JP2021130656A - Peptide synthesis - Google Patents
Peptide synthesis Download PDFInfo
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- JP2021130656A JP2021130656A JP2021024252A JP2021024252A JP2021130656A JP 2021130656 A JP2021130656 A JP 2021130656A JP 2021024252 A JP2021024252 A JP 2021024252A JP 2021024252 A JP2021024252 A JP 2021024252A JP 2021130656 A JP2021130656 A JP 2021130656A
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Images
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Abstract
Description
本発明は新規のペプチド合成法に関わる。より具体的には、チオカルボン酸中間体を経由するＮ末端からＣ末端へとペプチド鎖を伸長させる方法に関わる。 The present invention relates to a novel peptide synthesis method. More specifically, it relates to a method of extending a peptide chain from the N-terminal to the C-terminal via a thiocarboxylic acid intermediate.
中分子ペプチド化合物は次世代の医薬候補として注目を集めている。探索段階では固相合成によって供給されるが、大規模試験や市販を見据えた場合に供給量が不足するため、液相合成による量的供給が必要になる。 Medium-molecular-weight peptide compounds are attracting attention as next-generation pharmaceutical candidates. In the exploration stage, it is supplied by solid-phase synthesis, but when considering large-scale tests and commercialization, the supply amount is insufficient, so quantitative supply by liquid-phase synthesis is required.
非天然アミノ酸を含む様々なアミノ酸を自在に組み込める化学合成法は、Ｃ末端からＮ末端へと伸長させる手法（Ｃ→Ｎ末端伸長法）が従来からの主流であるが、当量以上の縮合剤や、高価で原子効率の良くない保護アミノ酸素子を利用することが必須であった。このため、主鎖アミノ基の保護基と縮合剤由来の廃棄物が大量に産生するという問題があり、ペプチド供給プロセスのコストや環境負荷の上昇につながっていた（図１参照）。 As a chemical synthesis method that can freely incorporate various amino acids including unnatural amino acids, the method of extending from the C-terminal to the N-terminal (C → N-terminal extension method) has been the mainstream from the past, but a condensing agent of an equivalent amount or more and It was essential to use a protected amino acid element, which is expensive and has poor atomic efficiency. For this reason, there is a problem that a large amount of waste derived from the protecting group of the main chain amino group and the condensing agent is produced, which leads to an increase in the cost of the peptide supply process and the environmental load (see FIG. 1).
一方、逆方向のＮ末端からＣ末端へと伸長させる手法（Ｎ→Ｃ末端伸長法）は、この問題を解決できる可能性を持つが、エピメリ化が併発してしまうため、生成物の純度に優れる合成法の実現はこれまで困難であった。 On the other hand, the method of extending from the N-terminal to the C-terminal in the opposite direction (N-> C-terminal extension method) has the potential to solve this problem, but since epimerization also occurs, the purity of the product is increased. It has been difficult to realize an excellent synthesis method.
本発明は、ペプチド供給プロセスにおける縮合剤及び主鎖保護基の使用を回避することができ、エピメリ化を抑制することができ、更に、無保護のアミノ酸素子の溶解度を向上し、反応を加速することができる、新規なペプチド合成法を提供することを目的とする。 The present invention can avoid the use of condensing agents and main chain protecting groups in the peptide feeding process, suppress epimerization, improve the solubility of unprotected amino acid devices, and accelerate the reaction. It is an object of the present invention to provide a novel peptide synthesis method capable of this.
本発明者らは、鋭意検討した結果、以下の３要素からなる新規ペプチド合成法により、上記課題を解決できることを見出し、本発明を完成した。
(1)ペプチドチオカルボン酸を経由するＮ→Ｃ末端方向へのペプチド鎖伸長法。
(2)新たに見出したエピメリ化抑制剤。
(3)主鎖無保護アミノ酸素子の可溶化剤。
As a result of diligent studies, the present inventors have found that the above problems can be solved by a novel peptide synthesis method consisting of the following three elements, and have completed the present invention.
(1) Peptide chain extension method from N to C terminal via peptide thiocarboxylic acid.
(2) A newly discovered epimerization inhibitor.
(3) Solubilizer for main chain unprotected amino acid device.
即ち、本発明は、
［１］以下の式（I）で表される化合物と、以下の式（II）で表されるアミノ酸を、以下の式（III）で表される化合物及び以下の式（IV）で表される化合物の存在下で反応させることにより、式（V）の化合物を調製する方法。
保護基を表すか、Ｎ末端が保護基で保護されたアミノ酸又はペプチドを表し、
Ｒ１は、α−アミノ酸の側鎖であり、当該側鎖は保護基で保護されていてもよい。）
Ｌは、エステル基（−ＣＯ２Ｒ；Ｒは炭素数１〜３０のアルキル基）、置換又は無置換のアリ−ル基、置換又は無置換のベンゾイル基、及びアミノ基で置換されていてもよいアミド基（−ＣＯＮＲ’Ｒ’’；Ｒ’、Ｒ’’は、各々独立に、水素原子又は炭素数１〜４の置換又は無置換のアルキル基であり、Ｒ’及びＲ’’の少なくとも一方はアミノ基で置換されているアルキル基である）、及びアシル基（−ＣＯＲａ’； Ｒａ’は炭素数１〜４のアルキル基）からなる群から選択され、但し、Ｒ３及びＲ４の少なくとも１つがエステル基（−ＣＯ２Ｒａ；Ｒａは炭素数１〜４のアルキル基）である場合は、Ｌは水素（Ｈ）であってもよく、
Ｒ３及びＲ４は、各々独立に、水素、炭素数１〜４のアルキル基、ハロゲン又はエステル基（−ＣＯ２Ｒａ；Ｒａは炭素数１〜４のアルキル基）を表す。）
Ｒ５及びＲ６は一緒になってＲ５及びＲ６が結合しているリン原子を含む４〜７員の置換又は無置換のヘテロシクリルを形成してもよい。）
［２］反応溶媒として、ＤＭＳＯとトルエンの混合溶媒を用いる、［１］に記載の方法。
［３］前記カップリング反応の前に、以下の式（VI）で表される化合物を、以下の式（１）の化合物と反応させることにより、式（I）で表される化合物を調製する工程を含む、［１］又は［２］に記載の方法。
［４］ＰＧ−（ＡＡ）ｎ−ＳＨ：
（ＰＧは、Ｎ末端の保護基を表し、
ＡＡは、任意のアミノ酸残基を表し、各出現において同一又は異なっていてもよく、アミノ酸残基の一部又は全ての側鎖は保護基で保護されていてもよく、
ｎは、１〜４の整数である。）
で表される化合物と、
Ｈ−（ＡＡ’）ｍ−ＯＨ：
（ＡＡは、任意のアミノ酸残基を表し、各出現において同一又は異なっていてもよく、アミノ酸残基の一部又は全ての側鎖は保護基で保護されていてもよく、
ｍは、１〜４の整数である。）
で表されるアミノ酸又はペプチドを、
以下の式（III）で表される化合物及び以下の式（IV）で表される化合物の存在下で反応させることにより、ＰＧ−（ＡＡ）ｎ（ＡＡ’）ｍ−ＯＨで表される化合物を調製する方法。
Ｌは、エステル基（−ＣＯ２Ｒ；Ｒは炭素数１〜３０のアルキル基）、置換又は無置換のアリ−ル基、置換又は無置換のベンゾイル基、及びアミノ基で置換されていてもよいアミド基（−ＣＯＮＲ’Ｒ’’；Ｒ’、Ｒ’’は、各々独立に、水素原子又は炭素数１〜４の置換又は無置換のアルキル基であり、Ｒ’及びＲ’’の少なくとも一方はアミノ基で置換されているアルキル基である）、及びアシル基（−ＣＯＲａ’； Ｒａ’は炭素数１〜４のアルキル基）からなる群から選択され、但し、Ｒ３及びＲ４の少なくとも１つがエステル基（−ＣＯ２Ｒａ；Ｒａは炭素数１〜４のアルキル基）である場合は、Ｌは水素（Ｈ）であってもよく、
Ｒ３及びＲ４は、各々独立に、炭素数１〜４のアルキル基、ハロゲン又はエステル基（−ＣＯ２Ｒａ；Ｒａは炭素数１〜４のアルキル基）を表す。）
Ｒ５及びＲ６は一緒になってＲ５及びＲ６が結合しているリン原子を含む４〜７員の置換又は無置換のヘテロシクリルを形成してもよい。）
［５］［１］〜［３］のいずれか１項に記載の方法に続けて、
（ｉ）以下の式（V）で表される化合物を、以下の式（１）の化合物と反応させることにより、式（VII）で表される化合物を調製する工程：
を含む、式（VIIII）の化合物を調製する方法。
Ｌは、エステル基（−ＣＯ２Ｒ；Ｒは炭素数１〜３０のアルキル基）、置換又は無置換のアリ−ル基、置換又は無置換のベンゾイル基、及びアミノ基で置換されていてもよいアミド基（−ＣＯＮＲ’Ｒ’’；Ｒ’、Ｒ’’は、各々独立に、水素原子又は炭素数１〜４の置換又は無置換のアルキル基であり、Ｒ’及びＲ’’の少なくとも一方はアミノ基で置換されているアルキル基である）、及びアシル基（−ＣＯＲａ’； Ｒａ’は炭素数１〜４のアルキル基）からなる群から選択され、但し、Ｒ３及びＲ４の少なくとも１つがエステル基（−ＣＯ２Ｒａ；Ｒａは炭素数１〜４のアルキル基）である場合は、Ｌは水素（Ｈ）であってもよく、
Ｒ３及びＲ４は、各々独立に、炭素数１〜４のアルキル基、ハロゲン又はエステル基（−ＣＯ２Ｒａ；Ｒａは炭素数１〜４のアルキル基）を表す。）
Ｒ５及びＲ６は一緒になってＲ５及びＲ６が結合しているリン原子を含む４〜７員の置換又は無置換のヘテロシクリルを形成してもよい。）
［６］以下の式（IV）で表される化合物をペプチド合成において可溶化剤として使用する方法。
Ｒ５及びＲ６は一緒になってＲ５及びＲ６が結合しているリン原子を含む４〜７員の置換又は無置換のヘテロシクリルを形成してもよい。）
を提供するものである。
That is, the present invention
[1] The compound represented by the following formula (I) and the amino acid represented by the following formula (II) are represented by the compound represented by the following formula (III) and the following formula (IV). A method for preparing a compound of formula (V) by reacting in the presence of a compound.
Represents a protecting group or represents an amino acid or peptide with the N-terminus protected by a protecting group.
R 1 is a side chain of α-amino acid, and the side chain may be protected by a protecting group. )
L may be substituted with an ester group (-CO 2 R; R is an alkyl group having 1 to 30 carbon atoms), a substituted or unsubstituted allyl group, a substituted or unsubstituted benzoyl group, and an amino group. A good amide group (-CONR'R'';R',R', respectively, is a hydrogen atom or a substituted or unsubstituted alkyl group having 1 to 4 carbon atoms, and at least of R'and R''. One is selected from the group consisting of an alkyl group substituted with an amino group) and an acyl group (-COR a '; Ra ' is an alkyl group having 1 to 4 carbon atoms), except that R 3 and R When at least one of 4 is an ester group (-CO 2 Ra ; Ra is an alkyl group having 1 to 4 carbon atoms), L may be hydrogen (H).
R 3 and R 4 independently represent hydrogen, an alkyl group having 1 to 4 carbon atoms, a halogen or an ester group (-CO 2 Ra ; Ra is an alkyl group having 1 to 4 carbon atoms). )
R 5 and R 6 may be combined to form a 4- to 7-membered substituted or unsubstituted heterocyclyl containing a phosphorus atom to which R 5 and R 6 are attached. )
[2] The method according to [1], wherein a mixed solvent of DMSO and toluene is used as the reaction solvent.
[3] Prior to the coupling reaction, the compound represented by the following formula (VI) is reacted with the compound represented by the following formula (1) to prepare the compound represented by the formula (I). The method according to [1] or [2], which comprises a step.
[4] PG- (AA) n- SH:
(PG represents an N-terminal protecting group,
AA represents any amino acid residue, which may be the same or different at each appearance, and some or all side chains of the amino acid residue may be protected by a protecting group.
n is an integer of 1 to 4. )
And the compound represented by
H- (AA') m- OH:
(AA represents any amino acid residue, which may be the same or different at each appearance, and some or all side chains of the amino acid residue may be protected by a protecting group.
m is an integer of 1 to 4. )
Amino acids or peptides represented by
A compound represented by PG- (AA) n (AA') m- OH by reacting in the presence of a compound represented by the following formula (III) and a compound represented by the following formula (IV). How to prepare.
L may be substituted with an ester group (-CO 2 R; R is an alkyl group having 1 to 30 carbon atoms), a substituted or unsubstituted allyl group, a substituted or unsubstituted benzoyl group, and an amino group. A good amide group (-CONR'R'';R',R', respectively, is a hydrogen atom or a substituted or unsubstituted alkyl group having 1 to 4 carbon atoms, and at least of R'and R''. One is selected from the group consisting of an alkyl group substituted with an amino group) and an acyl group (-COR a '; Ra ' is an alkyl group having 1 to 4 carbon atoms), except that R 3 and R When at least one of 4 is an ester group (-CO 2 Ra ; Ra is an alkyl group having 1 to 4 carbon atoms), L may be hydrogen (H).
R 3 and R 4 independently represent an alkyl group having 1 to 4 carbon atoms, a halogen or an ester group (-CO 2 Ra ; Ra is an alkyl group having 1 to 4 carbon atoms). )
R 5 and R 6 may be combined to form a 4- to 7-membered substituted or unsubstituted heterocyclyl containing a phosphorus atom to which R 5 and R 6 are attached. )
[5] Following the method according to any one of [1] to [3],
(I) A step of preparing a compound represented by the formula (VII) by reacting the compound represented by the following formula (V) with the compound represented by the following formula (1):
A method for preparing a compound of formula (VIIII), comprising:
L may be substituted with an ester group (-CO 2 R; R is an alkyl group having 1 to 30 carbon atoms), a substituted or unsubstituted allyl group, a substituted or unsubstituted benzoyl group, and an amino group. A good amide group (-CONR'R'';R',R', respectively, is a hydrogen atom or a substituted or unsubstituted alkyl group having 1 to 4 carbon atoms, and at least of R'and R''. One is selected from the group consisting of an alkyl group substituted with an amino group) and an acyl group (-COR a '; Ra ' is an alkyl group having 1 to 4 carbon atoms), except that R 3 and R When at least one of 4 is an ester group (-CO 2 Ra ; Ra is an alkyl group having 1 to 4 carbon atoms), L may be hydrogen (H).
R 3 and R 4 independently represent an alkyl group having 1 to 4 carbon atoms, a halogen or an ester group (-CO 2 Ra ; Ra is an alkyl group having 1 to 4 carbon atoms). )
R 5 and R 6 may be combined to form a 4- to 7-membered substituted or unsubstituted heterocyclyl containing a phosphorus atom to which R 5 and R 6 are attached. )
[6] A method of using a compound represented by the following formula (IV) as a solubilizer in peptide synthesis.
R 5 and R 6 may be combined to form a 4- to 7-membered substituted or unsubstituted heterocyclyl containing a phosphorus atom to which R 5 and R 6 are attached. )
Is to provide.
本発明により、ペプチド供給プロセスにおける縮合剤及び主鎖保護基の使用を回避することができ、エピメリ化を抑制することができ、更に、無保護のアミノ酸素子の溶解度を向上し、反応を加速することができる、新規ペプチド合成法を提供することができる。 According to the present invention, the use of a condensing agent and a main chain protecting group in the peptide feeding process can be avoided, epimerization can be suppressed, the solubility of the unprotected amino acid element can be improved, and the reaction can be accelerated. It is possible to provide a novel peptide synthesis method which can be used.
１．ペプチド合成法
本発明の１つの実施態様は、式（I）で表される化合物と、式（II）で表されるアミノ酸を、式（III）で表される化合物及び式（IV）で表される化合物の存在下でカップリング反応させることにより、式（V）の化合物を調製する方法である（以下「本発明のペプチド合成法」ともいう）。
1. 1. Peptide Synthesis Method In one embodiment of the present invention, the compound represented by the formula (I) and the amino acid represented by the formula (II) are represented by the compound represented by the formula (III) and the formula (IV). This is a method for preparing a compound of formula (V) by subjecting it to a coupling reaction in the presence of the compound (hereinafter, also referred to as “the peptide synthesis method of the present invention”).
本発明のペプチド合成法の基本的な反応スキ-ムを図２に示す。本発明においては、(1)ペプチドチオカルボン酸を経由するＮ→Ｃ末端方向へのペプチド鎖伸長法、(2)新たに見出したエピメリ化抑制剤、(3)主鎖無保護アミノ酸素子の可溶化剤の３つの要素を備えることが重要である。以下、これら３つの要素の詳細を含めて本発明のペプチド合成法について説明する。 The basic reaction skim of the peptide synthesis method of the present invention is shown in FIG. In the present invention, (1) a peptide chain extension method from N to C terminal via peptide thiocarboxylic acid, (2) a newly found epimerization inhibitor, and (3) solubilization of a main chain unprotected amino acid element. It is important to have three components of the agent. Hereinafter, the peptide synthesis method of the present invention will be described including the details of these three elements.
（１）チオカルボン酸化合物を経由するＮ→Ｃ末端方向へのペプチド鎖伸長法
本発明のペプチド合成法は、式（I）で表される化合物であるペプチドチオカルボン酸を経由するＮ→Ｃ末端方向へのペプチド鎖伸長法を用いる。
式（I）において、
保護基を表すか、Ｎ末端が保護基で保護されたアミノ酸又はペプチドを表す。
In equation (I)
It represents a protecting group or represents an amino acid or peptide whose N-terminus is protected by a protecting group.
