularly limited, but can be synthesized as follows. For example, cyanopyridine derivatives such as 2-amino-5-cyanopyridine and 5-amino-2-cyanopyridine are used as starting materials, and optionally reacted with iodomethane to obtain a dimethylamino compound, followed by hydrogenation It reacts with diisobutylaminium to produce an aldehyde form. Alternatively, a bromopyrazine derivative such as 2-amino-5-bromo-pyrazine is used as a starting material and, if desired, reacted with iodomethane to obtain a dimethylamino compound, which is then reacted with alkyllithium such as normal butyllithium. Then, it is reacted with an amide compound such as N, N-dimethylformamide to produce an aldehyde form. Next, the aldehyde form is reacted with triethyl 4-phosphonocrotonate to obtain an ethyl ester form, which is then converted into a carboxyl form in an aqueous sodium hydroxide solution. On the other hand, a D-cysteine-S-trityl compound is reacted in a methanol solution in the presence of hydrogen chloride and a 1,4-dioxane solution to prepare a methyl ester form, and then the methyl ester form and the carboxyl form are prepared. React to produce an amide form. Next, the amide is cyclized with an acid anhydride such as trifluoroacetic anhydride or trifluoromethanesulfonic anhydride to produce a heterocyclic ring, thereby obtaining a thiazoline. Next, if desired, the methyl ester portion of the thiazoline body is hydrolyzed to obtain a carboxyl body having a thiazoline ring. In addition, a desired heterocyclic compound can be obtained by appropriately changing the starting material, introducing various substituents, or utilizing other synthetic routes.
上述した一般式(I)で表される複素環式化合物の中でも、pHが中性付近の緩衝液への溶解性、発光効率、生体内深部の可視化、入手容易性の観点から、上記化学式(II)、(III)又は(IV)で表される複素環式化合物が好ましく、化学式(III)又は(IV)で表わされる化合物が特に好ましい。なお、上記化学式(III)で表わされる化合物は、pHが中性付近の緩衝液への溶解性が非常に高い上、発光波長が長く、生体内深部の可視化に特に有用である。また、上記化学式(IV)で表わされる化合物は、pHが中性付近の緩衝液への溶解性が極めて高い。 Among the heterocyclic compounds represented by the general formula (I) described above, from the viewpoints of solubility in a buffer solution having a pH near neutrality, luminous efficiency, visualization of deep in vivo, and availability, the above chemical formula ( A heterocyclic compound represented by II), (III) or (IV) is preferred, and a compound represented by chemical formula (III) or (IV) is particularly preferred. In addition, the compound represented by the chemical formula (III) has a very high solubility in a buffer solution having a pH near neutral, has a long emission wavelength, and is particularly useful for visualization in the deep part of the living body. In addition, the compound represented by the chemical formula (IV) has extremely high solubility in a buffer solution having a pH near neutral.
<一般式(I)で表される複素環式化合物の塩>
上記一般式(I)で表される複素環式化合物は、塩とすることもできる。ここで、本発明の複素環式化合物の塩は、酸との付加塩でも、塩基との付加塩でもよい。例えば、本発明の複素環式化合物と酸との付加塩における酸としては、塩酸、臭化水素酸、ヨウ化水素酸、硫酸、スルファミン酸、リン酸、硝酸、亜リン酸、亜硝酸、クエン酸、ギ酸、酢酸、シュウ酸、マレイン酸、乳酸、酒石酸、フマル酸、安息香酸、マンデル酸、ケイ皮酸、パモ酸、ステアリン酸、グルタミン酸、アスパラギン酸、メタンスルホン酸、エタンジスルホン酸、p−トルエンスルホン酸、サリチル酸、コハク酸、トリフルオロ酢酸等が挙げられ、また、酸付加塩としては、塩酸塩、臭化水素酸塩、ヨウ化水素酸塩、硫酸塩、スルファミン酸塩、リン酸塩、硝酸塩、亜リン酸塩、亜硝酸塩、クエン酸塩、ギ酸塩、酢酸塩、シュウ酸塩、マレイン酸塩、乳酸塩、酒石酸塩、フマル酸塩、安息香酸塩、マンデル酸塩、ケイ皮酸塩、パモ酸塩、ステアリン酸塩、グルタミン酸塩、アスパラギン酸塩、メタンスルホン酸塩、エタンジスルホン酸塩、p−トルエンスルホン酸塩、サリチル酸塩、コハク酸塩、トリフルオロ酢酸塩等が挙げられる。一方、本発明の複素環式化合物と塩基との付加塩における塩基としては、水酸化ナトリウム、水酸化カリウム、水酸化カルシウム等が挙げられ、また、塩基付加塩としては、ナトリウム塩、カリウム塩、カルシウム塩等が挙げられる。
<Salt of heterocyclic compound represented by general formula (I)>
The heterocyclic compound represented by the general formula (I) may be a salt. Here, the salt of the heterocyclic compound of the present invention may be an addition salt with an acid or an addition salt with a base. For example, the acid in the addition salt of the heterocyclic compound of the present invention and an acid includes hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, phosphorous acid, nitrous acid, citric acid. Acid, formic acid, acetic acid, oxalic acid, maleic acid, lactic acid, tartaric acid, fumaric acid, benzoic acid, mandelic acid, cinnamic acid, pamoic acid, stearic acid, glutamic acid, aspartic acid, methanesulfonic acid, ethanedisulfonic acid, p- Examples include toluenesulfonic acid, salicylic acid, succinic acid, trifluoroacetic acid, and acid addition salts include hydrochloride, hydrobromide, hydroiodide, sulfate, sulfamate, phosphate , Nitrate, phosphite, nitrite, citrate, formate, acetate, oxalate, maleate, lactate, tartrate, fumarate, benzoate, mandelate, cinnamic acid salt, Mo, stearate, glutamate, aspartate, methanesulfonate, ethanedisulfonate, p- toluenesulfonate, salicylate, succinate, trifluoroacetate, and the like. On the other hand, examples of the base in the addition salt of the heterocyclic compound and the base of the present invention include sodium hydroxide, potassium hydroxide, calcium hydroxide and the like, and examples of the base addition salt include sodium salt, potassium salt, A calcium salt etc. are mentioned.
上記一般式(I)で表される複素環式化合物の塩も、pHが中性付近の緩衝液への溶解性に優れ、ホタル生物発光系における発光基質として機能する。上述のように、上記一般式(I)で表される複素環式化合物はpHが中性付近の緩衝液への溶解性に優れるため、上記一般式(I)で表される複素環式化合物の塩をpHが中性付近の緩衝液に溶解させても、上記一般式(I)で表される複素環式化合物の析出を抑制することができる。 The salt of the heterocyclic compound represented by the general formula (I) is also excellent in solubility in a buffer solution having a pH near neutral, and functions as a luminescent substrate in a firefly bioluminescence system. As described above, since the heterocyclic compound represented by the general formula (I) is excellent in solubility in a buffer solution having a pH near neutral, the heterocyclic compound represented by the general formula (I) Even when the salt is dissolved in a buffer solution having a pH near neutral, precipitation of the heterocyclic compound represented by the general formula (I) can be suppressed.
<発光基質組成物>
本発明の発光基質組成物は、上述した一般式(I)で表される複素環式化合物又はその塩を含み、上述した一般式(I)で表される複素環式化合物又はその塩のみからなってもよい。本発明の発光基質組成物は、pHが中性付近でも、上記一般式(I)で表される複素環式化合物の析出が抑制されているため、安定であり、また、発光酵素のホタルルシフェラーゼと共にホタル生物発光系を構成できる。
<Luminescent substrate composition>
The luminescent substrate composition of the present invention includes the heterocyclic compound represented by the above general formula (I) or a salt thereof, and only from the heterocyclic compound represented by the above general formula (I) or a salt thereof. It may be. The luminescent substrate composition of the present invention is stable because the precipitation of the heterocyclic compound represented by the general formula (I) is suppressed even when the pH is near neutral, and the luminescent enzyme firefly luciferase A firefly bioluminescence system can be constructed together.
上述した本発明の複素環式化合物及びその塩は、発光甲虫ルシフェラーゼ、アデノシン三リン酸(ATP)及びマグネシウムイオン(Mg2+)の存在する系に添加することによって、発光甲虫ルシフェラーゼにより酸化して発光する。なお、本発明の複素環式化合物及びその塩は、ATP及びMg2+と共に発光検出キット(発光基質組成物)として提供することもでき、また、該発光検出キットには、他の発光基質や適切なpHに調整した溶液を含めてもよい。 The above-described heterocyclic compound of the present invention and its salt are oxidized by the luminescent beetle luciferase by adding to the system in which the luminescent beetle luciferase, adenosine triphosphate (ATP) and magnesium ion (Mg 2+ ) are present. Emits light. The heterocyclic compound and its salt of the present invention can also be provided as a luminescence detection kit (luminescence substrate composition) together with ATP and Mg 2+ , and the luminescence detection kit includes other luminescence substrates and A solution adjusted to an appropriate pH may be included.
本発明の複素環式化合物及びその塩を発光系に応用する場合、好適な発光強度を得るためには、本発明の複素環式化合物及びその塩を1μM以上の濃度で使用することが好ましく、5μM以上の濃度で使用することが更に好ましい。即ち、本発明の発光基質組成物は、上述した一般式(I)で表される複素環式化合物又はその塩を1μM以上の濃度で含むことが好ましく、5μM以上の濃度で含むことが更に好ましい。また、本発明の発光基質組成物のpH、並びに、発光系のpHは、好ましくは4〜10、より好ましくは6〜8であり、必要に応じて、pHを安定化するために、リン酸カリウム、トリス塩酸、グリシン、HEPES等の緩衝剤を含んでもよい。 When applying the heterocyclic compound of the present invention and a salt thereof to a light emitting system, it is preferable to use the heterocyclic compound of the present invention and a salt thereof at a concentration of 1 μM or more in order to obtain suitable emission intensity. More preferably, it is used at a concentration of 5 μM or more. That is, the luminescent substrate composition of the present invention preferably contains the above-described heterocyclic compound represented by the general formula (I) or a salt thereof at a concentration of 1 μM or more, more preferably 5 μM or more. . In addition, the pH of the luminescent substrate composition of the present invention and the pH of the luminescent system are preferably 4 to 10, more preferably 6 to 8. If necessary, phosphoric acid is used to stabilize the pH. Buffers such as potassium, Tris-HCl, glycine, HEPES may be included.
また、本発明の複素環式化合物及びその塩は、ホタル発光甲虫ルシフェラーゼ発光系において、種々の酸化酵素によって発光させることができる。ルシフェラーゼは、北アメリカ産ホタル(Photinus pyralis)、鉄道虫(Railroad worm)等から単離されており、いずれも使用できる。また、使用可能な酸化酵素としては、ヒカリコメツキムシルシフェラーゼ、イリオモテボタルルシフェラーゼ、フラビン含有モノオキシゲナーゼ等も挙げられる。 Moreover, the heterocyclic compound and its salt of this invention can be light-emitted by various oxidase in a firefly light emission beetle luciferase light emission system. Luciferase has been isolated from North American firefly (Photinus pyralis), railroad worm (Railroad worm), etc., and any of them can be used. Examples of the oxidase that can be used include Hikari Komekimushiluciferase, Iriomote luciferase, and flavin-containing monooxygenase.
本発明の複素環式化合物及びその塩を発光基質とする生物発光は、発光系にコエンザイムA(CoA)、ピロリン酸又はマグネシウムイオン(Mg2+)が存在すると、その発光が増強される。これらの化合物の発光増強効果は、発光系におけるCoA、ピロリン酸又はMg2+の濃度がそれぞれ5μM以上において顕著であり、濃度の増加にしたがって発光が増強される。 Bioluminescence using the heterocyclic compound of the present invention and a salt thereof as a luminescent substrate is enhanced when coenzyme A (CoA), pyrophosphate or magnesium ion (Mg 2+ ) is present in the luminescent system. The luminescence enhancement effect of these compounds is remarkable when the concentration of CoA, pyrophosphate or Mg 2+ in the luminescence system is 5 μM or more, respectively, and the luminescence is enhanced as the concentration increases.
