JP7264344B2

JP7264344B2 - Hybrid fluorescent probes that recognize biopolymers

Info

Publication number: JP7264344B2
Application number: JP2019034085A
Authority: JP
Inventors: 隆雄日▲び▼; 凌渡辺; 亮太中; 玄伍柏▲崎▼; 隆北山
Original assignee: FUKUI PREFECTURAL UNIVERSITY; Kinki University
Current assignee: FUKUI PREFECTURAL UNIVERSITY; Kinki University
Priority date: 2019-02-27
Filing date: 2019-02-27
Publication date: 2023-04-25
Anticipated expiration: 2039-02-27
Also published as: JP2023110919A; JP2020139782A

Description

本発明は、糖類を含む生体高分子を高感度に蛍光認識できるハイブリッド型蛍光プローブ、それを用いた解析方法、およびそれを調製するための合成中間体に関する。 TECHNICAL FIELD The present invention relates to a hybrid fluorescent probe capable of highly sensitive fluorescence recognition of biopolymers containing sugars, an analytical method using the same, and a synthetic intermediate for preparing the same.

糖類は生体関連分子に広く組み込まれる重要な分子単位であり、特に単糖が２～１０個結合した糖鎖であるオリゴ糖は、腸内環境の整備など、プレバイオティクスの観点からこれまでに多くの研究が報告されており、生理活性を利用した農薬、医薬品、食品添加物等への応用が期待されている。 Saccharides are important molecular units that are widely incorporated into bio-related molecules. In particular, oligosaccharides, which are sugar chains in which 2 to 10 monosaccharides are linked, have been used from the viewpoint of prebiotics, such as the maintenance of the intestinal environment. Many studies have been reported, and application to agricultural chemicals, pharmaceuticals, food additives, etc. using physiological activity is expected.

糖類の定量分析は、従来、（１）フェノール－硫酸法のような、強酸と処理し色素化合物に導く比色定量法（非特許文献１）、（２）Somogyi-Nelson法のような、糖による還元で色素を発色させる比色定量法（非特許文献２および３）、（３）グルコースバイオセンサーとして広く用いられている酵素法、（４）ＨＰＬＣなどクロマトグラフィー法を示差屈折率（ＲＩ）検出法など各種検出法と組み合わせた分離検出法が用いられてきたが、感度や選択的な検出の点で課題があり、いずれもハイスループットアッセイには適していななど問題があった。 Quantitative analysis of saccharides has been conventionally performed by (1) a colorimetric method (Non-Patent Document 1) that treats with a strong acid such as the phenol-sulfuric acid method and leads to a pigment compound, (2) the Somogyi-Nelson method. (3) an enzymatic method widely used as a glucose biosensor, (4) a chromatographic method such as HPLC with a differential refractive index (RI) Separation detection methods combined with various detection methods such as detection methods have been used, but there are problems in terms of sensitivity and selective detection, and none of them are suitable for high-throughput assays.

また、糖を蛍光誘導体化して検出する蛍光検出法として、リン酸－フェニルヒドラジン法やアルギニン試薬法も実施されているが、糖による誘導体化の反応性の違いや反応収率の影響で再現性のある解析が難しいという課題があった。
他に、糖鎖アレイ解析も実施されているが、アレイ解析によく用いられる蛍光色素Ｃｙ３を糖類の定量に用いた場合は、定量性には優れるが、量子収率が低く、蛍光色素としては不十分なため、検出感度に難があった。 Phosphate-phenylhydrazine method and arginine reagent method have also been used as fluorescence detection methods for fluorescent derivatization of sugars. There was a problem that it was difficult to analyze the
In addition, sugar chain array analysis has also been performed, but when the fluorescent dye Cy3, which is often used for array analysis, is used to quantify sugars, it has excellent quantification, but the quantum yield is low. Due to insufficient detection sensitivity, detection sensitivity was difficult.

一方、タンパク質の疎水性表面検出用の蛍光プローブとして、N. Dorhらが開発したHydrophobicity (HP) Sensorは、下記の母骨格ＢＯＤＩＰＹを有する蛍光プローブで、非極性溶媒中で高い量子収率と蛍光強度を示し、水溶液中では十分に消光するという特徴を有していた（非特許文献４）。しかしながら、糖類を解析するにあたっては、溶液中での蛍光の安定性に難があり、精密な蛍光定量には適さず、またプローブとしての選択性も不十分であった。

On the other hand, the Hydrophobicity (HP) Sensor developed by N. Dorh et al. is a fluorescent probe for detecting hydrophobic surfaces of proteins. It was characterized by exhibiting high intensity and sufficiently quenching in an aqueous solution (Non-Patent Document 4). However, in analyzing saccharides, the stability of fluorescence in solution is difficult, and it is not suitable for precise fluorescence quantification, and the selectivity as a probe is also insufficient.

Dubois, M. et al.: Anal. Chem., 28, 350 (1956)Dubois, M. et al.: Anal. Chem., 28, 350 (1956) P. A. Shaffer, M. Somogyi: J. Biol. Chem., 100, 695 (1933)P. A. Shaffer, M. Somogyi: J. Biol. Chem., 100, 695 (1933) Nelson, N.: J. Biol. Chem., 153, 375 (1944)Nelson, N.: J. Biol. Chem., 153, 375 (1944) N. Dorh, et al., Sci. Reports, 5, 18337 (2015)N. Dorh, et al., Sci. Reports, 5, 18337 (2015)

本発明の目的は、糖類、特にオリゴ糖を含む生体高分子を定量的に、且つ高感度に解析できる方法を提供することにある。 An object of the present invention is to provide a method capable of quantitatively and highly sensitively analyzing biopolymers containing saccharides, especially oligosaccharides.

本発明者らは、前記課題を解決するために鋭意検討したところ、タンパク質の解析に用いる蛍光色素の一種であるＢＯＤＩＰＹ（boron-dipyrromethene）の母骨格に水溶性を増大する置換基を導入し、更にジピロメテン骨格の５位に置換アリール基を導入することで、水溶液中ではほとんど蛍光を発しないことで知られたＢＯＤＩＰＹが、オリゴ糖と結合することで分子周辺で極性低下が生じ、その極性低下に応じて、水溶液中で強く飛躍的に安定な蛍光を発することを発見し、またアリール基の置換基として糖類選択的な官能基を導入することが可能となり、高感度で且つ各種糖類に対して選択的となり得るハイブリッド型蛍光プローブが得られることを見出し、本発明を完成するに至った。 The present inventors have made intensive studies to solve the above problems, and have found that a substituent that increases water solubility is introduced into the backbone of BODIPY (boron-dipyrromethene), which is a type of fluorescent dye used for protein analysis, Furthermore, by introducing a substituted aryl group at the 5-position of the dipyrromethene skeleton, BODIPY, which is known to emit almost no fluorescence in aqueous solution, reduces the polarity around the molecule by binding to the oligosaccharide. It was discovered that, depending on the The present inventors have found that a hybrid-type fluorescent probe that can be selective can be obtained by the method, and have completed the present invention.

すなわち、本発明は以下のとおりである。
［項１］
式（１）：

［式中、
Ｒ^１～Ｒ^４は、それぞれ独立して、水素原子、ハロゲン原子、ニトロ、シアノ、ヒドロキシ、アミン、チオール、エポキシ、カルボン酸、カルボン酸エステル、Ｃ_１－６アルキル、Ｃ_２－６アルケニル、Ｃ_３－８シクロアルキル、アリール、ヘテロアリール、ヘテロシクリル、－Ｏ－Ｃ_１－６アルキル、－Ｏ－Ｃ_２－６アルケニル、－Ｏ－Ｃ_３－８シクロアルキル、－Ｏ－アリール、－Ｏ－ヘテロアリール、－Ｏ－ヘテロシクリル、－ＮＨ－Ｃ_１－６アルキル、－Ｎ（Ｃ_１－６アルキル）_２、－ＮＨ－Ｃ_２－６アルケニル、－ＮＨ－Ｃ_３－８シクロアルキル、－ＮＨ－アリール、－ＮＨ－ヘテロアリール、－ＮＨ－ヘテロシクリル、－ＮＨＣＯ－Ｃ_１－６アルキル、－ＮＨＣＯ－Ｃ_２－６アルケニル、－ＮＨＣＯ－Ｃ_３－８シクロアルキル、－ＮＨＣＯ－アリール、－ＮＨＣＯ－ヘテロアリール、－ＮＨＣＯ－ヘテロシクリル、－ＣＯＮＨ－Ｃ_１－６アルキル、－ＣＯＮＨ－Ｃ_２－６アルケニル、－ＣＯＮＨ－Ｃ_３－８シクロアルキル、－ＣＯＮＨ－アリール、－ＣＯＮＨ－ヘテロアリール、－ＣＯＮＨ－ヘテロシクリル、－ＣＯ－Ｃ_１－６アルキル、－ＣＯ－Ｃ_２－６アルケニル、－ＣＯ－Ｃ_３－８シクロアルキル、－ＣＯ－アリール、－ＣＯ－ヘテロアリール、または－ＣＯ－ヘテロシクリルであり、ここでアルキル、アルケニル、シクロアルキル、アリール、ヘテロアリール、およびヘテロシクリルは、それぞれ独立して、ハロゲン原子、ニトロ、シアノ、ヒドロキシ、アミン、－ＮＨ－Ｃ_１－６アルキル、－Ｎ（Ｃ_１－６アルキル）_２、カルボン酸、Ｃ_１－６アルキル、Ｃ_１－６アルコキシ、Ｃ_３－８シクロアルキル、アリール、ヘテロアリール、およびヘテロシクリルからなる群から選択される１または２以上の置換基で置換されていてもよく、あるいはＲ^１とＲ^２、および／またはＲ^３とＲ^４が、それぞれの結合可能ないずれかの部位が結合して環を形成してもよく、および
Ｒ^５は、水素原子、ハロゲン原子、ニトロ、シアノ、ヒドロキシ、アミン、チオール、エポキシ、カルボン酸、カルボン酸エステル、Ｃ_１－６アルキル、Ｃ_２－６アルケニル、Ｃ_２－６アルキニル、－Ｏ－Ｃ_１－６アルキル、－Ｏ－Ｃ_２－６アルケニル、－Ｏ－Ｃ_２－６アルキニル、－Ｏ－Ｃ_３－８シクロアルキル、－Ｏ－アリール、－Ｏ－ヘテロアリール、－Ｏ－ヘテロシクリル、－ＮＨ－Ｃ_１－６アルキル、－Ｎ（Ｃ_１－６アルキル）_２、－ＮＨ－Ｃ_２－６アルケニル、－ＮＨ－Ｃ_２－６アルキニル、－ＮＨ－Ｃ_３－８シクロアルキル、－ＮＨ－アリール、－ＮＨ－ヘテロアリール、－ＮＨ－ヘテロシクリル、－ＮＨＣＯ－Ｃ_１－６アルキル、－ＮＨＣＯ－Ｃ_２－６アルケニル、－ＮＨＣＯ－Ｃ_２－６アルキニル、－ＮＨＣＯ－Ｃ_３－８シクロアルキル、－ＮＨＣＯ－アリール、－ＮＨＣＯ－ヘテロアリール、－ＮＨＣＯ－ヘテロシクリル、－ＣＯＮＨ－Ｃ_１－６アルキル、－ＣＯＮＨ－Ｃ_２－６アルケニル、－ＣＯＮＨ－Ｃ_２－６アルキニル、－ＣＯＮＨ－Ｃ_３－８シクロアルキル、－ＣＯＮＨ－アリール、－ＣＯＮＨ－ヘテロアリール、－ＣＯＮＨ－ヘテロシクリル、－ＣＯ－Ｃ_１－６アルキル、－ＣＯ－Ｃ_２－６アルケニル、－ＣＯ－Ｃ_２－６アルキニル、－ＣＯ－Ｃ_３－８シクロアルキル、－ＣＯ－アリール、－ＣＯ－ヘテロアリール、または－ＣＯ－ヘテロシクリルであり、ここでアルキル、アルケニル、アルキニル、シクロアルキル、アリール、ヘテロアリール、およびヘテロシクリルは、それぞれ独立して、ハロゲン原子、ニトロ、シアノ、ヒドロキシ、アミン、－ＮＨ－Ｃ_１－６アルキル、－Ｎ（Ｃ_１－６アルキル）_２、カルボン酸、Ｃ_１－６アルキル、Ｃ_１－６アルコキシ、Ｃ_３－８シクロアルキル、アリール、ヘテロアリール、およびヘテロシクリル（ここで、シクロアルキル、アリール、ヘテロアリール、およびヘテロシクリルは、更に適宜置換されていてもよいＣ_１－１２アルキル、適宜置換されていてもよいベンジル、適宜置換されていてもよいアリール、適宜置換されていてもよいヘテロアリール、または適宜置換されていてもよいヘテロシクリルで置換されていてもよい）からなる群から選択される１または２以上の置換基で置換されていてもよく、あるいは
Ｒ^５は、下式：

（式中、Ｒ^６はオリゴ糖認識構造であり、トリアゾール環の１位または３位の窒素原子に結合する）である］
の化合物またはその塩を含むプローブで、糖類をターゲットとした蛍光色素であるプローブ。 That is, the present invention is as follows.
[Section 1]
Formula (1):

[In the formula,
R ¹ to R ⁴ are each independently hydrogen atom, halogen atom, nitro, cyano, hydroxy, amine, thiol, epoxy, carboxylic acid, carboxylic acid ester, C _1-6 alkyl, C _2-6 alkenyl, C _3-8 cycloalkyl, aryl, heteroaryl, heterocyclyl, —O—C _1-6 alkyl, —O—C _2-6 alkenyl, —O—C _3-8 cycloalkyl, —O-aryl, —O-hetero Aryl, —O-heterocyclyl, —NH—C _1-6 alkyl, —N(C _1-6 alkyl) ₂ , —NH—C _2-6 alkenyl, —NH—C _3-8 cycloalkyl, —NH-aryl , —NH-heteroaryl, —NH-heterocyclyl, —NHCO—C _1-6 alkyl, —NHCO—C _2-6 alkenyl, —NHCO—C _3-8 cycloalkyl, —NHCO-aryl, —NHCO-heteroaryl , —NHCO-heterocyclyl, —CONH-C _1-6 alkyl, —CONH-C _2-6 alkenyl, —CONH-C _3-8 cycloalkyl, —CONH-aryl, —CONH-heteroaryl, —CONH-heterocyclyl, —CO—C _1-6 alkyl, —CO—C _2-6 alkenyl, —CO—C _3-8 cycloalkyl, —CO-aryl, —CO-heteroaryl, or —CO-heterocyclyl, wherein alkyl , alkenyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl are each independently a halogen atom, nitro, cyano, hydroxy, amine, —NH—C _1-6 alkyl, —N(C _1-6 alkyl) ₂ , carboxylic acid, C _1-6 alkyl, C _1-6 alkoxy, C _3-8 cycloalkyl, aryl, heteroaryl, and heterocyclyl. Alternatively, R ¹ and R ² and/or R ³ and R ⁴ may be bonded at any of their bondable sites to form a ring, and R ⁵ is a hydrogen atom or a halogen atom , nitro, cyano, hydroxy, amine, thiol, epoxy, carboxylic acid, carboxylic acid ester, C _1-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl, —O—C _1-6 alkyl, —O— C _2-6 alkenyl, —O—C _2-6 alkynyl, —O—C _3-8 cycloalkyl, —O-aryl, —O-heteroaryl, —O-heterocyclyl, —NH—C _1-6 alkyl, —N(C _1-6 alkyl) ₂ , —NH—C _2-6 alkenyl, —NH—C _2-6 alkynyl, —NH—C _3-8 cycloalkyl, —NH-aryl, —NH-heteroaryl, -NH-heterocyclyl, -NHCO-C _1-6 alkyl, -NHCO-C _2-6 alkenyl, -NHCO-C _2-6 alkynyl, -NHCO-C _3-8 cycloalkyl, -NHCO-aryl, -NHCO- heteroaryl, -NHCO-heterocyclyl, -CONH-C _1-6 alkyl, -CONH-C _2-6 alkenyl, -CONH-C _2-6 alkynyl, -CONH-C _3-8 cycloalkyl, -CONH-aryl, —CONH-heteroaryl, —CONH-heterocyclyl, —CO—C _1-6 alkyl, —CO—C _2-6 alkenyl, —CO—C _2-6 alkynyl, —CO—C _3-8 cycloalkyl, —CO -aryl, -CO-heteroaryl, or -CO-heterocyclyl, where alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl are each independently a halogen atom, nitro, cyano, hydroxy , amine, —NH—C _1-6 alkyl, —N(C _1-6 alkyl) ₂ , carboxylic acid, C _1-6 alkyl, C _1-6 alkoxy, C _3-8 cycloalkyl, aryl, heteroaryl, and heterocyclyl (wherein cycloalkyl, aryl, heteroaryl, and heterocyclyl are further optionally substituted C _1-12 alkyl, optionally substituted benzyl, optionally substituted aryl, optionally substituted heteroaryl or optionally substituted heterocyclyl), or ^R5 has the formula:

(Wherein, R ⁶ is an oligosaccharide recognition structure and binds to the nitrogen atom at the 1- or 3-position of the triazole ring)]
A probe that contains a compound of or a salt thereof and is a fluorescent dye that targets sugars.

［項２］
式（１）の化合物が式（２）：

の化合物である、項１のプローブ。 [Section 2]
The compound of formula (1) is represented by formula (2):

The probe of item 1, which is a compound of

［項３］
Ｒ^１およびＲ^３が水素原子である、項１または２のプローブ。 [Section 3]
3. The probe of paragraph ¹ or ² , wherein R1 and R3 are hydrogen atoms.

［項４］
Ｒ^２およびＲ^４がそれぞれ独立して、ハロゲン原子、ヒドロキシ、アミン、カルボン酸、－Ｏ－Ｃ_１－６アルキル、－Ｏ－Ｃ_２－６アルケニル、－ＮＨ－Ｃ_１－６アルキル、－Ｎ（Ｃ_１－６アルキル）_２、－ＮＨ－Ｃ_２－６アルケニル、－ＮＨＣＯ－Ｃ_１－６アルキル、－ＮＨＣＯ－Ｃ_２－６アルケニル、－ＣＯＮＨ－Ｃ_１－６アルキル、－ＣＯＮＨ－Ｃ_２－６アルケニル、－ＣＯ－Ｃ_１－６アルキル、または－ＣＯ－Ｃ_２－６アルケニルであり、ここでアルキルおよびアルケニルは、それぞれ独立して、ハロゲン原子、ニトロ、シアノ、ヒドロキシ、アミン、－ＮＨ－Ｃ_１－６アルキル、－Ｎ（Ｃ_１－６アルキル）_２、カルボン酸、およびＣ_１－６アルコキシからなる群から選択される１または２以上の置換基で置換されていてもよい、項１～３のいずれかのプローブ。 [Section 4]
R ² and R ⁴ are each independently a halogen atom, hydroxy, amine, carboxylic acid, —O—C _1-6 alkyl, —O—C _2-6 alkenyl, —NH—C _1-6 alkyl, —N (C _1-6 alkyl) ₂ , —NH—C _2-6 alkenyl, —NHCO—C _1-6 alkyl, —NHCO—C _2-6 alkenyl, —CONH-C _1-6 alkyl, —CONH-C _{2 -6} alkenyl, -CO-C _1-6 alkyl, or -CO-C _2-6 alkenyl, wherein alkyl and alkenyl are each independently a halogen atom, nitro, cyano, hydroxy, amine, -NH optionally substituted with one or more substituents selected from the group consisting of —C _1-6 alkyl, —N(C _1-6 alkyl) ₂ , carboxylic acid, and C _1-6 alkoxy, the item Any probe of 1-3.

［項５］
Ｒ^２およびＲ^４がそれぞれ独立して、－ＮＨ－Ｃ_１－６アルキレン－Ｏ－Ｃ_１－６アルキルである、項３のプローブ。 [Section 5]
4. The probe of clause 3, wherein R ² and R ⁴ are each independently -NH-C _1-6 alkylene-O-C _1-6 alkyl.

［項６］
Ｒ^２およびＲ^４が、－ＮＨ－ＣＨ_２ＣＨ_２－Ｏ－ＣＨ_３である、項５のプローブ。 [Section 6]
6. The probe of clause 5, wherein R ² and R ⁴ are -NH-CH ₂ CH ₂ -O-CH ₃ .