保護基としては、アミノ酸のＮ末端の保護基として慣用されている保護基、例えば、ｔｅｒｔ−ブトキシカルボニル基（Ｂｏｃ）、ベンジルオキシカルボニル基（Ｃｂｚ）、９−フルオレニルメトキシカルボニル基（Ｆｍｏｃ）、アセチル基（Ａｃ）、ベンゾイル基（Ｂｚ）等が挙げられる。 As the protecting group, a protecting group commonly used as an N-terminal protecting group of an amino acid, for example, a tert-butoxycarbonyl group (Boc), a benzyloxycarbonyl group (Cbz), a 9-fluorenylmethoxycarbonyl group (Fmoc) , Acetyl group (Ac), benzoyl group (Bz) and the like.
Ｎ末端が保護基で保護されたアミノ酸とは、上記の保護基でＮ末端が保護されたアミノ酸を意味する。
アミノ酸としては、特に限定なく使用することができる。例としては、α-アミノ酸、β−アミノ酸があげられる。α−アミノ酸として、例えば、ロイシン、イソロイシン、バリン、リジン、トレオニン、アルギニン、アスパラギン、アスパラギン酸、グルタミン、グルタミン酸、セリン、ヒスチジン、フェニルアラニン、アラニン、グリシン、トリプトファン、チロシン、システイン、メチオニン、プロリン、ヒドロキシプロリン、オルニチン、シトルリン、Ｎ-メチルグリシン（サルコシン）、Ｎ-メチルロイシン、２，３−ジアミノプロパン酸、２，４−ジアミノ酪酸、α−ヒドロキシロイシン、ホモセリン、ホモシステイン、ｔｅｒｔ−ロイシン、α−アミノイソ酪酸などが挙げられるが、これらに限定されない。また、β−アミノ酸として、β−アラニンなどが挙げられるがこれに限定されない。
上記のアミノ酸は、Ｌ型及びＤ型のいずれの光学異性体であってもよい。
これらアミノ酸の側鎖が官能基を有する場合は、Ｒ１について後述する保護基で保護してもよい。
An amino acid whose N-terminal is protected by a protecting group means an amino acid whose N-terminal is protected by the above-mentioned protecting group.
The amino acid can be used without particular limitation. Examples include α-amino acids and β-amino acids. As α-amino acids, for example, leucine, isoleucine, valine, lysine, threonine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, serine, histidine, phenylalanine, alanine, glycine, tryptophan, tyrosine, cysteine, methionine, proline, hydroxyproline. , Ornitine, citrulin, N-methylglycine (sarcosin), N-methylleucine, 2,3-diaminopropanoic acid, 2,4-diaminobutyric acid, α-hydroxyleucine, homoserine, homocysteine, tert-leucine, α-aminoiso Examples include, but are not limited to, butyric acid. Further, examples of the β-amino acid include, but are not limited to, β-alanine.
The above amino acids may be either L-type or D-type optical isomers.
When the side chains of these amino acids have functional groups, R 1 may be protected with a protecting group described later.
Ｎ末端が保護基で保護されたペプチドとは、上記の保護基でＮ末端が保護されたペプチドを意味する。ここで、ペプチドは、上記のアミノ酸の残基が２〜９、好ましくは、２〜６、更に好ましくは２〜４個結合した構造を有する。 A peptide having an N-terminal protected by a protecting group means a peptide having an N-terminal protected by the above-mentioned protecting group. Here, the peptide has a structure in which 2 to 9, preferably 2 to 6, and more preferably 2 to 4 amino acid residues are linked.
Ｒ１は、α−アミノ酸の側鎖である。Ｒ１は、ＳＨに結合しているα−アミノ酸残基の種類によって決まる。例えば、当該アミノ酸残基がグリシンの場合、Ｒ１は水素であり、アラニンの場合はメチル基である。
即ち、式（I）におけるＳＨに結合しているα−アミノ酸残基；ＮＨ−ＣＨ（Ｒ１）−Ｃ（＝Ｏ）は、フェニルグリシン以外の任意のα−アミノ酸の残基でよい。ここで、α−アミノ酸として、例えば、ロイシン、イソロイシン、バリン、リジン、トレオニン、アルギニン、アスパラギン、アスパラギン酸、グルタミン、グルタミン酸、セリン、ヒスチジン、フェニルアラニン、アラニン、グリシン、トリプトファン、チロシン、システイン、メチオニン、プロリン、ヒドロキシプロリン、オルニチン、シトルリン、Ｎ−メチルグリシン（サルコシン）、Ｎ−メチルロイシン、２，３−ジアミノプロパン酸、２，４−ジアミノ酪酸、α−ヒドロキシロイシン、ホモセリン、ホモシステイン、ｔｅｒｔ−ロイシン、α−アミノイソ酪酸などが挙げられるが、これらに限定されない。Ｒ１は、これらから選択されるアミノ酸の側鎖である。
上記のα−アミノ酸は、Ｌ型及びＤ型のいずれの光学異性体であってもよい。
R 1 is a side chain of α-amino acid. R 1 is determined by the type of α-amino acid residue bound to SH. For example, if the amino acid residue is glycine, R 1 is hydrogen, and if it is alanine, it is a methyl group.
That is, the α-amino acid residue bound to SH in the formula (I); NH-CH (R 1 ) -C (= O) may be a residue of any α-amino acid other than phenylglycine. Here, as α-amino acids, for example, leucine, isoleucine, valine, lysine, threonine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, serine, histidine, phenylalanine, alanine, glycine, tryptophan, tyrosine, cysteine, methionine, proline. , Hydroxyproline, ornithine, citrulin, N-methylglycine (sarcosin), N-methylleucine, 2,3-diaminopropanoic acid, 2,4-diaminobutyric acid, α-hydroxyleucine, homoserine, homocysteine, tert-leucine, Examples include, but are not limited to, α-aminoisobutyric acid. R 1 is a side chain of amino acids selected from these.
The α-amino acid may be either an L-type or D-type optical isomer.
Ｒ１で表される上記α−アミノ酸残基の側鎖は、保護基で保護されていてもよい。
例えば、Ｒ１がリジン側鎖（アミノ基を有する）である場合は、保護基として、ｔｅｒｔ−ブトキシカルボニル基（Ｂｏｃ）、ベンジルオキシカルボニル基（Ｃｂｚ）、９−フルオレニルメトキシカルボニル基（Ｆｍｏｃ）を用いることができる。
Ｒ１がグルタミン酸側鎖やアスパラギン酸側鎖でありカルボキシ基を有する場合は、保護基として、ベンジルエステル（Ｂｚｌ）、ｔｅｒｔ−ブチルエステル（ｔ−Ｂｕ）を用いることができる。
また、Ｒ１がセリンやトレオニン側鎖（ヒドロキシ基を有する）である場合は、保護基として、ベンジル基やｔｅｒｔ−ブチル基を用いることができる。
Ｒ１がチロシン側鎖（フェノ−ル性ヒドロキシ基を有する）である場合は、保護基として、２−ブロモベンジルオキシカルボニル（Ｚ（２Ｂｒ））やｔｅｒｔ−ブチル基を用いることができる。
Ｒ１がシステイン側鎖（スルフヒドリル基を有する）である場合は、保護基として、４−メチルベンジル基（Ｂｚｌ（４Ｍｅ））、トリチル基 （Ｔｒｔ）、ｔｅｒｔ−ブチル基、Ｎ−（アセチル）アミノメチル基（Ａｃｍ）を用いることができる。
Ｒ１がアルギニン側鎖（グアニジノ基を有する）である場合は、保護基として、ｐ−トルエンスルホニル基（ｐ−Ｔｓ）などを用いることができる。
また、Ｒ１がヒスチジン側鎖（イミダゾ−ル環を有する）である場合は、π−窒素をベンジルオキシメチル基（Ｂｏｍ）やｔｅｒｔ−ブトキシメチル基（Ｂｕｍ）で保護することができ、τ−窒素を２，４−ジニトロフェニル基（Ｄｎｐ）、トリチル基などで保護することができる。
The side chain of the α-amino acid residue represented by R 1 may be protected by a protecting group.
For example, when R 1 is a lysine side chain (having an amino group), the protecting group includes a tert-butoxycarbonyl group (Boc), a benzyloxycarbonyl group (Cbz), and a 9-fluorenylmethoxycarbonyl group (Fmoc). ) Can be used.
When R 1 is a glutamic acid side chain or an aspartic acid side chain and has a carboxy group, a benzyl ester (Bzl) or a tert-butyl ester (t-Bu) can be used as a protecting group.
When R 1 is a serine or threonine side chain (having a hydroxy group), a benzyl group or a tert-butyl group can be used as a protecting group.
When R 1 is a tyrosine side chain (having a phenolic hydroxy group), 2-bromobenzyloxycarbonyl (Z (2Br)) or a tert-butyl group can be used as the protecting group.
When R 1 is a cysteine side chain (having a sulfhydryl group), the protecting group is 4-methylbenzyl group (Bzl (4Me)), trityl group (Trt), tert-butyl group, N- (acetyl) amino. A methyl group (Acm) can be used.
When R 1 is an arginine side chain (having a guanidine group), a p-toluenesulfonyl group (p-Ts) or the like can be used as a protecting group.
When R 1 is a histidine side chain (having an imidazole ring), π-nitrogen can be protected with a benzyloxymethyl group (Bom) or a tert-butoxymethyl group (Bum), and τ- Nitrogen can be protected with a 2,4-dinitrophenyl group (Dnp), a trityl group, or the like.
式（I）で表されるチオカルボン酸化合物は、本発明者らのグル-プが報告した１工程変換条件（Chem. Commun. 2018, 54, 1222）、または、既存のいかなる手法を用いて得ることができる。 The thiocarboxylic acid compound represented by the formula (I) can be obtained by using the one-step conversion conditions (Chem. Commun. 2018, 54, 1222) reported by the group of the present inventors, or any existing method. be able to.
本発明の１つの好ましい側面においては、式（I）で表される化合物は、以下の式（VI）で表される化合物を、以下の式（１）の化合物と反応させて得ることができる。
式（VI）において、
保護基を表すか、Ｎ末端が保護基で保護されたアミノ酸又はペプチドを表し、Ｒ１は、α−アミノ酸の側鎖であり、これらの詳細については式（I）において説明したのと同様である。
In equation (VI)
Representing a protecting group or representing an amino acid or peptide with the N-terminus protected by a protecting group, R 1 is the side chain of the α-amino acid, the details of which are the same as described in formula (I). be.
上記の式（I）で表される化合物を得る方法の反応スキ-ムの概要を図３に示す。この反応では、触媒としてアセチルスルフィドを用いる。
この反応は、溶媒としてＤＭＦを用い、反応温度は、通常０℃〜室温で行う。
FIG. 3 shows an outline of the reaction skim of the method for obtaining the compound represented by the above formula (I). In this reaction, acetyl sulfide is used as a catalyst.
This reaction uses DMF as a solvent and the reaction temperature is usually 0 ° C. to room temperature.
本発明のペプチド合成法では、式（I）で表されるチオカルボン酸化合物と、以下の式（II）表される化合物とを反応させる。
式（II）表される化合物は、無保護のα−アミノ酸であり、α−アミノ酸として、天然アミノ酸に加えて、任意のα−アミノ酸を用いることができる。例えば、ロイシン、イソロイシン、バリン、リジン、トレオニン、アルギニン、アスパラギン、アスパラギン酸、グルタミン、グルタミン酸、セリン、ヒスチジン、フェニルアラニン、アラニン、グリシン、トリプトファン、チロシン、システイン、メチオニン、オルニチン、２，３−ジアミノプロパン酸、２，４−ジアミノ酪酸、α−ヒドロキシロイシン、ｔｅｒｔ−ロイシン、フェニルグリシン、α−アミノイソ酪酸などが挙げられるが、これらに限定されない。
上記のα−アミノ酸は、Ｌ型及びＤ型のいずれの光学異性体であってもよい。
The compound represented by the formula (II) is an unprotected α-amino acid, and any α-amino acid can be used as the α-amino acid in addition to the natural amino acid. For example, leucine, isoleucine, valine, lysine, threonine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, serine, histidine, phenylalanine, alanine, glycine, tryptophan, tyrosine, cysteine, methionine, ornithine, 2,3-diaminopropanoic acid. , 2,4-Diaminobutyric acid, α-hydroxyleucine, tert-leucine, phenylglycine, α-aminoisobutyric acid and the like, but are not limited thereto.
The α-amino acid may be either an L-type or D-type optical isomer.
式（II）におけるＲ２は、α−アミノ酸の側鎖であり、α−アミノ酸の種類によって決まる。例えば、当該アミノ酸がグリシンの場合、Ｒ２は水素であり、アラニンの場合はメチル基である。 R 2 in formula (II) is a side chain of α-amino acid and is determined by the type of α-amino acid. For example, if the amino acid is glycine, R 2 is hydrogen, and if it is alanine, it is a methyl group.
Ｒ２で表される上記α−アミノ酸の側鎖は、保護基で保護されていてもよい。保護基については、式（I）のＲ１について説明したのと同様のものを用いることができる。 The side chain of the α-amino acid represented by R 2 may be protected by a protecting group. As the protecting group, the same protecting group as described for R 1 of the formula (I) can be used.
本発明のペプチド合成法では、伸長させるペプチド又はアミノ酸を式（I）で表されるようにチオカルボン酸へと変換しておくことで、原料である式（I）の化合物のＣ末端と式（II）の無保護アミノ酸素子のＣ末端を必要最小限に区別可能になるとともに、原料化合物のＣ末端の選択的な活性化によって一アミノ酸ずつペプチド伸長させることができる。
また、チオカルボン酸を経由するＮ→Ｃ末端方向へのペプチド鎖伸長法を用いることで、等量以上の縮合剤を必要とせず、伸長過程で生じる廃棄物は溶媒と硫黄単体だけに原理上低減可能であり、安価な無保護アミノ酸を合成素子として用いることができるため、従来型ペプチド伸長法に比べて、大幅な廃棄物の低減およびコストの低減が期待できる。
また、生成物としては、Ｃ末端無保護のペプチド鎖が得られるため、チオカルボン酸変換→ペプチド鎖連結の繰り返しによって、速やかにペプチド鎖を伸長することが可能である。
In the peptide synthesis method of the present invention, the peptide or amino acid to be extended is converted into a thiocarboxylic acid as represented by the formula (I), whereby the C-terminal of the compound of the formula (I) as a raw material and the formula ( The C-terminal of the unprotected amino acid element of II) can be distinguished to the minimum necessary, and the peptide can be extended one amino acid at a time by selective activation of the C-terminal of the raw material compound.
In addition, by using the peptide chain extension method in the N → C terminal direction via thiocarboxylic acid, an equal amount or more of condensing agent is not required, and the waste generated in the extension process is reduced in principle to only the solvent and sulfur alone. Since a possible and inexpensive unprotected amino acid can be used as a synthetic element, a significant reduction in waste and cost can be expected as compared with the conventional peptide extension method.
Further, since a C-terminal unprotected peptide chain is obtained as a product, the peptide chain can be rapidly extended by repeating thiocarboxylic acid conversion → peptide chain linkage.
（２）エピメリ化抑制剤
本発明のペプチド合成法においては、以下の式（III）で表される化合物である新規なエピメリ化抑制剤を用いる。
(2) Epimerization inhibitor In the peptide synthesis method of the present invention, a novel epimerization inhibitor which is a compound represented by the following formula (III) is used.
従来通りの考え方でＮ→Ｃ末端伸長を行うと、環化中間体を経るエピメリ化（立体化学の消失）が引き起こされ、純度に優れる生成物の供給が不可能であった。本発明者らは、エピメリ化抑制能期待される様々な添加剤を合成・スクリ-ニングした結果、Ｎ−ヒドロキシピリドン型添加剤が本発明のペプチド合成法でのエピメリ化の問題を解決することを見出した。 When the N-→ C-terminal extension is carried out in the conventional way of thinking, epimerization (disappearance of stereochemistry) via the cyclization intermediate is caused, and it is impossible to supply a product having excellent purity. As a result of synthesizing and screening various additives expected to have an ability to suppress epimerization, the present inventors have solved the problem of epimerization in the peptide synthesis method of the present invention with the N-hydroxypyridone-type additive. I found.
式（III）において、Ｌは、エステル基（−ＣＯ２Ｒ；Ｒは炭素数１〜３０のアルキル基）、置換又は無置換のアリ−ル基、置換又は無置換のベンゾイル基、アミノ基で置換されていてもよいアミド基（−ＣＯＮＲ’Ｒ’’；Ｒ’、Ｒ’’は、各々独立に、水素原子又は炭素数１〜４の置換又は無置換のアルキル基であり、Ｒ’及びＲ’’の少なくとも一方はアミノ基で置換されているアルキル基である）、及びアシル基（−ＣＯＲａ’； Ｒａ’は炭素数１〜４のアルキル基）からなる群から選択される。 In formula (III), L is an ester group (-CO 2 R; R is an alkyl group having 1 to 30 carbon atoms), a substituted or unsubstituted allyl group, a substituted or unsubstituted benzoyl group, or an amino group. The optionally substituted amide group (-CONR'R'';R',R'' is an independently substituted or unsubstituted alkyl group having a hydrogen atom or 1 to 4 carbon atoms, and R'and 'at least one of an alkyl group substituted with an amino group), and an acyl group (-COR a' R '; R a' is selected from the group consisting of alkyl group) having 1 to 4 carbon atoms.
エステル基は、−ＣＯ２Ｒで表され、Ｒは炭素数１〜３０のアルキル基である。アルキル基は、無置換であってもよく、置換基を有していてもよい。
アルキル基は、直鎖アルキル基、分岐アルキル基のいずれでもよい。分岐アルキル基の場合、炭素総数が３０程度に大きくなっても、再結晶により生成物からの添加剤の回収がし易くなるという利点がある。
分岐アルキル基としては、−（ＣＨ２ＣＨ２ＣＨ（ＣＨ３）ＣＨ２）ｓ−Ｈ（ｓは、１〜４）の分岐アルキル基が、再結晶による生成物からの添加剤の回収率が高くなる点から好ましい。 直鎖アルキル基の場合は、炭素数が大きくなっても収率、エピメリ化度は良好であるが、結晶化度が上がり、生成物からの回収がしにくくなる場合がある。直鎖アルキルでは、炭素数１〜４のアルキルが、収率、エピメリ化度、生成物からの添加剤の回収のしやすさの点から好ましく、特に好ましくは、メチル基（即ち、エステル基としてはメチルエステル基（−ＣＯ２ＣＨ３））である。
The ester group is represented by −CO 2 R, where R is an alkyl group having 1 to 30 carbon atoms. The alkyl group may be unsubstituted or may have a substituent.