ホタル生物発光系を測定/検出に使用するためには、酵素の失活を防止してプラトーな発光挙動を示すように、発光を安定化させることが好ましく、例えば、発光系にマグネシウムイオンを存在させることが好ましく、マグネシウムイオンとピロリン酸を共存させることが更に好ましい。なお、マグネシウムイオン単独の場合、発光安定化の観点から、発光系のマグネシウムイオン濃度は、0.5mM以上が好ましく、濃度の増加に従って発光の安定性が向上する。また、ピロリン酸マグネシウムを使用する場合、発光安定化の観点から、発光系のピロリン酸マグネシウム濃度は、10μM以上が好ましく、100μM以上が更に好ましい。なお、ピロリン酸とマグネシウムイオンとの割合は、当量比でなくてもよい。また、好適なマグネシウム塩としては、硫酸マグネシウム、塩化マグネシウム等の無機酸塩、酢酸マグネシウム等の有機酸塩が挙げられる。また、好適なピロリン酸塩として、ナトリウム、カリウム等のアルカリ金属のピロリン酸塩、マグネシウム、カルシウム等のアルカリ土類金属のピロリン酸塩、鉄のピロリン酸塩が挙げられる。 In order to use the firefly bioluminescence system for measurement / detection, it is preferable to stabilize the luminescence so as to prevent the inactivation of the enzyme and show a plateau luminescence behavior, for example, the presence of magnesium ions in the luminescence system It is preferable to coexist magnesium ions and pyrophosphate. In the case of magnesium ion alone, from the viewpoint of light emission stabilization, the magnesium ion concentration of the light emitting system is preferably 0.5 mM or more, and the stability of light emission improves as the concentration increases. When using magnesium pyrophosphate, the concentration of the magnesium pyrophosphate in the luminescent system is preferably 10 μM or more, and more preferably 100 μM or more, from the viewpoint of stabilization of light emission. In addition, the ratio of pyrophosphate and magnesium ion may not be an equivalent ratio. Suitable magnesium salts include inorganic acid salts such as magnesium sulfate and magnesium chloride, and organic acid salts such as magnesium acetate. Suitable pyrophosphates include alkali metal pyrophosphates such as sodium and potassium, alkaline earth metal pyrophosphates such as magnesium and calcium, and iron pyrophosphate.
本発明の複素環式化合物及びその塩は、生物学的測定/検出における発光標識として利用でき、例えば、アミノ酸、ポリペプチド、タンパク質、核酸等を標識するために使用できる。なお、本発明の複素環式化合物又はその塩をこれらの物質に結合させる方法は、当業者に周知であり、例えば、当業者に周知の方法を使用して、目的の物質のカルボキシル基やアミノ基に対して本発明の複素環式化合物又はその塩を結合させることができる。 The heterocyclic compounds and salts thereof of the present invention can be used as luminescent labels in biological measurement / detection, and can be used, for example, for labeling amino acids, polypeptides, proteins, nucleic acids and the like. In addition, the method for binding the heterocyclic compound of the present invention or a salt thereof to these substances is well known to those skilled in the art. For example, a method known to those skilled in the art can be used to form a carboxyl group or amino group of the target substance. The heterocyclic compound of the present invention or a salt thereof can be bonded to the group.
また、本発明の複素環式化合物及びその塩は、発光基質の発光によって発光甲虫ルシフェラーゼ活性を検出することを利用した測定/検出に利用することができる。例えば、ルシフェラーゼ遺伝子を導入した細胞又は動物に対して本発明の複素環式化合物又はその塩を投与することにより、インビボにおける標的遺伝子又はタンパク質の発現などを測定/検出することができる。なお、波長の長い光は、光透過性が高く、組織透過性も高い。従って、本発明の複素環式化合物及びその塩の中でも、長波長の発光を有する複素環式化合物及びその塩は、生体内深部を可視化するための標識材料として有用である。 Further, the heterocyclic compound and the salt thereof of the present invention can be used for measurement / detection utilizing detection of luminescent beetle luciferase activity by luminescence of a luminescent substrate. For example, by administering the heterocyclic compound of the present invention or a salt thereof to a cell or animal into which a luciferase gene has been introduced, the expression or the like of a target gene or protein in vivo can be measured / detected. Note that light having a long wavelength has high light permeability and high tissue permeability. Therefore, among the heterocyclic compounds and salts thereof of the present invention, the heterocyclic compounds having a long wavelength emission and salts thereof are useful as labeling materials for visualizing the deep part in the living body.
以下に、実施例を挙げて本発明を更に詳しく説明するが、本発明は下記の実施例に何ら限定されるものではない。 Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to the following examples.
(1)機器分析及び測定装置
・1H核磁気共鳴スペクトル(1H NMR)
日本電子社製ECA500(500MHz)を使用して測定し、“1H NMR(測定周波数,測定溶媒):ケミカルシフト値(水素の数,多重度,スピン結合定数)”と記載した。ケミカルシフト値(δ)はテトラメチルシラン(δ=0)を内部基準とし、ppmで表記した。多重度は、s(単一線)、d(二重線)、t(三重線)、q(四重線)、m(多重線あるいは複雑に重なったシグナル)で表示し、幅広いシグナルについては、brと付記した。スピン結合定数(J)は、Hzで記載した。
(1) Instrumental Analysis and measurement device · 1 H nuclear magnetic resonance spectra (1 H NMR)
It was measured using ECA500 (500 MHz) manufactured by JEOL Ltd. and described as “ 1 H NMR (measurement frequency, measurement solvent): chemical shift value (number of hydrogen, multiplicity, spin coupling constant)”. The chemical shift value (δ) is expressed in ppm with tetramethylsilane (δ = 0) as an internal standard. The multiplicity is displayed as s (single line), d (double line), t (triple line), q (quadruple line), m (multiple line or complex overlapping signals). It was noted as br. The spin coupling constant (J) is described in Hz.
・13C核磁気共鳴スペクトル(13C NMR)
日本電子社製ECA 500(125MHz)を使用して測定し、“13C NMR(測定周波数,測定溶媒):ケミカルシフト値(多重度)”と記載した。ケミカルシフト値(δ)はテトラメチルシラン(δ=0)を内部基準とし、ppmで表記した。多重度は、s(単一線)、d(二重線)、t(三重線)、q(四重線)、m(多重線あるいは複雑に重なったシグナル)で表示した。
・13 C nuclear magnetic resonance spectrum ( 13 C NMR)
It was measured using ECA 500 (125 MHz) manufactured by JEOL Ltd. and described as “ 13 C NMR (measurement frequency, measurement solvent): chemical shift value (multiplicity)”. The chemical shift value (δ) is expressed in ppm with tetramethylsilane (δ = 0) as an internal standard. The multiplicity was expressed as s (single line), d (double line), t (triple line), q (quadruple line), m (multiple line or complicated overlapping signals).
・質量スペクトル(MS):電子衝撃法(EI)
日本電子社製JMS−600H型質量分析計を用い、電子衝撃法(EI、イオン化エネルギー:70eV)により測定した。“MS(EI)m/z 質量数(相対強度)”と記載した。
Mass spectrum (MS): Electron impact method (EI)
It measured by the electron impact method (EI, ionization energy: 70 eV) using the JMS-600H type | mold mass spectrometer by JEOL. It was described as “MS (EI) m / z mass number (relative intensity)”.
・質量スペクトル(MS):エレクトロンスプレーイオン法(ESI)
日本電子社製JMS−T100LC型TOF質量分析計AccuTOFを用い、エレクトロンスプレーイオン化法(ESI)により測定した。なお、装置の設定は、脱溶媒ガス250℃、オリフィス1温度80℃、ニードル電圧2000V、リングレンズ電圧10V、オリフィス1電圧85V、オリフィス2電圧5Vとした。サンプル送液は、インフュージョン法で行い、流速30μl/minとした。“MS(ESI)m/z 質量数(M+付加イオン)”と記載した。
Mass spectrum (MS): Electron spray ion method (ESI)
It measured by the electron spray ionization method (ESI) using JEOL Co., Ltd. JMS-T100LC type TOF mass spectrometer AccuTOF. The apparatus was set to a solvent removal gas of 250 ° C., an
(2)クロマトグラフィー
・分析用薄層クロマトグラフィー(TLC)
E.Merck社製のTLCプレート、シリカゲル60F254(Art.5715)厚さ0.25mmを使用した。TLC上の化合物の検出はUV照射(254nm又は365nm)及び発色剤に浸した後に加熱して発色させることによって行った。発色剤としてはp−アニスアルデヒド(9.3ml)と酢酸(3.8ml)をエタノール(340ml)に溶解し、濃硫酸(12.5ml)を添加したものを使用した。
(2) Thin-layer chromatography (TLC) for chromatography and analysis
E. A Merck TLC plate, silica gel 60F 254 (Art. 5715) thickness 0.25 mm was used. Detection of the compound on TLC was performed by irradiating with UV irradiation (254 nm or 365 nm) and a color former, followed by heating to cause color development. As the color former, p-anisaldehyde (9.3 ml) and acetic acid (3.8 ml) dissolved in ethanol (340 ml) and concentrated sulfuric acid (12.5 ml) were added.
・分取用薄層クロマトグラフィー(PTLC)
E.Merck社製のTLCプレート、シリカゲル60F254(Art.5744)厚さ0.5mmを用いるか、又はE.Merck社製の薄層クロマトグラフィー用シリカゲル60GF254(Art.7730)を20cm×20cmのガラスプレート上に、厚さ1.75mmに調整したものを使用して行い、“[使用したガラスプレートの縦の長さ×横の長さ×厚さ×枚数;展開溶媒]”と記載した。
・ Preparative thin layer chromatography (PTLC)
E. A Merck TLC plate, silica gel 60F 254 (Art. 5744) 0.5 mm thick is used, or E.I. This is performed using Merck silica gel 60GF 254 (Art. 7730) for thin layer chromatography on a 20 cm × 20 cm glass plate adjusted to a thickness of 1.75 mm. Length × horizontal length × thickness × number of sheets; developing solvent] ”.
・高速液体クロマトグラフィー(HPLC)
Agilent社製の1100seriesを用いて測定した。
・ High performance liquid chromatography (HPLC)
Measurement was performed using Agilent 1100 series.
・シリカゲルカラムクロマトグラフィー
E.Merck社製のシリカゲル60F254(Art.7734)を使用して行い、“使用したシリカゲルの重さ、展開溶媒”と記載した。
・ Silica gel column chromatography This was performed using Merck silica gel 60F 254 (Art. 7734), and was described as “weight of silica gel used, developing solvent”.
(3)基本操作
・反応後の抽出溶液の乾燥は、飽和食塩水にて洗浄後、無水硫酸ナトリウムを加えることで行った。
(3) Drying of the extracted solution after the basic operation and reaction was performed by adding anhydrous sodium sulfate after washing with saturated saline.
・反応後の中和を樹脂で行ったものについては、オルガノ株式会社製陽イオン交換樹脂アンバーライトIR120B NA、又は陰イオン交換樹脂アンバーライトIRA400 OH AGを用いた。 -About what performed neutralization after reaction with resin, the cation exchange resin Amberlite IR120B NA or the anion exchange resin Amberlite IRA400 OH AG made from Organo Corporation was used.
・溶液の減圧濃縮は、アスピレーターの減圧下(20〜30mmHg)、ロータリーエバポレーターを用いて行った。痕跡量の溶媒の除去は、液体窒素浴で冷却したトラップを装着させた真空ポンプ(約1mmHg)を用いて行った。 The solution was concentrated under reduced pressure using a rotary evaporator under reduced pressure of an aspirator (20 to 30 mmHg). The trace amount of the solvent was removed using a vacuum pump (about 1 mmHg) equipped with a trap cooled in a liquid nitrogen bath.
・溶媒の混合比はすべて体積比で表した。 -All the mixing ratios of the solvent were expressed by volume ratio.
(4)溶媒
・蒸留水
アドバンテック東洋株式会社製GS−200型蒸留水製造装置を用いて蒸留、及びイオン交換処理したものを使用した。
(4) Solvent / Distilled Water Distilled and ion-exchanged using a GS-200 type distilled water production apparatus manufactured by Advantech Toyo Co., Ltd. was used.
・トルエン、メタノール、エタノール、イソプロパノール、ジクロロメタン、テトラヒドロフラン、N,N−ジメチルホルムアミド、アセトニトリル
関東化学株式会社製の有機合成用脱水溶媒、又は特級溶媒を、モレキュラーシーブス(4A)を用いて乾燥させて使用した。
・ Toluene, methanol, ethanol, isopropanol, dichloromethane, tetrahydrofuran, N, N-dimethylformamide, acetonitrile Dehydrated solvent for organic synthesis or special grade solvent manufactured by Kanto Chemical Co., Ltd., dried using molecular sieves (4A) did.
・NMR測定用溶媒
以下に示すものをそのまま用いた。
CDCl3:ISOTEC Inc.製 99.7 ATOM%D、0.03% TMS
CD3OD:ISOTEC Inc.製 99.8 ATOM%D(〜0.7 ATOM%13C)、0.05% TMS
-Solvent for NMR measurement The following were used as they were.
CDCl 3 : 99.7 ATOM% D, 0.03% TMS, manufactured by ISOTEC Inc.