［項７］
Ｒ^５が、水素原子、ハロゲン原子、ニトロ、シアノ、ヒドロキシ、アミン、チオール、エポキシ、－Ｏ－Ｃ_１－６アルキル、－Ｏ－Ｃ_２－６アルケニル、－Ｏ－Ｃ_２－６アルキニル、－Ｏ－Ｃ_３－８シクロアルキル、－Ｏ－アリール、－Ｏ－ヘテロアリール、－Ｏ－ヘテロシクリル、－ＮＨ－Ｃ_１－６アルキル、－Ｎ（Ｃ_１－６アルキル）_２、－ＮＨ－Ｃ_２－６アルケニル、－ＮＨ－Ｃ_２－６アルキニル、－ＮＨ－Ｃ_３－８シクロアルキル、－ＮＨ－アリール、－ＮＨ－ヘテロアリール、－ＮＨ－ヘテロシクリル、－ＮＨＣＯ－Ｃ_１－６アルキル、－ＮＨＣＯ－Ｃ_２－６アルケニル、－ＮＨＣＯ－Ｃ_２－６アルキニル、－ＮＨＣＯ－Ｃ_３－８シクロアルキル、－ＮＨＣＯ－アリール、－ＮＨＣＯ－ヘテロアリール、または－ＮＨＣＯ－ヘテロシクリル、であり、ここでアルキル、アルケニル、シクロアルキル、アリール、ヘテロアリール、およびヘテロシクリルは、それぞれ独立して、ハロゲン原子、ニトロ、シアノ、ヒドロキシ、アミン、－ＮＨ－Ｃ_１－６アルキル、－Ｎ（Ｃ_１－６アルキル）_２、カルボン酸、Ｃ_１－６アルキル、Ｃ_１－６アルコキシ、Ｃ_３－８シクロアルキル、アリール、ヘテロアリール、およびヘテロシクリル（ここで、シクロアルキル、アリール、ヘテロアリール、およびヘテロシクリルは、更に適宜置換されていてもよいＣ_１－１２アルキル、適宜置換されていてもよいベンジル、適宜置換されていてもよいアリール、適宜置換されていてもよいヘテロアリール、または適宜置換されていてもよいヘテロシクリルで置換されていてもよい）からなる群から選択される１または２以上の置換基で置換されていてもよい、項１～６のいずれかのプローブ。 [Section 7]
R ⁵ is hydrogen atom, halogen atom, nitro, cyano, hydroxy, amine, thiol, epoxy, —O—C _1-6 alkyl, —O—C _2-6 alkenyl, —O—C _2-6 alkynyl, — O—C _3-8 cycloalkyl, —O-aryl, —O-heteroaryl, —O-heterocyclyl, —NH—C _1-6 alkyl, —N(C _1-6 alkyl) ₂ , —NH—C _{2 -6} alkenyl, -NH-C _2-6 alkynyl, -NH-C _3-8 cycloalkyl, -NH-aryl, -NH-heteroaryl, -NH-heterocyclyl, -NHCO-C _1-6 alkyl, -NHCO —C _2-6 alkenyl, —NHCO—C _2-6 alkynyl, —NHCO—C _3-8 cycloalkyl, —NHCO-aryl, —NHCO-heteroaryl, or —NHCO-heterocyclyl, wherein alkyl, Alkenyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl are each independently a halogen atom, nitro, cyano, hydroxy, amine, —NH—C _1-6 alkyl, —N(C _1-6 alkyl) ₂ , carboxylic acids, C _1-6 alkyl, C _1-6 alkoxy, C _3-8 cycloalkyl, aryl, heteroaryl and heterocyclyl (wherein cycloalkyl, aryl, heteroaryl and heterocyclyl are further optionally substituted optionally substituted C _1-12 alkyl, optionally substituted benzyl, optionally substituted aryl, optionally substituted heteroaryl, or optionally substituted heterocyclyl 7. The probe of any one of items 1 to 6, optionally substituted with one or more substituents selected from the group consisting of:

［項８］
Ｒ^５が、水素原子、ニトロ、ヒドロキシ、アミン、チオール、エポキシ、－Ｏ－Ｃ_１－６アルキル、－Ｏ－Ｃ_２－６アルケニル、－Ｏ－Ｃ_２－６アルキニル、－Ｏ－Ｃ_３－８シクロアルキル、－Ｏ－アリール、－Ｏ－ヘテロアリール、－Ｏ－ヘテロシクリル、－ＮＨＣＯ－Ｃ_１－６アルキル、－ＮＨＣＯ－Ｃ_２－６アルケニル、－ＮＨＣＯ－Ｃ_２－６アルキニル、－ＮＨＣＯ－Ｃ_３－８シクロアルキル、－ＮＨＣＯ－アリール、－ＮＨＣＯ－ヘテロアリール、または－ＮＨＣＯ－ヘテロシクリル、であり、ここでアルキル、アルケニル、シクロアルキル、アリール、ヘテロアリール、およびヘテロシクリルは、それぞれ独立して、ハロゲン原子、ニトロ、シアノ、ヒドロキシ、アミン、－ＮＨ－Ｃ_１－６アルキル、－Ｎ（Ｃ_１－６アルキル）_２、カルボン酸、Ｃ_１－６アルキル、Ｃ_１－６アルコキシ、Ｃ_３－８シクロアルキル、アリール、ヘテロアリール、およびヘテロシクリル（ここで、シクロアルキル、アリール、ヘテロアリール、およびヘテロシクリルは、更に適宜置換されていてもよいＣ_１－１２アルキル、適宜置換されていてもよいベンジル、適宜置換されていてもよいアリール、適宜置換されていてもよいヘテロアリール、または適宜置換されていてもよいヘテロシクリルで置換されていてもよい）からなる群から選択される１または２以上の置換基で置換されていてもよい、項１～７のいずれかのプローブ。 [Item 8]
R ⁵ is a hydrogen atom, nitro, hydroxy, amine, thiol, epoxy, —O—C _1-6 alkyl, —O—C _2-6 alkenyl, —O—C _2-6 alkynyl, —O—C _{3- 8} cycloalkyl, -O-aryl, -O-heteroaryl, -O-heterocyclyl, -NHCO-C _1-6 alkyl, -NHCO-C _2-6 alkenyl, -NHCO-C _2-6 alkynyl, -NHCO- C _3-8 cycloalkyl, —NHCO-aryl, —NHCO-heteroaryl, or —NHCO-heterocyclyl, wherein alkyl, alkenyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl are each independently halogen atom, nitro, cyano, hydroxy, amine, -NH-C _1-6 alkyl, -N(C _1-6 alkyl) ₂ , carboxylic acid, C _1-6 alkyl, C _1-6 alkoxy, C _3-8 Cycloalkyl, aryl, heteroaryl, and heterocyclyl (wherein cycloalkyl, aryl, heteroaryl, and heterocyclyl are further optionally substituted C _1-12 alkyl, optionally substituted benzyl, optionally optionally substituted aryl, optionally substituted heteroaryl, or optionally substituted heterocyclyl) with one or more substituents selected from the group consisting of The probe of any of clauses 1-7, optionally substituted.

［項９］
Ｒ^５が、Ｃ_２－６アルキニル、－Ｏ－Ｃ_２－６アルキニル、－ＮＨ－Ｃ_２－６アルキニル、－ＮＨＣＯ－Ｃ_２－６アルキニル、または－ＣＯ－Ｃ_２－６アルキニルである、項１～６のいずれかのプローブ。 [Item 9]
The term wherein R ⁵ is C _2-6 alkynyl, —O—C _2-6 alkynyl, —NH—C _2-6 alkynyl, —NHCO—C _2-6 alkynyl, or —CO—C _2-6 alkynyl Any probe of 1-6.

［項１０］
Ｒ^５が下式：

（式中、Ｒ^６はオリゴ糖認識構造であり、トリアゾール環の１位または３位の窒素原子に結合する）
である、項１～６のいずれかのプローブ。 [Item 10]
R ⁵ is of the formula:

(wherein ^R6 is an oligosaccharide recognition structure and binds to the nitrogen atom at the 1- or 3-position of the triazole ring)
7. The probe of any one of items 1-6.

［項１１］
Ｒ^６が

のいずれかである、項１０のプローブ。 [Item 11]
^R6 is

11. The probe of clause 10, which is any of

［項１２］
式（１）の化合物が、下記のいずれかである項１のプローブ。

[Item 12]
Item 1. The probe of item 1, wherein the compound of formula (1) is any of the following.

［項１３］
式（１）の化合物が、下記のいずれかである項１のプローブ。

[Item 13]
Item 1. The probe of item 1, wherein the compound of formula (1) is any of the following.

［項１４］
項１０または１１のいずれかのプローブを製造するために用いる式（３）または式（４）：

の合成中間体化合物。 [Item 14]
Formula (3) or Formula (4) used to manufacture the probe of either Section 10 or 11:

Synthetic intermediate compounds of

［項１５］
ターゲットの糖類がオリゴ糖である、項１～１３のいずれかのプローブ。 [Item 15]
14. The probe of any one of items 1 to 13, wherein the target saccharide is an oligosaccharide.

［項１６］
ターゲットの糖類が生体高分子中の糖類である、項１～１３のいずれかのプローブ。 [Item 16]
14. The probe of any one of items 1 to 13, wherein the target saccharide is a saccharide in a biopolymer.

［項１７］
下記のいずれかの化合物。

[Item 17]
A compound of any of the following.

［項１８］
項１～１３のいずれかに記載のプローブを用いることを特徴とする、糖類を含む生体高分子の解析方法。 [Item 18]
Item 14. A method for analyzing biopolymers containing sugars, characterized by using the probe according to any one of items 1 to 13.

本発明のプローブを酵素の多糖類分解活性や糖転移活性の高感度アッセイ系に利用することで、これまで困難であった糖代謝酵素のハイスループットアッセイを汎用的に実用化することが期待される。また、本発明の蛍光プローブにアミノ基、チオール基もしくはエポキシ基を導入することで、ガラスプレート上への固定化することで、糖結合タンパク質の特異性を評価するための高感度な糖鎖蛍光アレイを構築することができれば、糖鎖による生体認識機構の基礎的な解明だけでなく、遺伝子やアミノ酸配列に比べて変異が入りにくい糖鎖をバイオマーカーとする、プレシジョン・メディスンへの応用により、がん治療や生活習慣病予防への応用が期待される。 By using the probe of the present invention in a high-sensitivity assay system for polysaccharide-degrading activity and transglycosylation activity of enzymes, it is expected that high-throughput assays for sugar-metabolizing enzymes, which have been difficult so far, will be put to practical use for general purposes. be. In addition, by introducing an amino group, a thiol group or an epoxy group into the fluorescent probe of the present invention and immobilizing it on a glass plate, highly sensitive sugar chain fluorescence can be obtained for evaluating the specificity of a sugar binding protein. If we can construct an array, we will not only be able to fundamentally elucidate the mechanism of biorecognition by sugar chains, but also apply it to precision medicine by using sugar chains, which are less likely to mutate than genes and amino acid sequences, as biomarkers. It is expected to be applied to cancer treatment and prevention of lifestyle-related diseases.

試験例１の結果を示す。グラフ中ｅｘは励起スペクトルの結果を示し、ｅｍは蛍光スペクトルの結果を示す。The results of Test Example 1 are shown. In the graph, ex indicates the result of excitation spectrum, and em indicates the result of fluorescence spectrum. 試験例３の結果を示す。ｘ軸の数値は化合物番号を示す。各溶媒溶液の蛍光強度を測定（励起波長 565 nm)し、各蛍光色素濃度＝1 μMのときの蛍光強度に補正した上で、アセトン溶液の蛍光強度に対して、エチレングリコール中（黒）および水溶液中（白）の蛍光強度変化を化合物１０（後述）のアセトン溶液中の蛍光強度を100として比較した結果を示す。The results of Test Example 3 are shown. Numbers on the x-axis indicate compound numbers. The fluorescence intensity of each solvent solution was measured (excitation wavelength 565 nm), corrected to the fluorescence intensity at each fluorochrome concentration = 1 μM, and compared to the fluorescence intensity of the acetone solution in ethylene glycol (black) and The result of comparing the change in fluorescence intensity in an aqueous solution (white) with the fluorescence intensity in an acetone solution of compound 10 (described later) as 100 is shown. 試験例４の結果を示す。The results of Test Example 4 are shown. 試験例５の結果を示す。The results of Test Example 5 are shown. 試験例６の結果を示す。The results of Test Example 6 are shown.

本発明において「ハロゲン原子」の具体例としては、フッ素原子、塩素原子、臭素原子またはヨウ素原子が挙げられる。 Specific examples of the "halogen atom" in the present invention include fluorine atom, chlorine atom, bromine atom and iodine atom.

本発明において「Ｃ_１－６アルキル」は、炭素数１～６個を有する直鎖状もしくは分枝状の飽和炭化水素基を意味する。好ましくは、「Ｃ_１－４アルキル基」である。「Ｃ_１－６アルキル基」の具体例としては、例えば、メチル、エチル、プロピル、イソプロピル、ブチル、イソブチル、ｓｅｃ－ブチル、ｔｅｒｔ－ブチル、ペンチル、イソペンチル、ネオペンチル、１－エチルプロピル、ヘキシル、イソヘキシル、１，１－ジメチルブチル、２，２－ジメチルブチル、３，３－ジメチルブチル、２－エチルブチル等が挙げられる。 In the present invention, "C _1-6 alkyl" means a linear or branched saturated hydrocarbon group having 1-6 carbon atoms. Preferably, it is a “C _1-4 alkyl group”. Specific examples of the "C _1-6 alkyl group" include, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, 1-ethylpropyl, hexyl, isohexyl , 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 2-ethylbutyl and the like.

本発明において「Ｃ_２－６アルケニル」とは、少なくとも１つの炭素－炭素二重結合を含有し、鎖中に含まれる炭素原子数が２～６の脂肪族炭化水素基を意味する。「Ｃ_２－６アルケニル」の具体例としては、例えば、ビニル、アリル、１－プロペニル、１－ブテニルなどが挙げられる。 In the present invention, "C _2-6 alkenyl" means an aliphatic hydrocarbon group containing at least one carbon-carbon double bond and having 2-6 carbon atoms in the chain. Specific examples of “C _2-6 alkenyl” include vinyl, allyl, 1-propenyl, 1-butenyl and the like.

本発明において「Ｃ_２－６アルキニル」とは、炭素原子数２～６個を有し、三重結合を１個含む直鎖状又は分枝鎖状の不飽和炭化水素基を意味する。「Ｃ_２－６アルキニル」として、好ましくは「Ｃ_２－４アルキニル」が挙げられる。 In the present invention, “C _2-6 alkynyl” means a linear or branched unsaturated hydrocarbon group having 2 to 6 carbon atoms and containing one triple bond. “C _2-6 alkynyl” preferably includes “C _2-4 alkynyl”.

本発明において「Ｃ_３－８シクロアルキル」は、３員～８員の単環式の飽和または部分不飽和の炭化水素基を意味する。好ましくは、「Ｃ_３－６シクロアルキル」である。「Ｃ_３－８シクロアルキル」の具体例としては、例えば、シクロプロピル、シクロブチル、シクロペンチル、シクロヘキシル、シクロペンテニル、シクロヘキセニル、シクロオクチル等が挙げられる。 In the present invention, “C _3-8 cycloalkyl” means a 3- to 8-membered monocyclic saturated or partially unsaturated hydrocarbon group. Preferred is “C _3-6 cycloalkyl”. Specific examples of “C _3-8 cycloalkyl” include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopentenyl, cyclohexenyl, cyclooctyl and the like.

本発明において「Ｃ_１－６アルコキシ」の「Ｃ_１－６アルキル」部分は、前記「Ｃ_１－６アルキル」と同義である。好ましくは、「Ｃ_１－４アルコキシ」である。「Ｃ_１－６アルコキシ」の具体例としては、例えば、メトキシ、エトキシ、プロポキシ、イソプロポキシ、ブトキシ、イソブトキシ、ｓｅｃ－ブトキシ、ｔｅｒｔ－ブトキシ等が挙げられる。 In the present invention, the “C _1-6 alkyl” portion of “C _1-6 alkoxy” has the same meaning as the aforementioned “C _1-6 alkyl”. Preferably, it is “C _1-4 alkoxy”. Specific examples of “C _1-6 alkoxy” include methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy and the like.

本発明において「アリール」は、通常炭素数６～１０個を有する芳香族炭化水素基を意味する。好ましくは「Ｃ_６アリール」（フェニル）である。「Ｃ_６－１０アリール」の具体例としては、例えば、フェニル、１－ナフチルまたは２－ナフチル等が挙げられる。 In the present invention, "aryl" means an aromatic hydrocarbon group usually having 6-10 carbon atoms. Preferred is " _C6 aryl" (phenyl). Specific examples of “C _6-10 aryl” include phenyl, 1-naphthyl, 2-naphthyl and the like.

本発明において「ヘテロアリール」としては、例えば、５員～１０員の単環式もしくは二環式の芳香族ヘテロ環基等が挙げられ、該基は、窒素原子、硫黄原子および酸素原子から選択される同種または異種のヘテロ原子を１個以上（例えば１～４個）含有する。二環式のヘテロアリール基には、前記単環式のへテロアリール基と芳香族環（ベンゼン、ピリジン等）または非芳香族環（シクロヘキサン、ピペリジン等）とが縮環したものも含む。 In the present invention, "heteroaryl" includes, for example, a 5- to 10-membered monocyclic or bicyclic aromatic heterocyclic group, and the group is selected from a nitrogen atom, a sulfur atom and an oxygen atom. contains one or more (eg, 1-4) heteroatoms of the same or different type. Bicyclic heteroaryl groups also include condensed monocyclic heteroaryl groups with aromatic rings (benzene, pyridine, etc.) or non-aromatic rings (cyclohexane, piperidine, etc.).

本発明において「ヘテロシクリル」としては、例えば、窒素原子、酸素原子および硫黄原子から選択される同種または異種の原子を１～３個有する４員～１０員の単環式もしくは多環式の飽和ヘテロ環基等が挙げられる。前記窒素原子、酸素原子および硫黄原子はいずれも環を構成する原子である。該ヘテロ環基は、飽和または部分不飽和のいずれであってもよい。好ましくは飽和ヘテロ環基であり、さらに好ましくは５員もしくは６員の飽和ヘテロ環基である。具体的には、ピラニル、ジヒドロピラニル、テトラヒドロピラニル、テトラヒドロフリル、アゼチジニル、ピロリジニル、イミダゾリジニル、ピペリジニル、ピペラジニル、アゼパニル、モルホリニル、チオモルホリニル、ジオキソチオモルホリニル、ヘキサメチレンイミニル、オキサゾリジニル、チアゾリジニル、オキソオキサゾリジル、ジオキソオキサゾリジニル、ジオキソチアゾリジニル、５－オキソ－１,２,４－オキサジアゾール－３－イル、５－オキソ－１,２,４－チアジアゾール－３－イル、５－チオキソ－１,２,４－オキサジアゾール－３－イル、１，２，３－トリアゾリル、１，２，４－トリアゾリル等が挙げられる。該基の結合手は、環を構成する炭素原子および窒素原子のいずれであってもよい。 In the present invention, "heterocyclyl" includes, for example, a 4- to 10-membered monocyclic or polycyclic saturated heterocycle having 1 to 3 same or different atoms selected from a nitrogen atom, an oxygen atom and a sulfur atom. A cyclic group and the like can be mentioned. The nitrogen atom, oxygen atom and sulfur atom are all ring-constituting atoms. The heterocyclic group may be either saturated or partially unsaturated. A saturated heterocyclic group is preferred, and a 5- or 6-membered saturated heterocyclic group is more preferred. Specifically, pyranyl, dihydropyranyl, tetrahydropyranyl, tetrahydrofuryl, azetidinyl, pyrrolidinyl, imidazolidinyl, piperidinyl, piperazinyl, azepanyl, morpholinyl, thiomorpholinyl, dioxothiomorpholinyl, hexamethyleneiminyl, oxazolidinyl, thiazolidinyl, oxoxazolidyl, dioxoxazolidinyl, dioxothiazolidinyl, 5-oxo-1,2,4-oxadiazol-3-yl, 5-oxo-1,2,4-thiadiazole-3 -yl, 5-thioxo-1,2,4-oxadiazol-3-yl, 1,2,3-triazolyl, 1,2,4-triazolyl and the like. The bond of the group may be either a carbon atom or a nitrogen atom that constitutes the ring.

本発明において「Ｃ_１－６アルキレン」は、炭素数１～６個を有する直鎖状もしくは分枝状の二価の飽和炭化水素基、または炭素数３～６個を有する環状構造を含む二価の飽和炭化水素基を意味する。 In the present invention, "C _1-6 alkylene" means a linear or branched divalent saturated hydrocarbon group having 1 to 6 carbon atoms, or a divalent divalent hydrocarbon group having 3 to 6 carbon atoms. valent saturated hydrocarbon group.

本発明において「カルボン酸エステル」としては、アルキルエステル、アルケニルエステルなど、種々のエステルを意味し、またここでのアルキル基やアルケニル基は更に種々置換されていてもよい。 In the present invention, "carboxylic acid ester" means various esters such as alkyl esters and alkenyl esters, and the alkyl group and alkenyl group may be further substituted variously.