The alkyl group may be either a linear alkyl group or a branched alkyl group. In the case of a branched alkyl group, even if the total number of carbons is as large as about 30, there is an advantage that the additive can be easily recovered from the product by recrystallization.
As the branched alkyl group, the branched alkyl group of − (CH 2 CH 2 CH (CH 3 ) CH 2 ) s −H (s is 1 to 4) has a recovery rate of the additive from the product by recrystallization. It is preferable because it becomes expensive. In the case of a linear alkyl group, the yield and the degree of epimerization are good even if the number of carbon atoms is large, but the degree of crystallinity is increased and it may be difficult to recover from the product. In the linear alkyl, an alkyl having 1 to 4 carbon atoms is preferable from the viewpoint of yield, degree of epimerization, and ease of recovery of the additive from the product, and particularly preferably as a methyl group (that is, an ester group). Is a methyl ester group (-CO 2 CH 3 )).
アミノ基で置換されていてもよいアミド基としては好ましくは、ジメチルアミノエチルアミド基（-ＣＯＮＨＣ２Ｈ４Ｎ（ＣＨ３）２）である。 The amide group which may be substituted with an amino group is preferably a dimethylaminoethylamide group (-CONHC 2 H 4 N (CH 3 ) 2 ).
アリ−ル基としては、フェニル基が挙げられ、置換されていても無置換でもよい。アリ−ル基の置換基としては、アルキル基、エステル基等が挙げられるが、好ましくはエステル基（例えば、メチルエステル基（-ＣＯ２ＣＨ３））である。 Examples of the aryl group include a phenyl group, which may be substituted or unsubstituted. Examples of the substituent of the allyl group include an alkyl group and an ester group, but an ester group (for example, a methyl ester group (-CO 2 CH 3 )) is preferable.
ベンゾイル基の置換基としては、アルキル基、アミノ基等が挙げられるが、好ましくはアミノ基（例えば、ジメチルアミノ基）である。 Examples of the substituent of the benzoyl group include an alkyl group and an amino group, and an amino group (for example, a dimethylamino group) is preferable.
アシル基は、−ＣＯＲａ’（Ｒａ’は炭素数１〜４のアルキル基）で表され、好ましくは、アセチル基である。 Acyl group, -COR a '(R a' is an alkyl group having 1 to 4 carbon atoms) represented by, preferably, acetyl.
Ｌとして、上記した基から選択されることが好ましいが、Ｒ３及びＲ４の少なくとも１つがエステル基（−ＣＯ２Ｒａ；Ｒａは炭素数１〜４のアルキル基）である場合は、Ｌは水素（Ｈ）であってもよい。この場合には、Ｌとしてエステル基などの基がある場合に比べて、エピメリ度が若干高くなる傾向があるものの、高い収率で生成物を得ることが可能である。 L is preferably selected from the groups described above, but when at least one of R 3 and R 4 is an ester group (-CO 2 Ra ; Ra is an alkyl group having 1 to 4 carbon atoms), it is preferable. L may be hydrogen (H). In this case, the product can be obtained in a high yield, although the degree of epimery tends to be slightly higher than that in the case where L has a group such as an ester group.
式（III）において、Ｒ３及びＲ４は、各々独立に、水素原子、炭素数１〜４のアルキル基、ハロゲン又はエステル基（−ＣＯ２Ｒａ；Ｒａは炭素数１〜４のアルキル基）を表す。ハロゲンとしては、塩素が好ましい。エステル基としてはメチルエステル基（−ＣＯ２ＣＨ３）が好ましい。 In formula (III), R 3 and R 4 are each independently a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, a halogen or an ester group (-CO 2 Ra ; Ra is an alkyl having 1 to 4 carbon atoms. Group). Chlorine is preferable as the halogen. As the ester group, a methyl ester group (-CO 2 CH 3 ) is preferable.
本発明の１つの好ましい側面において、Ｒ３及びＲ４の何れもが水素原子である。
また、本発明の別の好ましい側面において、Ｒ３及びＲ４の一方が塩素であり、他方が水素原子である。
また、本発明の別の好ましい側面において、Ｒ３及びＲ４の一方がメチルエステル基であり、他方が水素原子である。
In one preferred aspect of the invention, both R 3 and R 4 are hydrogen atoms.
Further, in another preferable aspect of the present invention, one of R 3 and R 4 is chlorine and the other is a hydrogen atom.
Further, in another preferable aspect of the present invention, one of R 3 and R 4 is a methyl ester group and the other is a hydrogen atom.
（３）主鎖無保護アミノ酸の可溶化剤
本発明のペプチド合成法においては、式（IV）で表される化合物である可溶化剤を用いる。
(3) Solubilizer for main chain unprotected amino acid In the peptide synthesis method of the present invention, a solubilizer which is a compound represented by the formula (IV) is used.
主鎖が無保護のアミノ酸素子（原料）は、一般に有機溶媒で不要であるため、これを用いるペプチド伸長法は反応効率が悪く、一工程完結までに長時間を要していた。本発明者らは、無保護アミノ酸素子を可溶化する添加剤の探索を行ったところ、ホスファイト系添加剤が無保護アミノ酸素子の溶解度向上に寄与し、液相での反応効率を向上させることで反応時間短縮を実現することが可能となる。 Since an amino acid element (raw material) having an unprotected main chain is generally unnecessary in an organic solvent, the peptide extension method using this has poor reaction efficiency, and it takes a long time to complete one step. The present inventors searched for an additive that solubilizes the unprotected amino acid element, and found that the phosphite-based additive contributes to the improvement of the solubility of the unprotected amino acid element and improves the reaction efficiency in the liquid phase. It is possible to shorten the reaction time.
式（IV）において、Ｒ５及びＲ６は、各々独立に、炭素数１〜４のアルキル基、炭素数１〜４のアルコキシ基、置換基を有していてもよいベンジルオキシ基又はヒドロキシ基を表す。但し、Ｒ５及びＲ６の両方がヒドロキシ基になることはない。
アルキル基としては、ｎ−ブチル基が好ましい。アルコキシ基としては、メトキシ基、エトキシ基、ｉ−プロポキシ基、ｎ−ブトキシ基が好ましい。
In formula (IV), R 5 and R 6 independently have an alkyl group having 1 to 4 carbon atoms, an alkoxy group having 1 to 4 carbon atoms, and a benzyloxy group or a hydroxy group which may have a substituent. Represents. However, both R 5 and R 6 do not become hydroxy groups.
As the alkyl group, an n-butyl group is preferable. As the alkoxy group, a methoxy group, an ethoxy group, an i-propoxy group and an n-butoxy group are preferable.
本発明の１つの好ましい側面においては、式（IV）の化合物は、以下の式（IVa）で表される。
式（IVa）において、Ｒ５’及びＲ６’は、各々独立に、炭素数１〜４のアルキル基、置換基を有していてもよいベンジル基又は水素を表す。但し、Ｒ５’及びＲ６’の両方が水素になることはない。 In formula (IVa), R 5 'and R 6' each independently represent an alkyl group having 1 to 4 carbon atoms, an optionally benzyl group or hydrogen may have a substituent. However, R 5 'and R 6' both are not a hydrogen.
本発明の１つの好ましい側面においては、Ｒ５’及びＲ６’の両方がメチル基である。
また、本発明の１つの好ましい側面においては、Ｒ５’及びＲ６’の両方がエチル基である。
また、本発明の１つの好ましい側面においては、Ｒ５’及びＲ６’の両方がｉ−プロピル基である。
また、本発明の１つの好ましい側面においては、Ｒ５’及びＲ６’の両方がｎ−ブチル基である。
また、本発明の１つの好ましい側面においては、Ｒ５’及びＲ６’の両方がベンジル基である。
In one preferred aspect of the present invention, both R 5 'and R 6' is a methyl group.
Further, in one preferred aspect of the present invention, both R 5 'and R 6' is an ethyl group.
Further, in one preferred aspect of the present invention, both R 5 'and R 6' is i- propyl.
Further, in one preferred aspect of the present invention, both R 5 'and R 6' is an n- butyl group.
Further, in one preferred aspect of the present invention, both R 5 'and R 6' is a benzyl group.
式（IV）において、Ｒ５及びＲ６は一緒になってＲ５及びＲ６が結合しているリン原子を含む４〜７員の置換又は無置換のヘテロシクリルを形成してもよい。
このようなヘテロシクリルとしては、５員環又は６員環の構造が好ましく、６員環の構造がより好ましい。
置換基としては、１〜４のアルキル基が挙げられ、置換基は１つでも２以上であってもよい。アルキル基としては、好ましくはメチル基、エチル基である。
In formula (IV), R 5 and R 6 may be combined to form a 4- to 7-membered substituted or unsubstituted heterocyclyl containing a phosphorus atom to which R 5 and R 6 are attached.
As such a heterocyclyl, a 5-membered ring or a 6-membered ring structure is preferable, and a 6-membered ring structure is more preferable.
Examples of the substituent include 1 to 4 alkyl groups, and the number of substituents may be one or two or more. The alkyl group is preferably a methyl group or an ethyl group.
本発明の方法で用いられる式（IV）の非限定的例を以下に示すが、これらに限定されるものではない。 Non-limiting examples of formula (IV) used in the method of the present invention are shown below, but are not limited thereto.
本発明のペプチド合成法により、以下の式（V）で表される化合物を得ることができる。
式（V）において、
本発明のペプチド合成法においては、通常、式（I）の化合物のモル量に対して、式（II）の化合物を１〜２等量、式（ＩＩＩ）の化合物を１等量、式（ＩＶ）の化合物を１等量で反応させる。 In the peptide synthesis method of the present invention, the compound of the formula (II) is usually equal to 1 to 2 equal to the molar amount of the compound of the formula (I), and the compound of the formula (III) is usually equal to the molar amount of the compound of the formula (III). The compound of IV) is reacted in an equal amount.
本発明のペプチド合成法は、ペプチドの液相合成に通常用いられる溶媒を使用することができるが、ＤＭＳＯ系の溶媒を用いるとチオ酸の活性化を効率化できて好ましい。更に、ＤＭＳＯとトルエン等の混合溶媒を用いると、極性を下げることができ、エピメリ化を有効に抑制することができる点から好ましい。
本発明の方法の１つの好ましい側面において、ＤＭＳＯ：トルエン＝１：１（体積比）の混合溶媒が用いられる。
In the peptide synthesis method of the present invention, a solvent usually used for liquid phase synthesis of peptides can be used, but it is preferable to use a DMSO-based solvent because activation of thioic acid can be made more efficient. Further, it is preferable to use a mixed solvent such as DMSO and toluene because the polarity can be lowered and epimerization can be effectively suppressed.
In one preferred aspect of the method of the invention, a mixed solvent of DMSO: toluene = 1: 1 (volume ratio) is used.
本発明のペプチド合成法は、通常、室温程度で、３〜６時間反応させることにより行われる。 The peptide synthesis method of the present invention is usually carried out by reacting at about room temperature for 3 to 6 hours.
本発明のペプチド合成法は、上記で説明したように、ペプチド又はアミノ酸のチオカルボン酸化合物を経由するＮ→Ｃ末端方向へのペプチド鎖伸長法に基づくが、本発明の方法を、ペプチドのフラグメントカップリング、反復カップリングに適用することができる。 As described above, the peptide synthesis method of the present invention is based on the method of extending the peptide chain in the N → C terminal direction via the thiocarboxylic acid compound of the peptide or amino acid, but the method of the present invention is based on the peptide fragment cup. It can be applied to rings and repetitive couplings.
２．フラグメントカップリング
本発明のもう１つの実施態様は、
ＰＧ-（ＡＡ）ｎ-ＳＨ：
（ＰＧは、Ｎ末端の保護基を表し、
ＡＡは、任意のアミノ酸残基を表し、各出現において同一又は異なっていてもよく、アミノ酸残基の一部又は全ての側鎖は保護基で保護されていてもよく、
ｎは、１〜４の整数である。）
で表される化合物と、
Ｈ-（ＡＡ’）ｍ-ＯＨ：
（ＡＡ’は、任意のアミノ酸残基を表し、各出現において同一又は異なっていてもよく、アミノ酸残基の一部又は全ての側鎖は保護基で保護されていてもよく、
ｍは、１〜４の整数である。）
で表されるペプチドを、
以下の式（III）で表される化合物及び以下の式（IV）で表される化合物の存在下で反応させることにより、ＰＧ-（ＡＡ）ｎ（ＡＡ’）ｍ-ＯＨで表される化合物を調製する方法である（以下「本発明のフラグメントカップリング」ともいう）。
Ｌは、エステル基（−ＣＯ２Ｒ；Ｒは炭素数１〜３０のアルキル基）、置換又は無置換のアリ-ル基、置換又は無置換のベンゾイル基、アミノ基で置換されていてもよいアミド基（−ＣＯＮＲ’Ｒ’’；Ｒ’、Ｒ’’は、各々独立に、水素原子又は炭素数１〜４の置換又は無置換のアルキル基であり、Ｒ’及びＲ’’の少なくとも一方はアミノ基で置換されているアルキル基である）、アシル基（−ＣＯＲａ’； Ｒａ’は炭素数１〜４のアルキル基）からなる群から選択され、但し、Ｒ３及びＲ４の少なくとも１つがエステル基（−ＣＯ２Ｒａ；Ｒａは炭素数１〜４のアルキル基）である場合は、Ｌは水素（Ｈ）であってもよく、
Ｒ３及びＲ４は、各々独立に、炭素数１〜４のアルキル基、ハロゲン又はエステル基（-ＣＯ２Ｒａ；Ｒａは炭素数１〜４のアルキル基）を表す。）
Ｒ５及びＲ６は一緒になってＲ５及びＲ６が結合しているリン原子を含む４〜７員の置換又は無置換のヘテロシクリルを形成してもよい
2. Fragment Coupling Another embodiment of the present invention is
PG- (AA) n -SH:
(PG represents an N-terminal protecting group,
AA represents any amino acid residue, which may be the same or different at each appearance, and some or all side chains of the amino acid residue may be protected by a protecting group.
n is an integer of 1 to 4. )
And the compound represented by
H- (AA') m -OH:
(AA'represents any amino acid residue, which may be the same or different at each appearance, and some or all side chains of the amino acid residue may be protected by a protecting group.
m is an integer of 1 to 4. )
The peptide represented by
A compound represented by PG- (AA) n (AA') m- OH by reacting in the presence of a compound represented by the following formula (III) and a compound represented by the following formula (IV). (Hereinafter, also referred to as "fragment coupling of the present invention").
L may be substituted with an ester group (-CO 2 R; R is an alkyl group having 1 to 30 carbon atoms), a substituted or unsubstituted allyl group, a substituted or unsubstituted benzoyl group, or an amino group. The amide group (-CONR'R ";R',R" is an independently substituted or unsubstituted alkyl group having a hydrogen atom or 1 to 4 carbon atoms, and at least one of R'and R ". Is an alkyl group substituted with an amino group), selected from the group consisting of an acyl group (-COR a '; Ra ' is an alkyl group having 1 to 4 carbon atoms), except that R 3 and R 4 When at least one is an ester group (-CO 2 Ra ; Ra is an alkyl group having 1 to 4 carbon atoms), L may be hydrogen (H).
R 3 and R 4 each independently represent an alkyl group having 1 to 4 carbon atoms, a halogen or an ester group (-CO 2 Ra ; Ra is an alkyl group having 1 to 4 carbon atoms). )
R 5 and R 6 may be combined to form a 4- to 7-membered substituted or unsubstituted heterocyclyl containing a phosphorus atom to which R 5 and R 6 are attached.
ＰＧのＮ末端の保護基は、本発明のペプチド合成法で説明したのと同様のＮ末端の保護基を用いることができる。
ＡＡ及びＡＡ’の任意のアミノ酸残基としては、α−アミノ酸、β−アミノ酸の残基があげられ、α−アミノ酸の残基として、ロイシン、イソロイシン、バリン、リジン、トレオニン、アルギニン、アスパラギン、アスパラギン酸、グルタミン、グルタミン酸、セリン、ヒスチジン、フェニルアラニン、アラニン、グリシン、トリプトファン、チロシン、システイン、メチオニン、プロリン、オルニチン、Ｎ−メチルロイシン、２，３−ジアミノプロパン酸、２，４−ジアミノ酪酸、α−ヒドロキシロイシンなどのアミノ酸残基が挙げられる。また、β−アミノ酸として、β−アラニンなどのアミノ酸残基である。但し、ＡＡのアミノ酸残基のうちＳＨに結合しているアミノ酸残基は、フェニルグリシン以外の任意のアミノ酸残基であり、ＡＡ’のアミノ酸残基のうちＮ末端のアミノ酸残基はプロリン、Ｎ−メチルアミノ酸以外のアミノ酸残基である。
これらアミノ酸残基の側鎖が保護されている場合は、本発明のペプチド合成法で説明したのと同様の保護基を用いることができる。
ＡＡ及びＡＡ’の任意のアミノ酸残基は、Ｌ型及びＤ型のいずれの光学異性体であってもよい。
As the N-terminal protecting group of PG, the same N-terminal protecting group as described in the peptide synthesis method of the present invention can be used.
Arbitrary amino acid residues of AA and AA'include α-amino acid and β-amino acid residues, and α-amino acid residues include leucine, isoleucine, valine, lysine, threonine, arginine, aspartic acid, and aspartic acid. Acid, glutamine, glutamine, serine, histidine, phenylalanine, alanine, glycine, tryptophan, tyrosine, cysteine, methionine, proline, ornithine, N-methylleucine, 2,3-diaminopropanoic acid, 2,4-diaminobutyric acid, α- Amino acid residues such as hydroxyleucine can be mentioned. Further, as β-amino acid, it is an amino acid residue such as β-alanine. However, among the amino acid residues of AA, the amino acid residue bound to SH is an arbitrary amino acid residue other than phenylglycine, and among the amino acid residues of AA', the N-terminal amino acid residue is proline or N. -Amino acid residues other than methyl amino acids.