CD 3 OD: manufactured by ISOTEC Inc. 99.8 ATOM% D (˜0.7 ATOM% 13 C), 0.05% TMS
<ピリジン環含有発光基質の合成1>
(2−ジメチルアミノ−5−シアノピリジン(1)の合成)
2−アミノ−5−シアノピリジン(0)(1,185mg、10mmol)、ヨードメタン(MeI)(2.83ml、45mmol)をテトラヒドロフラン(THF)(50ml)に溶解させた。この溶液を0℃にし、60%水素化ナトリウム(NaH)(1,437mg、35mmol)をゆっくり加え、混合物を30分間撹拌した。反応混合物を室温に昇温し、12時間撹拌した。反応混合物に少量のメタノールを加え、過剰のヨードメタン、水素化ナトリウムを分解させた。続いて水(10ml)を加え、酢酸エチル(3×30ml)で抽出した。有機層を合わせ、無水硫酸ナトリウムで乾燥後、減圧濃縮した。得られた残渣をカラムクロマトグラフィー[シリカゲル62.9g;ヘキサン−酢酸エチル(1:1)]にて精製すると、2−ジメチルアミノ−5−シアノピリジン(1)(1,121mg、7.62mmol、76.2%)が茶褐色粉末固体として得られた。
(Synthesis of 2-dimethylamino-5-cyanopyridine (1))
2-Amino-5-cyanopyridine (0) (1,185 mg, 10 mmol) and iodomethane (MeI) (2.83 ml, 45 mmol) were dissolved in tetrahydrofuran (THF) (50 ml). The solution was brought to 0 ° C., 60% sodium hydride (NaH) (1,437 mg, 35 mmol) was added slowly and the mixture was stirred for 30 minutes. The reaction mixture was warmed to room temperature and stirred for 12 hours. A small amount of methanol was added to the reaction mixture to decompose excess iodomethane and sodium hydride. Subsequently, water (10 ml) was added and extracted with ethyl acetate (3 × 30 ml). The organic layers were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The obtained residue was purified by column chromatography [silica gel 62.9 g; hexane-ethyl acetate (1: 1)] to give 2-dimethylamino-5-cyanopyridine (1) (1,121 mg, 7.62 mmol, 76.2%) was obtained as a brown powder solid.
・2−ジメチルアミノ−5−シアノピリジン(1)の同定結果
1H NMR(500 MHz, CDCl3):δ 3.15 (6 H, s), 6.48 (1 H, d, J = 9.1 Hz), 7.58 (1 H, dd, J = 9.1, 2.3 Hz), 8.41 (1 H, d, J = 2.3 Hz)
13C NMR(125 MHz, CDCl3):δ 38.0 (q) × 2, 95.3 (d), 105.2 (s), 119.1 (s), 139.4 (s), 152.8 (s), 159.7 (s)
MS(EI) m/z 147 (M+・,97%),132 (100%), 118 (78%)
・ Identification result of 2-dimethylamino-5-cyanopyridine (1)
1 H NMR (500 MHz, CDCl 3 ): δ 3.15 (6 H, s), 6.48 (1 H, d, J = 9.1 Hz), 7.58 (1 H, dd, J = 9.1, 2.3 Hz), 8.41 ( (1 H, d, J = 2.3 Hz)
13 C NMR (125 MHz, CDCl 3 ): δ 38.0 (q) × 2, 95.3 (d), 105.2 (s), 119.1 (s), 139.4 (s), 152.8 (s), 159.7 (s)
MS (EI) m / z 147 (M +・, 97%), 132 (100%), 118 (78%)
(5−アルデヒド−2−ジメチルアミノピリジン(2)の合成)
5−シアノ−2−ジメチルアミノピリジン(1)(1,121mg、7.62mmol)を脱水テトラヒドロフラン(THF)(10ml)に溶解し、アルゴン雰囲気下とした。この溶液を0℃まで冷却し、1.0M水素化ジイソブチルアルミニウム(DIBAL−H)−トルエン溶液(12ml)をゆっくり加え、30分間撹拌した。続いてこの混合溶液を室温に昇温させ、1時間撹拌した。その後この反応混合物に少量のアセトン、水を加え過剰の還元剤を分解させた。さらにこの混合溶液に飽和酒石酸カリウムナトリウム水溶液(50ml)、酢酸エチル(10ml)を加え、一晩攪拌した。その後、酢酸エチル(2×30ml)で抽出した。有機層を合わせ、無水硫酸ナトリウムで乾燥後、減圧濃縮した。得られた残渣をシリカゲルカラムクロマトグラフィー[シリカゲル60g;ヘキサン−酢酸エチル(1:1→0:1)]にて精製すると、5−アルデヒド−2−ジメチルアミノピリジン(2)(699.5mg、4.67mmol、61.0%)が淡黄色粉末固体として得られた。
5-Cyano-2-dimethylaminopyridine (1) (1,121 mg, 7.62 mmol) was dissolved in dehydrated tetrahydrofuran (THF) (10 ml) and placed in an argon atmosphere. The solution was cooled to 0 ° C., 1.0 M diisobutylaluminum hydride (DIBAL-H) -toluene solution (12 ml) was slowly added, and the mixture was stirred for 30 minutes. Subsequently, the mixed solution was warmed to room temperature and stirred for 1 hour. Thereafter, a small amount of acetone and water were added to the reaction mixture to decompose the excess reducing agent. Furthermore, saturated potassium sodium tartrate aqueous solution (50 ml) and ethyl acetate (10 ml) were added to this mixed solution and stirred overnight. Then extracted with ethyl acetate (2 × 30 ml). The organic layers were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography [silica gel 60 g; hexane-ethyl acetate (1: 1 → 0: 1)] to give 5-aldehyde-2-dimethylaminopyridine (2) (699.5 mg, 4 .67 mmol, 61.0%) was obtained as a pale yellow powdered solid.
・5−アルデヒド−2−ジメチルアミノピリジン(2)の同定結果
1H NMR(500 MHz, CDCl3):δ 3.21 (6 H, s), 6.55 (1 H, d, J = 9.2 Hz), 7.91 (1 H, dd, J = 9.2, 2.3 Hz), 8.55 (1 H, d, J = 2.3 Hz), 9.77 (1 H, s)
13C NMR (125 MHz, CDCl3):δ 38.3 (q) × 2, 105.8 (d), 121.8 (s), 136.1 (d), 154.7 (d), 161.5 (s), 189.3 (d)
MS(EI) m/z 150 (M+・, 100%), 135 (74%), 121 (60%)
・ Identification result of 5-aldehyde-2-dimethylaminopyridine (2)
1 H NMR (500 MHz, CDCl 3 ): δ 3.21 (6 H, s), 6.55 (1 H, d, J = 9.2 Hz), 7.91 (1 H, dd, J = 9.2, 2.3 Hz), 8.55 ( 1 H, d, J = 2.3 Hz), 9.77 (1 H, s)
13 C NMR (125 MHz, CDCl 3 ): δ 38.3 (q) × 2, 105.8 (d), 121.8 (s), 136.1 (d), 154.7 (d), 161.5 (s), 189.3 (d)
MS (EI) m / z 150 (M + ・, 100%), 135 (74%), 121 (60%)
(エチルエステル体(3)の合成)
5−アルデヒド−2−ジメチルアミノピリジン(2)(600mg、4mmol)、4−ホスホノクロトン酸トリエチル(1.5ml、12mmol)を、脱水テトラヒドロフラン(THF)(3ml)に溶解し、アルゴン雰囲気下とした。この溶液を0℃まで冷却し、水素化ナトリウム(NaH)(342mg、14.3mmol)をゆっくり加え、30分間撹拌した。この反応混合物に水(10ml)を加えて過剰の水素化ナトリウムを分解し、この溶液を室温に昇温させた。続いて、酢酸エチル(3×30ml)で抽出した。有機層を合わせ、無水硫酸ナトリウムで乾燥後、減圧濃縮した。得られた残渣をシリカゲルカラムクロマトグラフィー[シリカゲル60.1g;ヘキサン−酢酸エチル(2:1)]にて精製すると、エチルエステル体(3)(433mg、1.76mmol、44.0%)が黄色粉末固体として得られた。
5-Aldehyde-2-dimethylaminopyridine (2) (600 mg, 4 mmol) and triethyl 4-phosphonocrotonate (1.5 ml, 12 mmol) were dissolved in dehydrated tetrahydrofuran (THF) (3 ml) and placed under an argon atmosphere. did. The solution was cooled to 0 ° C. and sodium hydride (NaH) (342 mg, 14.3 mmol) was added slowly and stirred for 30 minutes. Water (10 ml) was added to the reaction mixture to decompose excess sodium hydride and the solution was allowed to warm to room temperature. Subsequently, it was extracted with ethyl acetate (3 × 30 ml). The organic layers were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography [silica gel 60.1 g; hexane-ethyl acetate (2: 1)], and the ethyl ester (3) (433 mg, 1.76 mmol, 44.0%) was yellow. Obtained as a powdered solid.
・エチルエステル体(3)の同定結果
1H NMR(500 MHz, CDCl3):δ 1.30 (3 H, t, J = 7.2 Hz), 3.12 (6 H, s), 4.21 (2 H, q, J = 7.2 Hz), 5.88 (1 H, d, J = 14.9 Hz), 6.50 (1 H, d, J = 9.2 Hz), 6.66 (1 H, dd, J = 10.9, 16.0 Hz), 6.79 (1 H, d, J = 16.0 Hz), 7.42 (1 H, dd, J = 14.9, 10.9 Hz), 7.62 (1 H, dd, J = 2.3, 9.2 Hz), 8.19 (1 H, d, J = 2.3 Hz)
13C NMR(125 MHz, CDCl3):δ 14.4 (q), 38.2 (q) × 2, 60.2 (t), 105.9 (d), 119.0 (d), 120.1 (s), 122.4 (d), 134.4 (d), 138.0 (d), 145.3 (d), 148.9 (d), 159.2 (s), 167.4 (s)
MS(ESI) [M+H]+ ; m/z 247.11
・ Identification result of ethyl ester (3)
1 H NMR (500 MHz, CDCl 3 ): δ 1.30 (3 H, t, J = 7.2 Hz), 3.12 (6 H, s), 4.21 (2 H, q, J = 7.2 Hz), 5.88 (1 H , d, J = 14.9 Hz), 6.50 (1 H, d, J = 9.2 Hz), 6.66 (1 H, dd, J = 10.9, 16.0 Hz), 6.79 (1 H, d, J = 16.0 Hz), 7.42 (1 H, dd, J = 14.9, 10.9 Hz), 7.62 (1 H, dd, J = 2.3, 9.2 Hz), 8.19 (1 H, d, J = 2.3 Hz)
13 C NMR (125 MHz, CDCl 3 ): δ 14.4 (q), 38.2 (q) × 2, 60.2 (t), 105.9 (d), 119.0 (d), 120.1 (s), 122.4 (d), 134.4 (d), 138.0 (d), 145.3 (d), 148.9 (d), 159.2 (s), 167.4 (s)
MS (ESI) [M + H] + ; m / z 247.11
(カルボキシル体(4)の合成)
エチルエステル体(3)(340mg、1.38mmol)をイソプロパノール(iPrOH)(10ml)に溶解し、5.0M水酸化ナトリウム水溶液(560μl)を加え、その溶液を5時間加熱還流した。反応混合物に5.0M塩酸(560μl)を加え中和した後、THF(3×50ml)で抽出した。有機層を合わせ、無水硫酸ナトリウムで乾燥後、減圧濃縮した。カルボキシル体(4)(240.2mg、1.10mmol、79.7%)が薄黄色固体として得られた。
The ethyl ester (3) (340 mg, 1.38 mmol) was dissolved in isopropanol (iPrOH) (10 ml), 5.0 M aqueous sodium hydroxide solution (560 μl) was added, and the solution was heated to reflux for 5 hours. The reaction mixture was neutralized with 5.0 M hydrochloric acid (560 μl), and extracted with THF (3 × 50 ml). The organic layers were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The carboxyl body (4) (240.2 mg, 1.10 mmol, 79.7%) was obtained as a pale yellow solid.
・カルボキシル体(4)の同定結果
1H NMR(500 MHz, CDCl3):δ 3.13 (6 H, s), 5.88 (1 H, d, J = 15.4 Hz), 6.51 (1 H, d, J = 9.2 Hz), 6.69 (1 H, dd, J = 11.4, 15.2 Hz), 6.83 (1 H, d, J = 15.4 Hz), 7.50 (1 H, dd, J = 11.4, 15.2 Hz), 7.63 (1 H, dd, J = 2.3, 9.2 Hz), 8.20 (1 H, d, J = 2.3 Hz)
13C NMR(125MHz, DMSO-D6):δ 38.1(q) × 2, 106.6(d), 120.1(d), 122.9(s), 135.0(d), 138.0(d), 138.3(d), 145.6(d), 149.1(d), 159.3(s), 168.3(s)
MS(ESI) [M+H]+ ; m/z 219.08
・ Identification result of carboxyl body (4)
1 H NMR (500 MHz, CDCl 3 ): δ 3.13 (6 H, s), 5.88 (1 H, d, J = 15.4 Hz), 6.51 (1 H, d, J = 9.2 Hz), 6.69 (1 H , dd, J = 11.4, 15.2 Hz), 6.83 (1 H, d, J = 15.4 Hz), 7.50 (1 H, dd, J = 11.4, 15.2 Hz), 7.63 (1 H, dd, J = 2.3, 9.2 Hz), 8.20 (1 H, d, J = 2.3 Hz)
13 C NMR (125 MHz, DMSO-D6): δ 38.1 (q) × 2, 106.6 (d), 120.1 (d), 122.9 (s), 135.0 (d), 138.0 (d), 138.3 (d), 145.6 (d), 149.1 (d), 159.3 (s), 168.3 (s)
MS (ESI) [M + H] + ; m / z 219.08
(アミド体(5)の合成)
カルボキシル体(4)(126mg、0.58mmol)とS−トリチル−D−システインメチルエステル(261mg、0.70mmol)をN,N−ジメチルホルムアミド(DMF)(5.0ml)に溶解した。この溶液に1−エチル−3−(3−ジメチルアミノプロピル)カルボジイミド塩酸塩(EDC)(278mg、1.45mmol)、N,N−ジメチル−4−アミノピリジン(DMAP)(141mg、1.15mmol)を加え、この反応混合物を室温で15時間撹拌した。反応混合物に水(50ml)を加え、酢酸エチル(3×50ml)で抽出した。有機層を合わせ、無水硫酸ナトリウムで乾燥後、減圧濃縮した。得られた残渣をシリカゲルカラムクロマトグラフィー[シリカゲル50g;酢酸エチル−ヘキサン(1:1)]にて精製すると、アミド体(5)(264mg、0.46mmol、79.0%)が黄色固体として得られた。
Carboxyl derivative (4) (126 mg, 0.58 mmol) and S-trityl-D-cysteine methyl ester (261 mg, 0.70 mmol) were dissolved in N, N-dimethylformamide (DMF) (5.0 ml). To this solution was added 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) (278 mg, 1.45 mmol), N, N-dimethyl-4-aminopyridine (DMAP) (141 mg, 1.15 mmol). And the reaction mixture was stirred at room temperature for 15 hours. Water (50 ml) was added to the reaction mixture and extracted with ethyl acetate (3 × 50 ml). The organic layers were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography [silica gel 50 g; ethyl acetate-hexane (1: 1)] to obtain amide compound (5) (264 mg, 0.46 mmol, 79.0%) as a yellow solid. It was.