本発明において「塩」とは、式（１）で表される化合物が酸性基を有する場合は、例えば、ナトリウム塩、カリウム塩等のアルカリ金属塩；カルシウム塩、マグネシウム塩等のアルカリ土類金属塩；亜鉛塩等の無機金属塩；トリエチルアミン、トリエタノールアミン、トリヒドロキシメチルアミノメタン、アミノ酸等の有機塩基塩等が挙げられる。
式（１）で表される化合物が塩基性基を有する場合は、例えば、塩酸塩、臭化水素酸塩、硫酸塩、リン酸塩、硝酸塩等の無機酸塩；および酢酸塩、プロピオン酸塩、コハク酸塩、乳酸塩、リンゴ酸塩、酒石酸塩、クエン酸塩、マレイン酸塩、フマル酸塩、メタンスルホン酸塩、ｐ－トルエンスルホン酸塩、ベンゼンスルホン酸塩、アスコルビン酸塩等の有機酸塩等が挙げられる。 In the present invention, the term "salt" means, when the compound represented by formula (1) has an acidic group, examples thereof include alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as calcium salts and magnesium salts; salts; inorganic metal salts such as zinc salts; organic base salts such as triethylamine, triethanolamine, trihydroxymethylaminomethane, and amino acids;
When the compound represented by formula (1) has a basic group, for example, inorganic acid salts such as hydrochloride, hydrobromide, sulfate, phosphate, nitrate; and acetate, propionate , succinate, lactate, malate, tartrate, citrate, maleate, fumarate, methanesulfonate, p-toluenesulfonate, benzenesulfonate, ascorbate, etc. and acid salts.

本発明において「オリゴ糖認識構造」とは、オリゴ糖に選択的に相互作用できる構造であり、本発明のＢＯＤＩＰＹ骨格からなる蛍光プローブをオリゴ糖と結びつけるはたらきを有する。本発明ではこれらに限定されないが、例えば下記の構造を有する部分構造がオリゴ糖認識構造として挙げられる。

In the present invention, the "oligosaccharide recognition structure" is a structure that can selectively interact with oligosaccharides, and has the function of binding the fluorescent probe comprising the BODIPY skeleton of the present invention to oligosaccharides. Although the present invention is not limited to these, for example, partial structures having the following structures are examples of oligosaccharide recognition structures.

本発明において「糖類」とは、単糖類、二糖類、多糖類など、種々のサイズの糖類を意図し、また構成される各単糖は、これらに限定されないが、グルコース、アロース、マンノース、ガラクトース、フルクトースなどの六炭糖、リボースなどの五炭糖など、種々の単糖が含まれる。
本発明において「オリゴ糖」とは、単糖が２～１０個、好ましくは３～１０個、または３～６個で構成され、それぞれがα－またはβ－グリコシド結合した糖鎖をいう。なお、構成する単糖に還元糖が必ずしも含まれる必要はない。 In the present invention, "sugar" intends sugars of various sizes, such as monosaccharides, disaccharides, polysaccharides, and each monosaccharide composed of, but not limited to, glucose, allose, mannose, galactose. , hexoses such as fructose, and pentoses such as ribose.
In the present invention, the term "oligosaccharide" refers to a sugar chain composed of 2 to 10, preferably 3 to 10, or 3 to 6 monosaccharides, each of which is α- or β-glycoside-linked. Note that the constituent monosaccharides do not necessarily contain reducing sugars.

本発明の化合物の合成は、下記の実施例の方法やそれを一部最適化して変更した方法によって行うことができる。具体的には、各種ベンズアルデヒド誘導体を酸性条件下ピロールと反応させ、適宜クロル化して三フッ化ホウ素と反応させることで得ることができる。 The synthesis of the compounds of the present invention can be carried out by the methods of the following examples and partially optimized and modified methods thereof. Specifically, it can be obtained by reacting various benzaldehyde derivatives with pyrrole under acidic conditions, appropriately chlorinating them, and reacting them with boron trifluoride.

また、下記の実施例で得られた下記の式（３）のアセチレン部分を有する本発明化合物（実施例における化合物５ｄ）を製造中間体として、オリゴ糖認識部位が導入されたアジ化物を反応させることで、アセチレン部分とアジド部分が環化してトリアゾール環を形成させ、オリゴ糖認識部位を有した本発明化合物を合成することができる。また、式（３）の化合物の代わりに、式（４）の化合物（実施例における化合物４ｄ）から同様の環化反応を行い、次いで置換基の変換を行い、最終化合物へと合成してもよい。これらの合成方法により、種々のオリゴ糖に選択的なオリゴ糖認識部位を導入することができ、これにより種々のオリゴ糖に適用可能なプローブを調製することが可能である。

In addition, the compound of the present invention (compound 5d in Examples) having an acetylene moiety of the following formula (3) obtained in the Examples below is used as a production intermediate, and an azide into which an oligosaccharide recognition site has been introduced is reacted. Thus, the acetylene moiety and the azide moiety are cyclized to form a triazole ring, and the compound of the present invention having an oligosaccharide recognition site can be synthesized. Further, instead of the compound of formula (3), the compound of formula (4) (compound 4d in the example) is subjected to the same cyclization reaction, and then the substituent is converted to synthesize the final compound. good. By these synthetic methods, it is possible to introduce selective oligosaccharide recognition sites into various oligosaccharides, thereby making it possible to prepare probes applicable to various oligosaccharides.

以下に本発明を、実施例および試験例により、さらに具体的に説明するが、本発明はこれに限定されるものではない。なお、以下の実施例において示された化合物名は、必ずしもＩＵＰＡＣ命名法に従うものではない。 EXAMPLES The present invention will be described in more detail below with reference to examples and test examples, but the present invention is not limited thereto. The compound names shown in the following examples do not necessarily follow the IUPAC nomenclature.

４－プロパルギルオキシベンズアルデヒド：化合物１ｄの合成

500 mLナスフラスコに、化合物１ｃ（3.00 g、24.6 mmol）、炭酸カリウム（13.58 g、98.3 mmol、4.00 eq.）、臭化プロパルギル（11.69 g、98.3 mmol、4.00 eq.）、アセトン（250 mL）を加え、還流下４時間撹拌した。その後室温まで冷却し、水を加えた後、反応液をエバポレーターで濃縮し、残渣をジクロロメタンで３回抽出した。その後、無水硫酸ナトリウムで乾燥させ、ロータリーエバポレーターで減圧濃縮し、真空乾燥した。得られた生成物をシリカゲル中圧カラムクロマトグラフィー（ヘキサン／酢酸エチル＝１５／１）で分離精製し、白色固体として化合物１ｄを得た（2.94 g、収率74.7 %）。
Mp: 85.0-86.0℃.
¹H NMR (CDCl₃, 400 MHz): δ 9.91 (1H, s, CHO at C1), 7.86 (2H, d, J = 8.8 Hz, CH at C4 and C6), 7.09 (2H, d, J = 8.8 Hz, CH at C3 and C7), 4.78 (2H, d, J = 2.4 Hz, CH₂ at C8), 2.58 (1H, s, CH at C10). 4-propargyloxybenzaldehyde: synthesis of compound 1d

In a 500 mL eggplant flask, compound 1c (3.00 g, 24.6 mmol), potassium carbonate (13.58 g, 98.3 mmol, 4.00 eq.), propargyl bromide (11.69 g, 98.3 mmol, 4.00 eq.), acetone (250 mL). was added and stirred under reflux for 4 hours. After cooling to room temperature and adding water, the reaction solution was concentrated by an evaporator, and the residue was extracted three times with dichloromethane. After that, it was dried over anhydrous sodium sulfate, concentrated under reduced pressure using a rotary evaporator, and dried in vacuum. The resulting product was separated and purified by silica gel medium pressure column chromatography (hexane/ethyl acetate=15/1) to obtain compound 1d as a white solid (2.94 g, yield 74.7%).
MP: 85.0-86.0℃.
¹ H NMR (CDCl ₃ , 400 MHz): δ 9.91 (1H, s, CHO at C1), 7.86 (2H, d, J = 8.8 Hz, CH at C4 and C6), 7.09 (2H, d, J = 8.8 Hz, CH at C3 and C7), 4.78 (2H, d, J = 2.4 Hz, CH ₂ at C8), 2.58 (1H, s, CH at C10).

５－フェニルジピロメタン：化合物２ａの合成

100 mL 三口ナスフラスコに、0.18M ＨＣｌ水溶液（25 mL）を加え、ピロール（0.98 mL、14.1 mmol、3.0 eq.）および化合物１ａ（500 mg、4.7 mmol）をそれぞれ滴下で加え、室温で４時間撹拌した。ＴＬＣ（ヘキサン／酢酸エチル＝８／１）で反応終了を確認し、吸引濾過し、水とヘキサンで洗浄した。その後、得られた固体生成物をシリカゲルオープンカラムクロマトグラフィー（ヘキサン／酢酸エチル＝１５／１）で分離精製し、黄色固体として化合物２ａを得た（555 mg、収率53.0 %）。
Mp: 100.0-101.0℃.
¹H NMR (CDCl₃, 400 MHz): δ 7.91 (2H, brs, NH at N1 and N2), 7.20-7.34 (5H, m, CH at C11, C12, C13, C14 and C15) , 6.70 (2H, m, CH at C1 and C9), 6.17 (2H, dd, J = 5.9, 2.8 Hz, CH at C2 and C8), 5.92 (2H, s, CH at C3 and C7), 5.48 (1H, s, CH at C5). 5-Phenyldipyrromethane: Synthesis of Compound 2a

0.18M HCl aqueous solution (25 mL) was added to a 100 mL three-necked round-bottomed flask, pyrrole (0.98 mL, 14.1 mmol, 3.0 eq.) and compound 1a (500 mg, 4.7 mmol) were added dropwise, respectively, and incubated at room temperature for 4 hours. Stirred. The completion of the reaction was confirmed by TLC (hexane/ethyl acetate=8/1), suction filtration was performed, and the mixture was washed with water and hexane. Thereafter, the resulting solid product was separated and purified by silica gel open column chromatography (hexane/ethyl acetate=15/1) to obtain compound 2a as a yellow solid (555 mg, yield 53.0%).
MP: 100.0-101.0℃.
¹ H NMR (CDCl ₃ , 400 MHz): δ 7.91 (2H, brs, NH at N1 and N2), 7.20-7.34 (5H, m, CH at C11, C12, C13, C14 and C15), 6.70 (2H, m, CH at C1 and C9), 6.17 (2H, dd, J = 5.9, 2.8 Hz, CH at C2 and C8), 5.92 (2H, s, CH at C3 and C7), 5.48 (1H, s, CH at C5).

５－（４－メトキシフェニル）ジピロメタン：化合物２ｂの合成

100 mL 三口ナスフラスコに、0.18M ＨＣｌ水溶液（75 mL）を加え、ピロール（2.29 mL, 33.0 mmol、3.0 eq.）および化合物１ｂ（1.50 g、11.0 mmol）をそれぞれ滴下し、室温で１５時間撹拌した。ＴＬＣ（ヘキサン／酢酸エチル＝４／１）で反応終了を確認し、吸引濾過し、水とヘキサンで洗浄した。その後、得られた固体生成物をシリカゲルオープンカラムクロマトグラフィー（ヘキサン／酢酸エチル＝８／１）で分離精製し、黄色固体として化合物２ｂを得た（1.54 g、収率55.5 %）。
Mp: 99.0-100.0℃.
¹H NMR (CDCl₃, 400 MHz): δ 7.92 (2H, brs, NH, at N1 and N2), 7.13 (2H, d, J = 8.6 Hz, CH at C12 and C14), 6.85 (2H, d, J = 8.6 Hz, CH at C11 and C15), 6.70 (2H, m, CH at C1 and C9), 6.16 (2H, dd, J = 5.9, 2.8 Hz, CH at C2 and C8), 5.91 (2H, s, CH at C3 and C7), 5.44 (1H, s, CH at C5), 3.80 (3H, s, CH₃ at C16). 5-(4-Methoxyphenyl)dipyrromethane: synthesis of compound 2b

Add 0.18M HCl aqueous solution (75 mL) to a 100 mL three-neck eggplant flask, add pyrrole (2.29 mL, 33.0 mmol, 3.0 eq.) and compound 1b (1.50 g, 11.0 mmol) dropwise, and stir at room temperature for 15 hours. bottom. The completion of the reaction was confirmed by TLC (hexane/ethyl acetate=4/1), suction filtration was performed, and the mixture was washed with water and hexane. Thereafter, the resulting solid product was separated and purified by silica gel open column chromatography (hexane/ethyl acetate=8/1) to obtain compound 2b as a yellow solid (1.54 g, yield 55.5%).
Mp: 99.0-100.0℃.
¹ H NMR (CDCl ₃ , 400 MHz): δ 7.92 (2H, brs, NH, at N1 and N2), 7.13 (2H, d, J = 8.6 Hz, CH at C12 and C14), 6.85 (2H, d, J = 8.6 Hz, CH at C11 and C15), 6.70 (2H, m, CH at C1 and C9), 6.16 (2H, dd, J = 5.9, 2.8 Hz, CH at C2 and C8), 5.91 (2H, s , CH at C3 and C7), 5.44 (1H, s, CH at C5), 3.80 (3H, s, CH ₃ at C16).

５－（４－ヒドロキシフェニル）ジピロメタン：化合物２ｃの合成

100 mL 三口ナスフラスコに、0.18 M ＨＣｌ水溶液（25 mL）、ピロール（0.85 mL、12.3 mmol、3.0 eq.）、ＥｔＯＨ（10 mL）に溶解した化合物１ｃ（500 mg、4.09 mmol）を加え、室温で４時間撹拌した。ＴＬＣ（ヘキサン／酢酸エチル＝２／１）で反応終了を確認し、飽和炭酸水素ナトリウム水溶液で反応停止した。反応液を酢酸エチルで3回抽出し、飽和食塩水溶液で3回洗浄した。その後、無水硫酸ナトリウムで乾燥させ、ロータリーエバポレーターで減圧濃縮し、真空乾燥した。得られた生成物をシリカゲルオープンカラムクロマトグラフィー（ヘキサン／酢酸エチル＝２／１）で分離精製し、橙色固体として化合物２ｃを得た（250 mg、収率25.6 %）。
Mp: 107.5-109.5℃.
¹H NMR (CDCl₃, 400 MHz): δ 7.92 (2H, brs, NH at N1 and N2), 7.09 (2H, d, J = 8.5 Hz, CH at C12 and C14), 6.78 (2H, d, J = 8.5 Hz, CH at C11 and C15), 6.70 (2H, m, CH at C1 and C9), 6.16 (2H, dd, J = 5.9, 2.8 Hz, CH at C2 and C8), 5.91 (2H, s, CH at C3 and C7), 5.43 (1H, s, CH at C5), 4.66 (1H, s, OH at C13).
HRMS m/z [M+Na]⁺ Calcd for C₁₅H₁₄N₂NaO⁺ 261.0998; Found 261.1003. 5-(4-hydroxyphenyl)dipyrromethane: synthesis of compound 2c

Compound 1c (500 mg, 4.09 mmol) dissolved in 0.18 M HCl aqueous solution (25 mL), pyrrole (0.85 mL, 12.3 mmol, 3.0 eq.), and EtOH (10 mL) was added to a 100 mL three-necked eggplant flask, and the mixture was stirred at room temperature. and stirred for 4 hours. The completion of the reaction was confirmed by TLC (hexane/ethyl acetate=2/1), and the reaction was stopped with a saturated sodium bicarbonate aqueous solution. The reaction solution was extracted with ethyl acetate three times and washed with saturated saline solution three times. After that, it was dried over anhydrous sodium sulfate, concentrated under reduced pressure using a rotary evaporator, and dried in vacuum. The resulting product was separated and purified by silica gel open column chromatography (hexane/ethyl acetate=2/1) to obtain compound 2c as an orange solid (250 mg, yield 25.6%).
MP: 107.5-109.5℃.
¹ H NMR (CDCl ₃ , 400 MHz): δ 7.92 (2H, brs, NH at N1 and N2), 7.09 (2H, d, J = 8.5 Hz, CH at C12 and C14), 6.78 (2H, d, J = 8.5 Hz, CH at C11 and C15), 6.70 (2H, m, CH at C1 and C9), 6.16 (2H, dd, J = 5.9, 2.8 Hz, CH at C2 and C8), 5.91 (2H, s, CH at C3 and C7), 5.43 (1H, s, CH at C5), 4.66 (1H, s, OH at C13).
HRMS m/z [M+Na] ⁺ _Calcd for _C15H14N2NaO ⁺ 261.0998; Found _261.1003 .

５－（４－プロパルギルオキシフェニル）ジピロメタン：化合物２ｄの合成

300 mL 三口ナスフラスコに、0.18 M ＨＣｌ水溶液（100 mL）を加え、ピロール（2.59 mL、37.5 mmol、3.0 eq.)、化合物１ｄ（2.00 g、12.5 mmol）を加え、室温で１５時間撹拌した。ＴＬＣ（ヘキサン／酢酸エチル＝４／１)で反応終了を確認し、飽和ＮａＨＣＯ_３水溶液で反応停止した。反応液を酢酸エチルで３回抽出し、飽和食塩水溶液で洗浄した。その後、無水硫酸ナトリウムで乾燥させ、ロータリーエバポレーターで減圧濃縮し、真空乾燥した。得られた生成物をシリカゲルオープンカラムクロマトグラフィー（ヘキサン／酢酸エチル＝８／１）で分離精製し、化合物２ｄ含有の黄色ピロール／酢酸エチル溶液を得た（5.21 g（うち化合物２ｄ 2.19 g）、収率63.4%）。
¹H NMR (CDCl₃, 400 MHz): δ 7.89 (2H, brs, NH at N1 and N2), 7.12 (2H, d, J = 8.8 Hz, CH at C12 and C14), 6.91 (2H, d, J = 8.8 Hz, CH at C11 and C15), 6.67 (2H, m, CH at C1 and C9), 6.15 (2H, q, J = 2.8 Hz, CH at C2 and C8), 5.90 (2H, s, CH at C3 and C7), 5.43 (1H, s, CH at C5), 4.66 (2H, d, J = 2.6 Hz, CH₂ at C16), 2.50 (1H, t, J = 2.5 Hz, CH at C18). 5-(4-propargyloxyphenyl)dipyrromethane: synthesis of compound 2d

A 0.18 M HCl aqueous solution (100 mL) was added to a 300 mL three-neck eggplant flask, pyrrole (2.59 mL, 37.5 mmol, 3.0 eq.) and compound 1d (2.00 g, 12.5 mmol) were added, and the mixture was stirred at room temperature for 15 hours. After confirming the completion of the reaction by TLC (hexane/ethyl acetate=4/1), the reaction was quenched with saturated NaHCO ₃ aqueous solution. The reaction solution was extracted three times with ethyl acetate and washed with saturated saline solution. After that, it was dried over anhydrous sodium sulfate, concentrated under reduced pressure using a rotary evaporator, and dried in vacuum. The obtained product was separated and purified by silica gel open column chromatography (hexane/ethyl acetate = 8/1) to obtain a yellow pyrrole/ethyl acetate solution containing compound 2d (5.21 g (including 2.19 g of compound 2d), Yield 63.4%).
¹ H NMR (CDCl ₃ , 400 MHz): δ 7.89 (2H, brs, NH at N1 and N2), 7.12 (2H, d, J = 8.8 Hz, CH at C12 and C14), 6.91 (2H, d, J = 8.8 Hz, CH at C11 and C15), 6.67 (2H, m, CH at C1 and C9), 6.15 (2H, q, J = 2.8 Hz, CH at C2 and C8), 5.90 (2H, s, CH at C3 and C7), 5.43 (1H, s, CH at C5), 4.66 (2H, d, J = 2.6 Hz, CH ₂ at C16), 2.50 (1H, t, J = 2.5 Hz, CH at C18).

５－（４－ニトロフェニル）ジピロメタン：化合物２ｅの合成

300 mLナスフラスコに、0.18 M ＨＣｌ水溶液（50 mL）を加え、ピロール（1.37 mL, 19.9 mmol、3.0 eq.）、化合物１ｅ（1.00 g、6.62 mmol）を加え、室温で２時間撹拌した。ＴＬＣ（ヘキサン／酢酸エチル＝５／１）で反応終了を確認し、吸引ろ過し、水とヘキサンで洗浄し、黄色固体として化合物２ｅを得た（1.73 g、収率98.1 %）。
Mp: 157-160℃.
¹H NMR (CDCl₃, 400 MHz): δ 8.16 (2H, d, J = 8.7 Hz, CH at C12 and C14), 8.01 (2H, brs, NH at N1 and N2), 7.36 (2H, d, J = 8.7 Hz, CH at C11 and C15), 6.75 (2H, m, CH at C1 and C9), 6.17 (2H, m, CH at C2 and C8), 5.87 (2H, m, CH at C3 and C7), 5.58 (1H, s, CH at C5). 5-(4-Nitrophenyl)dipyrromethane: synthesis of compound 2e

A 0.18 M HCl aqueous solution (50 mL) was added to a 300 mL eggplant flask, pyrrole (1.37 mL, 19.9 mmol, 3.0 eq.) and compound 1e (1.00 g, 6.62 mmol) were added, and the mixture was stirred at room temperature for 2 hours. The completion of the reaction was confirmed by TLC (hexane/ethyl acetate=5/1), suction filtration was performed, and the residue was washed with water and hexane to obtain compound 2e as a yellow solid (1.73 g, yield 98.1%).
MP: 157-160℃.
¹ H NMR (CDCl ₃ , 400 MHz): δ 8.16 (2H, d, J = 8.7 Hz, CH at C12 and C14), 8.01 (2H, brs, NH at N1 and N2), 7.36 (2H, d, J = 8.7 Hz, CH at C11 and C15), 6.75 (2H, m, CH at C1 and C9), 6.17 (2H, m, CH at C2 and C8), 5.87 (2H, m, CH at C3 and C7), 5.58 (1H, s, CH at C5).