When the side chains of these amino acid residues are protected, protecting groups similar to those described in the peptide synthesis method of the present invention can be used.
Any amino acid residue of AA and AA'may be either an L-type or D-type optical isomer.
本発明のフラグメントカップリングにおいては、好ましくはｎとｍは同じである。ｎ及びｍは、好ましくは、２又は３である。 In the fragment coupling of the present invention, n and m are preferably the same. n and m are preferably 2 or 3.
本発明のフラグメントカップリングにおいては、本発明のペプチド合成法で説明した溶媒、温度及び時間などの反応条件を同様に用いることができる。 In the fragment coupling of the present invention, the reaction conditions such as the solvent, temperature and time described in the peptide synthesis method of the present invention can be similarly used.
３．反復カップリング
本発明のもう１つの実施態様は、上記で説明した本発明のペプチド合成法を実施し、これに続けて、
（ｉ）以下の式（V）で表される化合物を、以下の式（１）の化合物と反応させることにより、式（VII）で表される化合物を調製する工程：
を含む、式（VIIII）の化合物を調製する方法である（以下「本発明の反復カップリング」ともいう）。
(I) A step of preparing a compound represented by the formula (VII) by reacting the compound represented by the following formula (V) with the compound represented by the following formula (1):
This is a method for preparing a compound of the formula (VIIII), which comprises (hereinafter, also referred to as “repeated coupling of the present invention”).
式（VIII）におけるＲ３ａはα−アミノ酸の側鎖であるが、当該α−アミノ酸については、式（II）のアミノ酸について説明したのと同様である。また、当該側鎖は保護基で保護されていてもよく、保護基についても式（II）について説明したのと同様である。 R 3a in the formula (VIII) is a side chain of the α-amino acid, and the α-amino acid is the same as that described for the amino acid in the formula (II). Further, the side chain may be protected by a protecting group, and the protecting group is the same as described in the formula (II).
本発明の反復カップリングは、上記で記載した方法を繰り返し用いて、ペプチド鎖を伸長させることができる。ペプチド鎖の伸長は、通常２〜５個のアミノ酸残基を有するペプチドを得るまで行うことが可能である。 The repeated coupling of the present invention can extend the peptide chain by repeatedly using the method described above. Elongation of the peptide chain can usually be carried out until a peptide having 2 to 5 amino acid residues is obtained.
本発明の反復カップリングにおいては、本発明のペプチド合成法で説明した溶媒、温度及び時間などの反応条件を同様に用いることができる。 In the repeated coupling of the present invention, the reaction conditions such as the solvent, temperature and time described in the peptide synthesis method of the present invention can be similarly used.
上記した本発明のペプチド合成法、本発明のフラグメントカップリング、本発明の反復カップリングは、ペプチドの液相合成に適用することが可能である。 The peptide synthesis method of the present invention, the fragment coupling of the present invention, and the repeated coupling of the present invention described above can be applied to the liquid phase synthesis of peptides.
また、本発明のもう１つの態様は、以下の式（IV）で表される化合物をペプチド合成において可溶化剤として使用する方法である。
Ｒ５及びＲ６は一緒になってＲ５及びＲ６が結合しているリン原子を含む４〜７員の置換又は無置換のヘテロシクリルを形成してもよい。）
Another aspect of the present invention is a method of using a compound represented by the following formula (IV) as a solubilizer in peptide synthesis.
R 5 and R 6 may be combined to form a 4- to 7-membered substituted or unsubstituted heterocyclyl containing a phosphorus atom to which R 5 and R 6 are attached. )
以下本発明を実施例によりさらに詳細に説明するが、本発明はこれらの実施例に限定されるものではない。 Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.
１．一般的方法
（１）一般論
エレクトロスプレイイオン化（ＥＳＩ）質量スペクトルをＳｈｉｍａｄｚｕ ＬＣＭＳ-２０２０スペクトロメ-タ（ＬＲＭＳの場合）で測定した。分析ＨＰＬＣチャ-トは、ＵＶ-２０７５分光計、ＰＵ-２０８０ポンプ、ＤＧ-２０８０-５４脱ガス装置、及びＭＸ-２０８０-３２ミキサ-を備えたＪＡＳＣＯ ＨＰＬＣシステムを用いて得られた。
1. 1. General method (1) General theory Electrospray ionization (ESI) mass spectrum was measured by Shimadzu LCMS-2020 spectrometer (in the case of LRMS). Analytical HPLC charts were obtained using a JASCO HPLC system equipped with a UV-2075 spectrometer, a PU-2080 pump, a DG-2080-54 degassing device, and an MX-2080-32 mixer.
（２）分析用ＨＰＬＣ
ペプチド組成を、アセトニトリル対０．１％トリフルオロ酢酸（ＴＦＡ）の水溶液の勾配を用いる分析逆相ＨＰＬＣによって評価した。
逆相分析ＨＰＬＣは以下のようにして行った：ＹＭＣ-Ｔｒｉａｒｔ-Ｃ１８（内径４．６ｍｍＩ．Ｄ．×１５０ｍｍ）カラムを用い、０．１％ＴＦＡ水溶液中０〜１００％アセトニトリルの直線勾配を用い、室温で４０分間以上、流速１ｍＬ／分-１で行った。溶離液を２３０ｎｍの吸光度でモニタ-した。
(2) HPLC for analysis
Peptide composition was evaluated by analytical reverse phase HPLC using an aqueous gradient of acetonitrile vs. 0.1% trifluoroacetic acid (TFA).
Reversed phase analysis HPLC was performed as follows: Using a YMC-Triart-C 18 (inner diameter 4.6 mm ID x 150 mm) column, a linear gradient of 0-100% acetonitrile in 0.1% TFA aqueous solution. It was used at room temperature for 40 minutes or more at a flow rate of 1 mL / min- 1 . The eluate was monitored with an absorbance of 230 nm.
（３）分取ＨＰＬＣ
アセトニトリル対０．１％ＴＦＡ水溶液の勾配を用いた分取逆相ＨＰＬＣにより、より長いペプチドを精製した。分取ＨＰＬＣは以下のように行った：４０℃で流速１０．０ｍＬｍｉｎ-１でのＹＭＣ-Ｔｒｉａｒｔ Ｃ１８（内径２０ｎｍ×２５０ｍｍ）カラム。勾配条件は各ペプチドで修正した。溶離液を２３０ｎｍでの吸光度によりモニタ-した。
(3) Preparative HPLC
Longer peptides were purified by preparative reverse phase HPLC using a gradient of acetonitrile vs. 0.1% TFA aqueous solution. Preparative HPLC was performed as follows: YMC-Triart C18 (inner diameter 20 nm x 250 mm) column at 40 ° C. and a flow rate of 10.0 mL min- 1. Gradient conditions were modified for each peptide. The eluate was monitored by absorbance at 230 nm.
（４）ペプチドについての検量線の作成
真正ペプチド（５μｍｏｌ）をＤＭＳＯ（２５０μＬ）に溶解して、２０ｍＭのペプチド溶液を作製した。この溶液を１５ｍＭ，１０ｍＭ，８ｍＭ，５ｍＭ，２ｍＭに希釈した。Ｔｒｔ又はＰｂｆを含むペプチドについては、真正ペプチド（１μｍｏｌ）をＤＭＳＯ（５００μＬ）に溶解して２ｍＭのペプチド溶液とし、この溶液を１ｍＭ、０．５ｍＭ、０．２５ｍＭ、０．１２５ｍＭに希釈した後、これらの溶液を２３０ｎｍでの吸光度による分析ＨＰＬＣで分析した。各溶液について、ペプチドに対応するピ-ク面積を測定し、これらの値から検量線を作成した。
(4) Preparation of Calibration Curve for Peptide A 20 mM peptide solution was prepared by dissolving a genuine peptide (5 μmol) in DMSO (250 μL). This solution was diluted to 15 mM, 10 mM, 8 mM, 5 mM, 2 mM. For peptides containing Trt or Pbf, a genuine peptide (1 μmol) is dissolved in DMSO (500 μL) to make a 2 mM peptide solution, and this solution is diluted to 1 mM, 0.5 mM, 0.25 mM, 0.125 mM, and then. These solutions were analyzed by analysis HPLC by absorbance at 230 nm. For each solution, the peak area corresponding to the peptide was measured and a calibration curve was prepared from these values.
（５）チオ酸のエピマ-化レベルの計算方法
ペプチドチオ酸をｐ-メトキシベンジルクロリドによりｐ-メトキシベンジルチオエステルに変換した。そして、チオエステルを含む反応混合物を、順相キラルＨＰＬＣにより分析し、チオエステルの異性体に対応するピ-ク面積を測定し、次式によりエピマ-化速度を算出した。
（６）カップリング反応後のペプチドのエピマ-化レベルの算出方法
ペプチドカップリング反応後、反応混合物を逆相ＨＰＬＣで分析した。ＬＬＬ異性体及びＬＤＬ異性体に対応するピ-ク面積を測定し、見かけのエピマ-化レベル（ＡＥＬ）を次式に基づいて算出した。
＜合成実施例＞
１．ペプチドチオ酸の調製
ペプチドチオ酸はChem. Commun., 2018,54, 12222に記載の方法により得た。以下に各ペプチドチオ酸の調製方法について記載する。
<Synthesis Example>
1. 1. Preparation of Peptide Thioic Acid Peptide thioic acid was obtained by the method described in Chem. Commun., 2018, 54, 12222. The method for preparing each peptide thioic acid is described below.
（１）Ｃｂｚ-Ｐｈｅ-Ｖａｌ-ＳＨの合成
フレ-ム乾燥フラスコに、Ｃｂｚ−Ｐｈｅ−Ｖａｌ−ＯＨ（１．１９ｇ、３．０ミリモル）及びＤＭＦ（２０ｍＭ）をアルゴン大気下で添加した。次いで，ＡｃＳＫ（３．４３ｇ、３０ミリモル）とＡｃ２Ｓ（１６１μＬ、１．５ミリモル）を溶液に添加した。得られた混合物を０℃で３時間攪拌した後、反応混合物を酢酸エチルで抽出し、合わせた有機層を減圧下で濃縮した。残渣をアセトニトリルに溶解し、自動ＲＰ-カラムクロマトグラフィ-により精製して、黄色固体としてＣｂｚ−Ｐｈｅ−Ｖａｌ−ＳＨ（８７０ｍｇ、７０％）を得た。ＭＳ（ＥＳＩ）：ｍ／ｚ ４１５．５３（ｃａｌｃｄ ［Ｍ＋Ｈ］＋＝４１５．１０）。保存時間：２９．７５分。
(1) Synthesis of Cbz-Phe-Val-SH Cbz-Phe-Val-OH (1.19 g, 3.0 mmol) and DMF (20 mM) were added to a frame drying flask under an argon atmosphere. It was then added AcSK (3.43 g, 30 mmol) and Ac 2 S (161μL, 1.5 mmol) to the solution. The resulting mixture was stirred at 0 ° C. for 3 hours, the reaction mixture was extracted with ethyl acetate, and the combined organic layers were concentrated under reduced pressure. The residue was dissolved in acetonitrile and purified by automatic RP-column chromatography-to give Cbz-Phe-Val-SH (870 mg, 70%) as a yellow solid. MS (ESI): m / z 415.53 (calcd [M + H] + = 415.10). Storage time: 29.75 minutes.
（２）Ｃｂｚ-Ｐｈｅ-Ｐｈｅ-ＳＨの合成
フレ-ム乾燥フラスコに、Ｃｂｚ−Ｐｈｅ−Ｐｈｅ−ＯＨ（４４６ｍｇ、１．０ｍｍｏｌ）及びＤＭＦ（１０ｍＭ）をアルゴン大気下で添加した。次いで、ＡｃＳＫ（３４２ｍｇ、３．０ｍｍｏｌ）とＡｃ２Ｓ（２１μＬ、０．２ミリモル）を溶液に添加した。得られた混合物を０℃で３時間攪拌した後、反応混合物を酢酸エチルで抽出し、合わせた有機層を減圧下で濃縮した。残渣をアセトニトリルに溶解し、自動ＲＰ-カラムクロマトグラフィ-により精製して、白色固体としてＣｂｚ−Ｐｈｅ−Ｐｈｅ−ＳＨ（２５４ｍｇ、５５％）を得た。ＭＳ（ＥＳＩ）：ｍ／ｚ ４８５．１５（ｃａｌｃｄ ［Ｍ＋Ｎａ］＋＝４８５．５５）。保存時間：３１．６７分。
(2) Synthesis of Cbz-Phe-Phe-SH Cbz-Phe-Phe-OH (446 mg, 1.0 mmol) and DMF (10 mM) were added to a frame drying flask under an argon atmosphere. It was then added AcSK (342 mg, 3.0 mmol) and Ac 2 S (21μL, 0.2 mmol) to the solution. The resulting mixture was stirred at 0 ° C. for 3 hours, the reaction mixture was extracted with ethyl acetate, and the combined organic layers were concentrated under reduced pressure. The residue was dissolved in acetonitrile and purified by automatic RP-column chromatography-to give Cbz-Phe-Phe-SH (254 mg, 55%) as a white solid. MS (ESI): m / z 485.15 (calcd [M + Na] + = 485.55). Storage time: 31.67 minutes.
（３）Ｂｏｃ-Ｐｈｅ-Ｖａｌ-ＳＨの合成
フレ-ム乾燥フラスコに、Ｂｏｃ−Ｐｈｅ−Ｖａｌ−ＯＨ（１０９ｍｇ、０．３ｍｍｏｌ）及びＤＭＦ（３ｍＭ）をアルゴン大気下で添加した。次いで、ＡｃＳＫ（１７１ｍｇ、１．５ｍｍｏｌ）とＡｃ２Ｓ（１８μＬ、０．１５ミリモル）を溶液に添加した。得られた混合物を０℃で３時間攪拌した後、反応混合物を酢酸エチルで抽出し、合わせた有機層を減圧下で濃縮した。残渣をアセトニトリルに溶解し、分取ＨＰＬＣにより精製して、Ｂｏｃ-Ｐｈｅ-Ｖａｌ-ＳＨ（５７ｍｇ、５０％）を白色固体として得た。ＭＳ（ＥＳＩ）：ｍ／ｚ ４０３．１５（ｃａｌｃｄ ［Ｍ＋Ｎａ］＋＝４０３．４９）。保存時間：３０．１６分。
(3) Synthesis of Boc-Phe-Val-SH Boc-Phe-Val-OH (109 mg, 0.3 mmol) and DMF (3 mM) were added to a frame drying flask under an argon atmosphere. It was then added AcSK (171 mg, 1.5 mmol) and Ac 2 S (18μL, 0.15 mmol) to the solution. The resulting mixture was stirred at 0 ° C. for 3 hours, the reaction mixture was extracted with ethyl acetate, and the combined organic layers were concentrated under reduced pressure. The residue was dissolved in acetonitrile and purified by preparative HPLC to give Boc-Phe-Val-SH (57 mg, 50%) as a white solid. MS (ESI): m / z 403.15 (calcd [M + Na] + = 403.49). Storage time: 30.16 minutes.
（４）Ｃｂｚ-Ｐｈｅ-Ｃｙｓ（Ｔｒｔ）-ＳＨの合成
フレ-ム乾燥試験管に、Ｃｂｚ−Ｐｈｅ−Ｃｙｓ（Ｔｒｔ）−ＯＨ（１２９ｍｇ、０．２ｍｍｏｌ）とＤＭＦ（２ｍＭ）をアルゴン大気下で添加した。次いで、ＡｃＳＫ（１１４ｍｇ、１ｍｍｏｌ）とＡｃ２Ｓ（１０μＬ、０．１ミリモル）を溶液に添加した。得られた混合物を０℃で３時間攪拌した後、反応混合物を酢酸エチルで抽出し、合わせた有機層を減圧下で濃縮した。残渣をアセトニトリルに溶解し、自動ＲＰ-カラムクロマトグラフィ-により精製して、黄色固体としてＣｂｚ−Ｐｈｅ−Ｃｙｓ（Ｔｒｔ）−ＳＨ（２５ｍｇ、１９％）を得た。ＭＳ（ＥＳＩ）：ｍ／ｚ ６８３．１５（ｃａｌｃｄ ［Ｍ＋Ｎａ］＋＝６８３．８４）。保存時間：３７．８９分。
(4) Synthesis of Cbz-Phe-Cys (Trt) -SH Cbz-Phe-Cys (Trt) -OH (129 mg, 0.2 mmol) and DMF (2 mM) were placed in a frame drying test tube under an argon atmosphere. Added. It was then added AcSK (114 mg, 1 mmol) and Ac 2 S (10μL, 0.1 mmol) to the solution. The resulting mixture was stirred at 0 ° C. for 3 hours, the reaction mixture was extracted with ethyl acetate, and the combined organic layers were concentrated under reduced pressure. The residue was dissolved in acetonitrile and purified by automatic RP-column chromatography-to give Cbz-Phe-Cys (Trt) -SH (25 mg, 19%) as a yellow solid. MS (ESI): m / z 683.15 (calcd [M + Na] + = 683.84). Storage time: 37.89 minutes.