・アミド体(5)の同定結果
1H NMR(500 MHz, CDCl3):δ 2.69 (2 H, m), 3.13 (6 H, s), 3.70 (3 H, s), 4.73 (1 H, m), 5.84 (1 H, d, J = 15.0 Hz), 6.50 (1 H, d, J = 9.2 Hz), 6.64 (1 H, dd, J = 10.9, 15.5 Hz), 6.76 (1 H, d, J = 15.5 Hz), 7.18-7.38 (17 H, comp), 7.60 (1 H, dd, J = 2.3, 9.2 Hz), 8.19 (1 H, d, J = 2.3 Hz)
13C NMR(125 MHz, CDCl3):δ 34.2 (t), 38.2 (q) × 2, 51.2 (q), 52.7 (d), 67.0 (s), 105.9 (d), 120.3 (s), 121.0 (d), 122.4 (d), 127.0 (d), 128.1 (d) × 2, 129.6 (d) × 2, 134.5 (d), 137.4 (d), 142.5 (d), 144.4 (s), 148.6 (d), 159.1 (s), 165.8 (s), 171.2 (s)
MS(ESI) [(M+H)+] m/z : 578.22
・ Identification result of amide (5)
1 H NMR (500 MHz, CDCl 3 ): δ 2.69 (2 H, m), 3.13 (6 H, s), 3.70 (3 H, s), 4.73 (1 H, m), 5.84 (1 H, d , J = 15.0 Hz), 6.50 (1 H, d, J = 9.2 Hz), 6.64 (1 H, dd, J = 10.9, 15.5 Hz), 6.76 (1 H, d, J = 15.5 Hz), 7.18- 7.38 (17 H, comp), 7.60 (1 H, dd, J = 2.3, 9.2 Hz), 8.19 (1 H, d, J = 2.3 Hz)
13 C NMR (125 MHz, CDCl 3 ): δ 34.2 (t), 38.2 (q) × 2, 51.2 (q), 52.7 (d), 67.0 (s), 105.9 (d), 120.3 (s), 121.0 (d), 122.4 (d), 127.0 (d), 128.1 (d) × 2, 129.6 (d) × 2, 134.5 (d), 137.4 (d), 142.5 (d), 144.4 (s), 148.6 ( d), 159.1 (s), 165.8 (s), 171.2 (s)
MS (ESI) [(M + H) + ] m / z: 578.22
(チアゾリン体(6)の合成)
アミド体(5)(160mg、0.28mmol)をアルゴン雰囲気下にし、ジクロロメタン(5.0ml)を加えた。この溶液を0℃に冷却し、トリフルオロ酢酸無水物(TFAA)(0.2ml、1.44mmol)をゆっくり加え、30分間撹拌した。続いて室温に昇温させ、24時間撹拌した。原料が消失したのを確認した後、陰イオン交換樹脂IRA400 OH AGを用いて中和した。樹脂を洗浄して濾別し、得られた洗液を減圧濃縮した。得られた残渣を分取薄層クロマトグラフィー[20cm×20cm×1.75mm×3枚;ヘキサン−酢酸エチル(1:1)]で精製すると、チアゾリン体(6)(13.4mg、0.042mmol、15.0%)が黄色固体として得られた。
The amide compound (5) (160 mg, 0.28 mmol) was placed under an argon atmosphere, and dichloromethane (5.0 ml) was added. The solution was cooled to 0 ° C. and trifluoroacetic anhydride (TFAA) (0.2 ml, 1.44 mmol) was added slowly and stirred for 30 minutes. Then, it heated up to room temperature and stirred for 24 hours. After confirming the disappearance of the raw material, it was neutralized using an anion exchange resin IRA400 OH AG. The resin was washed and filtered off, and the resulting wash was concentrated under reduced pressure. The obtained residue was purified by preparative thin layer chromatography [20 cm × 20 cm × 1.75 mm × 3; hexane-ethyl acetate (1: 1)] to obtain thiazoline (6) (13.4 mg, 0.042 mmol). 15.0%) was obtained as a yellow solid.
・チアゾリン体(6)の同定結果
1H NMR(500 MHz, CDCl3):δ 3.11 (6 H, s), 3.56 (2 H, m), 3.81 (3 H, s), 5.15 (1 H, t, J = 9.2 Hz), 6.49 (1 H, d, J = 9.2 Hz), 6.53 (1 H, d, J = 14.9 Hz), 6.72 (1 H, d, J = 15.5 Hz), 6.92 (1 H, dd, J = 9.8, 15.5 Hz), 7.37 (1 H, dd, J = 9.8, 14.9 Hz), 7.61 (1 H, dd, J = 2.3, 9.2 Hz), 8.16 (1 H, d, J = 2.3 Hz)
13C NMR(125 MHz, CDCl3):δ 34.6 (t), 38.2 (q) × 2, 52.8 (q), 77.9 (d), 106.0 (d), 120.2 (s), 123.1 (d), 123.3 (d), 134.1 (d), 136.3 (d), 143.3 (d), 148.8 (d), 159.0 (s), 170.2 (s), 171.4 (s)
MS(ESI) m/z: [(M+H)+] 318.08
・ Identification result of thiazoline (6)
1 H NMR (500 MHz, CDCl 3 ): δ 3.11 (6 H, s), 3.56 (2 H, m), 3.81 (3 H, s), 5.15 (1 H, t, J = 9.2 Hz), 6.49 (1 H, d, J = 9.2 Hz), 6.53 (1 H, d, J = 14.9 Hz), 6.72 (1 H, d, J = 15.5 Hz), 6.92 (1 H, dd, J = 9.8, 15.5 Hz), 7.37 (1 H, dd, J = 9.8, 14.9 Hz), 7.61 (1 H, dd, J = 2.3, 9.2 Hz), 8.16 (1 H, d, J = 2.3 Hz)
13 C NMR (125 MHz, CDCl 3 ): δ 34.6 (t), 38.2 (q) × 2, 52.8 (q), 77.9 (d), 106.0 (d), 120.2 (s), 123.1 (d), 123.3 (d), 134.1 (d), 136.3 (d), 143.3 (d), 148.8 (d), 159.0 (s), 170.2 (s), 171.4 (s)
MS (ESI) m / z: [(M + H) + ] 318.08
(ピリジン環含有発光基質(7)の合成)
チアゾリン体(6)(10.7mg、0.034mmol)をテトラヒドロフラン(THF)(5ml)に溶解し、5.0M塩酸(300μl)を加え、その溶液を24時間室温で撹拌した。反応混合物に重曹を加え中和した後、酢酸エチル(3×50ml)で抽出した。ピリジン環含有発光基質(7)[即ち、上記化学式(II)で表される化合物](9.2mg、0.030mmol、88.2%)が粗生成物として得られた。
The thiazoline (6) (10.7 mg, 0.034 mmol) was dissolved in tetrahydrofuran (THF) (5 ml), 5.0 M hydrochloric acid (300 μl) was added, and the solution was stirred for 24 hours at room temperature. The reaction mixture was neutralized with sodium bicarbonate, and extracted with ethyl acetate (3 × 50 ml). A pyridine ring-containing luminescent substrate (7) [that is, a compound represented by the above chemical formula (II)] (9.2 mg, 0.030 mmol, 88.2%) was obtained as a crude product.
・発光基質(7)の同定結果
1H NMR(500 MHz, CD3OD):δ 3.29 (6 H, s), 3.76-3.68 (2 H, comp.), 5.25 (1 H, m), 6.69 (1 H, d, J = 15.5 Hz), 7.10 (1 H, dd, J = 10.9, 15.5 Hz), 7.22 (1 H, d, J = 9.8 Hz), 8.05 (1 H, s), 7.23 (1 H, m), 8.25 (1 H, d, J = 9.7 Hz)
13C NMR(125 MHz, CD3CD):δ 35.5 (t), 37.2 (q) × 2, 80.8 (d), 106.5 (d), 120.7 (d), 123.3 (d), 123.6 (d), 134.4 (s), 135.3 (d), 142.5 (d), 147.7 (d), 158.9 (s), 169.5 (s), 176.9 (s)
MS(ESI) m/z: [(M+H)+] 304.10
・ Identification result of luminescent substrate (7)
1 H NMR (500 MHz, CD 3 OD): δ 3.29 (6 H, s), 3.76-3.68 (2 H, comp.), 5.25 (1 H, m), 6.69 (1 H, d, J = 15.5 Hz), 7.10 (1 H, dd, J = 10.9, 15.5 Hz), 7.22 (1 H, d, J = 9.8 Hz), 8.05 (1 H, s), 7.23 (1 H, m), 8.25 (1 (H, d, J = 9.7 Hz)
13 C NMR (125 MHz, CD 3 CD): δ 35.5 (t), 37.2 (q) × 2, 80.8 (d), 106.5 (d), 120.7 (d), 123.3 (d), 123.6 (d), 134.4 (s), 135.3 (d), 142.5 (d), 147.7 (d), 158.9 (s), 169.5 (s), 176.9 (s)
MS (ESI) m / z: [(M + H) + ] 304.10
<ピリジン環含有発光基質の合成2>
(2−シアノ−5−ジメチルアミノピリジン(11)の合成)
5−アミノ−2−シアノピリジン(10)(1,308mg、11mmol)、ヨードメタン(CH3I)(2.0ml、33mmol)をテトラヒドロフラン(THF)(100ml)に溶解させた。この溶液を0℃にし、60%水素化ナトリウム(NaH)(1308 mg、33mmol)をゆっくり加え、混合物を50時間撹拌した。反応混合物に少量のメタノールを加え、過剰のヨードメタン、水素化ナトリウムを分解させた。続いて水(10ml)を加え、酢酸エチル(3×30ml)で抽出した。有機層を合わせ、無水硫酸ナトリウムで乾燥後、減圧濃縮した。得られた残渣をカラムクロマトグラフィー[シリカゲル59.2g;ヘキサン−酢酸エチル(1:2)]にて精製すると、2−シアノ−5−ジメチルアミノピリジン(11)(1,303.8mg、8.86mmol、80%)が茶褐色粉末固体として得られた。
(Synthesis of 2-cyano-5-dimethylaminopyridine (11))
5-Amino-2-cyanopyridine (10) (1,308 mg, 11 mmol) and iodomethane (CH 3 I) (2.0 ml, 33 mmol) were dissolved in tetrahydrofuran (THF) (100 ml). The solution was brought to 0 ° C., 60% sodium hydride (NaH) (1308 mg, 33 mmol) was added slowly and the mixture was stirred for 50 h. A small amount of methanol was added to the reaction mixture to decompose excess iodomethane and sodium hydride. Subsequently, water (10 ml) was added and extracted with ethyl acetate (3 × 30 ml). The organic layers were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The obtained residue was purified by column chromatography [silica gel 59.2 g; hexane-ethyl acetate (1: 2)] to give 2-cyano-5-dimethylaminopyridine (11) (1,303.8 mg, 8. 86 mmol, 80%) was obtained as a brown powder solid.