１，９－ジクロロ－５－フェニルジピリン：化合物３ａの合成

窒素雰囲気下の300 mL 三口ナスフラスコに、化合物２ａ（400 mg、1.8 mmol）を加え、ＴＨＦ（20 mL）を加えて撹拌し、－７８℃に冷却した後、ＴＨＦ（20 mL）中のＮＣＳ（577 mg, 4.3 mmol、2.4 eq.）を滴下し、－７８℃のまま２時間撹拌した。その後、室温で更に２時間撹拌し、ＴＬＣ（ヘキサン／酢酸エチル＝４／１)で反応終了を確認し、更に、ＴＨＦ（15 mL）中のＤＤＱ（490 mg、2.2 mmol、1.2 eq.）を滴下し、室温で２時間撹拌した。ＴＬＣ（ヘキサン／酢酸エチル＝８／１）で反応終了を確認し、反応停止した。反応液をＣＨ_２Ｃｌ_２で３回抽出し、飽和食塩水溶液で３回洗浄した。その後、無水硫酸ナトリウムで乾燥させ、ロータリーエバポレーターで減圧濃縮し、真空乾燥した。得られた生成物をシリカゲルオープンカラムクロマトグラフィー（ヘキサン／酢酸エチル＝８／１、ヘキサン／酢酸エチル＝１５／１）で、２回分離精製し、橙色固体として化合物３ａを得た（412.6 mg、収率79.3 %）。
Mp: 157-160℃.
¹H NMR (CDCl₃, 400 MHz): δ 12.36 (1H, brs, NH at N1), 7.52-7.41 (5H, m, CH at C11, C12, C13, C14 and C15), 6.52 (2H, d, J = 4.3 Hz, CH at C2 and C8), 6.25 (2H, d, J = 4.3 Hz, CH at C3 and C7). 1,9-dichloro-5-phenyldipyrine: synthesis of compound 3a

Compound 2a (400 mg, 1.8 mmol) was added to a 300 mL three-neck eggplant flask under a nitrogen atmosphere, THF (20 mL) was added and stirred, cooled to −78° C., NCS in THF (20 mL) was (577 mg, 4.3 mmol, 2.4 eq.) was added dropwise, and the mixture was stirred at -78°C for 2 hours. After that, the reaction was further stirred at room temperature for 2 hours, and the completion of the reaction was confirmed by TLC (hexane/ethyl acetate=4/1). It was added dropwise and stirred at room temperature for 2 hours. After confirming the completion of the reaction by TLC (hexane/ethyl acetate=8/1), the reaction was terminated. The reaction was extracted with CH ₂ Cl ₂ three times and washed with saturated brine three times. After that, it was dried over anhydrous sodium sulfate, concentrated under reduced pressure using a rotary evaporator, and dried in vacuum. The resulting product was separated and purified twice by silica gel open column chromatography (hexane/ethyl acetate = 8/1, hexane/ethyl acetate = 15/1) to obtain compound 3a as an orange solid (412.6 mg, Yield 79.3%).
MP: 157-160℃.
¹ H NMR (CDCl ₃ , 400 MHz): δ 12.36 (1H, brs, NH at N1), 7.52-7.41 (5H, m, CH at C11, C12, C13, C14 and C15), 6.52 (2H, d, J = 4.3 Hz, CH at C2 and C8), 6.25 (2H, d, J = 4.3 Hz, CH at C3 and C7).

３，７－ジクロロ－５，５－ジフルオロ－１０－フェニル－５Ｈ－４λ ^４，５λ ^４－ジピロロ［１，２－ｃ：２’，１’－ｆ］［１，３，２］ジアザボリニン：化合物４ａの合成

窒素雰囲気下の100 mL 三口ナスフラスコに、化合物３ａ（400 mg、1.4 mmol）を加え、ＣＨ_２Ｃｌ_２（40 mL）を加えて撹拌し、トリエチルアミン（1.2 mL、8.5 mmol、6.0 eq.）を滴下し、室温で１０分間撹拌した。その後、三フッ化ホウ素ジエチルエーテル錯塩（1.2 mL、8.5 mmol、6.0 eq.）を滴下し、室温で２４時間撹拌した。ＴＬＣ（ヘキサン／酢酸エチル＝１０／１）で反応終了を確認し、反応停止した。反応液をＣＨ_２Ｃｌ_２で３回抽出し、飽和食塩水溶液で３回洗浄した。その後、無水硫酸ナトリウムで乾燥させ、ロータリーエバポレーターで減圧濃縮し、真空乾燥した。得られた生成物をシリカゲルオープンカラムクロマトグラフィー（ヘキサン／酢酸エチル＝１５／１）で分離精製し、赤色固体として化合物４ａを得た（368 mg、収率78.9 %）。
Mp: 68-71℃.
¹H NMR (CDCl₃, 400 MHz): δ 7.50-7.61 (5H, m, CH at C11, C12, C13, C14 and C15), 6.86 (2H, d, J = 4.4 Hz, CH at C2 and C8), 6.45 (2H, d, J = 4.4 Hz, CH at C3 and C7). 3,7-dichloro-5,5-difluoro-10-phenyl-5H-4λ ⁴ ,5λ ⁴ -dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborinine: compounds Synthesis of 4a

Compound 3a (400 mg, 1.4 mmol) was added to a 100 mL three-neck eggplant flask under a nitrogen atmosphere, CH ₂ Cl ₂ (40 mL) was added and stirred, and triethylamine (1.2 mL, 8.5 mmol, 6.0 eq.) was added. It was added dropwise and stirred at room temperature for 10 minutes. After that, boron trifluoride diethyl etherate (1.2 mL, 8.5 mmol, 6.0 eq.) was added dropwise and stirred at room temperature for 24 hours. After confirming the completion of the reaction by TLC (hexane/ethyl acetate=10/1), the reaction was stopped. The reaction was extracted with CH ₂ Cl ₂ three times and washed with saturated brine three times. After that, it was dried over anhydrous sodium sulfate, concentrated under reduced pressure using a rotary evaporator, and dried in vacuum. The resulting product was separated and purified by silica gel open column chromatography (hexane/ethyl acetate=15/1) to obtain compound 4a as a red solid (368 mg, yield 78.9%).
MP: 68-71℃.
¹ H NMR (CDCl ₃ , 400 MHz): δ 7.50-7.61 (5H, m, CH at C11, C12, C13, C14 and C15), 6.86 (2H, d, J = 4.4 Hz, CH at C2 and C8) , 6.45 (2H, d, J = 4.4 Hz, CH at C3 and C7).

３，７－ジクロロ－５，５－ジフルオロ－１０－（４－メトキシフェニル）－５Ｈ－４λ ^４，５λ ^４－ジピロロ［１，２－ｃ：２’，１’－ｆ］［１，３，２］ジアザボリニン：化合物４ｂの合成

窒素雰囲気下の300 mL 三口ナスフラスコに、化合物２ｂ（1.50 g、5.94 mmol）を加え、ＴＨＦ（30 mL）を加えて撹拌し、－７８℃に冷却した後、ＴＨＦ（70 mL）中のＮ－クロロコハク酸イミド（1.59 g、11.89 mmol、2.0 eq.）を滴下し、－７８℃のまま１時間撹拌した。その後、－２０℃で更に３時間撹拌し、ＴＬＣ（ヘキサン／酢酸エチル＝４／１)で反応終了を確認し、反応停止した。反応液をＣＨ_２Ｃｌ_２で３回抽出し、水で３回洗浄した。その後、無水硫酸ナトリウムで乾燥させ、ロータリーエバポレーターで減圧濃縮し、真空乾燥した。得られた残渣を更に窒素雰囲気下の300 mL 三口ナスフラスコに加え、ＣＨ_２Ｃｌ_２（50 mL）を加えて撹拌し、ＣＨ_２Ｃｌ_２（150 mL）中の２，３－ジクロロ－５，６－ジシアノ－ｐ－ベンゾキノン（1.62 g、7.13 mmol、1.2 eq.）を滴下し、室温で２時間撹拌した。ＴＬＣ（ヘキサン／酢酸エチル＝６／１）で反応終了を確認した。その後、Ｎ，Ｎ－ジイソプロピルエチルアミン（10 mL、57.4/1 mmol、9.7 eq.）を滴下し、更に三フッ化ホウ素ジエチルエーテル錯塩（10 mL、79.33 mmol、13.4 eq.）を滴下し、室温で１時間撹拌した。ＴＬＣ（ヘキサン／酢酸エチル＝６／１）で反応終了を確認し、反応停止した。反応液をＣＨ_２Ｃｌ_２で３回抽出し、飽和食塩水溶液で３回洗浄した。その後、無水硫酸ナトリウムで乾燥させ、ロータリーエバポレーターで減圧濃縮し、真空乾燥した。得られた生成物をシリカゲルオープンカラムクロマトグラフィー（ヘキサン／酢酸エチル＝４／１）で分離精製し、更にヘキサン／ＣＨ_２Ｃｌ_２で再結晶し、赤褐色固体として化合物４ｂを得た（1.49 g、収率 78.9 %）。
Mp: 157-160℃.
¹H NMR (CDCl₃, 400 MHz): δ 7.46 (2H, d, J = 8.8 Hz, CH at C12 and C14), 7.04 (2H, d, J = 8.8 Hz, CH at C11 and C15), 6.89 (2H, d, J = 4.5 Hz, CH at C2 and C8), 6.44 (2H, d, J = 4.5 Hz, CH at C3 and C7), 3.91 (s, 3H, CH₃ at C16). 3,7-dichloro-5,5-difluoro-10-(4-methoxyphenyl)-5H-4λ ⁴ ,5λ ⁴ -dipyrrolo[1,2-c:2′,1′-f][1,3, 2] Diazaborinine: synthesis of compound 4b

Compound 2b (1.50 g, 5.94 mmol) was added to a 300 mL three-neck eggplant flask under a nitrogen atmosphere, THF (30 mL) was added and stirred, cooled to −78° C., and N in THF (70 mL) was added. -Chlorosuccinimide (1.59 g, 11.89 mmol, 2.0 eq.) was added dropwise and stirred at -78°C for 1 hour. After that, the mixture was stirred at -20°C for 3 hours, and after confirming the completion of the reaction by TLC (hexane/ethyl acetate = 4/1), the reaction was terminated. The reaction was extracted with CH ₂ Cl ₂ three times and washed with water three times. After that, it was dried over anhydrous sodium sulfate, concentrated under reduced pressure using a rotary evaporator, and dried in vacuum. The resulting residue was further added to a 300 mL three-neck eggplant flask under a nitrogen _{atmosphere, CH 2 Cl 2} ₍ ₅₀ mL) was added and stirred, and 2,3-dichloro- ₅ , 6-Dicyano-p-benzoquinone (1.62 g, 7.13 mmol, 1.2 eq.) was added dropwise and stirred at room temperature for 2 hours. The completion of the reaction was confirmed by TLC (hexane/ethyl acetate=6/1). After that, N,N-diisopropylethylamine (10 mL, 57.4/1 mmol, 9.7 eq.) was added dropwise, and boron trifluoride diethyl etherate (10 mL, 79.33 mmol, 13.4 eq.) was added dropwise. Stirred for 1 hour. After confirming the completion of the reaction by TLC (hexane/ethyl acetate=6/1), the reaction was stopped. The reaction was extracted with CH ₂ Cl ₂ three times and washed with saturated brine three times. After that, it was dried over anhydrous sodium sulfate, concentrated under reduced pressure using a rotary evaporator, and dried in vacuum. The resulting product was separated and purified by silica gel open column chromatography (hexane/ethyl acetate = 4/1) and recrystallized with hexane/CH ₂ Cl ₂ to obtain compound 4b as a reddish brown solid (1.49 g, Yield 78.9%).
MP: 157-160℃.
¹ H NMR (CDCl ₃ , 400 MHz): δ 7.46 (2H, d, J = 8.8 Hz, CH at C12 and C14), 7.04 (2H, d, J = 8.8 Hz, CH at C11 and C15), 6.89 ( 2H, d, J = 4.5 Hz, CH at C2 and C8), 6.44 (2H, d, J = 4.5 Hz, CH at C3 and C7), 3.91 (s, 3H, CH ₃ at C16).

４－（３，７－ジクロロ－５，５－ジフルオロ－５Ｈ－４λ ^４，５λ ^４－ジピロロ［１，２－ｃ：２’，１’－ｆ］［１，３，２］ジアザボリニン－１０－イル）フェノール：化合物４ｃの合成

窒素雰囲気下の300 mL 三口ナスフラスコに、化合物２ｃ（600 mg、5.94 mmol）を加え、ＴＨＦ（15 mL）を加えて撹拌し、－７８℃に冷却した後、ＴＨＦ（40 mL）中のＮ－クロロコハク酸イミド（740 mg、5.54 mmol、2.2 eq.）を滴下し、－７８℃のまま２時間撹拌した。その後、室温で更に３時間撹拌し、ＴＬＣ（ヘキサン／酢酸エチル＝２／１)で反応終了を確認し、反応停止した。反応液をＣＨ_２Ｃｌ_２で３回抽出した。その後、無水硫酸ナトリウムで乾燥させ、ロータリーエバポレーターで減圧濃縮し、真空乾燥した。得られた残渣を更に窒素雰囲気下の300 mL 一口ナスフラスコに加え、ＣＨ_２Ｃｌ_２（30 mL）を加えて撹拌し、ＣＨ_２Ｃｌ_２（50 mL）中の２，３－ジクロロ－５，６－ジシアノ－ｐ－ベンゾキノン（686 mg、3.02 mmol、1.2 eq.）を滴下し、室温で２時間撹拌した。ＴＬＣ（ヘキサン／酢酸エチル＝６／１)で反応終了を確認し、その後Ｎ，Ｎ－ジイソプロピルエチルアミン（4.3 mL, 25.18 mmol、10.0 eq.）を滴下し、更に、三フッ化ホウ素ジエチルエーテル錯塩（4.4 mL, 35.25 mmol、14.0 eq.）を滴下し、室温で２４時間撹拌した。ＴＬＣ（ヘキサン／酢酸エチル＝１／１）で反応終了を確認し、反応停止した。反応液をＣＨ_２Ｃｌ_２で３回抽出した。その後、無水硫酸ナトリウムで乾燥させ、ロータリーエバポレーターで減圧濃縮し、真空乾燥した。得られた生成物をシリカゲル中圧カラムクロマトグラフィー（ヘキサン／酢酸エチル＝３／１）で分離精製し、赤褐色固体として標題化合物を得た（308.4 mg、収率34.7 %）。
Mp: 223-227℃.
¹H NMR (CDCl₃, 400 MHz): δ 7.40 (2H, d, J = 8.6 Hz, CH at C12 and C14), 7.00 (2H, d, J = 8.6 Hz, CH at C11 and C15), 6.89 (2H, d, J = 4.2 Hz, CH at C2 and C8), 6.44 (2H, d, J = 4.2 Hz, CH at C3 and C7).
HRMS m/z [M+Na]⁺ Calcd for C₁₅H₉BCl₂F₂N₂NaO⁺ 375.0045; Found 375.0032. 4-(3,7-dichloro-5,5-difluoro-5H-4λ 4 ^, 5λ ⁴ -dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborinine-10- yl) Phenol: Synthesis of Compound 4c

Compound 2c (600 mg, 5.94 mmol) was added to a 300 mL three-neck eggplant flask under a nitrogen atmosphere, THF (15 mL) was added and stirred, cooled to −78° C., and N in THF (40 mL) was added. -Chlorosuccinimide (740 mg, 5.54 mmol, 2.2 eq.) was added dropwise and stirred at -78°C for 2 hours. After that, the mixture was stirred at room temperature for another 3 hours, the completion of the reaction was confirmed by TLC (hexane/ethyl acetate=2/1), and the reaction was stopped. The reaction was extracted with _CH2Cl2 _three times. After that, it was dried over anhydrous sodium sulfate, concentrated under reduced pressure using a rotary evaporator, and dried in vacuum. The resulting residue was further added to a 300 mL single-neck eggplant flask under a nitrogen atmosphere _, CH ₂ Cl ₂ (30 mL) was added and stirred, and 2,3-dichloro- ₅ , 6-Dicyano-p-benzoquinone (686 mg, 3.02 mmol, 1.2 eq.) was added dropwise and stirred at room temperature for 2 hours. After confirming the completion of the reaction by TLC (hexane/ethyl acetate = 6/1), N,N-diisopropylethylamine (4.3 mL, 25.18 mmol, 10.0 eq.) was added dropwise, and boron trifluoride diethyl ether complex ( 4.4 mL, 35.25 mmol, 14.0 eq.) was added dropwise and stirred at room temperature for 24 hours. After confirming the completion of the reaction by TLC (hexane/ethyl acetate=1/1), the reaction was stopped. The reaction was extracted with _CH2Cl2 _three times. After that, it was dried over anhydrous sodium sulfate, concentrated under reduced pressure using a rotary evaporator, and dried in vacuum. The obtained product was separated and purified by silica gel medium pressure column chromatography (hexane/ethyl acetate=3/1) to obtain the title compound as a reddish brown solid (308.4 mg, yield 34.7%).
MP: 223-227℃.
¹ H NMR (CDCl ₃ , 400 MHz): δ 7.40 (2H, d, J = 8.6 Hz, CH at C12 and C14), 7.00 (2H, d, J = 8.6 Hz, CH at C11 and C15), 6.89 ( 2H, d, J = 4.2 Hz, CH at C2 and C8), 6.44 (2H, d, J = 4.2 Hz, CH at C3 and C7).
_HRMS m/ _z [M+Na] ⁺ Calcd for _{C15H9BCl2F2N2NaO} ⁺ _375.0045 ; _Found 375.0032.

３，７－ジクロロ－５，５－ジフルオロ－１０－（４－（プロパ－２－イン－１－イルオキシ）フェニル－５Ｈ－４λ ^４，５λ ^４－ジピロロ［１，２－ｃ：２’，１’－ｆ］［１，３，２］ジアザボリニン：化合物４ｄの合成

窒素雰囲気下の300 mL 三口ナスフラスコに、化合物２ｄ溶液（1.58 g、5.72 mmol）を加え、ＴＨＦ（50 mL）を加えて撹拌し、－７８℃に冷却した後、ＴＨＦ（100 mL）中のＮ－クロロコハク酸イミド（1.68 g、12.6 mmol、2.2 eq.）を滴下し、－７８℃のまま１５分間撹拌した。その後、室温で更に２時間撹拌し、ＴＬＣ（ヘキサン／酢酸エチル＝４／１）で反応終了を確認し、反応停止した。反応液をＣＨ_２Ｃｌ_２で３回抽出した。その後、無水硫酸ナトリウムで乾燥させ、ロータリーエバポレーターで減圧濃縮し、真空乾燥した。得られた残渣を更に窒素雰囲気下の500 mL 一口ナスフラスコに加え、ＣＨ_２Ｃｌ_２（50 mL）を加えて撹拌し、ＣＨ_２Ｃｌ_２（150 mL）中の２，３－ジクロロ－５，６－ジシアノ－ｐ－ベンゾキノン（1.56 g、6.87 mmol、1.2 eq.）を滴下し、室温で２時間撹拌した。ＴＬＣ（ヘキサン／酢酸エチル＝４／１)で反応終了を確認しその後、Ｎ，Ｎ－ジイソプロピルエチルアミン (7.9 mL、45.8 mmol、8.0 eq.）を滴下し、更に、三フッ化ホウ素ジエチルエーテル錯塩（7.3 mL、57.2 mmol、10.0 eq.）を滴下し、室温で２４時間撹拌した。ＴＬＣ（ヘキサン／酢酸エチル＝１／１）で反応終了を確認し、反応停止した。反応液をＣＨ_２Ｃｌ_２で３回抽出した。その後、無水硫酸ナトリウムで乾燥させ、ロータリーエバポレーターで減圧濃縮し、真空乾燥した。得られた生成物をシリカゲル中圧カラムクロマトグラフィー（ヘキサン／酢酸エチル＝５／１）で分離精製し、赤褐色固体として化合物４ｄを得た（1.78 g、収率79.5 %）。
Mp: 201-204℃.
¹H NMR (CDCl₃, 400 MHz): δ 7.46 (2H, d, J = 8.7 Hz, CH at C12 and C14), 7.12 (2H, d, J = 8.7 Hz, CH at C11 and C15), 6.89 (2H, d, J = 4.2 Hz, CH at C2 and C8), 6.44 (2H, d, J = 4.2 Hz, CH at C3 and C7), 4.79 (2H, d, J = 2.4 Hz, CH₂ at C16), 2.60 (1H, t, J = 2.4 Hz, CH at C18). 3,7-dichloro-5,5-difluoro-10-(4-(prop-2-yn-1-yloxy)phenyl-5H-4λ ⁴ ,5λ ⁴ -dipyrrolo[1,2-c:2′,1 '-f][1,3,2]diazaborinine: synthesis of compound 4d

Compound 2d solution (1.58 g, 5.72 mmol) was added to a 300 mL three-neck eggplant flask under a nitrogen atmosphere, THF (50 mL) was added and stirred, cooled to −78° C., and dissolved in THF (100 mL). N-chlorosuccinimide (1.68 g, 12.6 mmol, 2.2 eq.) was added dropwise and stirred at -78°C for 15 minutes. After that, the mixture was stirred at room temperature for another 2 hours, the completion of the reaction was confirmed by TLC (hexane/ethyl acetate=4/1), and the reaction was stopped. The reaction was extracted with _CH2Cl2 _three times. After that, it was dried over anhydrous sodium sulfate, concentrated under reduced pressure using a rotary evaporator, and dried in vacuum. The resulting residue was further added to a 500 mL single-neck eggplant flask under a nitrogen _atmosphere , CH ₂ Cl ₂ (50 mL) was added and stirred, and 2,3-dichloro- ₅ , 6-Dicyano-p-benzoquinone (1.56 g, 6.87 mmol, 1.2 eq.) was added dropwise and stirred at room temperature for 2 hours. After confirming the completion of the reaction by TLC (hexane/ethyl acetate = 4/1), N,N-diisopropylethylamine (7.9 mL, 45.8 mmol, 8.0 eq.) was added dropwise, and boron trifluoride diethyl ether complex ( 7.3 mL, 57.2 mmol, 10.0 eq.) was added dropwise and stirred at room temperature for 24 hours. After confirming the completion of the reaction by TLC (hexane/ethyl acetate=1/1), the reaction was stopped. The reaction was extracted with _CH2Cl2 _three times. After that, it was dried over anhydrous sodium sulfate, concentrated under reduced pressure using a rotary evaporator, and dried in vacuum. The resulting product was separated and purified by silica gel medium pressure column chromatography (hexane/ethyl acetate=5/1) to obtain compound 4d as a reddish brown solid (1.78 g, yield 79.5%).
MP: 201-204℃.
¹ H NMR (CDCl ₃ , 400 MHz): δ 7.46 (2H, d, J = 8.7 Hz, CH at C12 and C14), 7.12 (2H, d, J = 8.7 Hz, CH at C11 and C15), 6.89 ( 2H, d, J = 4.2 Hz, CH at C2 and C8), 6.44 (2H, d, J = 4.2 Hz, CH at C3 and C7), 4.79 (2H, d, J = 2.4 Hz, CH ₂ at C16) , 2.60 (1H, t, J = 2.4 Hz, CH at C18).