（５）Ｃｂｚ-Ｐｈｅ-Ｌｙｓ（Ｂｏｃ）-ＳＨの合成
フレ-ム乾燥試験管に、Ｃｂｚ−Ｐｈｅ−Ｌｙｓ（Ｂｏｃ）−ＯＨ（１０５ｍｇ、０．２ｍｍｏｌ）とＤＭＦ（２ｍＭ）をアルゴン大気下で添加した。次いで、ＡｃＳＫ（６８ｍｇ、０．６ｍｍｏｌ）とＡｃ２Ｓ（５μＬ、４０μｍｏｌ）を溶液に添加した。得られた混合物を０℃で３時間攪拌した後、反応混合物を酢酸エチルで抽出し、合わせた有機層を還元圧力下で濃縮した。残渣をアセトニトリルに溶解し、自動ＲＰ-カラムクロマトグラフィ-により精製して、黄色固体としてＣｂｚ−Ｐｈｅ−Ｌｙｓ（Ｂｏｃ）−ＳＨ（３０ｍｇ，２８％）を得た。ＭＳ（ＥＳＩ）：ｍ／ｚ ５６６．１０（ｃａｌｃｄ ［Ｍ＋Ｎａ］＋＝５６６．６７）。保存時間：３１．８０分。
(5) Synthesis of Cbz-Phe-Lys (Boc) -SH In a frame drying test tube, Cbz-Phe-Lys (Boc) -OH (105 mg, 0.2 mmol) and DMF (2 mM) were placed in an argon atmosphere. Added. Then AcSK (68 mg, 0.6 mmol) and Ac 2 S (5 μL, 40 μmol) were added to the solution. The resulting mixture was stirred at 0 ° C. for 3 hours, the reaction mixture was extracted with ethyl acetate, and the combined organic layers were concentrated under reducing pressure. The residue was dissolved in acetonitrile and purified by automatic RP-column chromatography-to give Cbz-Phe-Lys (Boc) -SH (30 mg, 28%) as a yellow solid. MS (ESI): m / z 566.10 (calcd [M + Na] + = 566.67). Storage time: 31.80 minutes.
（６）Ｃｂｚ-Ｐｈｅ-Ａｌａ-Ｖａｌ-ＳＨの合成
フレ-ム乾燥試験管に、Ｃｂｚ−Ｐｈｅ−Ａｌａ−Ｖａｌ−ＯＨ（４７ｍｇ、０．１ ｍｍｏｌ）及びＤＭＦ（１ｍＭ）をアルゴン大気下で添加した。次いで，ＡｃＳＫ（５７ｍｇ、０．５ ｍｍｏｌ）とＡｃ２Ｓ（５μＬ、５０μｍｏｌ）を溶液に添加した。得られた混合物を０℃で３時間攪拌した後、反応混合物を酢酸エチルで抽出し、合わせた有機層を還元圧力下で濃縮した。残渣をアセトニトリルに溶解し、分取ＨＰＬＣにより精製して、黄色固体としてＣｂｚ−Ｐｈｅ−Ａｌａ−Ｖａｌ−ＳＨ（１１ｍｇ、２５％）を得た。ＭＳ（ＥＳＩ）：ｍ／ｚ ５０８．１５（ｃａｌｃｄ ［Ｍ＋Ｎａ］＋＝５０８．５９）。保存時間：２９．３４分。
(6) Synthesis of Cbz-Phe-Ala-Val-SH Cbz-Phe-Ala-Val-OH (47 mg, 0.1 mmol) and DMF (1 mM) were added to a frame drying test tube under an argon atmosphere. bottom. Then, AcSK (57mg, 0.5 mmol) was added and Ac 2 S (5μL, 50μmol) to the solution. The resulting mixture was stirred at 0 ° C. for 3 hours, the reaction mixture was extracted with ethyl acetate, and the combined organic layers were concentrated under reducing pressure. The residue was dissolved in acetonitrile and purified by preparative HPLC to give Cbz-Phe-Ala-Val-SH (11 mg, 25%) as a yellow solid. MS (ESI): m / z 508.15 (calcd [M + Na] + = 508.59). Storage time: 29.34 minutes.
２．Ｎ-ヒドロキシ-ピリドン添加剤の調製
（１）メチル１−ヒドロキシ−６−オキソ−１，６−ジヒドロピリジン−３−カルボキシレ−ト（化合物１）
以下の合成スキ-ムに則り、化合物１を合成した。
この化合物を文献（Ando, M.; Sato, N.; Nagase, T.; Nagai, K.; Ishikawa, S.; Takahashi, H.; Ohtake, N.; Ito, J.; Hirayama, M.; Mitobe, Y.; et al. Bioorganic Med. Chem. 2009, 17, 6106-6122.）に記載の方法で合成した。Ｓ１（１０．２ｇ、６０ミリモル）とＤＣＭ（８０ｍＬ）の攪拌溶液に、尿素過酸化水素（１２．０ｇ、１２２ミリモル）とＴＦＡＡ（１７．１ｍＬ、１２０ｍｍｏｌ）を０℃で連続的に滴下し、反応混合物を室温に温め、３時間攪拌した。０℃に冷却した後、反応混合物にＮａ２Ｓ２Ｏ３水溶液を添加し、濃縮した。混合物をＤＣＭで抽出し、ＮａＨＣＯ３水溶液及び食塩水で洗浄し、Ｎａ２ＳＯ４上で乾燥した。有機層を濃縮して粗Ｓ２を得た。粗混合物をフラッシュカラムクロマトグラフィ-（酢酸エチル／ヘキサン＝３：１）で精製して、黄色固体としてＳ２を得た（７．８ｇ、７０％）。 This compound is described in the literature (Ando, M .; Sato, N .; Nagase, T .; Nagai, K .; Ishikawa, S .; Takahashi, H .; Ohtake, N .; Ito, J .; Hirayama, M .; It was synthesized by the method described in Mitobe, Y .; et al. Bioorganic Med. Chem. 2009, 17, 6106-6122.). Urea hydrogen peroxide (12.0 g, 122 mmol) and TFAA (17.1 mL, 120 mmol) were continuously added dropwise at 0 ° C. to a stirred solution of S1 (10.2 g, 60 mmol) and DCM (80 mL). The reaction mixture was warmed to room temperature and stirred for 3 hours. After cooling to 0 ° C., an aqueous Na 2 S 2 O 3 solution was added to the reaction mixture and concentrated. The mixture was extracted with DCM, washed with aqueous NaHCO 3 solution and brine, and dried over Na 2 SO 4 . The organic layer was concentrated to give crude S2. The crude mixture was purified by flash column chromatography (ethyl acetate / hexane = 3: 1) to give S2 as a yellow solid (7.8 g, 70%).
Ｓ２（７．８ｇ、４１．７ミリモル）及びＭｅＣＮ（３ｍＬ）の撹拌溶液に、ＴＦＡＡ（７ｍｌ、５０ｍｍｏｌ）を室温で添加し、混合物を３時間撹拌した。反応混合物を減圧下で濃縮した。残渣に固体ＮａＨＣＯ３及びメタノ-ルを添加した。ろ過後、ろ液を濃縮して粗生成物１を得た。粗生成物をフラッシュカラムクロマトグラフィ-（ＤＣＭ／ＭｅＯＨ＝１９：１）で精製して、淡黄色固体として１（３．０ｇ、４３％）を得た。
1H NMR (500 MHz, CD3OD): δ 3.86 (3H, s), 6.62 (1H. d, J = 9.2 Hz), 7.92 (1H, dd, J = 2.4, 9.2 Hz), 8.56 (1H, d, J = 2.4 Hz); MS (ESI): m/z 170.10 (calcd [M+H] += 170.14).
TFAA (7 ml, 50 mmol) was added to a stirred solution of S2 (7.8 g, 41.7 mmol) and MeCN (3 mL) at room temperature and the mixture was stirred for 3 hours. The reaction mixture was concentrated under reduced pressure. Solid NaHCO 3 and methanol were added to the residue. After filtration, the filtrate was concentrated to obtain
1 H NMR (500 MHz, CD 3 OD): δ 3.86 (3H, s), 6.62 (1H. D, J = 9.2 Hz), 7.92 (1H, dd, J = 2.4, 9.2 Hz), 8.56 (1H, 1H, d, J = 2.4 Hz); MS (ESI): m / z 170.10 (calcd [M + H] + = 170.14).
（２）１−ヒドロキシ−５−フェニルピリジン−２（１Ｈ）−オン（化合物２）
以下の合成スキ-ムに則り、化合物２を合成した。
Ｓ３（２４１μＬ、２ｍｍｏｌ）及びＤＣＭ（１０ｍＬ）の撹拌溶液に、ｍＣＰＢＡ（９８６ｍｇ、４ｍｍｏｌ、３０％Ｈ２Ｏを含む）を０℃で添加し、混合物を２時間撹拌した。１ＭのＮａ２Ｓ２Ｏ３水溶液を反応混合物に添加し、混合物をＤＣＭで抽出した。合わせた有機層を、ＮａＨＣＯ３水溶液及び塩水で洗浄し、Ｎａ２ＳＯ４上で乾燥した。濾過後、有機層を濃縮して粗生成物Ｓ４を得た。粗生成物Ｓ４をフラッシュカラムクロマトグラフィ−（ＤＣＭ／ＭｅＯＨ＝１９：１）で精製して、白色固体としてＳ４（３２０ｍｇ，７９％）を得た。 S3 (241μL, 2mmol) was added to a stirred solution of and DCM (10 mL), was added mCPBA (986mg, 4mmol, including 30% H 2 O) at 0 ° C., the mixture was stirred for 2 hours. A 1 M aqueous Na 2 S 2 O 3 solution was added to the reaction mixture and the mixture was extracted with DCM. The combined organic layers were washed with aqueous NaHCO 3 solution and brine and dried over Na 2 SO 4 . After filtration, the organic layer was concentrated to give the crude product S4. The crude product S4 was purified by flash column chromatography (DCM / MeOH = 19: 1) to give S4 (320 mg, 79%) as a white solid.
Ｓ４（５０ｍｇ、０．２５ ｍｍｏｌ）及びＤＭＦ（５００μＬ）の撹拌溶液に、フェニルボロン酸（４５ｍｇ、０．３７５ ｍｍｏｌ）、Ｐｄ（ＰＰｈ３）４（１４ｍｇ、２．５μｍｏｌ）及びＫ２ＣＯ３（６９ｍｇ、０．５ ｍｍｏｌ）を加え、１００℃で一晩撹拌し、反応混合物をＤＣＭで抽出し、合わせた有機層を食塩水で洗浄し、Ｎａ２ＳＯ４上で乾燥し、濃縮して粗生成物Ｓ５を得た。粗混合物をフラッシュカラムクロマトグラフィ−（ＤＣＭ／ＭｅＯＨ＝１９：１）で精製して、白色固体としてＳ５を得た（３４ｍｇ，６７％）。 S4 (50mg, 0.25 mmol) to a stirred solution of and DMF (500 [mu] L), phenylboronic acid (45mg, 0.375 mmol), Pd (PPh 3) 4 (14mg, 2.5μmol) and K 2 CO 3 ( 69 mg (0.5 mmol) was added, stirred overnight at 100 ° C., the reaction mixture was extracted with DCM, the combined organic layers were washed with brine, dried over Na 2 SO 4 , concentrated and crude. Product S5 was obtained. The crude mixture was purified by flash column chromatography (DCM / MeOH = 19: 1) to give S5 as a white solid (34 mg, 67%).
Ｓ５（２０ｍｇ、０．１ ｍｍｏｌ）及び乾燥ＤＣＭ（５００μＬ）の撹拌溶液に、ＢＣｌ３（１Ｍヘプタン溶液、１５０μＬ、０．１５ｍｍｏｌ）をアルゴン大気下で、-１０℃で滴下し、反応混合物を室温に温め、一晩撹拌した。ＭｅＯＨ（１ｍＬ）を添加した後、反応混合物を濃縮して粗生成物２を得た。粗生成物をフラッシュカラムクロマトグラフィ-（酢酸エチル／ヘキサン＝１：３）で精製して、白色固体として２（５ｍｇ，２７％）を得た。
1H NMR (500 MHz, CDCl3): δ 6.84 (1H, d, J = 9.2), 7.36 (1H, m), 7.44 (4H, m), 7.67 (1H, d, J = 10.3), 8.02 (1H, s); MS (ESI): m/z 188.10 (calcd [M+H]+=188.21).
To a stirred solution of S5 (20 mg, 0.1 mmol) and dry DCM (500 μL), BCl 3 (1 M heptane solution, 150 μL, 0.15 mmol) was added dropwise at −10 ° C. under an argon atmosphere and the reaction mixture was added to room temperature. Warmed to and stirred overnight. After adding MeOH (1 mL), the reaction mixture was concentrated to give
1 1 H NMR (500 MHz, CDCl 3 ): δ 6.84 (1H, d, J = 9.2), 7.36 (1H, m), 7.44 (4H, m), 7.67 (1H, d, J = 10.3), 8.02 ( 1H, s); MS (ESI): m / z 188.10 (calcd [M + H] + = 188.21).
（３）メチル１−ヒドロキシル−２−オキソ−５−フェニル−１，２−ジヒドロピリジン−３−カルボキシレ−ト（化合物３）
以下の合成スキ-ムに則り、化合物３を合成した。
フレ-ム乾燥した５０ｍＬフラスコに、Ｎａ（３４０ｍｇ、１４ｍｍｏｌ）及びＭｅＯＨ（１０ｍＬ）をアルゴン大気下、０℃で添加し、溶液にＳ６（１．０ｇ、４ミリモル）を添加し、混合物を３時間攪拌した。酢酸を中和するまで反応混合物に添加し、混合物を減圧下で濃縮した。酢酸エチル及びＨ２Ｏを残渣に加え、生成物を酢酸エチルで抽出した。有機層をＨ２Ｏ及び食塩水で洗浄し、Ｎａ２ＳＯ４上で乾燥し、濃縮して粗生成物Ｓ７を得た。粗混合物をフラッシュカラムクロマトグラフィ-（酢酸エチル／ヘキサン＝１：３）で精製して、無色油状物としてＳ７を得た（８００ｍｇ，７７％）。 To a frame-dried 50 mL flask, Na (340 mg, 14 mmol) and MeOH (10 mL) were added under argon atmosphere at 0 ° C., S6 (1.0 g, 4 mmol) was added to the solution, and the mixture was added for 3 hours. Stirred. The acetic acid was added to the reaction mixture until neutralized and the mixture was concentrated under reduced pressure. Was added to the residue ethyl acetate and H 2 O, the product was extracted with ethyl acetate. The organic layer was washed with H 2 O and brine, dried over Na 2 SO 4 , and concentrated to give the crude product S7. The crude mixture was purified by flash column chromatography (ethyl acetate / hexane = 1: 3) to give S7 as a colorless oil (800 mg, 77%).
３０ｍＬのフラスコに、Ｓ７（３９０ｍｇ、１．５ ｍｍｏｌ）、フェニルボロン酸（２７２ｍｇ，２．２５ ｍｍｏｌ）、Ｐｄ（ＰＰｈ３）４（８６ｍｇ、７５μｍｏｌ）、Ｋ２ＣＯ３（４１４ｍｇ，３ｍｍｏｌ）、及びＤＭＦ（８ｍＬ）をアルゴン大気下で加え、混合物を１００℃で一晩撹拌した。酢酸エチル及びＨ２Ｏを反応混合物に加え、生成物を酢酸エチルで抽出した。合わせた有機層をＨ２Ｏで洗浄し、食塩水でＮａ２ＳＯ４上に乾燥し、粗生成物Ｓ８に濃縮した。粗混合物をフラッシュカラムクロマトグラフィ-（酢酸エチル／ヘキサン＝１：３）で精製して、白色固体としてＳ８を得た（２８０ｍｇ、７７％）。 In a 30 mL flask, S7 (390 mg, 1.5 mmol), phenylboronic acid (272 mg, 2.25 mmol), Pd (PPh 3 ) 4 (86 mg, 75 μmol), K 2 CO 3 (414 mg, 3 mmol), and. DMF (8 mL) was added under argon atmosphere and the mixture was stirred at 100 ° C. overnight. Ethyl acetate and H 2 O was added to the reaction mixture and the product was extracted with ethyl acetate. The combined organic layers were washed with H 2 O, dried over Na 2 SO 4 with saline and concentrated to crude product S8. The crude mixture was purified by flash column chromatography (ethyl acetate / hexane = 1: 3) to give S8 as a white solid (280 mg, 77%).
試験管にＳ８（１２０ｍｇ、０．５ｍｍｏｌ）、過酸化水素尿素（２９４ｍｇ、６．１ ｍｍｏｌ）、及びＭｅＣＮ（２ｍＬ）を添加した。ＴＦＡＡ（４５０μＬ、６ｍｍｏｌ）を０℃の溶液に滴下し、反応物を室温に温め、混合物を一晩攪拌した。反応混合物にＮａ２Ｓ２Ｏ３水溶液を添加し、混合物を濃縮した。酢酸エチル及びＨ２Ｏを残渣に加え、生成物を酢酸エチルで抽出した。有機層を食塩水で洗浄し、Ｎａ２ＳＯ４上で乾燥し、濃縮して粗生成物Ｓ９を得た。粗混合物をフラッシュカラムクロマトグラフィ-（酢酸エチル／ヘキサン＝３：１）で精製して、白色固体としてＳ９を得た（５０ｍｇ、３８％）。 S8 (120 mg, 0.5 mmol), urea hydrogen peroxide (294 mg, 6.1 mmol), and MeCN (2 mL) were added to the test tubes. TFAA (450 μL, 6 mmol) was added dropwise to a solution at 0 ° C., the reaction was warmed to room temperature and the mixture was stirred overnight. An aqueous Na 2 S 2 O 3 solution was added to the reaction mixture to concentrate the mixture. Was added to the residue ethyl acetate and H 2 O, the product was extracted with ethyl acetate. The organic layer was washed with brine, dried over Na 2 SO 4 , and concentrated to give the crude product S9. The crude mixture was purified by flash column chromatography (ethyl acetate / hexane = 3: 1) to give S9 as a white solid (50 mg, 38%).
試験管にＳ９（２５ｍｇ、０．１ｍｍｏｌ）及びＤＣＭ（５００μＬ）をアルゴン大気下で添加した。ＡｃＣｌ（７２μＬ、１ｍｍｏｌ）を溶液に添加し、混合物を１時間加熱還流した。反応混合物を濃縮した。メタノ-ルを加え、混合物を室温で一晩撹拌した。混合物を濃縮して粗生成物３を得た。粗混合物をフラッシュカラムクロマトグラフィ−（ＤＣＭ／ＭｅＯＨ＝１９：１）で精製して、白色固体として３を得た（１１ｍｇ、４５％）。
1H NMR (500 MHz, CDCl3): δ3.97 (3H, s), 7.39 (2H, m), 7.46 (3H, m), 8.26 (1H, s), 8.47 (1H, s); MS (ESI): m/z 246 (calcd [M+H]+= 246.24).