・2−シアノ−5−ジメチルアミノピリジン(11)の同定結果
1H NMR(500 MHz, CDCl3):δ 3.08 (6 H, s), 6.87 (1 H, dd, J = 9.2, 2.9 Hz), 7.48 (1 H, d, J = 9.2 Hz), 8.12 (1 H, d, J = 2.9 Hz)
13C NMR(125 MHz, CDCl3):δ 39.8 (q) × 2, 116.5 (s), 119.0 (d), 119.1 (s), 129.1 (d), 135.3 (d), 147.2 (s)
MS(EI) m/z 146 (M+・, 100%), 147 (81%)
・ Identification result of 2-cyano-5-dimethylaminopyridine (11)
1 H NMR (500 MHz, CDCl 3 ): δ 3.08 (6 H, s), 6.87 (1 H, dd, J = 9.2, 2.9 Hz), 7.48 (1 H, d, J = 9.2 Hz), 8.12 ( (1 H, d, J = 2.9 Hz)
13 C NMR (125 MHz, CDCl 3 ): δ 39.8 (q) × 2, 116.5 (s), 119.0 (d), 119.1 (s), 129.1 (d), 135.3 (d), 147.2 (s)
MS (EI) m / z 146 (M + ・, 100%), 147 (81%)
(2−アルデヒド−5−ジメチルアミノピリジン(12)の合成)
2−シアノ−5−ジメチルアミノピリジン(11)(723.0mg、4.9mmol)を脱水テトラヒドロフラン(THF)(20ml)に溶解し、アルゴン雰囲気下とした。この溶液を0℃まで冷却し、1.0M水素化ジイソブチルアルミニウム(DIBAL−H)−トルエン溶液(7.5ml)をゆっくり加え、15分間撹拌した。続いてこの混合溶液を室温に昇温させ、1時間撹拌した。その後この反応混合物に十分量のアセトン、水を加え過剰の還元剤を分解させた。さらにこの混合溶液に飽和酒石酸カリウムナトリウム水溶液(50ml)、酢酸エチル(10ml)を加え、一晩攪拌した。その後、酢酸エチル(2×30ml)で抽出した。有機層を合わせ、無水硫酸ナトリウムで乾燥後、減圧濃縮した。得られた残渣をシリカゲルカラムクロマトグラフィー[シリカゲル56.9g;クロロホルム−メタノール(10:1)]にて精製すると、2−アルデヒド−5−ジメチルアミノピリジン(12)(368.7mg、2.46mmol、50%)が淡黄色粉末固体として得られた。
2-Cyano-5-dimethylaminopyridine (11) (723.0 mg, 4.9 mmol) was dissolved in dehydrated tetrahydrofuran (THF) (20 ml) and placed in an argon atmosphere. The solution was cooled to 0 ° C., 1.0 M diisobutylaluminum hydride (DIBAL-H) -toluene solution (7.5 ml) was slowly added, and the mixture was stirred for 15 minutes. Subsequently, the mixed solution was warmed to room temperature and stirred for 1 hour. Thereafter, sufficient amounts of acetone and water were added to the reaction mixture to decompose excess reducing agent. Furthermore, saturated potassium sodium tartrate aqueous solution (50 ml) and ethyl acetate (10 ml) were added to this mixed solution and stirred overnight. Then extracted with ethyl acetate (2 × 30 ml). The organic layers were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography [silica gel 56.9 g; chloroform-methanol (10: 1)] to give 2-aldehyde-5-dimethylaminopyridine (12) (368.7 mg, 2.46 mmol, 50%) was obtained as a pale yellow powdered solid.
・2−アルデヒド−5−ジメチルアミノピリジン(12)の同定結果
1H NMR(500 MHz, CDCl3):δ 3.13 (6 H, s), 6.96 (1 H, dd, J = 8.6, 2.9 Hz), 7.86 (1 H, d, J = 8.6 Hz), 8.19 (1 H, d, J = 2.9 Hz), 9.88 (1 H, s)
13C NMR (125 MHz, CDCl3):δ 39.7 (q) × 2, 116.5 (d), 123.4 (d), 133.9 (d), 141.5 (s), 148.2 (s), 191.5(s)
MS(EI) m/z 150 (M+・, 100%)
・ Identification result of 2-aldehyde-5-dimethylaminopyridine (12)
1 H NMR (500 MHz, CDCl 3 ): δ 3.13 (6 H, s), 6.96 (1 H, dd, J = 8.6, 2.9 Hz), 7.86 (1 H, d, J = 8.6 Hz), 8.19 ( 1 H, d, J = 2.9 Hz), 9.88 (1 H, s)
13 C NMR (125 MHz, CDCl 3 ): δ 39.7 (q) × 2, 116.5 (d), 123.4 (d), 133.9 (d), 141.5 (s), 148.2 (s), 191.5 (s)
MS (EI) m / z 150 (M + ・, 100%)
(エチルエステル体(13)の合成)
2−アルデヒド−5−ジメチルアミノピリジン(12)(31.4mg、0.21mmol)、4−ホスホノクロトン酸トリエチル(80μl、0.36mmol)を、脱水テトラヒドロフラン(THF)(10ml)に溶解し、アルゴン雰囲気下とした。この溶液を0℃まで冷却し、60%水素化ナトリウム(NaH)(21.6mg、0.54mmol)をゆっくり加え、30分間撹拌した。この反応混合物に水(10ml)を加えて過剰の水素化ナトリウムを分解し、この溶液を室温に昇温させた。続いて、酢酸エチル(3×30ml)で抽出した。有機層を合わせ、無水硫酸ナトリウムで乾燥後、減圧濃縮した。得られた残渣を分取薄層クロマトグラフィー[20cm×20cm×1.75mm×2枚;ヘキサン−酢酸エチル(1:1)]にて精製すると、エチルエステル体(13)(38.7mg、0.157mmol、74%)が黄色粉末固体として得られた。
2-aldehyde-5-dimethylaminopyridine (12) (31.4 mg, 0.21 mmol), triethyl 4-phosphonocrotonate (80 μl, 0.36 mmol) was dissolved in dehydrated tetrahydrofuran (THF) (10 ml), Under an argon atmosphere. The solution was cooled to 0 ° C. and 60% sodium hydride (NaH) (21.6 mg, 0.54 mmol) was added slowly and stirred for 30 minutes. Water (10 ml) was added to the reaction mixture to decompose excess sodium hydride and the solution was allowed to warm to room temperature. Subsequently, it was extracted with ethyl acetate (3 × 30 ml). The organic layers were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The obtained residue was purified by preparative thin layer chromatography [20 cm × 20 cm × 1.75 mm × 2 sheets; hexane-ethyl acetate (1: 1)] to give an ethyl ester (13) (38.7 mg, 0 .157 mmol, 74%) was obtained as a yellow powdered solid.
・エチルエステル体(13)の同定結果
1H NMR(500 MHz, CDCl3):δ 1.31 (3 H, t, J = 7.5 Hz), 3.03 (6 H, s), 4.21 (2 H, q, J = 6.8 Hz), 5.98 (1 H, d, J = 14.9 Hz), 6.87 (1 H, d, J = 15.5 Hz), 6.90 (1 H, dd, J = 8.6, 2.8 Hz), 7.12 (1 H, dd, J = 11.4, 15.5 Hz), 7.22 (1 H, d, J = 8.6 Hz), 7.46 (1 H, dd, J = 11.5, 15.5 Hz), 8.14 (1 H, d, J = 2.8 Hz)
13C NMR(125 MHz, CDCl3):δ 14.4 (q), 40.0 (q) × 2, 60.3 (t), 118.0 (d), 120.7 (d), 123.8 (d), 125.4 (d), 135.0 (d), 139.8 (d), 142.3 (s), 144.9 (s), 145.6 (d), 167.4 (s)
MS(ESI) [M+H]+ ; m/z 247.15
・ Identification result of ethyl ester (13)
1 H NMR (500 MHz, CDCl 3 ): δ 1.31 (3 H, t, J = 7.5 Hz), 3.03 (6 H, s), 4.21 (2 H, q, J = 6.8 Hz), 5.98 (1 H , d, J = 14.9 Hz), 6.87 (1 H, d, J = 15.5 Hz), 6.90 (1 H, dd, J = 8.6, 2.8 Hz), 7.12 (1 H, dd, J = 11.4, 15.5 Hz ), 7.22 (1 H, d, J = 8.6 Hz), 7.46 (1 H, dd, J = 11.5, 15.5 Hz), 8.14 (1 H, d, J = 2.8 Hz)
13 C NMR (125 MHz, CDCl 3 ): δ 14.4 (q), 40.0 (q) × 2, 60.3 (t), 118.0 (d), 120.7 (d), 123.8 (d), 125.4 (d), 135.0 (d), 139.8 (d), 142.3 (s), 144.9 (s), 145.6 (d), 167.4 (s)
MS (ESI) [M + H] + ; m / z 247.15
(カルボキシル体(14)の合成)
エチルエステル体(13)(54.0mg、0.22mmol)をイソプロパノール(iPrOH)(10ml)に溶解し、5M水酸化ナトリウム水溶液(130μl)を加え、その溶液を26時間加熱還流した。反応混合物に4M塩酸を加え中和した後、減圧濃縮した。カルボキシル体(14)(240.2mg、1.10mmol、79.7%)が薄黄色個体として得られた。得られた残渣をシリカゲルカラムクロマトグラフィー[シリカゲル11.9g;クロロホルム−メタノール(5:1)]にて精製すると、カルボキシル体(14)(50.0mg、0.23mmol、115%)が淡緑色粉末固体として得られた。
The ethyl ester compound (13) (54.0 mg, 0.22 mmol) was dissolved in isopropanol (iPrOH) (10 ml), 5M aqueous sodium hydroxide solution (130 μl) was added, and the solution was heated to reflux for 26 hours. The reaction mixture was neutralized with 4M hydrochloric acid, and concentrated under reduced pressure. The carboxyl body (14) (240.2 mg, 1.10 mmol, 79.7%) was obtained as a pale yellow solid. When the obtained residue was purified by silica gel column chromatography [silica gel 11.9 g; chloroform-methanol (5: 1)], the carboxyl form (14) (50.0 mg, 0.23 mmol, 115%) was a pale green powder. Obtained as a solid.
・カルボキシル体(14)の同定結果
1H NMR(500 MHz, CD3OD):δ 3.02 (6 H, s), 5.99 (1 H, d, J = 15.4 Hz), 6.86 (1 H, d, J = 15.5 Hz), 7.06 (1 H, dd, J = 11.5, 15.4 Hz), 7.12 (1 H, dd, J = 2.9, 8.6 Hz), 7.37 (1 H, dd, J = 11.5, 15.4 Hz), 7.45 (1 H, d, J = 8.6 Hz), 8.00 (1 H, d, J = 2.9 Hz)
13C NMR(125MHz, CD3OD):δ 38.6 (q) × 2, 118.9 (d), 123.1 (d), 123.6 (d), 126.1 (d), 133.2 (d), 137.8 (d), 142.0 (s), 143.5 (s), 146.0 (d), 172.3 (s)
MS(ESI) [M+H]+ ; m/z 219.11
・ Identification result of carboxyl body (14)
1 H NMR (500 MHz, CD 3 OD): δ 3.02 (6 H, s), 5.99 (1 H, d, J = 15.4 Hz), 6.86 (1 H, d, J = 15.5 Hz), 7.06 (1 H, dd, J = 11.5, 15.4 Hz), 7.12 (1 H, dd, J = 2.9, 8.6 Hz), 7.37 (1 H, dd, J = 11.5, 15.4 Hz), 7.45 (1 H, d, J = 8.6 Hz), 8.00 (1 H, d, J = 2.9 Hz)
13 C NMR (125 MHz, CD 3 OD): δ 38.6 (q) × 2, 118.9 (d), 123.1 (d), 123.6 (d), 126.1 (d), 133.2 (d), 137.8 (d), 142.0 (s), 143.5 (s), 146.0 (d), 172.3 (s)
MS (ESI) [M + H] + ; m / z 219.11
(アミド体(15)の合成)
カルボキシル体(14)(104.8mg、0.48mmol)とS−トリチル−D−システインメチルエステル(386.8mg、1.06mmol)をN,N−ジメチルホルムアミド(DMF)(50ml)に溶解した。この溶液に1−エチル−3−(3−ジメチルアミノプロピル)カルボジイミド塩酸塩(EDC)(300.6mg、1.57mmol)、N,N−ジメチル−4−アミノピリジン(DMAP)(295.1mg、2.41mmol)を加え、この反応混合物を室温で2時間撹拌した。反応混合物に水(50ml)を加え、酢酸エチル(3×50ml)で抽出した。有機層を合わせ、無水硫酸ナトリウムで乾燥後、減圧濃縮した。得られた残渣をシリカゲルカラムクロマトグラフィー[シリカゲル50.5g;クロロホルム−メタノール(10:1)]にて精製したのち、さらに、分取薄層クロマトグラフィー[20cm×20cm×1.75mm×4枚;クロロホルム−メタノール(10:1)]にて精製すると、アミド体(15)(264mg、0.46mmol、79%)が黄色固体として得られた。
Carboxyl compound (14) (104.8 mg, 0.48 mmol) and S-trityl-D-cysteine methyl ester (386.8 mg, 1.06 mmol) were dissolved in N, N-dimethylformamide (DMF) (50 ml). To this solution was added 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) (300.6 mg, 1.57 mmol), N, N-dimethyl-4-aminopyridine (DMAP) (295.1 mg, 2.41 mmol) was added and the reaction mixture was stirred at room temperature for 2 hours. Water (50 ml) was added to the reaction mixture and extracted with ethyl acetate (3 × 50 ml). The organic layers were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography [silica gel 50.5 g; chloroform-methanol (10: 1)], and further preparative thin layer chromatography [20 cm × 20 cm × 1.75 mm × 4 pieces; Purification with chloroform-methanol (10: 1)] gave the amide compound (15) (264 mg, 0.46 mmol, 79%) as a yellow solid.