３，７－ジクロロ－５，５－ジフルオロ－１０－（４－ニトロフェニル）－５Ｈ－４λ ^４，５λ ^４－ジピロロ［１，２－ｃ：２’，１’－ｆ］［１，３，２］ジアザボリニン：化合物４ｅの合成

窒素雰囲気下の300 mL三口ナスフラスコに、ＴＨＦ（60 mL）に溶解した化合物２ｅ（2.00 g、7.48 mmol）を加え、－７８℃に冷却し、ＴＨＦ（70 mL）中のＮＣＳ（2.51 g、18.9 mmol、2.51 eq.）を滴下し、室温で３時間撹拌した。ＴＬＣ（ヘキサン／酢酸エチル＝５／１）で反応終了を確認し、反応停止した。反応液をジクロロメタンで３回抽出し、無水硫酸ナトリウムで乾燥させ、ロータリーエバポレーターで減圧濃縮し、真空乾燥した。得られた残渣を窒素雰囲気下のフラスコに加え、ジクロロメタン（50 mL）に溶解し、ジクロロメタン（50 mL）中のＤＤＱ（2.04 g、8.98 mmol、1.20 eq.）を滴下し、室温で２時間撹拌した。ＴＬＣ（ヘキサン／酢酸エチル＝５／１）で反応終了を確認し、更にＤＩＰＥＡ（7.42 mL、44.9 mmol、6.00 eq.）を滴下した後に、三フッ化ホウ素ジエチルエーテル錯塩（7.9 mL、74.8 mmol、10.0 eq.）を滴下し、室温で２時間撹拌した。ＴＬＣ（ヘキサン／酢酸エチル＝５／１）で反応終了を確認し、反応停止した。反応液をジクロロメタンで３回抽出し、無水硫酸ナトリウムで乾燥させ、ロータリーエバポレーターで減圧濃縮し、真空乾燥した。得られた生成物をシリカゲル中圧カラムクロマトグラフィー（ヘキサン／酢酸エチル／メタノール＝１０／１／０．１）で分離精製し、赤紫色固体として化合物４ｅを得た（1.57 g、収率55.1 %）。
Mp: 202-207℃.
IR (KBr): 3225, 1562, 1452, 1402, 1348, 1267, 1196, 1105 cm^-1.
¹H NMR (CDCl₃, 400 MHz): δ 8.41 (2H, d, J = 6.8 Hz, CH at C12 and C14), 7.70 (2H, d, J = 6.8 Hz, CH at C11 and C15), 6.76 (2H, d, J = 3.5 Hz, CH at C2 and C8), 6.49 (2H, d, J = 3.5 Hz, CH at C3 and C7). 3,7-dichloro-5,5-difluoro-10-(4-nitrophenyl)-5H-4λ ⁴ ,5λ ⁴ -dipyrrolo[1,2-c:2′,1′-f][1,3, 2] Diazaborinine: synthesis of compound 4e

Compound 2e (2.00 g, 7.48 mmol) dissolved in THF (60 mL) was added to a 300 mL three-neck eggplant flask under a nitrogen atmosphere, cooled to −78° C., and NCS (2.51 g, 18.9 mmol, 2.51 eq.) was added dropwise and stirred at room temperature for 3 hours. After confirming the completion of the reaction by TLC (hexane/ethyl acetate=5/1), the reaction was stopped. The reaction solution was extracted three times with dichloromethane, dried over anhydrous sodium sulfate, concentrated under reduced pressure using a rotary evaporator, and dried in vacuo. The resulting residue was added to a flask under nitrogen atmosphere, dissolved in dichloromethane (50 mL), DDQ (2.04 g, 8.98 mmol, 1.20 eq.) in dichloromethane (50 mL) was added dropwise and stirred at room temperature for 2 hours. bottom. The completion of the reaction was confirmed by TLC (hexane/ethyl acetate = 5/1), and DIPEA (7.42 mL, 44.9 mmol, 6.00 eq.) was added dropwise, followed by boron trifluoride diethyl etherate (7.9 mL, 74.8 mmol, 10.0 eq.) was added dropwise and stirred at room temperature for 2 hours. After confirming the completion of the reaction by TLC (hexane/ethyl acetate=5/1), the reaction was stopped. The reaction solution was extracted three times with dichloromethane, dried over anhydrous sodium sulfate, concentrated under reduced pressure using a rotary evaporator, and dried in vacuo. The resulting product was separated and purified by silica gel medium pressure column chromatography (hexane/ethyl acetate/methanol=10/1/0.1) to obtain compound 4e as a reddish purple solid (1.57 g, yield 55.1%). ).
MP: 202-207℃.
IR (KBr): 3225, 1562, 1452, 1402, 1348, 1267, 1196, 1105 cm ^-1 .
¹ H NMR (CDCl ₃ , 400 MHz): δ 8.41 (2H, d, J = 6.8 Hz, CH at C12 and C14), 7.70 (2H, d, J = 6.8 Hz, CH at C11 and C15), 6.76 ( 2H, d, J = 3.5 Hz, CH at C2 and C8), 6.49 (2H, d, J = 3.5 Hz, CH at C3 and C7).

５，５－ジフルオロ－Ｎ ^３，Ｎ ^７－ビス（２－メトキシエチル）－１０－フェニル－５Ｈ－４λ ^４，５λ ^４－ジピロロ［１，２－ｃ：２’，１’－ｆ］［１，３，２］ジアザボリニン－３，７－ジアミン：化合物５ａの合成

10.2 cmエース高耐圧チューブに、化合物４ａ（170 mg、0.50 mmol）を加え、ＣＨ_３ＣＮ（4 mL）を加えて撹拌し、さらに２－メトキシエチルアミン（176 μL、2.02 mmol、4.0 eq.）を加えて１００℃で２４時間撹拌した。ＴＬＣ（ヘキサン／酢酸エチル＝１／１）で反応終了を確認後、ロータリーエバポレーターで減圧濃縮し、真空乾燥した。得られた生成物をシリカゲル中圧カラムクロマトグラフィー（ヘキサン／酢酸エチル＝４／１）で分離精製し、青紫色固体として化合物５ａを得た（125 mg、収率60.0 %）。
Mp: 129-130℃.
¹H NMR (CDCl₃, 400 MHz): δ 7.38-7.48 (5H, m, CH at C11, C12, C13, C14 and C15), 6.54 (2H, d, J = 4.4 Hz, CH at C2 and C8), 5.74 (2H, d, J = 4.4 Hz, CH at C3 and C7), 5.77-5.70 (2H, brs, NH at N3 and N4), 3.59 (4H, t, J = 5.5 Hz, CH₂ at C17 and C20), 3.44 (4H, q, J = 5.5 Hz, CH₂ at C16 and C19), 3.40 (6H, s, CH₃ at C18 and C21).
¹³C NMR (CDCl₃, 100 MHz): δ 156.6 (C1 and C9), 135.0 (C10), 130.4 (CH at C11 and C15), 128.9 (C4 and C6), 131.3 (C5), 128.5 (CH at C13), 128.5 (CH at C2 and C8), 127.9 (CH at C12 and C14), 101.1 (CH at C3 and C7), 71.4 (CH₂ at C17 and C20), 59.1 (CH₃ at C18 and C21), 44.3 (CH₂ at C16 and C19).
HRMS m/z [M+Na]⁺ Calcd for C₂₁H₂₅BF₂N₄NaO₂ ⁺ 437.1931; Found 437.1931. 5,5-difluoro-N ³ ,N ⁷ -bis(2-methoxyethyl)-10-phenyl-5H-4λ ⁴ ,5λ ⁴ -dipyrrolo[1,2-c:2′,1′-f][1 ,3,2]diazaborinine-3,7-diamine: synthesis of compound 5a

Compound 4a (170 mg, 0.50 mmol) was added to a 10.2 cm Ace high pressure tube, CH ₃ CN (4 mL) was added and stirred, and 2-methoxyethylamine (176 μL, 2.02 mmol, 4.0 eq.) was added. In addition, the mixture was stirred at 100° C. for 24 hours. After confirming the completion of the reaction by TLC (hexane/ethyl acetate=1/1), the mixture was concentrated under reduced pressure using a rotary evaporator and dried in vacuum. The resulting product was separated and purified by silica gel medium pressure column chromatography (hexane/ethyl acetate=4/1) to obtain compound 5a as a bluish purple solid (125 mg, yield 60.0%).
MP: 129-130℃.
¹ H NMR (CDCl ₃ , 400 MHz): δ 7.38-7.48 (5H, m, CH at C11, C12, C13, C14 and C15), 6.54 (2H, d, J = 4.4 Hz, CH at C2 and C8) , 5.74 (2H, d, J = 4.4 Hz, CH at C3 and C7), 5.77-5.70 (2H, brs, NH at N3 and N4), 3.59 (4H, t, J = 5.5 Hz, CH ₂ at C17 and C20), 3.44 (4H, q, J = 5.5 Hz, CH ₂ at C16 and C19), 3.40 (6H, s, CH ₃ at C18 and C21).
¹³ C NMR (CDCl ₃ , 100 MHz): δ 156.6 (C1 and C9), 135.0 (C10), 130.4 (CH at C11 and C15), 128.9 (C4 and C6), 131.3 (C5), 128.5 (CH at C13 ), 128.5 (CH at C2 and C8), 127.9 (CH at C12 and C14), 101.1 (CH at C3 and C7), 71.4 (CH ₂ at C17 and C20), 59.1 (CH ₃ at C18 and C21), 44.3 ( _CH2 at C16 and C19).
_HRMS m/z _[ M+ _Na ] ⁺ Calcd for _{C21H25BF2N4NaO2} ⁺ 437.1931; _Found 437.1931.

５，５－ジフルオロ－Ｎ ^３，Ｎ ^７－ビス（２－メトキシエチル）－１０－（４－メトキシフェニル）－５Ｈ－４λ ^４，５λ ^４－ジピロロ［１，２－ｃ：２’，１’－ｆ］［１，３，２］ジアザボリニン－３，７－ジアミン：化合物５ｂの合成

10.2 cmエース高耐圧チューブに、化合物４ｂ（600 mg、1.63 mmol）を加え、ＣＮ_３ＣＮ（4 mL）を加えて撹拌し、２－メトキシエチルアミン（1.2 mL、13.08 mmol、8.0 eq.）を加えて１００℃で２４時間撹拌した。ＴＬＣ（ヘキサン／酢酸エチル＝１／１）で反応終了を確認した。その後、ロータリーエバポレーターで減圧濃縮し、真空乾燥した。得られた生成物をシリカゲルオープンカラムクロマトグラフィー（ヘキサン／ＣＨ_２Ｃｌ_２／酢酸エチル＝３／２／１）で分離精製し、青紫色固体として化合物５ｂを得た（563 mg、収率77.6 %）。
Mp: 169-170℃.
¹H NMR (CDCl₃, 400 MHz): δ 7.38 (2H, d, J = 8.7 Hz, CH at C12 and C14), 6.94 (2H, d, J = 8.7 Hz, CH at C11 and C15), 6.56 (2H, d, J = 4.2 Hz, CH at C2 and C8), 5.74 (2H, d, J = 4.2 Hz, CH at C3 and C7), 5.77-5.70 (2H, brs, NH at N3 and N4), 3.86 (3H, s, CH₃ at C22), 3.59 (4H, t, J = 5.5 Hz, CH₂ at C17 and C20), 3.44 (4H, q, J = 5.5 Hz, CH₂ at C16 and C19), 3.40 (s, 6H, CH₃ at C18 and C21).
¹³C NMR (CDCl₃, 100 MHz): δ 160.0 (C13), 156.4 (C1 and C9), 131.6 (CH at C12 and C14), 131.2 (C5), 129.0 (C4 and C6), 128.5 (CH at C2 and C8), 127.4 (C10), 113.4 (CH at C11 and C15), 100.9 (CH at C3 and C7), 71.2 (CH₂ at C17 and C20), 59.0 (CH₃ at C18 and C21), 55.3 (CH₃ at C22), 44.3 (CH₂ at C16 and C19).
HRMS m/z [M+Na]⁺ Calcd for C₂₂H₂₇BN₄O₃F₂Na⁺ 467.2036; Found 467.2050. 5,5-difluoro-N ³ ,N ⁷ -bis(2-methoxyethyl)-10-(4-methoxyphenyl)-5H-4λ ⁴ ,5λ ⁴ -dipyrrolo[1,2-c:2′,1′ -f][1,3,2]diazaborinine-3,7-diamine: synthesis of compound 5b

Compound 4b (600 mg, 1.63 mmol) was added to a 10.2 cm Ace high pressure tube, CN ₃ CN (4 mL) was added and stirred, and 2-methoxyethylamine (1.2 mL, 13.08 mmol, 8.0 eq.) was added. The mixture was stirred at 100° C. for 24 hours. The completion of the reaction was confirmed by TLC (hexane/ethyl acetate=1/1). After that, it was concentrated under reduced pressure using a rotary evaporator and dried in vacuum. The resulting product was separated and purified by silica gel open column chromatography (hexane/CH ₂ Cl ₂ /ethyl acetate = 3/2/1) to obtain compound 5b as a bluish purple solid (563 mg, yield 77.6%). ).
MP: 169-170℃.
¹ H NMR (CDCl ₃ , 400 MHz): δ 7.38 (2H, d, J = 8.7 Hz, CH at C12 and C14), 6.94 (2H, d, J = 8.7 Hz, CH at C11 and C15), 6.56 ( 2H, d, J = 4.2 Hz, CH at C2 and C8), 5.74 (2H, d, J = 4.2 Hz, CH at C3 and C7), 5.77-5.70 (2H, brs, NH at N3 and N4), 3.86 (3H, s, CH ₃ at C22), 3.59 (4H, t, J = 5.5 Hz, CH ₂ at C17 and C20), 3.44 (4H, q, J = 5.5 Hz, CH ₂ at C16 and C19), 3.40 (s, 6H, _CH3 at C18 and C21).
¹³ C NMR (CDCl ₃ , 100 MHz): δ 160.0 (C13), 156.4 (C1 and C9), 131.6 (CH at C12 and C14), 131.2 (C5), 129.0 (C4 and C6), 128.5 (CH at C2 and C8), 127.4 (C10), 113.4 (CH at C11 and C15), 100.9 (CH at C3 and C7), 71.2 (CH ₂ at C17 and C20), 59.0 (CH ₃ at C18 and C21), 55.3 (CH ₃ at C22), 44.3 ( _CH2 at C16 and C19).
_HRMS _m _/ z [M+Na] ⁺ _Calcd for _{C22H27BN4O3F2Na} ⁺ 467.2036; Found 467.2050.

４－（５，５－ジフルオロ－３，７－ビス（（２－メトキシエチル）アミン）－５Ｈ－４λ ^４，５λ ^４－ジピロロ［１，２－ｃ：２’，１’－ｆ］［１，３，２］ジアザボリニン－１０－イル）フェノール：化合物５ｃの合成

10.2 cmエース高耐圧チューブに、化合物４ｃ（26.8 mg、0.076 mmol）を加え、ＣＨ_３ＣＮ（4 mL）を加えて撹拌し、２－メトキシエチルアミン（0.2 mL、2.30 mmol、30.0 eq.）を加えて１００℃で２４時間撹拌した。ＴＬＣ（ヘキサン／酢酸エチル＝１／１）で反応終了を確認した。その後、ロータリーエバポレーターで減圧濃縮し、真空乾燥した。得られた生成物をシリカゲルオープンカラムクロマトグラフィー（ヘキサン／ＣＨ_２Ｃｌ_２／酢酸エチル＝１／２／１）で分離精製し、青紫色固体として化合物５ｃを得た（8.2 mg、収率25.1 %）。
Mp: 129-130℃.
¹H NMR (CDCl₃, 400 MHz): δ 7.29 (2H, d, J = 8.5 Hz, CH at C12 and C14), 6.84 (2H, d, J = 8.5 Hz, CH at C11 and C15), 6.55 (2H, d, J = 4.3 Hz, CH at C2 and C8), 5.73 (2H, d, J = 4.3 Hz, CH at C3 and C7), 5.80-5.64 (2H, brs, NH at N3 and N4), 3.60 (4H, t, J = 5.4 Hz, CH₂ at C17 and C20), 3.44 (4H, m, CH₂ at C16 and C19), 3.40 (6H, s, CH₃ at C18 and C21).
¹³C NMR (CDCl₃, 100 MHz): δ 156.4 (C1 and C9), 156.3 (C13), 131.7 (CH at C12 and C14), 131.2 (C5), 129.0 (C4 and C6), 128.4 (CH at C2 and C8), 127.4 (C10), 114.9 (CH at C11 and C15), 101.0 (CH at C3 and C7), 71.2 (CH₂ at C17 and C20), 59.0 (CH₃ at C18 and C21), 44.2 (CH₂ at C16 and C19).
HRMS m/z [M+Na]⁺ Calcd for C₂₁H₂₅BF₂N₄NaO₃ ⁺ 453.1880; Found 453.1841. 4-(5,5-difluoro-3,7-bis((2-methoxyethyl)amine)-5H-4λ ⁴ ,5λ ⁴ -dipyrrolo[1,2-c:2′,1′-f][1 ,3,2]diazaborin-10-yl)phenol: synthesis of compound 5c

Compound 4c (26.8 mg, 0.076 mmol) was added to a 10.2 cm Ace high pressure tube, CH ₃ CN (4 mL) was added and stirred, and 2-methoxyethylamine (0.2 mL, 2.30 mmol, 30.0 eq.) was added. The mixture was stirred at 100° C. for 24 hours. The completion of the reaction was confirmed by TLC (hexane/ethyl acetate=1/1). After that, it was concentrated under reduced pressure using a rotary evaporator and dried in vacuum. The resulting product was separated and purified by silica gel open column chromatography (hexane/CH ₂ Cl ₂ /ethyl acetate = 1/2/1) to obtain compound 5c as a bluish purple solid (8.2 mg, yield 25.1%). ).
MP: 129-130℃.
¹ H NMR (CDCl ₃ , 400 MHz): δ 7.29 (2H, d, J = 8.5 Hz, CH at C12 and C14), 6.84 (2H, d, J = 8.5 Hz, CH at C11 and C15), 6.55 ( 2H, d, J = 4.3 Hz, CH at C2 and C8), 5.73 (2H, d, J = 4.3 Hz, CH at C3 and C7), 5.80-5.64 (2H, brs, NH at N3 and N4), 3.60 (4H, t, J = 5.4 Hz, CH ₂ at C17 and C20), 3.44 (4H, m, CH ₂ at C16 and C19), 3.40 (6H, s, CH ₃ at C18 and C21).
¹³ C NMR (CDCl ₃ , 100 MHz): δ 156.4 (C1 and C9), 156.3 (C13), 131.7 (CH at C12 and C14), 131.2 (C5), 129.0 (C4 and C6), 128.4 (CH at C2 and C8), 127.4 (C10), 114.9 (CH at C11 and C15), 101.0 (CH at C3 and C7), 71.2 (CH ₂ at C17 and C20), 59.0 (CH ₃ at C18 and C21), 44.2 (CH ₂ at C16 and C19).
_HRMS ^{m/z [M+Na] + Calcd for C21H25BF2N4NaO3 +} _453.1880 ^; _Found _453.1841 _.