S9 (25 mg, 0.1 mmol) and DCM (500 μL) were added to the test tube under an atmosphere of argon. AcCl (72 μL, 1 mmol) was added to the solution and the mixture was heated to reflux for 1 hour. The reaction mixture was concentrated. Metall was added and the mixture was stirred at room temperature overnight. The mixture was concentrated to give
1 1 H NMR (500 MHz, CDCl 3 ): δ3.97 (3H, s), 7.39 (2H, m), 7.46 (3H, m), 8.26 (1H, s), 8.47 (1H, s); MS ( ESI): m / z 246 (calcd [M + H] + = 246.24).
（４）メチル２−（１−ヒドロキシ−６−オキソ−１，６−ジヒドロピリジン−３−イル）ベンゾエ−ト（化合物４）
以下の合成スキ-ムに則り、化合物４を合成した。
Compound 4 was synthesized according to the following synthetic framework.
１００ｍＬのフラスコに、Ｓ３（４８２ｍｇ、４ｍｍｏｌ）、２-（メトキシカルボニル）-フェニルボロン酸（１０７４ｍｇ、６ｍｍｏｌ）、Ｐｄ（ＰＰｈ３）４（２３１ｍｇ、０．２ｍｍｏｌ）、Ｋ２ＣＯ３（１１００ｍｇ、１２ｍｍｏｌ）及びＤＭＦ（２０ｍＬ）をアルゴン雰囲気下で加え、混合物を１００℃で一晩撹拌した。酢酸エチル及びＨ２Ｏを反応混合物に加え、生成物を酢酸エチルで抽出した。合わせた有機層をＨ２Ｏ及び食塩水で洗浄し、Ｎａ２ＳＯ４上で乾燥し、濃縮して粗生成物Ｓ１０を得た。粗混合物をフラッシュカラムクロマトグラフィ-（酢酸エチル／ヘキサン＝１：３）で精製して、白色固体としてＳ１０を得た（８００ｍｇ、８２％）。 In a 100 mL flask, S3 (482 mg, 4 mmol), 2- (methoxycarbonyl) -phenylboronic acid (1074 mg, 6 mmol), Pd (PPh 3 ) 4 (231 mg, 0.2 mmol), K 2 CO 3 (1100 mg, 12 mmol). ) And DMF (20 mL) were added under an argon atmosphere and the mixture was stirred at 100 ° C. overnight. Ethyl acetate and H 2 O was added to the reaction mixture and the product was extracted with ethyl acetate. The combined organic layers were washed with H 2 O and brine, dried over Na 2 SO 4 and concentrated to give the crude product S10. The crude mixture was purified by flash column chromatography (ethyl acetate / hexane = 1: 3) to give S10 as a white solid (800 mg, 82%).
５０ｍＬフラスコ中のＤＣＭ（１０ｍＬ）中のＳ１０（６００ｍｇ、２．５ｍｍｏｌ）に、ｍＣＰＢＡ（１．８５ｇ、７．５ｍｍｏｌ、３０％Ｈ２Ｏを含む）を０℃下で添加し、混合物を室温まで温め、一晩撹拌した。１Ｍ Ｎａ２Ｓ２Ｏ３水溶液を反応混合物に添加し、混合物をＤＣＭで抽出した。合わせた有機層を、ＮａＨＣＯ３水溶液及び食塩水で洗浄し、Ｎａ２ＳＯ４上で乾燥した。有機層を濃縮して粗生成物Ｓ１１を得た。粗混合物をフラッシュカラムクロマトグラフィ-（ＤＣＭ／ＭｅＯＨ＝１９：１）で精製して、白色固体としてＳ１１（５８０ｍｇ、９０％）を得た。 S10 (600mg, 2.5mmol) in DCM (10 mL) in 50mL flask, mCPBA a (1.85 g, 7.5 mmol, containing 30% H 2 O) was added under 0 ° C., the mixture to room temperature It was warmed and stirred overnight. A 1M Na 2 S 2 O 3 aqueous solution was added to the reaction mixture and the mixture was extracted with DCM. The combined organic layers were washed with aqueous NaHCO 3 solution and saline and dried over Na 2 SO 4 . The organic layer was concentrated to give the crude product S11. The crude mixture was purified by flash column chromatography (DCM / MeOH = 19: 1) to give S11 (580 mg, 90%) as a white solid.
反応混合物を濃縮し、メタノ-ルを加え、室温で一晩撹拌した。混合物を濃縮して粗生成物３を得た。粗混合物をフラッシュカラムクロマトグラフィ-（ＤＣＭ／ＭｅＯＨ＝１９：１）で精製して、白色固体として４を得た（１２５ｍｇ、５１％）。
1H NMR (500 MHz, CDCl3): δ 3.65 (3H, s), 6.79 (1H, s), 7.41 (4H, m), 7.84 (1H, d, J = 8.0), 8.10 (1H, s); MS (ESI): m/z 246 (calcd [M+H]+= 246.24).
The reaction mixture was concentrated, methanol was added, and the mixture was stirred at room temperature overnight. The mixture was concentrated to give
1 H NMR (500 MHz, CDCl 3 ): δ 3.65 (3H, s), 6.79 (1H, s), 7.41 (4H, m), 7.84 (1H, d, J = 8.0), 8.10 (1H, s) MS (ESI): m / z 246 (calcd [M + H] + = 246.24).
（５）メチル１−ヒドロキシ−２−オキソ−１，２−ジヒドロピリジン−３−カルビキシレ−ト（化合物５）
以下の合成スキ-ムに則り、化合物５を合成した。
Compound 5 was synthesized according to the following synthetic framework.
この化合物は、化合物１について記載した手順を用いて、黄色固体として調製した（７２ｍｇ、８５％）。
(500 MHz, (CD3)2SO): δ 3.74 (3H, s), 6.25 (1H, dd, J = 6.7, 7.4), 7.96 (1H, d, J = 7.4), 8.23 (1H, d, J = 6.7); MS (ESI): m/z 170.10 (calcd [M+H]+= 170.14).
This compound was prepared as a yellow solid (72 mg, 85%) using the procedure described for
(500 MHz, (CD 3 ) 2 SO): δ 3.74 (3H, s), 6.25 (1H, dd, J = 6.7, 7.4), 7.96 (1H, d, J = 7.4), 8.23 (1H, d, J = 6.7); MS (ESI): m / z 170.10 (calcd [M + H] + = 170.14).
（６）Ｎ−（２−（ジメチルアミノ）エチル）−１−ヒドロキシ−６−オキソ−１，６−ジヒドロピリジン−３−カルボキサミド（化合物６）
以下の合成スキ-ムに則り、化合物６を合成した。
Compound 6 was synthesized according to the following synthetic framework.
化合物１（３．０ｇ、１７．７５ミリモル）、Ｋ２ＣＯ３（７．３ｇ、５３．２５ミリモル）、ＤＭＦ（４０ｍＬ）の攪拌溶液に、ＢｎＢｒ（２．５ｍＬ、２１．３ｍｍｏｌ）を加え、８０℃で３時間攪拌し、反応混合物を濃縮した。得られた混合物に酢酸エチル及びＨ２Ｏを加え、生成物を酢酸エチルで抽出した。有機層を食塩水で洗浄し、Ｎａ２ＳＯ４上で乾燥し、濾過し、濃縮して粗生成物Ｓ１３を得た。粗混合物をフラッシュカラムクロマトグラフィ-（酢酸エチル／ヘキサン＝１：３）で精製して、白色固体としてＳ１３（２．０ｇ、４４％）を得た。 Add BnBr (2.5 mL, 21.3 mmol) to a stirred solution of compound 1 (3.0 g, 17.75 mmol), K 2 CO 3 (7.3 g, 53.25 mmol), DMF (40 mL). The reaction mixture was concentrated by stirring at 80 ° C. for 3 hours. The resulting mixture of ethyl acetate and H 2 O was added, the product was extracted with ethyl acetate. The organic layer was washed with brine, dried over Na 2 SO 4 , filtered and concentrated to give the crude product S13. The crude mixture was purified by flash column chromatography (ethyl acetate / hexane = 1: 3) to give S13 (2.0 g, 44%) as a white solid.
Ｓ１３（２．０ｇ、８ミリモル）及びＭｅＯＨ（８ｍＬ）の撹拌溶液に、３Ｍ ＮａＯＨ水溶液（８ｍＬ、２４ｍｍｏｌ）を添加し、混合物を一晩撹拌した。反応混合物に、１Ｍ ＨＣｌ水溶液を中和するまで添加した。生成物を濃縮した。得られた残渣に酢酸エチル及び１Ｍ ＨＣｌ水溶液を加え、生成物を酢酸エチルで抽出した。有機層を食塩水で洗浄し、Ｎａ２ＳＯ４上で乾燥し、濾過し、そして濃縮して白色固体としてＳ１４（１．７ｇ、８７％）を得た。 To a stirred solution of S13 (2.0 g, 8 mmol) and MeOH (8 mL) was added 3M aqueous NaOH solution (8 mL, 24 mmol) and the mixture was stirred overnight. A 1M aqueous HCl solution was added to the reaction mixture until neutralized. The product was concentrated. Ethyl acetate and 1M HCl aqueous solution were added to the obtained residue, and the product was extracted with ethyl acetate. The organic layer was washed with brine, dried over Na 2 SO 4 , filtered and concentrated to give S14 (1.7 g, 87%) as a white solid.
Ｓ１４（４９ｍｇ、０．２ｍｍｏｌ）、Ｎ，Ｎ−ジメチルエチレンジアミン（４３μＬ、０．４ミリモル）、及びＤＣＭ（１ｍＬ）の撹拌溶液に、ＷＳＣＩ（７６ｍｇ、０．４ｍｍｏｌ）、及びＨＯＢｔ（５４ｍｇ、０．４ｍｍｏｌ）を０℃で添加し、反応混合物を室温に温め、３時間撹拌した。得られた混合物に、ＤＣＭ及びＮａＨＣＯ３水溶液を添加した。生成物をＤＣＭで抽出し、有機層を食塩水で洗浄し、Ｎａ２ＳＯ４上で乾燥し、濾過し、濃縮して粗生成物Ｓ１５を得た。この粗混合物を、さらなる精製なしで、次の反応に使用した。 In a stirred solution of S14 (49 mg, 0.2 mmol), N, N-dimethylethylenediamine (43 μL, 0.4 mmol), and DCM (1 mL), WSCI (76 mg, 0.4 mmol), and HOBt (54 mg, 0. 4 mmol) was added at 0 ° C., the reaction mixture was warmed to room temperature and stirred for 3 hours. DCM and an aqueous NaHCO 3 solution were added to the resulting mixture. The product was extracted with DCM, the organic layer was washed with brine, dried over Na 2 SO 4 , filtered and concentrated to give the crude product S15. This crude mixture was used in the next reaction without further purification.
粗生成物Ｓ１５及びＤＣＭ（１ｍＬ）の撹拌溶液に、ＢＣｌ３（１Ｍヘプタン溶液２４０μＬ、０．２４ｍｍｏｌ）を添加し、混合物を一晩撹拌した。反応混合物を濃縮して粗生成物６を得た。粗混合物を分取ＨＰＬＣで精製して、白色固体として６（１１ｍｇ、２４％，２段階）を得た。
1H NMR (500 MHz, CDCl3): δ 2.25 (6H, s), 2.44 (2H, t, J = 5.7), 3.39 (2H, t, J = 5.7), 6.67 (1H, d, J = 9.7), 7.58 (1H, d, J = 9.7), 7.88 (1H, s); MS (ESI): m/z 259 (calcd [M+H]+= 259.28).
BCl 3 (240 μL, 0.24 mmol of 1 M heptane solution) was added to the stirred solution of crude product S15 and DCM (1 mL) and the mixture was stirred overnight. The reaction mixture was concentrated to give the crude product 6. The crude mixture was purified by preparative HPLC to give 6 (11 mg, 24%, 2 steps) as a white solid.
1 1 H NMR (500 MHz, CDCl 3 ): δ 2.25 (6H, s), 2.44 (2H, t, J = 5.7), 3.39 (2H, t, J = 5.7), 6.67 (1H, d, J = 9.7) ), 7.58 (1H, d, J = 9.7), 7.88 (1H, s); MS (ESI): m / z 259 (calcd [M + H] + = 259.28).
（７）５−（３−（ジメチルアミノ）ベンジル）−１−ヒドロキシピリジン−２（１Ｈ）−オン（化合物７）
以下の合成スキ-ムに則り、化合物７を合成した。
Compound 7 was synthesized according to the following synthetic framework.
ＤＣＭ（１０ｍＬ）中のＳ１４（４９０ｍｇ、２．４ｍｍｏｌ）及びｐ-トルエンチオ-ル（２９８ｍｇ、２．４ｍｍｏｌ）の撹拌溶液に、ＷＳＣＩ（４５８ｍｇ、２．４ｍｍｏｌ）及びＨＯＢｔ（２７０ｍｇ、２ｍｍｏｌ）を０℃で添加し、反応混合物を室温に温め、３時間撹拌した。得られた混合物を濃縮した。酢酸エチル及びＮａＨＣＯ３水溶液を加え、生成物を酢酸エチルで抽出した。有機層を食塩水で洗浄し、Ｎａ２ＳＯ４上に乾燥し、濾過し、そして濃縮して粗生成物Ｓ１６を得た。粗混合物をフラッシュカラムクロマトグラフィ−（酢酸エチル／ヘキサン＝１：３）によって精製して、白色固体としてＳ１６（４８０ｍｇ、６８％）を得た。 WSCI (458 mg, 2.4 mmol) and HOBt (270 mg, 2 mmol) were added to a stirred solution of S14 (490 mg, 2.4 mmol) and p-toluenethiol (298 mg, 2.4 mmol) in DCM (10 mL) at 0 ° C. The reaction mixture was warmed to room temperature and stirred for 3 hours. The resulting mixture was concentrated. Ethyl acetate and 3 aqueous NaHCO solutions were added and the product was extracted with ethyl acetate. The organic layer was washed with brine, dried over Na 2 SO 4 , filtered and concentrated to give the crude product S16. The crude mixture was purified by flash column chromatography (ethyl acetate / hexane = 1: 3) to give S16 (480 mg, 68%) as a white solid.
Ｓ１６（７０ｍｇ、０．２ｍｍｏｌ）及びＴＨＦ（２ｍＬ）の撹拌溶液に、２-ジメチルアミノフェニルボロン酸（９９ｍｇ、０．６ｍｍｏｌ）、Ｐｄ２（ｄｂａ）３・ＣＨＣｌ３（５ｍｇ、４μｍｏｌ）、トリ（２-フリル）ホスフィン（３．５ｍｇ、１５μｍｏｌ）、及びＣｕＴＣ（１１５ｍｇ、０．６ｍｍｏｌ）を添加した。反応混合物を加熱還流し、一晩撹拌した。セライトで濾過した後、濾液を濃縮してＳ１７を得た。この粗混合物をさらに精製することなく次の反応に用いた。 In a stirred solution of S16 (70 mg, 0.2 mmol) and THF (2 mL), 2-dimethylaminophenylboronic acid (99 mg, 0.6 mmol), Pd 2 (dba) 3. CHCl 3 (5 mg, 4 μmol), tri ( 2-Frill) phosphine (3.5 mg, 15 μmol) and CuTC (115 mg, 0.6 mmol) were added. The reaction mixture was heated to reflux and stirred overnight. After filtering with Celite, the filtrate was concentrated to obtain S17. This crude mixture was used in the next reaction without further purification.
Ｓ１７及びＤＣＭ（１ｍＬ）の撹拌溶液に、ＢＣｌ３（６９０μＬ、０．７ミリモル）を添加し、混合物を一晩撹拌した。反応溶液を濃縮して粗生成物７を得た。粗混合物を分取ＨＰＬＣで精製して、無色油状物として７（６ｍｇ、１２％、２段階）を得た。
1H NMR (500 MHz, CDCl3): δ 3.28 (6H, s), 6.73 (1H, s), 7.28-7.43 (4H, m), 7.75 (1H, d, J = 8.0), 7.99 (1H, d, J = 8.0); MS (ESI): m/z 259 (calcd [M+H]+= 259.28).
BCl 3 (690 μL, 0.7 mmol) was added to a stirred solution of S17 and DCM (1 mL) and the mixture was stirred overnight. The reaction solution was concentrated to give the crude product 7. The crude mixture was purified by preparative HPLC to give 7 (6 mg, 12%, 2 steps) as a colorless oil.
1 H NMR (500 MHz, CDCl 3 ): δ 3.28 (6H, s), 6.73 (1H, s), 7.28-7.43 (4H, m), 7.75 (1H, d, J = 8.0), 7.99 (1H, 1H, d, J = 8.0); MS (ESI): m / z 259 (calcd [M + H] + = 259.28).
（８）化合物８の合成
以下の合成スキ-ムに則り、化合物７を合成した。
Ｓ１８（４ｇ、２５．２ｍｍｏｌ）、ＷＳＣＩ（７．２ｇ、３７．８ｍｍｏｌ）、ＤＭＡＰ（４．６ｇ、３７．８ｍｍｏｌ）及びＤＣＭ（８０ｍＬ）の混合溶液に、３,７,１１,１５―テトラメチルヘキサデカン−１−オール（１０．８ｍＬ、３０．２ｍｍｏｌ）及びトリエチルアミン（７．２ｍＬ、３７．８ｍｍｏｌ）を室温で添加し、アルゴン雰囲気下、室温で２５時間撹拌し、溶媒を減圧除去した。残渣に酢酸エチル及び１ＭＨＣl水溶液を添加し、混合物を酢酸エチルで抽出した。有機層を飽和重曹水で洗浄を二回行い、食塩水で洗浄し、Ｎａ２ＳＯ４上で乾燥し、濾過し、溶媒を減圧除去して粗生成物Ｓ１９を得た。粗混合物をシリカゲルカラムクロマトグラフィ-（酢酸エチル／ヘキサン＝２０：８０ → ４０：６０）で精製して、Ｓ１９を得た（８．８５ｇ、８０％）。 3,7,11,15-Tetramethyl in a mixed solution of S18 (4 g, 25.2 mmol), WSCI (7.2 g, 37.8 mmol), DMAP (4.6 g, 37.8 mmol) and DCM (80 mL). Hexadecane-1-ol (10.8 mL, 30.2 mmol) and triethylamine (7.2 mL, 37.8 mmol) were added at room temperature, and the mixture was stirred at room temperature for 25 hours under an argon atmosphere to remove the solvent under reduced pressure. Ethyl acetate and 1 MHCl aqueous solution were added to the residue, and the mixture was extracted with ethyl acetate. The organic layer was washed twice with saturated aqueous sodium hydrogen carbonate, washed with brine, dried over Na2SO4, filtered, and the solvent was removed under reduced pressure to obtain crude product S19. The crude mixture was purified by silica gel column chromatography (ethyl acetate / hexane = 20:80 → 40:60) to give S19 (8.85 g, 80%).