・アミド体(15)の同定結果
1H NMR(500 MHz, CDCl3):δ 2.70 (2 H, m), 3.02 (6 H, s), 4.73 (1 H, m), 5.91 (1 H, d, J = 14.9 Hz), 5.96 (1 H, d, J = 8.0 Hz), 6.49 (1 H, m), 6.84 (1 H, d, J = 15.4 Hz), 6.90 (1H, dd, J = 2.9 Hz, 8.6 Hz), 7.12 (1 H, dd, J = 11.5, 15.4 Hz), 7.25-7.39 (15 H, m), 8.14 (1 H, d, J = 2.8 Hz)
13C NMR(125 MHz, CDCl3):δ 34.1 (t), 40.1 (q) × 2, 51.2 (s), 52.8 (d), 67.1 (s), 118.1 (d), 122.6 (d), 123.8 (d), 125.5 (d), 127.0 (t) × 3, 128.1 (d) × 6, 129.6 (d) × 6, 135.0 (d), 139.1 (n), 142.2 (d), 144.4 (s) × 3, 145.5 (s) × 3, 149.4 (s), 165.7 (s), 171.1 (s)
MS(ESI) [(M+H)+] m/z : 578.22
・ Identification result of amide compound (15)
1 H NMR (500 MHz, CDCl 3 ): δ 2.70 (2 H, m), 3.02 (6 H, s), 4.73 (1 H, m), 5.91 (1 H, d, J = 14.9 Hz), 5.96 (1 H, d, J = 8.0 Hz), 6.49 (1 H, m), 6.84 (1 H, d, J = 15.4 Hz), 6.90 (1H, dd, J = 2.9 Hz, 8.6 Hz), 7.12 ( 1 H, dd, J = 11.5, 15.4 Hz), 7.25-7.39 (15 H, m), 8.14 (1 H, d, J = 2.8 Hz)
13 C NMR (125 MHz, CDCl 3 ): δ 34.1 (t), 40.1 (q) × 2, 51.2 (s), 52.8 (d), 67.1 (s), 118.1 (d), 122.6 (d), 123.8 (d), 125.5 (d), 127.0 (t) × 3, 128.1 (d) × 6, 129.6 (d) × 6, 135.0 (d), 139.1 (n), 142.2 (d), 144.4 (s) × 3, 145.5 (s) × 3, 149.4 (s), 165.7 (s), 171.1 (s)
MS (ESI) [(M + H) + ] m / z: 578.22
(チアゾリン体(16)の合成)
アミド体(15)(145.1mg、0.25mmol)をアルゴン雰囲気下にし、ジクロロメタン(10.0ml)を加えた。この溶液を0℃に冷却し、トリフルオロメタンスルホン酸無水物(Tf2O)(105μl、0.62mmol)をゆっくり加え、50分間撹拌した。原料が消失したのを確認した後、陰イオン交換樹脂IRA400 OH AGを用いて中和した。樹脂を洗浄して濾別し、得られた洗液を減圧濃縮した。得られた残渣を分取薄層クロマトグラフィー[20cm×20cm×1.75mm×2枚;酢酸エチル]で精製すると、チアゾリン体(16)(15.6mg、0.049mmol、20%)が黄色油状物として得られた。
The amide compound (15) (145.1 mg, 0.25 mmol) was placed under an argon atmosphere, and dichloromethane (10.0 ml) was added. The solution was cooled to 0 ° C. and trifluoromethanesulfonic anhydride (Tf 2 O) (105 μl, 0.62 mmol) was added slowly and stirred for 50 minutes. After confirming the disappearance of the raw material, it was neutralized using an anion exchange resin IRA400 OH AG. The resin was washed and filtered off, and the resulting wash was concentrated under reduced pressure. The obtained residue was purified by preparative thin layer chromatography [20 cm × 20 cm × 1.75 mm × 2; ethyl acetate] to give thiazoline derivative (16) (15.6 mg, 0.049 mmol, 20%) as a yellow oil It was obtained as a product.
・チアゾリン体(16)の同定結果
1H NMR(500 MHz, CD3OD):δ 3.05 (6 H, s), 3.56 (2 H, m), 3.82 (3 H, s), 5.16 (1 H, t, J = 9.2 Hz), 6.64 (1 H, d, J = 15.5 Hz), 6.79 (1 H, d, J = 15.5 Hz), 6.89 (1 H, dd, J = 2.9, 8.6 Hz), 6.95 (1 H, dd, J = 10.9, 15.5 Hz), 7.13 (1 H, dd, J = 10.9, 15.5 Hz), 7.20 (1 H, d, J = 8.6 Hz), 8.12 (1 H, d, J = 2.9 Hz)
13C NMR(125 MHz, CDCl3):δ 34.6 (t), 40.0 (q) × 2, 52.9 (q), 78.0 (d), 118.1 (d), 123.7 (d), 125.1 (d), 126.3 (d), 135.0 (d), 138.1 (d), 142.6 (s), 142.9 (s), 145.5 (d), 170.2 (s), 171.5 (s)
MS(ESI) m/z: [(M+H)+] 318.12
・ Identification result of thiazoline (16)
1 H NMR (500 MHz, CD 3 OD): δ 3.05 (6 H, s), 3.56 (2 H, m), 3.82 (3 H, s), 5.16 (1 H, t, J = 9.2 Hz), 6.64 (1 H, d, J = 15.5 Hz), 6.79 (1 H, d, J = 15.5 Hz), 6.89 (1 H, dd, J = 2.9, 8.6 Hz), 6.95 (1 H, dd, J = 10.9, 15.5 Hz), 7.13 (1 H, dd, J = 10.9, 15.5 Hz), 7.20 (1 H, d, J = 8.6 Hz), 8.12 (1 H, d, J = 2.9 Hz)
13 C NMR (125 MHz, CDCl 3 ): δ 34.6 (t), 40.0 (q) × 2, 52.9 (q), 78.0 (d), 118.1 (d), 123.7 (d), 125.1 (d), 126.3 (d), 135.0 (d), 138.1 (d), 142.6 (s), 142.9 (s), 145.5 (d), 170.2 (s), 171.5 (s)
MS (ESI) m / z: [(M + H) + ] 318.12
(ピリジン環含有発光基質(17)の合成)
チアゾリン体(16)(15.6mg、0.049mmol)をH2O(1ml)に溶解し、4M塩酸(1ml)を加え、その溶液を40時間室温で撹拌した。反応混合物に重曹を加え中和した後、減圧濃縮した。ピリジン環含有発光基質(17)[即ち、上記化学式(III)で表される化合物](3mg、0.01mmol、20%)が粗生成物として得られた。
The thiazoline derivative (16) (15.6 mg, 0.049 mmol) was dissolved in H 2 O (1 ml), 4M hydrochloric acid (1 ml) was added, and the solution was stirred at room temperature for 40 hours. The reaction mixture was neutralized with sodium bicarbonate, and then concentrated under reduced pressure. A pyridine ring-containing luminescent substrate (17) [that is, a compound represented by the above chemical formula (III)] (3 mg, 0.01 mmol, 20%) was obtained as a crude product.
・ピリジン環含有発光基質(17)の同定結果
1H NMR(500 MHz, CD3OD):δ 3.02 (6 H, s), 3.52 (2 H, m), 5.95 (1 H, t, J = 9.2 Hz), 6.63 (1 H, d, J = 15.5 Hz), 6.81 (1 H, d, J = 15.5 Hz), 6.96 (1 H, dd, J = 10.9, 15.5 Hz), 7.08 (1 H, dd, J = 10.9, 15.5 Hz), 7.11 (1 H, dd, J = 2.8, 8.9 Hz), 7.44 (1 H, d, J = 8.9), 7.99 (1 H, d, J = 2.8 Hz)
13C NMR(125 MHz, CD3OD):δ 35.5 (t), 38.7 (q) × 2, 80.9 (d), 118.9 (d), 122.9 (d), 125.1 (d), 126.6 (d), 133.3 (d), 136.9 (d), 141.8 (s), 142.2 (s), 146.0 (d), 169.2 (s), 176.8 (s)
MS(ESI) m/z: [(M+H)+] 304.09
・ Identification result of luminescent substrate containing pyridine ring (17)
1 H NMR (500 MHz, CD 3 OD): δ 3.02 (6 H, s), 3.52 (2 H, m), 5.95 (1 H, t, J = 9.2 Hz), 6.63 (1 H, d, J = 15.5 Hz), 6.81 (1 H, d, J = 15.5 Hz), 6.96 (1 H, dd, J = 10.9, 15.5 Hz), 7.08 (1 H, dd, J = 10.9, 15.5 Hz), 7.11 ( 1 H, dd, J = 2.8, 8.9 Hz), 7.44 (1 H, d, J = 8.9), 7.99 (1 H, d, J = 2.8 Hz)
13 C NMR (125 MHz, CD 3 OD): δ 35.5 (t), 38.7 (q) × 2, 80.9 (d), 118.9 (d), 122.9 (d), 125.1 (d), 126.6 (d), 133.3 (d), 136.9 (d), 141.8 (s), 142.2 (s), 146.0 (d), 169.2 (s), 176.8 (s)
MS (ESI) m / z: [(M + H) + ] 304.09
<ピラジン環含有発光基質の合成>
(2−ジメチルアミノ−5−ブロモ−ピラジン(21)の合成)
2−アミノ−5−ブロモ−ピラジン(20)(1,170mg、6.72mmol)、ヨードメタン(CH3I)(1.2ml、19.5mmol)をテトラヒドロフラン(THF)(100ml)に溶解させた。この溶液を0℃にし、60%水素化ナトリウム(NaH)(1.5g、37.5mmol)をゆっくり加え、混合物を5時間撹拌した。反応混合物に少量のメタノールを加え、過剰のヨードメタン、水素化ナトリウムを分解させた。続いて水(10ml)を加え、酢酸エチル(3×200ml)で抽出した。有機層を合わせ、無水硫酸ナトリウムで乾燥後、減圧濃縮した。得られた残渣をカラムクロマトグラフィー[シリカゲル48.5g;ヘキサン−酢酸エチル(1:1)]にて精製すると、2−ジメチルアミノ−5−ブロモ−ピラジン(21)(1,096mg、5.4mmol、80%)が淡黄色粉末固体として得られた。
(Synthesis of 2-dimethylamino-5-bromo-pyrazine (21))
2-Amino-5-bromo-pyrazine (20) (1,170 mg, 6.72 mmol) and iodomethane (CH 3 I) (1.2 ml, 19.5 mmol) were dissolved in tetrahydrofuran (THF) (100 ml). The solution was brought to 0 ° C., 60% sodium hydride (NaH) (1.5 g, 37.5 mmol) was added slowly and the mixture was stirred for 5 hours. A small amount of methanol was added to the reaction mixture to decompose excess iodomethane and sodium hydride. Subsequently, water (10 ml) was added and extracted with ethyl acetate (3 × 200 ml). The organic layers were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The obtained residue was purified by column chromatography [silica gel 48.5 g; hexane-ethyl acetate (1: 1)] to give 2-dimethylamino-5-bromo-pyrazine (21) (1,096 mg, 5.4 mmol). 80%) was obtained as a pale yellow powdered solid.