５，５－ジフルオロ－Ｎ ^３，Ｎ ^７－ビス（２－メトキシエチル）－１０－（４－（プロパ－２－イン－１－イルオキシ）フェニル）－５Ｈ－４λ ^４，５λ ^４－ジピロロ［１，２－ｃ：２’，１’－ｆ］［１，３，２］ジアザボリニン－３，７－ジアミン：化合物５ｄの合成

10.2 cmエース高耐圧チューブに、化合物４ｄ（140 mg、0.036 mmol）を加え、ＣＨ_３ＣＮ（3 mL）を加えて撹拌し、２－メトキシエチルアミン（333 μL、2.30 mmol、10.0 eq.）を加えて１００℃で２４時間撹拌した。ＴＬＣ（ヘキサン／酢酸エチル＝１／１）で反応終了を確認した。その後、ロータリーエバポレーターで減圧濃縮し、真空乾燥した。得られた生成物をシリカゲルオープンカラムクロマトグラフィー（ヘキサン／ＣＨ_２Ｃｌ_２／酢酸エチル＝３／２／１）で分離精製し、青紫色固体として化合物５ｄを得た（83 mg、収率49.3 %）。
Mp: 159-162℃.
¹H NMR (CDCl₃, 400 MHz): δ 7.38 (2H, d, J = 8.7 Hz, CH at C12 and C14), 7.01 (2H, d, J = 8.7 Hz, CH at C11 and C15), 6.56 (2H, d, J = 4.2 Hz, CH at C2 and C8), 5.74 (2H, d, J = 4.2 Hz, CH at C3 and C7), 5.77-5.70 (2H, brs, NH at N3 and N4), 4.74 (2H, d, J = 2.4 Hz, CH₂ at C22), 3.59 (4H, t, J = 5.5 Hz, CH₂ at C17 and C20), 3.44 (4H, q, J = 5.5 Hz, CH₂ at C16 and C19), 3.40 (6H, s, CH₃ at C18 and C21), 2.57 (1H, s, CH at C24).
¹³C NMR (CDCl₃, 100 MHz): δ 158.0 (C13), 156.5 (C1 and C9), 131.6 (CH at C12 and C14), 131.5 (C5), 130.9 (C4 and C6), 129.0 (CH at C2 and C8), 128.4 (C10), 114.3 (CH at C11 and C15), 101.0 (CH at C3 and C7), 75.8 (CH₂ at C22), 71.1 (CH₂ at C17 and C20), 59.0 (CH₃ at C18 and C21), 55.9 (CH₃ at C22), 44.3 (CH₂ at C16 and C19), 29.7 (C24).
HRMS m/z [M+Na]⁺ Calcd for C₂₄H₂₇BF₂N₄NaO₃ ⁺ 491.2036; Found 491.2036. 5,5-difluoro-N ³ ,N ⁷ -bis(2-methoxyethyl)-10-(4-(prop-2-yn-1-yloxy)phenyl)-5H-4λ ⁴ ,5λ ⁴ -dipyrrolo[1 ,2-c: 2′,1′-f][1,3,2]diazaborinine-3,7-diamine: synthesis of compound 5d

Compound 4d (140 mg, 0.036 mmol) was added to a 10.2 cm Ace high pressure tube, CH ₃ CN (3 mL) was added and stirred, and 2-methoxyethylamine (333 μL, 2.30 mmol, 10.0 eq.) was added. The mixture was stirred at 100° C. for 24 hours. The completion of the reaction was confirmed by TLC (hexane/ethyl acetate=1/1). After that, it was concentrated under reduced pressure using a rotary evaporator and dried in vacuum. The resulting product was separated and purified by silica gel open column chromatography (hexane/CH ₂ Cl ₂ /ethyl acetate = 3/2/1) to obtain compound 5d as a bluish purple solid (83 mg, yield 49.3%). ).
MP: 159-162℃.
¹ H NMR (CDCl ₃ , 400 MHz): δ 7.38 (2H, d, J = 8.7 Hz, CH at C12 and C14), 7.01 (2H, d, J = 8.7 Hz, CH at C11 and C15), 6.56 ( 2H, d, J = 4.2 Hz, CH at C2 and C8), 5.74 (2H, d, J = 4.2 Hz, CH at C3 and C7), 5.77-5.70 (2H, brs, NH at N3 and N4), 4.74 (2H, d, J = 2.4 Hz, CH ₂ at C22), 3.59 (4H, t, J = 5.5 Hz, CH ₂ at C17 and C20), 3.44 (4H, q, J = 5.5 Hz, CH ₂ at C16 and C19), 3.40 (6H, s, _CH3 at C18 and C21), 2.57 (1H, s, CH at C24).
¹³ C NMR (CDCl ₃ , 100 MHz): δ 158.0 (C13), 156.5 (C1 and C9), 131.6 (CH at C12 and C14), 131.5 (C5), 130.9 (C4 and C6), 129.0 (CH at C2 and C8), 128.4 (C10), 114.3 (CH at C11 and C15), 101.0 (CH at C3 and C7), 75.8 ( _CH2 at C22), 71.1 ( _CH2 at C17 and C20), 59.0 ( _CH3 at C18 and C21), 55.9 (CH ₃ at C22), 44.3 (CH ₂ at C16 and C19), 29.7 (C24).
_HRMS m ^{/z [M+Na] + Calcd for C24H27BF2N4NaO3 +} _491.2036 ^; _Found _491.2036 _.

５，５－ジフルオロ－Ｎ ^３，Ｎ ^７－ビス（２－メトキシエチル）－１０－（４－ニトロフェニル）－５Ｈ－４λ ^４，５λ ^４－ジピロロ［１，２－ｃ：２’，１’－ｆ］［１，３，２］ジアザボリニン－３，７－ジアミン：化合物５ｅの合成

10.2 cm エース高耐圧チューブに、ＣＨ_３ＣＮ（5 mL）に溶解した化合物４ｅ（200 mg、0.524 mmol）を加え、２－メトキシエチルアミン（364 μL、4.19 mmol、8.0 eq.）を滴下し、１００℃で２４時間撹拌した。ＴＬＣ（ヘキサン／酢酸エチル／ＣＨ_２Ｃｌ_２＝１／１／１）で反応終了を確認した。その後、ロータリーエバポレーターで減圧濃縮し、真空乾燥した。得られた生成物をシリカゲルオープカラムクロマトグラフィー（ヘキサン／酢酸エチル／ＣＨ_２Ｃｌ_２＝１／１／１）で分離精製し、黒色固体として化合物５ｅをで得た（149 mg、収率62.1 %）。
Mp: 179-180℃.
¹H NMR (CDCl₃, 400 MHz): δ 8.23 (2H, d, J = 8.8 Hz, CH at C12 and C14), 7.62 (2H, d, J = 8.8 Hz, CH at C11 and C15), 6.45 (2H, d, J = 4.4 Hz, CH at C2 and C8), 5.81 (2H, br s, NH at N3 and N4), 5.79 (2H, d, J = 4.4 Hz, CH at C3 and C7), 3.60 (4H, t, J = 5.5 Hz, CH₂ at C17 and C20), 3.45 (4H, q, J = 5.5 Hz, CH₂ at C16 and C19), 3.40 (6H, s, CH₃ at C18 and C21).
¹³C NMR (CDCl₃, 100 MHz): δ 156.9 (C at C4 and C6), 148.0 (C at C13), 141.9 (C at C5), 131.2 (CH at C11 and C15), 128.5 (C at C1 and C9), 127.9 (CH at C2 and 8), 127.7 (CH at C10), 123.3 (CH at C12 and C14), 102.1 (CH at C3 and C7), 71.1 (CH₂ at C17 and C20), 59.1 (CH₃ at C18 and C21), 44.3 (CH₂ at C16 and C19).
HRMS m/z: [M+Na]⁺ Calcd for C₂₁H₂₄BN₅O₄F₂Na⁺ 482.1782; Found 482.1787. 5,5-difluoro-N ³ ,N ⁷ -bis(2-methoxyethyl)-10-(4-nitrophenyl)-5H-4λ ⁴ ,5λ ⁴ -dipyrrolo[1,2-c:2′,1′ -f][1,3,2]diazaborinine-3,7-diamine: synthesis of compound 5e

Compound 4e (200 mg, 0.524 mmol) dissolved in CH ₃ CN (5 mL) was added to a 10.2 cm Ace high pressure tube, and 2-methoxyethylamine (364 μL, 4.19 mmol, 8.0 eq.) was added dropwise. C. for 24 hours. The completion of the reaction was confirmed by TLC (hexane/ethyl acetate/CH ₂ Cl ₂ =1/1/1). After that, it was concentrated under reduced pressure using a rotary evaporator and dried in vacuum. The resulting product was separated and purified by silica gel open column chromatography (hexane/ethyl acetate/CH ₂ Cl ₂ =1/1/1) to obtain compound 5e as a black solid (149 mg, yield 62.1%). ).
MP: 179-180℃.
¹ H NMR (CDCl ₃ , 400 MHz): δ 8.23 (2H, d, J = 8.8 Hz, CH at C12 and C14), 7.62 (2H, d, J = 8.8 Hz, CH at C11 and C15), 6.45 ( 2H, d, J = 4.4 Hz, CH at C2 and C8), 5.81 (2H, br s, NH at N3 and N4), 5.79 (2H, d, J = 4.4 Hz, CH at C3 and C7), 3.60 ( 4H, t, J = 5.5 Hz, CH ₂ at C17 and C20), 3.45 (4H, q, J = 5.5 Hz, CH ₂ at C16 and C19), 3.40 (6H, s, CH ₃ at C18 and C21).
¹³ C NMR (CDCl ₃ , 100 MHz): δ 156.9 (C at C4 and C6), 148.0 (C at C13), 141.9 (C at C5), 131.2 (CH at C11 and C15), 128.5 (C at C1 and C9), 127.9 (CH at C2 and 8), 127.7 (CH at C10), 123.3 (CH at C12 and C14), 102.1 (CH at C3 and C7), 71.1 ( _CH2 at C17 and C20), 59.1 (CH ₃ at C18 and C21), 44.3 ( _CH2 at C16 and C19).
_HRMS m ^{/z: [M+Na] + Calcd for C21H24BN5O4F2Na +} _482.1782 _; ^Found _482.1787 _.

１０－（４－アミノフェニル）－５，５－ジフルオロ－Ｎ ^３，Ｎ ^７－ビス（２－メトキシエチル）－５Ｈ－４λ ^４，５λ ^４－ジピロロ［１，２－ｃ：２’，１’－ｆ］［１，３，２］ジアザボリニン－３，７－ジアミン：化合物６の合成

窒素雰囲気下の25 mLナスフラスコに、ＥｔＯＨ（10 mL）に溶解した化合物５ｅ（57.3 mg、0.125 mmol）を加え、１０分間撹拌し、ＮＨ_２ＮＨ_２・Ｈ_２Ｏ（72.8 μL、1.50 mmol、12 eq.）を滴下し、10% Ｐｄ／Ｃ（10 mol%）を加え、還流条件で３０分間撹拌した。ＴＬＣ（ヘキサン／酢酸エチル／ＣＨ_２Ｃｌ_２＝１／１／１）で反応終了を確認し、反応停止した。10% Ｐｄ／Ｃをセライトにより除去した後、ロータリーエバポレーターで減圧濃縮し、真空乾燥した。得られた生成物をシリカゲル中圧カラムクロマトグラフィー（ヘキサン／酢酸エチル／ＣＨ_２Ｃｌ_２＝１／１／１）で分離精製し、茶色固体として化合物６を得た（43.2 mg、収率81％）。
Mp: 69-70℃.
¹H NMR (CDCl₃, 400 MHz): δ 7.25 (2H, d, J = 8.4 Hz, CH at C12 and C14), 6.70 (2H, d, J = 8.4 Hz, CH at C11 and C15), 6.61 (2H, d, J = 4.4 Hz, CH at C2 and C8), 5.73 (2H, d, J = 4.3 Hz, CH at C3 and C7), 5.68 (2H, brs, NH at N3 and N4), 3.59 (4H, t, J = 5.4 Hz, CH₂ at C17 and C20), 3.43 (4H, m, CH₂ at C16 and C19), 3.40 (6H, s, CH₃ at C18 and C21).
¹³C NMR (CDCl₃, 100 MHz): δ 156.3 (C at C1 and C9), 147.0 (C at C13), 132.0 (C at C5), 131.6 (CH at C12 and C14), 128.9 (C at C4 and C6), 128.4 (CH at C2 and C8), 125.1 (C at C10), 114.4 (CH at C11 and C15), 100.7 (CH at C3 and C7), 71.2 (CH₂ at C17 and C20), 59.0 (CH₃ at C18 and C21), 44.2 (CH₂ at C16 and C19).
HRMS m/z [M+Na]⁺ Calcd for C₂₁H₂₆BF₂N₅NaO₂ ⁺ 452.2040; Found 452.2045. 10-(4-aminophenyl)-5,5-difluoro-N ³ ,N ⁷ -bis(2-methoxyethyl)-5H-4λ ⁴ ,5λ ⁴ -dipyrrolo[1,2-c:2′,1′ -f][1,3,2]diazaborinine-3,7-diamine: synthesis of compound 6

Compound 5e (57.3 mg, 0.125 mmol) dissolved in EtOH (10 mL) was added to a 25 mL eggplant flask under a nitrogen atmosphere, stirred for 10 minutes, and NH ₂ NH ₂ .H ₂ O (72.8 μL, 1.50 mmol, 12 eq.) was added dropwise, 10% Pd/C (10 mol%) was added, and the mixture was stirred under reflux conditions for 30 minutes. After confirming the completion of the reaction by TLC (hexane/ethyl acetate/CH ₂ Cl ₂ =1/1/1), the reaction was stopped. After removing 10% Pd/C with celite, the residue was concentrated under reduced pressure using a rotary evaporator and dried in vacuum. The resulting product was separated and purified by silica gel medium pressure column chromatography (hexane/ethyl acetate/CH ₂ Cl ₂ =1/1/1) to obtain compound 6 as a brown solid (43.2 mg, yield 81%). ).
MP: 69-70℃.
¹ H NMR (CDCl ₃ , 400 MHz): δ 7.25 (2H, d, J = 8.4 Hz, CH at C12 and C14), 6.70 (2H, d, J = 8.4 Hz, CH at C11 and C15), 6.61 ( 2H, d, J = 4.4 Hz, CH at C2 and C8), 5.73 (2H, d, J = 4.3 Hz, CH at C3 and C7), 5.68 (2H, brs, NH at N3 and N4), 3.59 (4H , t, J = 5.4 Hz, CH ₂ at C17 and C20), 3.43 (4H, m, CH ₂ at C16 and C19), 3.40 (6H, s, CH ₃ at C18 and C21).
¹³ C NMR (CDCl ₃ , 100 MHz): δ 156.3 (C at C1 and C9), 147.0 (C at C13), 132.0 (C at C5), 131.6 (CH at C12 and C14), 128.9 (C at C4 and C6), 128.4 (CH at C2 and C8), 125.1 (C at C10), 114.4 (CH at C11 and C15), 100.7 (CH at C3 and C7), 71.2 ( _CH2 at C17 and C20), 59.0 (CH ₃ at C18 and C21), 44.2 ( _CH2 at C16 and C19).
HRMS m/ _z _[ M+Na] ⁺ Calcd for _{C21H26BF2N5NaO2} ⁺ _452.2040 ; _Found 452.2045.

Ｎ－（４－（５，５－ジフルオロ－３，７－ビス（（２－メトキシエチル）アミノ）－５Ｈ－４λ ^４，５λ ^４－ジピロロ［１，２－ｃ：２’，１’－ｆ］［１，３，２］ジアザボリニン－１０－イル）フェニル）アセトアミド：化合物７の合成

20 mL の二口ナスフラスコに、ＣＨ_２Ｃｌ_２（20 mL）に溶解した化合物６（70 mg、0.163 mmol）を加えて氷浴にて０℃まで冷却した。溶液に無水酢酸（360 μL、3.26 mmol、20 eq.）、トリエチルアミン（341 μL、2.45 mmol、15 eq.）をそれぞれ滴下し、ＤＭＡＰ（10 mol%、2.0 mg、16.3 μmol）を加えて室温まで昇温し、２．５時間撹拌した。ＴＬＣ（ヘキサン／酢酸エチル／ＣＨ_２Ｃｌ_２＝１／２／１）で反応終了を確認し、反応停止した。溶液を酢酸エチルで３回抽出し、飽和塩化アンモニウム水溶液、飽和炭酸水素ナトリウム水溶液、飽和食塩水溶液でそれぞれ洗浄した。その後、無水硫酸ナトリウムで乾燥させ、ロータリーエバポレーターで減圧濃縮し、真空乾燥し、精製操作をせずに黒色固体として化合物７を得た（68.7 mg、収率89 %）。
Mp: 139-140℃.
¹H NMR (CDCl₃, 400 MHz): δ 7.52 (2H, d, J = 8.4 Hz, CH at C12 and C14), 7.35 (2H, d, J = 8.4 Hz, CH at C11 and C15), 7.23 (1H, br s, NH at N5), 6.51 (2H, d, J = 4.4 Hz, CH at C2 and C8), 5.75 (2H, d, J = 4.4 Hz, CH at C3 and C7 ), 5.73 (2H, brs, NH at N3 and N4), 3.58 (4H, t, J = 5.4 Hz, CH₂ at C17 and C20), 3.44 (4H, m, CH₂ at C16 and C19), 3.40 (6H, s, CH₃ at C18 and C20), 2.18 (3H, s, CH₃ at C23).
¹³C NMR (CDCl₃, 100 MHz): δ 168.3 (C at C1 and C9), 156.5 (C at C13), 138.3 (C at C5), 131.1 (CH at C12 and C14), 130.9 (C at C4 and C6), 128.9 (CH at C2 and C8), 128.3 (C at C10), 119.2 (CH at C11 and C15), 101.1 (CH at C3 and C7), 71.1 (CH₂ at C17 and C20), 59.0 (CH₃ at C18 and C21), 44.2 (CH₂ at C16 and C19), 24.7 (CH₃ at C23).
HRMS m/z [M+Na]⁺ Calcd for C₂₃H₂₈BF₂N₅NaO₃ ⁺ 494.2145; Found 494.2151. N-(4-(5,5-difluoro-3,7-bis((2-methoxyethyl)amino)-5H-4λ ⁴ ,5λ ⁴ -dipyrrolo[1,2-c:2′,1′-f ][1,3,2]diazaborinin-10-yl)phenyl)acetamide: synthesis of compound 7

Compound 6 (70 mg, 0.163 mmol) dissolved in CH ₂ Cl ₂ (20 mL) was added to a 20 mL two-neck eggplant flask and cooled to 0° C. in an ice bath. Acetic anhydride (360 μL, 3.26 mmol, 20 eq.) and triethylamine (341 μL, 2.45 mmol, 15 eq.) were added dropwise to the solution, DMAP (10 mol%, 2.0 mg, 16.3 μmol) was added, and the mixture was cooled to room temperature. The temperature was raised and stirred for 2.5 hours. After confirming the completion of the reaction by TLC (hexane/ethyl acetate/CH ₂ Cl ₂ =1/2/1), the reaction was stopped. The solution was extracted three times with ethyl acetate and washed with saturated aqueous ammonium chloride, saturated sodium bicarbonate and saturated brine, respectively. Then, it was dried over anhydrous sodium sulfate, concentrated under reduced pressure using a rotary evaporator, and dried in vacuum to obtain compound 7 as a black solid (68.7 mg, yield 89%) without purification.
MP: 139-140℃.
¹ H NMR (CDCl ₃ , 400 MHz): δ 7.52 (2H, d, J = 8.4 Hz, CH at C12 and C14), 7.35 (2H, d, J = 8.4 Hz, CH at C11 and C15), 7.23 ( 1H, br s, NH at N5), 6.51 (2H, d, J = 4.4 Hz, CH at C2 and C8), 5.75 (2H, d, J = 4.4 Hz, CH at C3 and C7), 5.73 (2H, brs, NH at N3 and N4), 3.58 (4H, t, J = 5.4 Hz, _CH2 at C17 and C20), 3.44 (4H, m, _CH2 at C16 and C19), 3.40 (6H, s, _CH3 at C18 and C20), 2.18 (3H, s, _CH3 at C23).
¹³ C NMR (CDCl ₃ , 100 MHz): δ 168.3 (C at C1 and C9), 156.5 (C at C13), 138.3 (C at C5), 131.1 (CH at C12 and C14), 130.9 (C at C4 and C6), 128.9 (CH at C2 and C8), 128.3 (C at C10), 119.2 (CH at C11 and C15), 101.1 (CH at C3 and C7), 71.1 ( _CH2 at C17 and C20), 59.0 (CH ₃ at C18 and C21), 44.2 ( _CH2 at C16 and C19), 24.7 ( _CH3 at C23).
_HRMS m/ ^{z [M+Na] + Calcd for C23H28BF2N5NaO3 +} _494.2145 ^; _Found _494.2151 _.