Ｓ１９（８．８５ｇ、２０．２ｍｍｏｌ）、尿素過酸化水素（３．９５ｇ、４２．４ｍｍｏｌ）及びＤＣＭ（５０ｍＬ）の混合溶液にトリフルオロ酢酸無水物（５．７ｍＬ、４０．４ｍｍｏｌ）を０℃で滴下し、アルゴン雰囲気下、室温で１６時間攪拌した。０℃に冷却し、反応溶液に飽和亜硫酸水素ナトリウム水溶液（５．５ｍＬ）を添加し、反応混合物を酢酸エチルで抽出した。有機層を飽和重曹水で洗浄を二回行い、食塩水で洗浄し、Ｎａ２ＳＯ４上で乾燥し、濾過し、溶媒を減圧除去して粗生成物Ｓ１９を得た。粗混合物をシリカゲルカラムクロマトグラフィ-（酢酸エチル／ヘキサン＝１０：９０ → １００：０）で精製して、Ｓ２０を得た（７．５１ｇ、８２％）。 Trifluoroacetic anhydride (5.7 mL, 40.4 mmol) at 0 ° C. in a mixed solution of S19 (8.85 g, 20.2 mmol), urea hydrogen peroxide (3.95 g, 42.4 mmol) and DCM (50 mL). And stirred at room temperature for 16 hours under an argon atmosphere. The mixture was cooled to 0 ° C., saturated aqueous sodium hydrogen sulfite solution (5.5 mL) was added to the reaction solution, and the reaction mixture was extracted with ethyl acetate. The organic layer was washed twice with saturated aqueous sodium hydrogen carbonate, washed with brine, dried over Na 2 SO 4 , filtered, and the solvent was removed under reduced pressure to obtain crude product S19. The crude mixture was purified by silica gel column chromatography (ethyl acetate / hexane = 10: 90 → 100: 0) to give S20 (7.51 g, 82%).
Ｓ２０（７．５１ｇ、１６．５ｍｍｏｌ）及びアセトニトリル（１５ｍＬ）の撹拌溶液に、ＴＦＡＡ（３０ｍｌ）を室温で添加し、アルゴン雰囲気下、室温で１４時間撹拌した。溶媒を減圧除去しを、残渣に固体ＮａＨＣＯ３及びクロロホルムを添加した。ろ過後、溶媒を減圧除去して粗生成物８を得た。粗生成物を中性シリカゲルカラムクロマトグラフィ-（酢酸エチル／ヘキサン＝４０：６０ → １００：０）で精製して黄色液体８（５．８２ｇ、８１％）を得た。
1H NMR (500 MHz, CD3OD): δ 0.83-0.89 (12H, m), 0.96 (3H, d, J = 6.3 Hz), 1.02-1.44 (20H, m), 1.45-1.66 (3H, m), 1.70-1.82 (1H, m), 4.24-4.40 (2H. m), 6.63 (1H, d, J = 9.2 Hz),7.92 (1H, dd, J = 2.3, 9.7 Hz), 8.52 (1H, d, J =2.9 Hz); MS (ESI): m/z 436.30 (calcd [M+H]+= 436.34).
TFAA (30 ml) was added to a stirred solution of S20 (7.51 g, 16.5 mmol) and acetonitrile (15 mL) at room temperature, and the mixture was stirred at room temperature for 14 hours under an argon atmosphere. The solvent was removed under reduced pressure, and solid NaHCO 3 and chloroform were added to the residue. After filtration, the solvent was removed under reduced pressure to obtain a crude product 8. The crude product was purified by neutral silica gel column chromatography (ethyl acetate / hexane = 40: 60 → 100: 0) to obtain a yellow liquid 8 (5.82 g, 81%).
1 1 H NMR (500 MHz, CD 3 OD): δ 0.83-0.89 (12H, m), 0.96 (3H, d, J = 6.3 Hz), 1.02-1.44 (20H, m), 1.45-1.66 (3H, m) ), 1.70-1.82 (1H, m), 4.24-4.40 (2H. M), 6.63 (1H, d, J = 9.2 Hz), 7.92 (1H, dd, J = 2.3, 9.7 Hz), 8.52 (1H, d, J = 2.9 Hz); MS (ESI): m / z 436.30 (calcd [M + H] + = 436.34).
（９）化合物９の合成
以下の合成スキ-ムに則り、化合物９を合成した。
この化合物は、化合物８について記載した手順を用いて、黄色液体９を得た（１．５３ｇ、９６％）。
1H NMR (500 MHz, CDCl3): δ0.87 (6H, d, J = 6.3 Hz), 0.94 (3H, d, J = 6.3 Hz), 1.08-1.20 (3H, m), 1.23-1.38 (3H, m), 1.44-1.64 (3H, m), 1.70-1.82 (1H, m), 4.28-4.38 (1H, m), 6.73 (1H, d, J =9.7 Hz), 7.96 (1H, dd, J =2.9, 9.7 Hz), 8.55 (1H, d, J =2.3 Hz); MS (ESI): m/z 296.20 (calcd [M+H]+= 296.19).
This compound gave a yellow liquid 9 (1.53 g, 96%) using the procedure described for compound 8.
1 1 H NMR (500 MHz, CDCl 3 ): δ0.87 (6H, d, J = 6.3 Hz), 0.94 (3H, d, J = 6.3 Hz), 1.08-1.20 (3H, m), 1.23-1.38 ( 3H, m), 1.44-1.64 (3H, m), 1.70-1.82 (1H, m), 4.28-4.38 (1H, m), 6.73 (1H, d, J = 9.7 Hz), 7.96 (1H, dd, J = 2.9, 9.7 Hz), 8.55 (1H, d, J = 2.3 Hz); MS (ESI): m / z 296.20 (calcd [M + H] + = 296.19).
（１０）化合物１０の合成
以下の合成スキ-ムに則り、化合物１０を合成した。
この化合物は、化合物８について記載した手順を用いて、黄色固体１０を得た（２０５ｍｇ、９２％）。
1H NMR (500 MHz, CD3OD): δ0.91 (3H, t, J =7.4 Hz), 1.30-1.38 (4H, m), 1.40-1.46 (2H, m),1.70-1.76 (2H, m), 4.26 (3H, t, J =6.9 Hz), 6.61 (1H, d, J = 9.2 Hz), 7.90-7.92 (1H, m), 8.48-8.54 (1H, m); MS (ESI): m/z 240.15 (calcd [M+H]+= 240.12).
This compound gave the yellow solid 10 (205 mg, 92%) using the procedure described for compound 8.
1 1 H NMR (500 MHz, CD 3 OD): δ0.91 (3H, t, J = 7.4 Hz), 1.30-1.38 (4H, m), 1.40-1.46 (2H, m), 1.70-1.76 (2H, m), 4.26 (3H, t, J = 6.9 Hz), 6.61 (1H, d, J = 9.2 Hz), 7.90-7.92 (1H, m), 8.48-8.54 (1H, m); MS (ESI): m / z 240.15 (calcd [M + H] + = 240.12).
（１１）化合物１１の合成
以下の合成スキ-ムに則り、化合物１１を合成した。
この化合物は、化合物８について記載した手順を用いて、黄色液体１１を得た（１３１ｍｇ、４８％）。
1H NMR (500 MHz, CD3OD): δ0.93 (6H, t, J = 7.4 Hz), 1.60-1.75 (4H, m), 4.90-4.95 (1H. m), 6.63 (1H, d, J = 9.2 Hz), 7.93 (1H, dd, J = 2.3 Hz, 9.2 Hz), 8.53 (1H, d, J = 2.3 Hz); MS (ESI): m/z 226.40 (calcd [M+H]+=226.11).
This compound gave Yellow Liquid 11 (131 mg, 48%) using the procedure described for Compound 8.
1 1 H NMR (500 MHz, CD 3 OD): δ0.93 (6H, t, J = 7.4 Hz), 1.60-1.75 (4H, m), 4.90-4.95 (1H. M), 6.63 (1H, d, J = 9.2 Hz), 7.93 (1H, dd, J = 2.3 Hz, 9.2 Hz), 8.53 (1H, d, J = 2.3 Hz); MS (ESI): m / z 226.40 (calcd [M + H] + = 226.11).
（１２）化合物１２の合成
以下の合成スキ-ムに則り、化合物１２を合成した。
Ｓ１８（６３２ｍｇ、４ｍｍｏｌ）、Ｂｏｃ２Ｏ（１．３１ｇ、６ｍｍｏｌ）、ＤＭＡＰ（４８．９ｍｇ、０．４ｍｍｏｌ）及びテトラヒドロフラン（１０ｍＬ）の混合溶液をアルゴン雰囲気下、加熱還流しながら３時間撹拌し、溶媒を減圧除去した。残渣にジエチルエーテル及び水を添加し、混合物をジエチルエーテルで抽出した。有機層を飽和重曹水、飽和食塩水の順に洗浄し、Ｎａ２ＳＯ４上で乾燥し、濾過し、溶媒を減圧除去して粗生成物Ｓ２７を得た。粗混合物をシリカゲルカラムクロマトグラフィ-（酢酸エチル／ヘキサン＝５：９５ → ３０：７０）で精製して、Ｓ２７を得た（６２２ｍｇ、７３％）。 A mixed solution of S18 (632 mg, 4 mmol), Boc 2 O (1.31 g, 6 mmol), DMAP (48.9 mg, 0.4 mmol) and tetrahydrofuran (10 mL) was stirred under an argon atmosphere for 3 hours while heating under reflux. The solvent was removed under reduced pressure. Diethyl ether and water were added to the residue, and the mixture was extracted with diethyl ether. The organic layer was washed in the order of saturated aqueous sodium hydrogen carbonate and saturated brine, dried over Na2SO4, filtered, and the solvent was removed under reduced pressure to obtain a crude product S27. The crude mixture was purified by silica gel column chromatography (ethyl acetate / hexane = 5:95 → 30:70) to give S27 (622 mg, 73%).
Ｓ２７（２８５ｍｇ、１．３３ｍｍｏｌ）、尿素過酸化水素（２６３ｍｇ、２．８０ｍｍｏｌ）及びＤＣＭ（１３．３ｍＬ）の混合溶液にトリフルオロ酢酸無水物（３７４μＬ、２．６６ｍｍｏｌ）を０℃で滴下し、アルゴン雰囲気下、室温で７時間攪拌した。０℃に冷却し、反応溶液に飽和亜硫酸水素ナトリウム水溶液及び水を添加し、反応混合物をクロロホルムで抽出した。有機層を飽和重曹水で洗浄を二回行い、食塩水で洗浄し、Ｎａ２ＳＯ４上で乾燥し、濾過し、溶媒を減圧除去して粗生成物Ｓ２８を得た。粗混合物をシリカゲルカラムクロマトグラフィ-（酢酸エチル／ヘキサン＝２０：９０ → １００：０）で精製して、Ｓ２８を得た（２８０ｍｇ、９２％）。 Trifluoroacetic anhydride (374 μL, 2.66 mmol) was added dropwise to a mixed solution of S27 (285 mg, 1.33 mmol), urea hydrogen peroxide (263 mg, 2.80 mmol) and DCM (13.3 mL) at 0 ° C. The mixture was stirred at room temperature for 7 hours under an argon atmosphere. The mixture was cooled to 0 ° C., saturated aqueous sodium hydrogen sulfite solution and water were added to the reaction solution, and the reaction mixture was extracted with chloroform. The organic layer was washed twice with saturated aqueous sodium hydrogen carbonate, washed with brine, dried over Na 2 SO 4 , filtered, and the solvent was removed under reduced pressure to give the crude product S28. The crude mixture was purified by silica gel column chromatography (ethyl acetate / hexane = 20: 90 → 100: 0) to give S28 (280 mg, 92%).
Ｓ２８（２８０ｍｇ、１．２２ｍｍｏｌ）及びアセトニトリル（１２．２ｍＬ）の撹拌溶液に、トリフルオロ酢酸無水物（３４４μＬ、２．４４ｍｍｏｌ）を室温で添加し、アルゴン雰囲気下、反応溶液を１時間２０分撹拌した。反応溶液にトリフルオロ酢酸無水物（１７５μＬ、１．２２ｍｍｏｌ）を室温で添加し、室温で１４時間３０分攪拌した。混合物を１時間２０分撹拌した。反応溶液にトリフルオロ酢酸無水物（１７５μＬ、１．２２ｍｍｏｌ）を室温で添加し、室温で２時間攪拌した。溶媒を減圧除去し、残渣に固体ＮａＨＣＯ３及びクロロホルムを添加した。ろ過後、溶媒を減圧除去して粗生成物１２を得た。粗生成物を中性シリカゲルカラムクロマトグラフィ-（酢酸エチル／メタノール＝１００：０ → ８５：１５）で精製して、白色固体１２（１１９ｍｇ、４６％）を得た
1H NMR (500 MHz, CD3OD): δ1.56 (9H,s), 6.60 (1H, d, J = 9.2 Hz), 7.84--7.90 (1H, m), 8.43 (1H, d, J =2.3 Hz); MS (ESI): m/z 240.12 (calcd [M+H]+= 240.15).
Trifluoroacetic anhydride (344 μL, 2.44 mmol) was added to a stirred solution of S28 (280 mg, 1.22 mmol) and acetonitrile (12.2 mL) at room temperature, and the reaction solution was stirred for 1 hour and 20 minutes under an argon atmosphere. bottom. Trifluoroacetic anhydride (175 μL, 1.22 mmol) was added to the reaction solution at room temperature, and the mixture was stirred at room temperature for 14 hours and 30 minutes. The mixture was stirred for 1 hour and 20 minutes. Trifluoroacetic anhydride (175 μL, 1.22 mmol) was added to the reaction solution at room temperature, and the mixture was stirred at room temperature for 2 hours. The solvent was removed under reduced pressure, and solid NaHCO 3 and chloroform were added to the residue. After filtration, the solvent was removed under reduced pressure to obtain a crude product 12. The crude product was purified by neutral silica gel column chromatography (ethyl acetate / methanol = 100: 0 → 85:15) to give a white solid 12 (119 mg, 46%).
1 H NMR (500 MHz, CD 3 OD): δ1.56 (9H, s), 6.60 (1H, d, J = 9.2 Hz), 7.84--7.90 (1H, m), 8.43 (1H, d, J) = 2.3 Hz); MS (ESI): m / z 240.12 (calcd [M + H] + = 240.15).
［合成実施例１］
ジペプチドチオ酸と種々のアミノ酸との縮合
以下の反応スキ-ムにより、ジペプチドチオ酸と種々のアミノ酸との縮合を行った。
Condensation of dipeptide thioic acid with various amino acids Condensation of dipeptide thioic acid with various amino acids was carried out by the following reaction skim.
Ｎ-ヒドロキシピリドン添加剤、二次亜リン酸及びペプチドチオ酸を原液として用いた。１．５ｍＬのマイクロチュ-ブに、Ｎ-ヒドロキシピリドン添加物、二次亜リン酸、アミノ酸を添加した。次いで、ペプチドチオ酸の溶液を添加し、３０℃で所定の反応時間撹拌した。反応混合物を１％ＴＦＡのＤＭＳＯ溶液で希釈し、ＨＰＬＣで分析した。収率は検量線により決定した。 N-Hydroxypyridone additive, hypophosphorous acid and peptide thioic acid were used as stock solutions. N-Hydroxypyridone additive, hypophosphorous acid, and amino acid were added to a 1.5 mL microtube. Then, a solution of peptide thioic acid was added, and the mixture was stirred at 30 ° C. for a predetermined reaction time. The reaction mixture was diluted with 1% TFA DMSO solution and analyzed by HPLC. The yield was determined by the calibration curve.