・2−ジメチルアミノ−5−ブロモ−ピラジン(21)の同定結果
1H NMR(500 MHz, CDCl3):δ 3.08 (6 H, s), 7.74 (1 H, s), 8.10 (1 H, s)
13C NMR(125 MHz, CDCl3):δ 37.9 (q) × 2, 124.7 (s), 129.2 (d), 143.8 (d), 154.0 (s)
MS(ESI) [M+H]+ ; m/z 201.96, 203.96
・ Identification result of 2-dimethylamino-5-bromo-pyrazine (21)
1 H NMR (500 MHz, CDCl 3 ): δ 3.08 (6 H, s), 7.74 (1 H, s), 8.10 (1 H, s)
13 C NMR (125 MHz, CDCl 3 ): δ 37.9 (q) × 2, 124.7 (s), 129.2 (d), 143.8 (d), 154.0 (s)
MS (ESI) [M + H] + ; m / z 201.96, 203.96
(5−アルデヒド−2−ジメチルアミノピラジン(22)の合成)
2−ジメチルアミノ−5−ブロモ−ピラジン(21)(1.37g、6.8mmol)を脱水テトラヒドロフラン(THF)(50ml)に溶解し、アルゴン雰囲気下とした。この溶液を、アセトンバスを用いて−80℃まで冷却し、1.57Mノルマルブチルリチウム(n−BuLi)−ヘキサン溶液(10.8ml)をゆっくり加え、1時間撹拌した。続いてこの混合溶液にN,N−ジメチルホルムアミド(DMF)(2.0ml、26mmol)をゆっくり加えた後、室温に昇温させ1時間撹拌した。その後この反応混合物に水を加え15分間撹拌し、酢酸エチル(2×200ml)で抽出した。有機層を合わせ、無水硫酸ナトリウムで乾燥後、減圧濃縮した。得られた残渣をシリカゲルカラムクロマトグラフィー[シリカゲル46.9g;ヘキサン−酢酸エチル(1:1)]にて精製すると、5−アルデヒド−2−ジメチルアミノピラジン(22)(975mg、6.5mmol、95%)が淡黄色粉末固体として得られた。
2-Dimethylamino-5-bromo-pyrazine (21) (1.37 g, 6.8 mmol) was dissolved in dehydrated tetrahydrofuran (THF) (50 ml) and placed under an argon atmosphere. The solution was cooled to −80 ° C. using an acetone bath, a 1.57 M normal butyl lithium (n-BuLi) -hexane solution (10.8 ml) was slowly added, and the mixture was stirred for 1 hour. Subsequently, N, N-dimethylformamide (DMF) (2.0 ml, 26 mmol) was slowly added to the mixed solution, and then the mixture was warmed to room temperature and stirred for 1 hour. Water was then added to the reaction mixture, stirred for 15 minutes, and extracted with ethyl acetate (2 × 200 ml). The organic layers were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography [silica gel 46.9 g; hexane-ethyl acetate (1: 1)] to give 5-aldehyde-2-dimethylaminopyrazine (22) (975 mg, 6.5 mmol, 95 %) Was obtained as a pale yellow powdered solid.
・5−アルデヒド−2−ジメチルアミノピラジン(22)の同定結果
1H NMR(500 MHz, CDCl3):δ 3.23 (6 H, s), 8.05 (1 H, d, J = 1.2 Hz), 8.64 (1 H, d, J = 1.2 Hz), 9.86 (1 H, s)
13C NMR (125 MHz, CDCl3):δ 38.0 (q) × 2, 129.4 (d), 136.8 (s), 144.4 (d), 155.7 (s), 190.8 (d)
MS(ESI) [M+Na]+ ; m/z 174.04
・ Identification result of 5-aldehyde-2-dimethylaminopyrazine (22)
1 H NMR (500 MHz, CDCl 3 ): δ 3.23 (6 H, s), 8.05 (1 H, d, J = 1.2 Hz), 8.64 (1 H, d, J = 1.2 Hz), 9.86 (1 H , s)
13 C NMR (125 MHz, CDCl 3 ): δ 38.0 (q) × 2, 129.4 (d), 136.8 (s), 144.4 (d), 155.7 (s), 190.8 (d)
MS (ESI) [M + Na] + ; m / z 174.04
(エチルエステル体(23)の合成)
5−アルデヒド−2−ジメチルアミノピラジン(22)(44.5mg、0.29mmol)、4−ホスホノクロトン酸トリエチル(150μl、0.58mmol)を、脱水テトラヒドロフラン(THF)(20ml)に溶解した。この溶液を0℃まで冷却し、60%水素化ナトリウム(NaH)(27.4mg、0.69mmol)をゆっくり加え、15分間撹拌した。この反応混合物にエタノール(EtOH)(5ml)を加えて過剰の水素化ナトリウムを分解し、この溶液を室温に昇温させた。続いて、酢酸エチル(3×30ml)で抽出した。有機層を合わせ、無水硫酸ナトリウムで乾燥後、減圧濃縮した。得られた残渣を分取薄層クロマトグラフィー[20cm×20cm×1.75mm×1枚;ヘキサン−酢酸エチル(1:1)]にて精製すると、エチルエステル体(23)(47.6mg、0.19mmol、65%)が黄色粉末固体として得られた。
5-Aldehyde-2-dimethylaminopyrazine (22) (44.5 mg, 0.29 mmol) and triethyl 4-phosphonocrotonate (150 μl, 0.58 mmol) were dissolved in dehydrated tetrahydrofuran (THF) (20 ml). The solution was cooled to 0 ° C. and 60% sodium hydride (NaH) (27.4 mg, 0.69 mmol) was added slowly and stirred for 15 minutes. Ethanol (EtOH) (5 ml) was added to the reaction mixture to decompose excess sodium hydride, and the solution was allowed to warm to room temperature. Subsequently, it was extracted with ethyl acetate (3 × 30 ml). The organic layers were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The obtained residue was purified by preparative thin layer chromatography [20 cm × 20 cm × 1.75 mm × 1 sheet; hexane-ethyl acetate (1: 1)] to obtain an ethyl ester (23) (47.6 mg, 0 .19 mmol, 65%) was obtained as a yellow powdered solid.
・エチルエステル体(23)の同定結果
1H NMR(500 MHz, CDCl3):δ 1.31 (3 H, t, J = 7.4 Hz), 3.17 (6 H, s), 4.22 (2 H, q, J = 7.4 Hz), 5.99 (1 H, d, J = 15.5 Hz), 6.83 (1 H, d, J = 15.5 Hz), 7.14 (1 H, dd, J = 11.5, 15.5 Hz), 7.45 (1 H, dd, J = 11.5, 15.5 Hz), 7.22 (1 H, d, J = 8.6 Hz), 8.04 (1 H, s), 8.05 (1 H, s)
13C NMR(125 MHz, CDCl3):δ 14.4 (q), 37.8 (q) × 2, 60.4 (t), 120.7 (s), 121.2 (d), 125.7 (d), 130.0 (d), 136.2 (s), 142.4 (d), 144.5 (d), 153.9 (s), 167.3 (s)
MS(ESI) [M+H]+ ; m/z 248.11
・ Identification result of ethyl ester (23)
1 H NMR (500 MHz, CDCl 3 ): δ 1.31 (3 H, t, J = 7.4 Hz), 3.17 (6 H, s), 4.22 (2 H, q, J = 7.4 Hz), 5.99 (1 H , d, J = 15.5 Hz), 6.83 (1 H, d, J = 15.5 Hz), 7.14 (1 H, dd, J = 11.5, 15.5 Hz), 7.45 (1 H, dd, J = 11.5, 15.5 Hz ), 7.22 (1 H, d, J = 8.6 Hz), 8.04 (1 H, s), 8.05 (1 H, s)
13 C NMR (125 MHz, CDCl 3 ): δ 14.4 (q), 37.8 (q) × 2, 60.4 (t), 120.7 (s), 121.2 (d), 125.7 (d), 130.0 (d), 136.2 (s), 142.4 (d), 144.5 (d), 153.9 (s), 167.3 (s)
MS (ESI) [M + H] + ; m / z 248.11
(カルボキシル体(24)の合成)
エチルエステル体(23)(47.6mg、0.19mmol)をイソプロパノール(iPrOH)(10ml)に溶解し、5M水酸化ナトリウム水溶液(120μl)を加え、その溶液を45分間加熱還流した。反応混合物に4M塩酸を加え中和した後、減圧濃縮した。得られた残渣をシリカゲルカラムクロマトグラフィー[シリカゲル16.5g;クロロホルム−メタノール(5:1)]にて精製すると、カルボキシル体(24)(36.8mg、0.16mmol、84%)が淡黄色粉末固体として得られた。
The ethyl ester compound (23) (47.6 mg, 0.19 mmol) was dissolved in isopropanol (iPrOH) (10 ml), 5M aqueous sodium hydroxide solution (120 μl) was added, and the solution was heated to reflux for 45 minutes. The reaction mixture was neutralized with 4M hydrochloric acid, and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography [silica gel 16.5 g; chloroform-methanol (5: 1)] to obtain a carboxyl form (24) (36.8 mg, 0.16 mmol, 84%) as a pale yellow powder. Obtained as a solid.
・カルボキシル体(24)の同定結果
1H NMR(500 MHz, CD3OD):δ 3.15 (6 H, s), 5.97 (1 H, d, J = 14.9 Hz), 6.87 (1 H, d, J = 14.9 Hz), 7.13 (1 H, dd, J = 11.5, 14.9 Hz), 7.39 (1 H, dd, J = 11.5, 14.9 Hz), 8.07 (1 H, s), 8.11 (1 H, s)
13C NMR(125MHz, CD3OD):δ 36.6 (q) × 2, 121.7 (d), 125.5 (d), 129.8 (d), 135.8 (d), 136.8 (d), 142.0 (d), 144.5 (s), 153.3 (s)
MS(ESI) [M+H]+ ; m/z 220.07
・ Identification result of carboxyl body (24)
1 H NMR (500 MHz, CD 3 OD): δ 3.15 (6 H, s), 5.97 (1 H, d, J = 14.9 Hz), 6.87 (1 H, d, J = 14.9 Hz), 7.13 (1 H, dd, J = 11.5, 14.9 Hz), 7.39 (1 H, dd, J = 11.5, 14.9 Hz), 8.07 (1 H, s), 8.11 (1 H, s)
13 C NMR (125 MHz, CD 3 OD): δ 36.6 (q) × 2, 121.7 (d), 125.5 (d), 129.8 (d), 135.8 (d), 136.8 (d), 142.0 (d), 144.5 (s), 153.3 (s)
MS (ESI) [M + H] + ; m / z 220.07
(アミド体(25)の合成)
カルボキシル体(24)(720.0mg、3.3mmol)とS−トリチル−D−システインメチルエステル(3,600mg、9.9mmol)をN,N−ジメチルホルムアミド(DMF)(50ml)に溶解した。この溶液に1−エチル−3−(3−ジメチルアミノプロピル)カルボジイミド塩酸塩(EDC)(2,035mg、6.6mmol)、N,N−ジメチル−4−アミノピリジン(DMAP)(1,270mg、6.6mmol)を加え、この反応混合物を室温で17時間撹拌した。反応混合物に水(50ml)を加え、酢酸エチル(3×50ml)で抽出した。有機層を合わせ、無水硫酸ナトリウムで乾燥後、減圧濃縮した。得られた残渣をシリカゲルカラムクロマトグラフィー[シリカゲル56.0g;ヘキサン−酢酸エチル(1:2)]にて精製すると、アミド体(25)(1,626mg、2.81mmol、84%)が黄色固体として得られた。
Carboxyl compound (24) (720.0 mg, 3.3 mmol) and S-trityl-D-cysteine methyl ester (3,600 mg, 9.9 mmol) were dissolved in N, N-dimethylformamide (DMF) (50 ml). To this solution was added 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) (2,035 mg, 6.6 mmol), N, N-dimethyl-4-aminopyridine (DMAP) (1,270 mg, (6.6 mmol) was added and the reaction mixture was stirred at room temperature for 17 hours. Water (50 ml) was added to the reaction mixture and extracted with ethyl acetate (3 × 50 ml). The organic layers were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography [silica gel 56.0 g; hexane-ethyl acetate (1: 2)] to give an amide compound (25) (1,626 mg, 2.81 mmol, 84%) as a yellow solid. As obtained.
・アミド体(25)の同定結果
1H NMR(500 MHz, CDCl3):δ 2.66-2.75 (2 H, m), 3.17 (6 H, s), 3.72 (3 H, s), 4.73 (1 H, m), 5.93 (1 H, dd, J = 7.5, 14.9 Hz), 6.81 (1 H, d, J = 14.9 Hz), 7.13 (1 H, dd, J = 11.4, 14.9 Hz), 7.20-7.43 (16 H, m), 8.05 (2 H, s)
13C NMR(125 MHz, CDCl3):δ 34.1 (t), 37.8 (q) × 2, 51.2 (q), 52.7 (d), 67.1 (s), 123.0 (d), 125.7 (d), 127.0 (d) × 3, 128.1 (d) × 4, 129.6 (d) × 4, 135.7 (d), 137.4 (d), 141.8 (d), 142.3 (d), 144.4 (s) × 3, 153.9 (s), 165.6 (s), 171.1 (s)
MS(ESI) [(M+Na)+] m/z : 601.13
・ Identification result of amide compound (25)
1 H NMR (500 MHz, CDCl 3 ): δ 2.66-2.75 (2 H, m), 3.17 (6 H, s), 3.72 (3 H, s), 4.73 (1 H, m), 5.93 (1 H , dd, J = 7.5, 14.9 Hz), 6.81 (1 H, d, J = 14.9 Hz), 7.13 (1 H, dd, J = 11.4, 14.9 Hz), 7.20-7.43 (16 H, m), 8.05 (2 H, s)
13 C NMR (125 MHz, CDCl 3 ): δ 34.1 (t), 37.8 (q) × 2, 51.2 (q), 52.7 (d), 67.1 (s), 123.0 (d), 125.7 (d), 127.0 (d) × 3, 128.1 (d) × 4, 129.6 (d) × 4, 135.7 (d), 137.4 (d), 141.8 (d), 142.3 (d), 144.4 (s) × 3, 153.9 (s ), 165.6 (s), 171.1 (s)
MS (ESI) [(M + Na) + ] m / z: 601.13
(チアゾリン体(26)の合成)
アミド体(25)(114.0mg、0.20mmol)をアルゴン雰囲気下にし、ジクロロメタン(10.0ml)を加えた。この溶液を0℃に冷却し、トリフルオロメタンスルホン酸無水物(Tf2O)(100μl、0.6mmol)をゆっくり加え、35分間撹拌した。原料が消失したのを確認した後、陰イオン交換樹脂IRA400 OH AGを用いて中和した。樹脂を洗浄して濾別し、得られた洗液を減圧濃縮した。得られた残渣を分取薄層クロマトグラフィー[20cm×20cm×1.75mm×1枚;ヘキサン−酢酸エチル(1:2)]で精製すると、チアゾリン体(26)(28.2mg、0.089mmol、44%)が黄色固体として得られた。
The amide (25) (114.0 mg, 0.20 mmol) was placed under an argon atmosphere, and dichloromethane (10.0 ml) was added. The solution was cooled to 0 ° C. and trifluoromethanesulfonic anhydride (Tf 2 O) (100 μl, 0.6 mmol) was added slowly and stirred for 35 minutes. After confirming the disappearance of the raw material, it was neutralized using an anion exchange resin IRA400 OH AG. The resin was washed and filtered off, and the resulting wash was concentrated under reduced pressure. The obtained residue was purified by preparative thin layer chromatography [20 cm × 20 cm × 1.75 mm × 1 sheet; hexane-ethyl acetate (1: 2)] to obtain thiazoline derivative (26) (28.2 mg, 0.089 mmol). 44%) as a yellow solid.