Ｎ－（４－（５，５－ジフルオロ－３，７－ビス（（２－メトキシエチル）アミノ）－５Ｈ－４λ ^４，５λ ^４－ジピロロ［１，２－ｃ：２’，１’－ｆ］［１，３，２］ジアザボリニン－１０－イル）フェニル）ヘキサナミド：化合物８の合成

200 mLの三口フラスコに、ＣＨ_２Ｃｌ_２（50 mL）に溶解した化合物６（100 mg、0.233 mmol）を加え、氷浴で０℃まで冷却した。溶液にトリエチルアミン（97.4 μL、0.699 mmol、3.0 eq.）、塩化ヘキサノイル（72.1 μL、0.466 mmol、2.0 eq.）をそれぞれ滴下し、１時間撹拌した。ＴＬＣ（ヘキサン／ＣＨ_２Ｃｌ_２／酢酸エチル＝２／２／１）で反応終了を確認し、反応停止した。反応液を0.18 M ＨＣｌ（5 mL）、0.5 M ＮａＯＨ（2 mL）、飽和食塩水溶液でそれぞれ３回洗浄した。その後、無水硫酸ナトリウムで乾燥させ、ロータリーエバポレーターで減圧濃縮し、真空乾燥した。得られた生成物をシリカゲル中圧カラムクロマトグラフィー（ヘキサン／酢酸エチル／ＣＨ_２Ｃｌ_２＝３／１／２）で分離精製し、黒色固体として化合物８を得た（110.7 mg、収率90.1 %）。
Mp: 99-100℃.
IR (KBr): 3416, 3306, 3177, 3111, 2928, 2868, 1667, 1603, 1555, 1468, 1427, 1427, 1341, 1306, 1244, 1192, 1165, 1094, 1057, 1013, 966, 889, 841, 781, 679, 561, 484, 415 cm^-1.
¹H NMR (CDCl₃, 400 MHz): δ 7.57 (2H, d, J = 8.5 Hz, CH at C12 and C14), 7.40 (2H, d, J = 8.5 Hz, CH at C11 and C15), 7.22 (1H, brs, NH at N5), 6.55 (2H, d, J = 4.4 Hz, CH at C2 and C8), 5.75-5.72 (4H, m, CH at C2 and C8, NH at N3 and N4), 3.59 (4H, t, J = 5.4 Hz, CH₂ at C17 and C20), 3.46 (4H, q, J = 5.6 Hz, CH at C16 and C19), 3.40 (6H, s, CH₃ at C18 and C20), 2.39 (2H, t, J = 7.4 Hz, CH₂ at C23), 1.76 (2H, m, CH₂ at C24), 1.40-1.36 (4H, m, CH₂ at C25 and C26), 0.92 (3H, m, CH₃ at C27).
¹³C NMR (CDCl₃, 100 MHz): δ 171.5 (C at C1 and C9), 156.5 (C at C13), 138.4 (C at C5), 131.1 (CH at C12 and C14), 130.8 (C at C4 and C6), 128.9 (CH at C2 and C8), 128.3 (C at C10), 119.1 (CH at C11 and C15), 101.1 (CH at C3 and C7), 71.1 (CH₂ at C17 and C20), 59.1 (CH₃ at C18 and C21), 44.2 (CH₂ at C16 and C19), 37.9 (CH₂ at C23), 31.4 (CH₂ at C25), 25.3 (CH₂ at C24), 22.4 (CH₂ at C26), 14.0 (CH₃ at C27).
HRMS m/z [M+Na]⁺ Calcd for C₂₇H₃₆BF₂N₅NaO₃ ⁺ 550.2771; Found 550.2777. N-(4-(5,5-difluoro-3,7-bis((2-methoxyethyl)amino)-5H-4λ ⁴ ,5λ ⁴ -dipyrrolo[1,2-c:2′,1′-f ][1,3,2]diazaborin-10-yl)phenyl)hexanamide: synthesis of compound 8

Compound 6 (100 mg, 0.233 mmol) dissolved in CH ₂ Cl ₂ (50 mL) was added to a 200 mL three-necked flask and cooled to 0° C. with an ice bath. Triethylamine (97.4 μL, 0.699 mmol, 3.0 eq.) and hexanoyl chloride (72.1 μL, 0.466 mmol, 2.0 eq.) were added dropwise to the solution and stirred for 1 hour. After confirming the completion of the reaction by TLC (hexane/CH ₂ Cl ₂ /ethyl acetate=2/2/1), the reaction was stopped. The reaction solution was washed with 0.18 M HCl (5 mL), 0.5 M NaOH (2 mL) and saturated saline solution three times each. After that, it was dried over anhydrous sodium sulfate, concentrated under reduced pressure using a rotary evaporator, and dried in vacuum. The resulting product was separated and purified by silica gel medium pressure column chromatography (hexane/ethyl acetate/CH ₂ Cl ₂ =3/1/2) to obtain compound 8 as a black solid (110.7 mg, yield 90.1%). ).
MP: 99-100℃.
IR (KBr): 3416, 3306, 3177, 3111, 2928, 2868, 1667, 1603, 1555, 1468, 1427, 1427, 1341, 1306, 1244, 1192, 1165, 1094, 1057, 106, 84, 896, 84 , 781, 679, 561, 484, 415 cm ^-1 .
¹ H NMR (CDCl ₃ , 400 MHz): δ 7.57 (2H, d, J = 8.5 Hz, CH at C12 and C14), 7.40 (2H, d, J = 8.5 Hz, CH at C11 and C15), 7.22 ( 1H, brs, NH at N5), 6.55 (2H, d, J = 4.4 Hz, CH at C2 and C8), 5.75-5.72 (4H, m, CH at C2 and C8, NH at N3 and N4), 3.59 ( 4H, t, J = 5.4 Hz, _CH2 at C17 and C20), 3.46 (4H, q, J = 5.6 Hz, CH at C16 and C19), 3.40 (6H, s, _CH3 at C18 and C20), 2.39 (2H, t, J = 7.4 Hz, CH ₂ at C23), 1.76 (2H, m, CH ₂ at C24), 1.40-1.36 (4H, m, CH ₂ at C25 and C26), 0.92 (3H, m, _CH3 at C27).
¹³ C NMR (CDCl ₃ , 100 MHz): δ 171.5 (C at C1 and C9), 156.5 (C at C13), 138.4 (C at C5), 131.1 (CH at C12 and C14), 130.8 (C at C4 and C6), 128.9 (CH at C2 and C8), 128.3 (C at C10), 119.1 (CH at C11 and C15), 101.1 (CH at C3 and C7), 71.1 ( _CH2 at C17 and C20), 59.1 (CH ₃ at C18 and C21), 44.2 (CH ₂ at C16 and C19), 37.9 (CH ₂ at C23), 31.4 (CH ₂ at C25), 25.3 (CH ₂ at C24), 22.4 (CH ₂ at C26), 14.0 ( _CH3 at C27).
^HRMS m/z _[ M+Na] ⁺ Calcd for _{C27H36BF2N5NaO3} ₊ _550.2771 ; _Found 550.2777.

３，７－ジクロロ－５，５－ジフルオロ－１０－（４－（（ヘプチル－１Ｈ－１，２，３－トリアゾル－４－イル）メトキシ）フェニル－５Ｈ－４λ ^４，５λ ^４－ジピロロ［１，２－ｃ：２’，１’－ｆ］［１，３，２］ジアザボリニン：化合物９の合成

10 mL一口ナスフラスコに、ヘプチルアジド（16 mg、0.12 mmol、1.5 eq.)を加え、化合物４ｄ（30 mg、0.077 mmol、1.50 eq.）、ＤＭＳＯ（4.0 mL）を加え撹拌し、水（1.0 mL）に溶解したＣｕＳＯ_４・５Ｈ_２Ｏ（1.0 mg、5 mol%）及びアスコルビン酸ナトリウム（1.5 mg、10 mol%）を滴下し、室温で１２時間撹拌した後、同量のＣｕＳＯ_４・５Ｈ_２Ｏ、アスコルビン酸ナトリウム混合の水溶液を加え、更に１２時間撹拌した。ＴＬＣ（ヘキサン／酢酸エチル＝１／１)で反応終了を確認し、反応停止した。反応液をＡｃＯＥｔで３回抽出し、飽和食塩水溶液で３回洗浄した。その後、無水硫酸ナトリウムで乾燥させ、ロータリーエバポレーターで減圧濃縮し、真空乾燥した。その後、得られた残渣をシリカゲルオープンカラムクロマトグラフィー（ヘキサン／酢酸エチル＝２／１）で分離精製し、橙油状物として標題化合物を得た（30 mg、収率72.7 %）。
¹H NMR (CDCl₃, 500 MHz): δ 7.68 (1H, s, CH at C17), 7.45 (2H, d, J = 8.4 Hz, CH at C12 and C14), 7.15 (2H, d, J = 8.4 Hz, CH at C11 and C15), 6.87 (2H, d, J = 4.2 Hz, CH at C2 and C8), 6.44 (2H, d, J = 4.2 Hz, CH at C3 and C7), 5.30 (2H, brs, NH at N3 and N4), 4.38 (2H, t, J = 7.2 Hz, CH₂ at C17), 1.93 (2H, m, CH₂ at C19), 1.35-1.20 (10H, m, (CH₂)₅ at C20-24), 0.90 (3H, m, CH₃ at C25).
¹³C NMR (CDCl₃, 125 MHz): δ 160.7 (C13), 144.2 (C1 and C9), 143.8 (C9), 143.2 (C15), 133.6 (C5), 132.3 (CH at C2 and C8), 131.4 (CH at C11 and C15), 125.2 (C10) , 122.7 (CH at C18), 118.6 (CH at C3 and C7), 62.1 (CH₂ at C16), 50.5 (CH₂ at C19), 31.5 (CH₂), 30.2 (CH₂), 28.6 (CH₂ at C20), 26.4 (CH₂), 22.5 (CH₂), 14.0 (CH₃ at C25).
HRMS m/z [M+Na]⁺ Calcd for C₂₅H₂₆BCl₂F₂N₅NaO⁺ 554.1468; Found 554.1453. 3,7-dichloro-5,5-difluoro-10-(4-((heptyl-1H-1,2,3-triazol-4-yl)methoxy)phenyl-5H-4λ ⁴ ,5λ ⁴ -dipyrrolo[1 , 2-c: 2′,1′-f][1,3,2]diazaborinine: synthesis of compound 9

Heptyl azide (16 mg, 0.12 mmol, 1.5 eq.) was added to a 10 mL single-neck eggplant flask, compound 4d (30 mg, 0.077 mmol, 1.50 eq.), DMSO (4.0 mL) were added and stirred, and water (1.0 mL) was added. ) dissolved in CuSO _4.5H ₂ O (1.0 mg, 5 mol%) and sodium ascorbate (1.5 mg, 10 mol%) were added dropwise, stirred at room temperature for 12 hours, and then added with the same amount of CuSO _4.5H ₂ An aqueous solution of a mixture of O and sodium ascorbate was added, and the mixture was further stirred for 12 hours. After confirming the completion of the reaction by TLC (hexane/ethyl acetate=1/1), the reaction was stopped. The reaction solution was extracted with AcOEt three times and washed with saturated saline solution three times. After that, it was dried over anhydrous sodium sulfate, concentrated under reduced pressure using a rotary evaporator, and dried in vacuum. Thereafter, the obtained residue was separated and purified by silica gel open column chromatography (hexane/ethyl acetate=2/1) to obtain the title compound as an orange oil (30 mg, yield 72.7%).
¹ H NMR (CDCl ₃ , 500 MHz): δ 7.68 (1H, s, CH at C17), 7.45 (2H, d, J = 8.4 Hz, CH at C12 and C14), 7.15 (2H, d, J = 8.4 Hz, CH at C11 and C15), 6.87 (2H, d, J = 4.2 Hz, CH at C2 and C8), 6.44 (2H, d, J = 4.2 Hz, CH at C3 and C7), 5.30 (2H, brs , NH at N3 and N4), 4.38 (2H, t, J = 7.2 Hz, CH ₂ at C17), 1.93 (2H, m, CH ₂ at C19), 1.35-1.20 (10H, m, (CH ₂ ) ₅ at C20-24), 0.90 (3H, m, _CH3 at C25).
¹³ C NMR (CDCl ₃ , 125 MHz): δ 160.7 (C13), 144.2 (C1 and C9), 143.8 (C9), 143.2 (C15), 133.6 (C5), 132.3 (CH at C2 and C8), 131.4 ( CH at C11 and C15), 125.2 (C10), 122.7 (CH at C18), 118.6 (CH at _C3 and C7), 62.1 (CH2 at C16), 50.5 ( _CH2 at C19), 31.5 ( _CH2 ), 30.2 ( _CH2 ), 28.6 ( _CH2 at C20), 26.4 ( _CH2 ), 22.5 ( _CH2 ), 14.0 ( _CH3 at C25).
HRMS m/ _z [ _M +Na] ⁺ Calcd for _{C25H26BCl2F2N5NaO} ⁺ 554.1468; _Found _554.1453 .

５，５－ジフルオロ－１０－（４－（（ヘプチル－１Ｈ－１，２，３－トリアゾル－４－イル）メトキシ）フェニル－Ｎ ^３，Ｎ ^７－ビス（２－メトキシエチル）－５Ｈ－４λ ^４，５λ ^４－ジピロロ［１，２－ｃ：２’，１’－ｆ］［１，３，２］ジアザボリニン－３，７－ジアミン：化合物１０の合成

10.2 cmエース高耐圧チューブに、化合物９（25 mg、0.047 mmol）を加え、ＣＨ_３ＣＮ（1 mL）を加えて撹拌し、２－メトキシエチルアミン（41 μL, 0.47 mmol、10.0 eq.）を加えて１００℃で２４時間撹拌した。ＴＬＣ（ヘキサン／酢酸エチル＝１／１）で反応終了を確認した。その後、ロータリーエバポレーターで減圧濃縮し、真空乾燥した。得られた生成物をシリカゲルオープンカラムクロマトグラフィー（ヘキサン／ＣＨ_２Ｃｌ_２／酢酸エチル＝１／１／１）で分離精製し、青紫色油状物として化合物１０を得た（15 mg、収率53.1 %）。
¹H NMR (CDCl₃, 400 MHz): δ 7.63 (1H, s, CH at C17), 7.38 (2H, d, J = 8.4 Hz, CH at C12 and C14), 7.03 (2H, d, J = 8.4 Hz, CH at C11 and C15), 6.55 (2H, d, J = 4.2 Hz, CH at C2 and C8), 5.74 (2H, d, J = 4.2 Hz, CH at C3 and C7), 5.71 (2H, br s, NH at N3 and N4), 5.26 (2H, s, CH₂ at C22), 4.36 (2H, t, J = 7.2 Hz, CH₂ at C17), 3.59 (4H, t, J = 5.5 Hz, CH₂ at C17 and C20), 3.43 (4H, d, J = 5.5 Hz, CH₂ at C16 and C19), 3.40 (s, 6H, CH₃ at C18 and C21), 1.93 (2H, m, CH₂ at C19), 1.35-1.20 (10H, m, (CH₂)₅ at C20-24), 0.90 (3H, m, CH₃ at C25).
¹³C NMR (CDCl₃, 100 MHz): δ 158.7 (C13), 156.5 (C1 and C9), 144.0 (C23), 131.7 (CH at C2 and C8), 131.6 (C12 and C14), 129.0 (C5), 128.9 (CH at C2 and C8), 127.9 (C10), 122.5 (CH at C24), 114.3 (CH at C11 and C15), 101.0 (CH at C3 and C7), 71.1 (CH₂ at C17 and C20), 62.2 (CH₂ at C22), 59.0 (CH₃ at C18 and C21), 50.5 (CH₂ at C25), 31.5 (CH₂), 30.2 (CH₂), 28.6 (CH₂ at C20), 26.4 (CH₂), 22.5 (CH₂), 14.0 (CH₃ at C31).
HRMS m/z [M+Na]⁺ Calcd for C₃₁H₄₂BF₂N₇NaO₃ ⁺ 632.3302; Found 632.3264. 5,5-difluoro-10-(4-((heptyl-1H-1,2,3-triazol-4-yl)methoxy)phenyl-N ³ ,N ⁷ -bis(2-methoxyethyl)-5H-4λ ^4,5λ 4 ^-Dipyrrolo [1,2-c:2′,1′-f][1,3,2]diazaborinine-3,7-diamine: synthesis of compound 10

Compound 9 (25 mg, 0.047 mmol) was added to a 10.2 cm Ace high pressure tube, CH ₃ CN (1 mL) was added and stirred, and 2-methoxyethylamine (41 μL, 0.47 mmol, 10.0 eq.) was added. The mixture was stirred at 100° C. for 24 hours. The completion of the reaction was confirmed by TLC (hexane/ethyl acetate=1/1). After that, it was concentrated under reduced pressure using a rotary evaporator and dried in vacuum. The resulting product was separated and purified by silica gel open column chromatography (hexane/CH ₂ Cl ₂ /ethyl acetate = 1/1/1) to obtain compound 10 as a bluish purple oil (15 mg, yield 53.1). %).
¹ H NMR (CDCl ₃ , 400 MHz): δ 7.63 (1H, s, CH at C17), 7.38 (2H, d, J = 8.4 Hz, CH at C12 and C14), 7.03 (2H, d, J = 8.4 Hz, CH at C11 and C15), 6.55 (2H, d, J = 4.2 Hz, CH at C2 and C8), 5.74 (2H, d, J = 4.2 Hz, CH at C3 and C7), 5.71 (2H, br s, NH at N3 and N4), 5.26 (2H, s, CH ₂ at C22), 4.36 (2H, t, J = 7.2 Hz, CH ₂ at C17), 3.59 (4H, t, J = 5.5 Hz, CH ₂ at C17 and C20), 3.43 (4H, d, J = 5.5 Hz, CH ₂ at C16 and C19), 3.40 (s, 6H, CH ₃ at C18 and C21), 1.93 (2H, m, CH ₂ at C19 ), 1.35-1.20 (10H, m, ( _CH2 ) ₅ at C20-24), 0.90 (3H, m, _CH3 at C25).
¹³ C NMR (CDCl ₃ , 100 MHz): δ 158.7 (C13), 156.5 (C1 and C9), 144.0 (C23), 131.7 (CH at C2 and C8), 131.6 (C12 and C14), 129.0 (C5), 128.9 (CH at C2 and C8), 127.9 (C10), 122.5 (CH at C24), 114.3 (CH at C11 and C15), 101.0 (CH at C3 and C7), 71.1 ( _CH2 at C17 and C20), 62.2 ( _CH2 at C22), 59.0 ( _CH3 at C18 and C21), 50.5 ( _CH2 at C25), 31.5 ( _CH2 ), 30.2 ( _CH2 ), 28.6 ( _CH2 at C20), 26.4 ( _CH2 ) , 22.5 ( _CH2 ), 14.0 ( _CH3 at C31).
_HRMS ^{m/z [M+Na] + Calcd for C31H42BF2N7NaO3 +} _632.3302 ^; _Found _632.3264 _.