・チアゾリン体(26)の同定結果
1H NMR(500 MHz, CDCl3):δ 3.16 (6 H, s), 3.53 (1 H, dd, J = 9.2, 11.5 Hz), 3.60 (1 H, dd, J = 9.2, 11.5 Hz), 3.82 (3 H, s), 5.17 (1 H, t, J = 9.2 Hz), 6.65 (1 H, d, J = 15.5 Hz), 6.77 (1 H, d, J = 15.5 Hz), 6.96 (1 H, dd, J = 11.2, 14.9 Hz), 7.16 (1 H, dd, J = 11.2, 14.9 Hz), 8.03 (2 H, s)
13C NMR(125 MHz, CDCl3):δ 34.6 (t), 37.8 (q) × 2, 52.9 (q), 78.1 (d), 125.4 (d), 126.5 (d), 130.0 (d), 134.6 (d), 137.4 (d), 142.3 (s), 142.5 (d), 153.8 (s), 170.1 (s), 171.4 (s)
MS(ESI) m/z: [(M+H)+] 319.05
・ Identification result of thiazoline (26)
1 H NMR (500 MHz, CDCl 3 ): δ 3.16 (6 H, s), 3.53 (1 H, dd, J = 9.2, 11.5 Hz), 3.60 (1 H, dd, J = 9.2, 11.5 Hz), 3.82 (3 H, s), 5.17 (1 H, t, J = 9.2 Hz), 6.65 (1 H, d, J = 15.5 Hz), 6.77 (1 H, d, J = 15.5 Hz), 6.96 (1 H, dd, J = 11.2, 14.9 Hz), 7.16 (1 H, dd, J = 11.2, 14.9 Hz), 8.03 (2 H, s)
13 C NMR (125 MHz, CDCl 3 ): δ 34.6 (t), 37.8 (q) × 2, 52.9 (q), 78.1 (d), 125.4 (d), 126.5 (d), 130.0 (d), 134.6 (d), 137.4 (d), 142.3 (s), 142.5 (d), 153.8 (s), 170.1 (s), 171.4 (s)
MS (ESI) m / z: [(M + H) + ] 319.05
(ピラジン環含有発光基質(27)の合成)
チアゾリン体(26)(11.2mg、0.035mmol)をH2O(2ml)に溶解し、4M塩酸(1.5ml)を加え、その溶液を19時間室温で撹拌した。反応混合物にアンモニア水を加え中和した後、減圧濃縮した。逆相カラムクロマトグラフィー(SupelcleanTM LC−18 SPE)で精製すると、ピラジン環含有発光基質(27)(4.1mg、0.013mmol、37%)が茶色固体として得られた。
The thiazoline (26) (11.2 mg, 0.035 mmol) was dissolved in H 2 O (2 ml), 4M hydrochloric acid (1.5 ml) was added, and the solution was stirred for 19 hours at room temperature. The reaction mixture was neutralized by adding aqueous ammonia, and then concentrated under reduced pressure. Purification by reverse phase column chromatography (Superclean ™ LC-18 SPE) gave the pyrazine ring-containing luminescent substrate (27) (4.1 mg, 0.013 mmol, 37%) as a brown solid.
・ピラジン環含有発光基質(27)の同定結果
1H NMR(500 MHz, CD3OD):δ 3.15 (6 H, s), 3.52-3.62 (2 H, m), 5.04 (1 H, t, J = 9.2 Hz), 6.60 (1 H, d, J = 15.4 Hz), 6.85 (1 H, d, J = 15.5 Hz), 7.02 (1 H, dd, J = 11.2, 15.5 Hz), 7.17 (1 H, dd, J = 11.2, 15.5 Hz), 8.08 (1 H, s), 8.11 (1H, s)
13C NMR(125 MHz, CD3OD):δ 35.1 (t), 36.6 (q) × 2, 79.1 (d), 124.5 (d), 126.3 (d), 129.8 (d), 133.8 (d), 134.5 (d), 137.1 (s), 141.8 (d), 142.2 (s), 153.9 (s)
MS(ESI) m/z: [(M+H)+] 305.06
-Identification results of pyrazine ring-containing luminescent substrate (27)
1 H NMR (500 MHz, CD 3 OD): δ 3.15 (6 H, s), 3.52-3.62 (2 H, m), 5.04 (1 H, t, J = 9.2 Hz), 6.60 (1 H, d , J = 15.4 Hz), 6.85 (1 H, d, J = 15.5 Hz), 7.02 (1 H, dd, J = 11.2, 15.5 Hz), 7.17 (1 H, dd, J = 11.2, 15.5 Hz), 8.08 (1 H, s), 8.11 (1H, s)
13 C NMR (125 MHz, CD 3 OD): δ 35.1 (t), 36.6 (q) × 2, 79.1 (d), 124.5 (d), 126.3 (d), 129.8 (d), 133.8 (d), 134.5 (d), 137.1 (s), 141.8 (d), 142.2 (s), 153.9 (s)
MS (ESI) m / z: [(M + H) + ] 305.06
<溶解度測定>
上記のようにして得たピリジン環含有発光基質(7)、ピリジン環含有発光基質(17)、ピラジン環含有発光基質(27)又は下記化学式(a):
A pyridine ring-containing luminescent substrate (7), a pyridine ring-containing luminescent substrate (17), a pyrazine ring-containing luminescent substrate (27) or the following chemical formula (a):
表1から、ピリジン環含有発光基質(7)は、アカルミネより、pHが中性付近の緩衝液への溶解性が5.7倍高く、また、ピリジン環含有発光基質(17)は、アカルミネより、pHが中性付近の緩衝液への溶解性が9.7倍高く、また、ピラジン環含有発光基質(27)は、アカルミネより、pHが中性付近の緩衝液への溶解性が14倍高いことが分かる。 From Table 1, the pyridine ring-containing luminescent substrate (7) is 5.7 times more soluble in a buffer near pH neutral than the Aka luminescence, and the pyridine ring-containing luminescent substrate (17) is from the Aka luminescence. In addition, the solubility in a buffer near neutral pH is 9.7 times higher, and the luminescent substrate (27) containing a pyrazine ring has 14 times the solubility in a buffer near neutral pH than Aka Lumine. I understand that it is expensive.
<発光測定>
上記のようにして得たピリジン環含有発光基質(7)又は(17)、或いは、ピラジン環含有発光基質(27)を用いて、発光経時変化及び発光強度、並びに、発光スペクトルの測定を行った。また、比較対照として、上記アカルミネ、並びに、下記化学式(b):
Using the pyridine ring-containing luminescent substrate (7) or (17) or the pyrazine ring-containing luminescent substrate (27) obtained as described above, luminescence change with time, emission intensity, and emission spectrum were measured. . Further, as a comparative control, the above-mentioned Akalumine, and the following chemical formula (b):
・発光経時変化及び発光強度測定
pH8.0で0.5Mのリン酸カリウム緩衝液(KPB溶液)を20μl、各発光基質の溶解液(発光基質濃度=100μM)を20μl、発光基質を分解する酵素である0.01mg/mlのphotinus pyralis(Promega社製)を20μl、0.2mMのMg−ATPを40μl混合し、発光測定装置(ATTO株式会社製、AB−2270)で60秒間発光経時変化の測定を行った。結果を図1、図2及び図3に示す。
また、同測定条件下、発光測定装置(ATTO株式会社製、AB−1850)で300秒間積算し、発光強度測定を行った(N=2)。なお、天然ホタルルシフェリンは570nm、アカルミネは675nm、ピリジン環含有発光基質(7)は625nm、ピリジン環含有発光基質(17)は670nm、ピラジン環含有発光基質(27)は625nmの発光強度で比較した。天然ホタルルシフェリンの発光強度を100とした相対発光強度の値を表2に示す。
・ Luminescence change and measurement of
Moreover, it integrated | accumulated for 300 second with the luminescence measuring apparatus (the ATTO Co., Ltd. make, AB-1850) on the same measurement conditions, and performed luminescence intensity measurement (N = 2). In addition, natural firefly luciferin was compared at 570 nm, red light intensity at 675 nm, pyridine ring-containing luminescent substrate (7) at 625 nm, pyridine ring-containing luminescent substrate (17) at 670 nm, and pyrazine ring-containing luminescent substrate (27) at 625 nm. . Table 2 shows values of relative luminescence intensity with the luminescence intensity of natural firefly luciferin as 100.
図1、図2及び図3から、ピリジン環含有発光基質(7)及び(17)、並びに、ピラジン環含有発光基質(27)は、ホタル生物発光系における発光基質として機能することが分かる。また、図1、図2及び図3から分かるように、ピリジン環含有発光基質(7)及び(17)、並びに、ピラジン環含有発光基質(27)の発光経時変化は、アカルミネの発光経時変化とほぼ同じであった。
また、表2から、ピリジン環含有発光基質(7)及び(17)、並びに、ピラジン環含有発光基質(27)の発光強度は、天然のホタルルシフェリンに比べ約1割の発光強度であるものの、アカルミネとほぼ同じ発光強度であることが分かる。
1, 2 and 3, it can be seen that the pyridine ring-containing luminescent substrates (7) and (17) and the pyrazine ring-containing luminescent substrate (27) function as luminescent substrates in the firefly bioluminescent system. In addition, as can be seen from FIGS. 1, 2 and 3, the luminescence time-dependent changes of the pyridine ring-containing luminescent substrates (7) and (17) and the pyrazine ring-containing luminescent substrate (27) It was almost the same.
Moreover, from Table 2, although the luminescence intensity of the pyridine ring-containing luminescent substrates (7) and (17) and the pyrazine ring-containing luminescent substrate (27) is about 10% of that of natural firefly luciferin, It can be seen that the emission intensity is almost the same as that of Aka Lumine.
・発光スペクトル測定
pH8.0で0.5Mのリン酸カリウム緩衝液(KPB溶液)を20μl、各発光基質の溶解液(発光基質濃度=100μM)を20μl、発光基質を分解する酵素である0.01mg/mlのphotinus pyralis(Promega社製)を20μl、0.2mMのMg−ATPを40μl混合し、発光測定装置(ATTO株式会社製、AB−1850)で180秒間発光スペクトルを測定した。結果を図4、図5及び図6に示す。
図4及び図6から分かるように、ピリジン環含有発光基質(7)及びピラジン環含有発光基質(27)の発光スペクトルにおける極大波長は625nmであり、天然のホタルルシフェリンの極大波長560nmに比べ、65nm長波長であり、また、アカルミネの極大波長675nmに比べ、50nm短波長であった。
また、図5から分かるように、ピリジン環含有発光基質(17)の発光スペクトルにおける極大波長は670nmであり、天然のホタルルシフェリンの極大波長560nmに比べ、110nm長波長であり、また、アカルミネの極大波長と同等であった。
As can be seen from FIGS. 4 and 6, the maximum wavelength in the emission spectrum of the pyridine ring-containing luminescent substrate (7) and the pyrazine ring-containing luminescent substrate (27) is 625 nm, which is 65 nm compared to the maximum wavelength 560 nm of natural firefly luciferin. It was a long wavelength, and it was a short wavelength of 50 nm as compared to the maximum wavelength of 675 nm of Aka Lumine.
Further, as can be seen from FIG. 5, the maximum wavelength in the emission spectrum of the pyridine ring-containing luminescent substrate (17) is 670 nm, which is 110 nm longer than the maximum wavelength 560 nm of natural firefly luciferin, and the maximum of red luminescence. It was equivalent to the wavelength.
本発明の複素環式化合物及びその塩は、pHが中性付近の緩衝液への溶解性に優れ、ホタル生物発光系における発光基質として利用できる。 The heterocyclic compound and salts thereof of the present invention are excellent in solubility in a buffer solution having a pH near neutral, and can be used as a luminescent substrate in a firefly bioluminescence system.
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