１０－（４－（（１－ベンジル－１Ｈ－１，２，３－トリアゾル－４－イル）メトキシ）フェニル）－３，７－ジクロロ－５，５－ジフルオロ－５Ｈ－４λ ^４，５λ ^４－ジピロロ［１，２－ｃ：２’，１’－ｆ］［１，３，２］ジアザボリニン：化合物１１の合成

10 mL一口ナスフラスコに、ジクロロメタン（10 mL）に溶解したヨウ化銅（3.8 mg、2 mol%）、ＤＩＰＥＡ（6.9μL、4 mol%）、酢酸（2.3μL、4 mol%）を入れ、化合物４ｄ（391 mg、1.0 mmol）、ベンジルアジド（160 mg、1.2 eq.）を加え、室温で２時間撹拌した。ＴＬＣで反応終了を確認し、反応液をロータリーエバポレーターで減圧濃縮し、真空乾燥した。その後、得られた残渣をシリカゲルオープンカラムクロマトグラフィー（ヘキサン／酢酸エチル＝２／１）で分離精製し、橙固体として標題化合物を得た（381 mg、収率72.6 %）。
¹H NMR (CDCl₃, 400 MHz): δ 7.63 (1H, s, CH at C17), 7.45-7.35 (4H, m, CH at C12 and C14), 7.31 (2H, m, CH at C11 and C15), 6.86 (2H, d, J = 4.2 Hz, CH at C2 and C8), 6.40 (2H, d, J = 4.2 Hz, CH at C3 and C7), 5.56 (2H, brs, NH at N3 and N4).
¹³C NMR (CDCl₃, 100 MHz): δ 160.7 (C13), 144.2 (C1 and C9), 143.9 (C5), 143.8 (C17), 134.4 (C13 and C20), 133.6 (C4 and C6), 132.3 (CH at C12 and C14), 131.5 (CH at C2 and C8), 129.2 (CH at C21 and C25), 128.9 (CH at C23), 128.2 (CH at C22 and C24), 125.2 (C10), 122.9 (CH at C18), 118.7 (CH at C3 and C7), 115.0 (CH at C11 and C15), 62.2 (CH₂ at C16), 54.4 (CH₂ at C19).
HRMS m/z [M+Na]⁺ Calcd for C₂₅H₁₈BCl₂F₂N₅NaO⁺ 546.0842; Found 546.0847. 10-(4-((1-benzyl-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-3,7-dichloro-5,5-difluoro-5H-4λ 4 ^, 5λ ⁴ - Dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborinine: synthesis of compound 11

Copper iodide (3.8 mg, 2 mol%), DIPEA (6.9 μL, 4 mol%), and acetic acid (2.3 μL, 4 mol%) dissolved in dichloromethane (10 mL) were placed in a 10 mL single-neck eggplant flask, and the compound 4d (391 mg, 1.0 mmol) and benzyl azide (160 mg, 1.2 eq.) were added and stirred at room temperature for 2 hours. After confirming the completion of the reaction by TLC, the reaction solution was concentrated under reduced pressure using a rotary evaporator and dried in vacuum. Thereafter, the obtained residue was separated and purified by silica gel open column chromatography (hexane/ethyl acetate=2/1) to obtain the title compound as an orange solid (381 mg, yield 72.6%).
^1H NMR ( _CDCl3 , 400 MHz): δ 7.63 (1H, s, CH at C17), 7.45-7.35 (4H, m, CH at C12 and C14), 7.31 (2H, m, CH at C11 and C15). , 6.86 (2H, d, J = 4.2 Hz, CH at C2 and C8), 6.40 (2H, d, J = 4.2 Hz, CH at C3 and C7), 5.56 (2H, brs, NH at N3 and N4).
¹³ C NMR (CDCl ₃ , 100 MHz): δ 160.7 (C13), 144.2 (C1 and C9), 143.9 (C5), 143.8 (C17), 134.4 (C13 and C20), 133.6 (C4 and C6), 132.3 ( CH at C12 and C14), 131.5 (CH at C2 and C8), 129.2 (CH at C21 and C25), 128.9 (CH at C23), 128.2 (CH at C22 and C24), 125.2 (C10), 122.9 (CH at C18), 118.7 (CH at C3 and C7), 115.0 (CH at C11 and C15), 62.2 (CH ₂ at C16), 54.4 (CH ₂ at C19).
_HRMS m/ _z _[ M+Na] ⁺ Calcd for _{C25H18BCl2F2N5NaO} ⁺ 546.0842; _Found 546.0847.

１０－（４－（（１－ベンジル－１Ｈ－１，２，３－トリアゾル－４－イル）メトキシ）フェニル）－５，５－ジフルオロ－Ｎ ^３，Ｎ ^７－ビス（２－メトキシエチル）－５Ｈ－４λ ^４，５λ ^４－ジピロロ［１，２－ｃ：２’，１’－ｆ］［１，３，２］ジアザボリニン－３，７－ジアミン：化合物１２の合成

10.2 cmエース高耐圧チューブに、化合物１１（200 mg、0.38 mmol）を加え、ＣＨ_３ＣＮ（3 mL）を加えて撹拌し、２－メトキシエチルアミン（332 μL、3.82 mmol、10.0 eq.）を加えて１００℃で２４時間撹拌した。ＴＬＣ（ヘキサン／酢酸エチル＝１／１)で反応終了を確認した。その後、ロータリーエバポレーターで減圧濃縮し、真空乾燥した。得られた生成物をシリカゲルオープンカラムクロマトグラフィー（ヘキサン／ＣＨ_２Ｃｌ_２／酢酸エチル＝３／２／１）で分離精製し、青紫色固体として化合物１２を得た（124 mg、収率54.0 %）。
Mp: 47-48℃.
¹H NMR (CDCl₃, 400 MHz): δ 7.56 (1H, s, CH at C17), 7.38-7.34 (5H, m, CH at C27-C31), 7.29 (2H, m, CH at C12 and C14), 7.00 (2H, d, J = 4.2 Hz, CH at C11 and C15), 6.53 (2H, d, J = 4.2 Hz, CH at C2 and C8), 5.72 (2H, d, J = 4.2 Hz, CH at C3 and C7), 5.73-5.70 (2H, brs, NH at N3 and N4), 5.22 (2H, s, CH₂ at C22), 3.58 (4H, t, J = 5.5 Hz, CH₂ at C17 and C20), 3.41 (4H, q, J = 5.5 Hz, CH₂ at C16 and C19), 3.39 (6H, s, CH₃ at C18 and C21).
¹³C NMR (CDCl₃, 100 MHz): δ 158.7 (C13), 156.5 (C1 and C9), 144.4 (C23), 134.4 (C26), 131.6 (CH at C2 and C8), 130.9 (C5), 129.2 (CH at C27 and C31), 128.9 (CH at C29), 128.9 (C4 and C6), 128.9 (C5), 128.4 (CH at C28 and C30), 128.1 (CH at C12 and C14) , 127.9 (C10), 122.7 (CH at C24), 114.3 (CH at C11 and C15), 101.0 (CH at C3 and C7), 71.1 (CH₂ at C17 and C20), 62.1 (CH₂ at C22), 59.0 (CH₃ at C18 and C21), 54.3 (CH₂ at C25), 44.2 (CH₂ at C16 and C19).
HRMS m/z [M+Na]⁺ Calcd for C₃₁H₃₄BF₂N₇NaO₃ ⁺ 624.2676; Found 624.2759. 10-(4-((1-benzyl-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-5,5-difluoro-N ³ ,N ⁷ -bis(2-methoxyethyl)- 5H-4λ ⁴ ,5λ ⁴ -dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborinine-3,7-diamine: synthesis of compound 12

Compound 11 (200 mg, 0.38 mmol) was added to a 10.2 cm Ace high pressure tube, CH ₃ CN (3 mL) was added and stirred, and 2-methoxyethylamine (332 μL, 3.82 mmol, 10.0 eq.) was added. The mixture was stirred at 100° C. for 24 hours. The completion of the reaction was confirmed by TLC (hexane/ethyl acetate=1/1). After that, it was concentrated under reduced pressure using a rotary evaporator and dried in vacuum. The resulting product was separated and purified by silica gel open column chromatography (hexane/CH ₂ Cl ₂ /ethyl acetate = 3/2/1) to obtain compound 12 as a blue-purple solid (124 mg, yield 54.0%). ).
MP: 47-48℃.
^1H NMR ( _CDCl3 , 400 MHz): δ 7.56 (1H, s, CH at C17), 7.38-7.34 (5H, m, CH at C27-C31), 7.29 (2H, m, CH at C12 and C14). , 7.00 (2H, d, J = 4.2 Hz, CH at C11 and C15), 6.53 (2H, d, J = 4.2 Hz, CH at C2 and C8), 5.72 (2H, d, J = 4.2 Hz, CH at C3 and C7), 5.73-5.70 (2H, brs, NH at N3 and N4), 5.22 (2H, s, CH ₂ at C22), 3.58 (4H, t, J = 5.5 Hz, CH ₂ at C17 and C20) , 3.41 (4H, q, J = 5.5 Hz, CH ₂ at C16 and C19), 3.39 (6H, s, CH ₃ at C18 and C21).
¹³ C NMR (CDCl ₃ , 100 MHz): δ 158.7 (C13), 156.5 (C1 and C9), 144.4 (C23), 134.4 (C26), 131.6 (CH at C2 and C8), 130.9 (C5), 129.2 ( CH at C27 and C31), 128.9 (CH at C29), 128.9 (C4 and C6), 128.9 (C5), 128.4 (CH at C28 and C30), 128.1 (CH at C12 and C14), 127.9 (C10), 122.7 (CH at C24), 114.3 (CH at C11 and C15), 101.0 (CH at C3 and C7), 71.1 (CH ₂ at C17 and C20), 62.1 (CH ₂ at C22), 59.0 (CH ₃ at C18 and C21 ), 54.3 ( _CH2 at C25), 44.2 ( _CH2 at C16 and C19).
_HRMS ^{m/z [M+Na] + Calcd for C31H34BF2N7NaO3 +} _624.2676 ^; _Found _624.2759 _.

試験例１．蛍光スペクトルの測定
化合物７、８、１２を吸光度が0.05以下となるように、アセトンまたは10 mM ＨＥＰＥＳ／ＮａＯＨ（pH 7.4）、および100 mM ＮａＣｌを用いて適宜希釈し、蛍光測定に用いた。蛍光測定には、日立分光蛍光光度計FL-7000を使用した。測定は全て25℃で行い、励起スペクトルについては、蛍光波長を578 nmとし、励起波長400－700 nmを掃引して測定を行い、一方、蛍光スペクトルについては、励起波長を564 nmとして蛍光波長570－700 nmを掃引して測定した。結果を図１に示す。 Test example 1. Measurement of Fluorescence Spectrum Compounds 7, 8 and 12 were appropriately diluted with acetone or 10 mM HEPES/NaOH (pH 7.4) and 100 mM NaCl so that the absorbance was 0.05 or less, and used for fluorescence measurement. Hitachi spectrofluorophotometer FL-7000 was used for fluorescence measurement. All measurements were performed at 25°C. Excitation spectra were measured with an excitation wavelength of 578 nm and an excitation wavelength of 400-700 nm. It was measured by sweeping -700 nm. The results are shown in FIG.

試験例２．相対量子収率の測定
化合物５ａ、５ｂ、５ｃ、５ｄ、５ｅ、６、７、８、１０、１２の相対量子収率を測定した。
化合物５ａ、５ｂ、５ｃ、５ｄ、５ｅ、６、７、８、１０、１２をそれぞれ10－20 mgずつ精秤し、アセトンに溶解し、50 ml溶液を調製した。さらに、100倍希釈液を調製し、日立ダブルビーム分光光度計UV-2900を用い吸収スペクトルを測定し、各吸収極大波長におけるモル吸光係数を求めた。以降の測定に使用する各溶液の濃度は、本モル吸光係数に基づいて決定した。
化合物５ａ、５ｂ、５ｃ、５ｄ、５ｅ、６、７、８、１０、１２を吸光度が0.05以下となるようにアセトンを用いて適宜希釈し、蛍光測定に用いた。蛍光測定には、日立分光蛍光光度計FL-7000を使用した。測定は全て25℃で行い蛍光スペクトルについては、励起波長を543 nmとして蛍光波長550－700 nmを掃引して測定した。量子収率既知の蛍光物質としてはローダミンＢのエタノール溶液を用い、蛍光スペクトル測定を行った。量子収率φは次式に従って算出した。結果を下表に示す。
φ＝φ_標準×（A_標準/A)×(F/F_標準)×(n_アセトン ²／n_{エタノール} ²)×（D/D_標準）
ここで、φ_標準、A_標準、F_標準、D_標準はそれぞれローダミンＢの量子収率、543 nmにおける吸光度、蛍光スペクトルのピーク半値幅の面積および希釈率を、A、F、Dはそれぞれ各色素の吸光度、蛍光スペクトルのピーク半値幅の面積および希釈率を、n_アセトンとn_{エタノール}はアセトンとエタノールの屈折率を表す。

Test example 2. Measurement of Relative Quantum Yields Relative quantum yields of

compounds

5a, 5b, 5c, 5d, 5e, 6, 7, 8, 10 and 12 were measured.
10 to 20 mg of each of

compounds

5a, 5b, 5c, 5d, 5e, 6, 7, 8, 10 and 12 was precisely weighed and dissolved in acetone to prepare a 50 ml solution. Furthermore, a 100-fold diluted solution was prepared, and the absorption spectrum was measured using a Hitachi double beam spectrophotometer UV-2900 to determine the molar extinction coefficient at each maximum absorption wavelength. The concentration of each solution used in subsequent measurements was determined based on this molar extinction coefficient.

Compounds

5a, 5b, 5c, 5d, 5e, 6, 7, 8, 10 and 12 were appropriately diluted with acetone so that the absorbance was 0.05 or less and used for fluorescence measurement. Hitachi spectrofluorophotometer FL-7000 was used for fluorescence measurement. All measurements were performed at 25° C., and the fluorescence spectrum was measured by sweeping the fluorescence wavelength from 550 to 700 nm with an excitation wavelength of 543 nm. An ethanol solution of Rhodamine B was used as a fluorescent substance with a known quantum yield, and a fluorescence spectrum was measured. The quantum yield φ was calculated according to the following formula. The results are shown in the table below.
φ = φ _standard x (A _standard /A) x (F/F _standard ) x (n _acetone ² /n _ethanol ² ) x (D/D _standard )
Here, φ _standard , A _standard , F _standard , and D _standard are the quantum yield of rhodamine B, the absorbance at 543 nm, the area of the peak half-width of the fluorescence spectrum, and the dilution ratio, and A, F, and D are the respective dyes. is the absorbance of the fluorescence spectrum, the area of the peak half-width of the fluorescence spectrum and the dilution ratio, and n- _acetone and n _-ethanol are the refractive indices of acetone and ethanol.

試験例３．溶媒極性の効果
予め50μMの化合物５ｄ、５ｅ、６、７、８、１０、１２のアセトン溶液を用意した。1 mlのアセトン、エチレングリコールまたは10 mM HEPES/NaOH (pH 7.4)、および100 mM NaClを25℃でインキュベートし、各アセトン溶液10μlを加え、蛍光測定に用いた。蛍光測定は、日立分光蛍光光度計FL-7000を使用し25℃で行い、励起波長を564 nmとする蛍光スペクトルを蛍光波長570－700 nmで掃引して測定し、各蛍光スペクトルに基づいて極大蛍光強度を求めた。予め測定しておいた溶液の吸光度から各濃度を求め、1μMの濃度の時の蛍光強度に補正し、アセトン溶媒中の蛍光強度を100%とした場合の、各溶媒中での相対蛍光強度を評価した。結果を図２に示す。
化合物１０と化合物１２は、黒バーと白バーが表す蛍光強度の差が最も大きく、極性変化に対する応答が鋭敏なことを示した。また、白バーが大きな負の値を示し、背景ノイズとなる水系での蛍光強度が小さいことを示した。 Test example 3. Effect of Solvent Polarity Acetone solutions of compounds 5d, 5e, 6, 7, 8, 10 and 12 at 50 μM were prepared in advance. 1 ml of acetone, ethylene glycol or 10 mM HEPES/NaOH (pH 7.4), and 100 mM NaCl were incubated at 25° C., 10 μl of each acetone solution was added and used for fluorescence measurements. Fluorescence measurements were performed at 25°C using a Hitachi spectrofluorophotometer FL-7000, and the fluorescence spectrum with an excitation wavelength of 564 nm was measured by sweeping the fluorescence wavelength from 570 to 700 nm. Fluorescence intensity was determined. Obtain each concentration from the absorbance of the solution measured in advance, correct the fluorescence intensity at a concentration of 1 μM, and calculate the relative fluorescence intensity in each solvent when the fluorescence intensity in the acetone solvent is set to 100%. evaluated. The results are shown in FIG.
Compound 10 and compound 12 had the largest difference in fluorescence intensity between the black bar and the white bar, indicating a sensitive response to polarity change. In addition, the white bar shows a large negative value, indicating that the fluorescence intensity in the water system, which is the background noise, is small.

試験例４．化合物６によるオリゴ糖定量
予め6μＭの化合物６のアセトン溶液を用意した。各濃度のβシクロデキストリンまたはグルコースを含む10 mM HEPES/NaOH (pH 7.4)、100 mM NaCl 1 mlを25℃でインキュベートし、化合物６のアセトン溶液10μlを加え、蛍光測定に用いた。蛍光測定は、日立分光蛍光光度計FL-7000を使用し25℃で行い、励起波長を564 nmとする蛍光スペクトルを蛍光波長570-700 nmで掃引して測定し、各蛍光スペクトルに基づいて極大蛍光強度を求め、検量線を作成した。βシクロデキストリンについては良好な検量線が得られたが、グルコースについては得られなかった。 Test example 4. Oligosaccharide Determination by Compound 6 A 6 μM acetone solution of Compound 6 was prepared in advance. 10 mM HEPES/NaOH (pH 7.4) containing various concentrations of β-cyclodextrin or glucose, 1 ml of 100 mM NaCl was incubated at 25° C., 10 μl of an acetone solution of compound 6 was added, and used for fluorescence measurement. Fluorescence measurements were performed at 25°C using a Hitachi spectrofluorophotometer FL-7000, and the fluorescence spectrum was measured with an excitation wavelength of 564 nm by sweeping the fluorescence wavelength from 570 to 700 nm. Fluorescence intensity was determined and a calibration curve was created. A good calibration curve was obtained for β-cyclodextrin, but not for glucose.

試験例５．化合物１０によるオリゴ糖定量
予め17μMの化合物１０アセトン溶液を用意した。各濃度のβシクロデキストリンまたはグルコースを含む10 mM HEPES/NaOH (pH 7.4), 100 mM NaCl 1 mlを25℃でインキュベートし、化合物１０アセトン溶液10μlを加え、蛍光測定に用いた。蛍光測定は、日立分光蛍光光度計FL-7000を使用し25℃で行い、励起波長を564 nmとする蛍光スペクトルを蛍光波長570-700 nmで掃引して測定し、各蛍光スペクトルに基づいて極大蛍光強度を求め、検量線を作成した。βシクロデキストリンについては良好な検量線が得られたが、グルコースについては得られなかった。 Test example 5. Oligosaccharide Determination by Compound 10 A 17 μM compound 10 acetone solution was prepared in advance. 1 ml of 10 mM HEPES/NaOH (pH 7.4), 100 mM NaCl containing various concentrations of β-cyclodextrin or glucose was incubated at 25° C., 10 μl of compound 10 acetone solution was added, and used for fluorescence measurement. Fluorescence measurements were performed at 25°C using a Hitachi spectrofluorophotometer FL-7000, and the fluorescence spectrum was measured with an excitation wavelength of 564 nm by sweeping the fluorescence wavelength from 570 to 700 nm. Fluorescence intensity was determined and a calibration curve was created. A good calibration curve was obtained for β-cyclodextrin, but not for glucose.

試験例６．化合物１２によるオリゴ糖定量
予め5μMの化合物１２アセトン溶液を用意した。各濃度のβシクロデキストリン（ナカライテスク）、マルトデキストリン(メルク)またはグルコース（和光純薬）を含む10 mM HEPES/NaOH (pH 7.4)、100 mM NaCl 1mlを25℃でインキュベートし、化合物１２アセトン溶液10μlを加え、蛍光測定に用いた。蛍光測定は、日立分光蛍光光度計FL-7000を使用し25℃で行い、励起波長を564 nmとする蛍光スペクトルを蛍光波長570-700 nmで掃引して測定し、各蛍光スペクトルに基づいて極大蛍光強度を求めた。また、タンパク質による蛍光強度への影響を確かめるため、塩化リゾチーム（ナカライテスク）を用い、同様の実験を行った。βシクロデキストリンについては良好な検量線が得られたが、グルコースについては得られなかった。リゾチームの影響は2 mg/mlでは無視できることを確認した。 Test example 6. Oligosaccharide Determination by Compound 12 A 5 μM compound 12 acetone solution was prepared in advance. 10 mM HEPES/NaOH (pH 7.4) containing various concentrations of β-cyclodextrin (Nacalai Tesque), maltodextrin (Merck) or glucose (Wako Pure Chemical Industries), 100 mM NaCl 1 ml was incubated at 25°C, compound 12 in acetone solution. 10 μl was added and used for fluorescence measurements. Fluorescence measurements were performed at 25°C using a Hitachi spectrofluorophotometer FL-7000, and the fluorescence spectrum was measured with an excitation wavelength of 564 nm by sweeping the fluorescence wavelength from 570 to 700 nm. Fluorescence intensity was determined. Also, in order to confirm the effect of protein on fluorescence intensity, a similar experiment was performed using lysozyme chloride (Nacalai Tesque). A good calibration curve was obtained for β-cyclodextrin, but not for glucose. We confirmed that the effect of lysozyme was negligible at 2 mg/ml.

Claims

A probe containing any of the following compounds or salts thereof, which is a fluorescent dye targeting sugars.

2. The probe of claim 1, wherein the compound contained in the probe is any one of the following compounds or salts thereof.

3. The probe of claim 1 or 2, wherein the target saccharide is an oligosaccharide.

4. The probe according to any one of claims 1 to 3, wherein the target saccharide is a saccharide in a biopolymer.

A method for analyzing biopolymers containing sugars, which comprises using the probe according to any one of claims 1 to 4.