得られた結果（収率、エピメリ度）を以下の表１に示す。
上記で得られたペプチドの特性を調べた結果を以下に記載する。
Cbz-Phe-Val-Gly-OH MS (ESI): m/z 478.15 (calcd [M+Na]+ = 478.50)
Purity: >95% (HPLC analysis at 230 nm) Retention time: 25.10 min
Cbz-Phe-Val-Ala-OH MS (ESI): m/z 470.10 (calcd [M+H]+ = 470.23)
Purity: >95% (HPLC analysis at 230 nm) Retention time: 24.69 min
Cbz-Phe-Val-Val-OH MS (ESI): m/z 520.10 (calcd [M+Na]+ = 520.24)
Purity: >95% (HPLC analysis at 230 nm) Retention time: 26.72 min
Cbz-Phe-Val-Ile-OH MS (ESI): m/z 512.20 (calcd [M+H] + = 512.28)
Purity: >95% (HPLC analysis at 230 nm) Retention time: 28.56 min
Cbz-Phe-Val-Phe-OH MS (ESI): m/z 568.15 (calcd [M+Na]+ = 568.24)
Purity: >95% (HPLC analysis at 230 nm) Retention time: 28.21 min
Cbz-Phe-Val-Met-OH MS (ESI): m/z 552.10 (calcd [M+Na]+ = 552.21)
Purity: >95% (HPLC analysis at 230 nm) Retention time: 27.59 min
Cbz-Phe-Val-Pro-OH MS (ESI): m/z 518.10 (calcd [M+Na]+ = 518.23)
Purity: >95% (HPLC analysis at 230 nm) Retention time: 25.52 min
Cbz-Phe-Val-Trp(Boc)-OH MS (ESI): m/z 707.20 (calcd [M+Na]+ = 707.31)
Purity: >95% (HPLC analysis at 230 nm) Retention time: 33.68 min
Cbz-Phe-Val-Lys(Boc)-OH MS (ESI): m/z 649.25 (calcd [M+Na]+ = 649.32)
Purity: >95% (HPLC analysis at 230 nm) Retention time: 28.58 min
Cbz-Phe-Val-Tyr(OtBu)-OH MS (ESI): m/z 584.40 (calcd [M+Na]+ = 584.24)
Purity: >95% (HPLC analysis at 230 nm) Retention time: 30.46 min
Cbz-Phe-Val-Cys(Trt)-OH MS (ESI): m/z 766.20 (calcd [M+Na]+ = 766.29)
Purity: >95% (HPLC analysis at 230 nm) Retention time: 34.59 min
Cbz-Phe-Val-Asp(tBu)-OH MS (ESI): m/z 592.10 (calcd [M+Na]+ = 592.64)
Purity: >95% (HPLC analysis at 230 nm) Retention time: 28.25 min
Cbz-Phe-Val-Asn(Trt)-OH MS (ESI): m/z 777.25 (calcd [M+Na]+ = 777.87)
Purity: >95% (HPLC analysis at 230 nm) Retention time: 33.25 min
Cbz-Phe-Val-Ser(OtBu)-OH MS (ESI): m/z 564.20 (calcd [M+Na]+ = 564.27)
Purity: >95% (HPLC analysis at 230 nm) Retention time: 29.08 min
Cbz-Phe-Val-Thr(OtBu)-OH MS (ESI): m/z 500.10 (calcd [M+Na] + = 500.24)
Purity: >95% (HPLC analysis at 230 nm) Retention time: 29.47 min
Cbz-Phe-Val-Arg(Pbf)-OH MS (ESI): m/z 807.05 (calcd [M+H]+ = 807.37)
Purity: >95% (HPLC analysis at 230 nm) Retention time: 29.87 min
Cbz-Phe-Val-His(Trt)-OH MS (ESI): m/z 778.20 (calcd [M+H]+ = 778.93)
Purity: >95% (HPLC analysis at 230 nm) Retention time: 30.87 min
Cbz-Phe-Val-tertLeu-OH MS (ESI): m/z 512.10 (calcd [M+H]+ = 512.63)
Purity: >95% (HPLC analysis at 230 nm) Retention time: 28.53 min
Cbz-Phe-Val-Aib-OH MS (ESI): m/z 484.15 (calcd [M+H]+ = 484.57)
Purity: >95% (HPLC analysis at 230 nm) Retention time: 25.31 min
Cbz-Phe-Val-Pg-OH MS (ESI): m/z 532.15 (calcd [M+H]+ = 532.62)
The results of examining the properties of the peptides obtained above are described below.
Cbz-Phe-Val-Gly-OH MS (ESI): m / z 478.15 (calcd [M + Na] + = 478.50)
Purity:> 95% (HPLC analysis at 230 nm) Retention time: 25.10 min
Cbz-Phe-Val-Ala-OH MS (ESI): m / z 470.10 (calcd [M + H] + = 470.23)
Purity:> 95% (HPLC analysis at 230 nm) Retention time: 24.69 min
Cbz-Phe-Val-Val-OH MS (ESI): m / z 520.10 (calcd [M + Na] + = 520.24)
Purity:> 95% (HPLC analysis at 230 nm) Retention time: 26.72 min
Cbz-Phe-Val-Ile-OH MS (ESI): m / z 512.20 (calcd [M + H] + = 512.28)
Purity:> 95% (HPLC analysis at 230 nm) Retention time: 28.56 min
Cbz-Phe-Val-Phe-OH MS (ESI): m / z 568.15 (calcd [M + Na] + = 568.24)
Purity:> 95% (HPLC analysis at 230 nm) Retention time: 28.21 min
Cbz-Phe-Val-Met-OH MS (ESI): m / z 552.10 (calcd [M + Na] + = 552.21)
Purity:> 95% (HPLC analysis at 230 nm) Retention time: 27.59 min
Cbz-Phe-Val-Pro-OH MS (ESI): m / z 518.10 (calcd [M + Na] + = 518.23)
Purity:> 95% (HPLC analysis at 230 nm) Retention time: 25.52 min
Cbz-Phe-Val-Trp (Boc) -OH MS (ESI): m / z 707.20 (calcd [M + Na] + = 707.31)
Purity:> 95% (HPLC analysis at 230 nm) Retention time: 33.68 min
Cbz-Phe-Val-Lys (Boc) -OH MS (ESI): m / z 649.25 (calcd [M + Na] + = 649.32)
Purity:> 95% (HPLC analysis at 230 nm) Retention time: 28.58 min
Cbz-Phe-Val-Tyr (O t Bu) -OH MS (ESI): m / z 584.40 (calcd [M + Na] + = 584.24)
Purity:> 95% (HPLC analysis at 230 nm) Retention time: 30.46 min
Cbz-Phe-Val-Cys (Trt) -OH MS (ESI): m / z 766.20 (calcd [M + Na] + = 766.29)
Purity:> 95% (HPLC analysis at 230 nm) Retention time: 34.59 min
Cbz-Phe-Val-Asp ( t Bu) -OH MS (ESI): m / z 592.10 (calcd [M + Na] + = 592.64)
Purity:> 95% (HPLC analysis at 230 nm) Retention time: 28.25 min
Cbz-Phe-Val-Asn (Trt)-OH MS (ESI): m / z 777.25 (calcd [M + Na] + = 777.87)
Purity:> 95% (HPLC analysis at 230 nm) Retention time: 33.25 min
Cbz-Phe-Val-Ser (O t Bu) -OH MS (ESI): m / z 564.20 (calcd [M + Na] + = 564.27)
Purity:> 95% (HPLC analysis at 230 nm) Retention time: 29.08 min
Cbz-Phe-Val-Thr (O t Bu) -OH MS (ESI): m / z 500.10 (calcd [M + Na] + = 500.24)
Purity:> 95% (HPLC analysis at 230 nm) Retention time: 29.47 min
Cbz-Phe-Val-Arg (Pbf) -OH MS (ESI): m / z 807.05 (calcd [M + H] + = 807.37)
Purity:> 95% (HPLC analysis at 230 nm) Retention time: 29.87 min
Cbz-Phe-Val-His (Trt) -OH MS (ESI): m / z 778.20 (calcd [M + H] + = 778.93)
Purity:> 95% (HPLC analysis at 230 nm) Retention time: 30.87 min
Cbz-Phe-Val-tertLeu-OH MS (ESI): m / z 512.10 (calcd [M + H] + = 512.63)
Purity:> 95% (HPLC analysis at 230 nm) Retention time: 28.53 min
Cbz-Phe-Val-Aib-OH MS (ESI): m / z 484.15 (calcd [M + H] + = 484.57)
Purity:> 95% (HPLC analysis at 230 nm) Retention time: 25.31 min
Cbz-Phe-Val-Pg-OH MS (ESI): m / z 532.15 (calcd [M + H] + = 532.62)
［合成実施例２］
保護基の検討
ペプチド合成にて汎用されるＦｍｏｃ、およびＢｏｃを有する末端Ｖａｌジペプチドを用いて検討を行った。結果を以下に示す。
[Synthesis Example 2]
Examination of protecting groups Fmoc, which is widely used in peptide synthesis, and terminal Val dipeptides having Boc were used for examination. The results are shown below.
上記で得られたペプチドの特性を調べた結果を以下に記載する。
Fmoc-Phe-Val-Ala-OH MS (ESI): m/z 580.15 (calcd [M+Na]+ = 580.64)
Purity: >95% (HPLC analysis at 230 nm) Retention time 29.83 min
Boc-Phe-Val-Ala-OH MS (ESI): m/z 458.15 (calcd [M+Na]+ = 458.51)
Purity: >95% (HPLC analysis at 230 nm) Retention time: 24.62 min
The results of examining the properties of the peptides obtained above are described below.
Fmoc-Phe-Val-Ala-OH MS (ESI): m / z 580.15 (calcd [M + Na] + = 580.64)
Purity:> 95% (HPLC analysis at 230 nm) Retention time 29.83 min
Boc-Phe-Val-Ala-OH MS (ESI): m / z 458.15 (calcd [M + Na] + = 458.51)
Purity:> 95% (HPLC analysis at 230 nm) Retention time: 24.62 min
表２で示されるように、Ｃｂｚを用いた場合と同様に反応は高収率および低エピメリ化率にて進行した。 As shown in Table 2, the reaction proceeded in high yield and low epimerization rate as in the case of using Cbz.
［合成実施例３］
Ｃ末端残基の検討
Ｃ末端残基がＶａｌ以外のジペプチドチオ酸を用いた検討を行った。結果を以下に示す。
[Synthesis Example 3]
Examination of C-terminal residue A study was conducted using a dipeptide thioic acid whose C-terminal residue was other than Val. The results are shown below.
上記で得られたペプチドの特性を調べた結果を以下に記載する。
Cbz-Phe-Phe-Ala-OH MS (ESI): m/z 540.05 (calcd [M+Na]+ = 540.21)
Purity: >95% (HPLC analysis at 230 nm) Retention time: 27.56 min
Cbz-Phe-Phe-Val-OH MS (ESI): m/z 568.10 (calcd [M+Na]+ = 568.24)
Purity: >95% (HPLC analysis at 230 nm) Retention time: 29.28 min
Cbz-Phe-Lys(Boc)-Ala-OH MS (ESI): m/z 621.20 (calcd [M+Na]+ = 621.69)
Purity: >95% (HPLC analysis at 230 nm) Retention time: 27.95 min
Cbz-Phe-Lys(Boc)-Val-OH MS (ESI): m/z 649.25 (calcd [M+Na]+ = 649.74)
Purity: >95% (HPLC analysis at 230 nm) Retention time: 29.53 min
Cbz-Phe-Cys(Trt)-Ala-OH MS (ESI): m/z 738.15 (calcd [M+Na]+ = 738.85)
Purity: >95% (HPLC analysis at 230 nm) Retention time: 34.15 min
Cbz-Phe-Cys(Trt)-Val-OH MS (ESI): m/z 766.10 (calcd [M+Na]+ = 766.91)
Purity: >95% (HPLC analysis at 230 nm) Retention time: 35.33 min
The results of examining the properties of the peptides obtained above are described below.
Cbz-Phe-Phe-Ala-OH MS (ESI): m / z 540.05 (calcd [M + Na] + = 540.21)
Purity:> 95% (HPLC analysis at 230 nm) Retention time: 27.56 min
Cbz-Phe-Phe-Val-OH MS (ESI): m / z 568.10 (calcd [M + Na] + = 568.24)
Purity:> 95% (HPLC analysis at 230 nm) Retention time: 29.28 min
Cbz-Phe-Lys (Boc)-Ala-OH MS (ESI): m / z 621.20 (calcd [M + Na] + = 621.69)
Purity:> 95% (HPLC analysis at 230 nm) Retention time: 27.95 min
Cbz-Phe-Lys (Boc)-Val-OH MS (ESI): m / z 649.25 (calcd [M + Na] + = 649.74)
Purity:> 95% (HPLC analysis at 230 nm) Retention time: 29.53 min
Cbz-Phe-Cys (Trt)-Ala-OH MS (ESI): m / z 738.15 (calcd [M + Na] + = 738.85)
Purity:> 95% (HPLC analysis at 230 nm) Retention time: 34.15 min
Cbz-Phe-Cys (Trt)-Val-OH MS (ESI): m / z 766.10 (calcd [M + Na] + = 766.91)
Purity:> 95% (HPLC analysis at 230 nm) Retention time: 35.33 min
Ｃ末端として、疎水性アミノ酸Ｐｈｅを用いた場合および親水性アミノ酸Ｌｙｓ（Ｂｏｃ）を用いた場合のどちらにおいても反応は高い収率で進行し、またエピメリ化率も１％未満に抑えられていた。一方、エピメリ化が起きやすいとされているＣｙｓ（Ｔｒｔ）を用いた場合では、Ａｌａとのカップリングで４％、バリンとのカップリングで２％程度のエピメリ化が認められた。 The reaction proceeded in high yield in both the case where the hydrophobic amino acid Ph was used and the case where the hydrophilic amino acid Lys (Boc) was used as the C-terminal, and the epimerization rate was suppressed to less than 1%. .. On the other hand, when Cys (Trt), which is considered to be prone to epimerization, was used, epimerization of about 4% was observed in the coupling with Ala and about 2% in the coupling with valine.
［合成実施例４］
添加剤（エピメリ化抑制剤）の検討（１）
Ｃｂｚ-Ｐｈｅ-Ｖａｌ-ＳＨとアラニンとの以下の縮合反応において、添加剤の種類を変更した。その結果を以下にまとめる。
[Synthesis Example 4]
Examination of Additives (Epimerization Inhibitors) (1)
In the following condensation reaction of Cbz-Phe-Val-SH and alanine, the type of additive was changed. The results are summarized below.
メチルエステルの部分をアミド構造にし、塩基性部位であるピリジンを持たせたものは、おそらくは活性エステルの段階で脱プロトン化が促進し、かつ活性エステルの反応性もエステル型より悪いために、収率/エピ化率ともに悪い結果を与えた。塩素置換体はヒドロキシ基の求核力が低く、活性エステル形成が適切な速度で行われないため、収率が低下する傾向にあった。 The methyl ester portion having an amide structure and having pyridine, which is a basic moiety, is likely to be deprotonated at the stage of the active ester, and the reactivity of the active ester is worse than that of the ester type. Both rate / epitonation rate gave bad results. The chlorine substituent has a low nucleophilic force of the hydroxy group, and the active ester formation is not performed at an appropriate rate, so that the yield tends to decrease.
［合成実施例５］
添加剤（エピメリ化抑制剤）の検討（２）
Ｃｂｚ-Ｐｈｅ-Ｖａｌ-ＳＨとアラニンとの以下の縮合反応において、添加剤の種類を変更した。その結果を以下にまとめる。
[Synthesis Example 5]
Examination of Additives (Epimerization Inhibitors) (2)
In the following condensation reaction of Cbz-Phe-Val-SH and alanine, the type of additive was changed. The results are summarized below.
反応時間を２２時間にして、各種添加剤（エピメリ化抑制剤）を用いた場合の結果（収率、エピメリ度）を以下に示す。
一般式(III)において、Ｒ４がエステル基であり、Ｌが水素である化合物５は、化合物１等に比べてエピメリ度が若干高いものの、高い収率で生成物を得ることができた。
In the general formula (III), the compound 5 in which R 4 is an ester group and L is hydrogen was able to obtain a product in a high yield, although the degree of epimery was slightly higher than that of
反応時間を６時間にして、各種添加剤を用いた場合の結果（収率、エピメリ度）を以下に示す。
Ｈ−Ａｌ−ＯＨを２当量に、添加剤を２当量にして、各種添加剤を用いた場合の結果（収率、エピメリ度）を以下に示す。
メチルエステルの部分のアルキル鎖を伸長したものは、収率、エピメリ度とも良好であった。ただし、エステル部分のアルキル鎖の炭素数が６である化合物１１では、結晶化度が高く、生成物２ｂａから分離するのは困難であった。これに対して、アルキル鎖の炭素数が化合物１１よりも大きいが分岐型のアルキル鎖を有する化合物１２、１３では、再結晶することにより、夫々、７６％、７３％の添加剤を回収することができた。 The extended alkyl chain of the methyl ester moiety had good yield and epimery. However, in the compound 11 having 6 carbon atoms in the alkyl chain of the ester portion, the crystallinity was high and it was difficult to separate from the product 2ba. On the other hand, in the compounds 12 and 13 having a branched alkyl chain having a larger carbon number than that of the compound 11, 76% and 73% of the additives can be recovered by recrystallization, respectively. Was made.
［合成実施例６］
ジペプチドチオ酸と種々のアミノ酸との縮合
以下の反応スキ-ムにより、ジペプチドチオ酸と種々のアミノ酸との縮合を行った。
Condensation of dipeptide thioic acid with various amino acids Condensation of dipeptide thioic acid with various amino acids was carried out by the following reaction skim.
試験管にＮ-ヒドロキシピリドン（７８．４ｍｇ、０．１８ｍｍｏｌ）、ジペプチドチオ酸（５０ｍｇ、０．１２ｍｍｏｌ）、Ｌ−バリン（２８．１ｍｇ、０．２４ｍｍｏｌ）を添加した。次いで、トルエン（６００μＬ）、ジメチルスルホキシド（６００μＬ）を添加し、反応溶液を３０℃で６時間撹拌した。反応溶液（１２μＬ）を１％トリフルオロ酢酸のジメチルスルホキシド溶液（６８μＬ）で希釈し、ＨＰＬＣで分析した。収率、エピメリ度は検量線により決定した（収率 <99%、エピメリ度 <1%）。残りの反応溶液の溶媒を減圧除去し、残渣に酢酸エチル及び１ＭＨＣｌ水溶液を添加し、酢酸エチルで抽出した。Ｎａ２ＳＯ４上で乾燥し、濾過し、溶媒を減圧除去して粗生成物トリペプチドを得た。粗生成物をシリカゲルカラムクロマトグラフィ-（酢酸エチル／ヘキサン＝３０：７０ → １００：０）で精製して、トリペプチド（５０ｍｇ、８４％）を得た。 N-Hydroxypyridone (78.4 mg, 0.18 mmol), dipeptide thioic acid (50 mg, 0.12 mmol) and L-valine (28.1 mg, 0.24 mmol) were added to the test tube. Then, toluene (600 μL) and dimethyl sulfoxide (600 μL) were added, and the reaction solution was stirred at 30 ° C. for 6 hours. The reaction solution (12 μL) was diluted with 1% trifluoroacetic acid in dimethyl sulfoxide (68 μL) and analyzed by HPLC. The yield and the degree of epimery were determined by the calibration curve (yield <99%, degree of epimery <1%). The solvent of the remaining reaction solution was removed under reduced pressure, ethyl acetate and 1M HCl aqueous solution were added to the residue, and the mixture was extracted with ethyl acetate. It was dried over Na2SO4, filtered and the solvent removed under reduced pressure to give the crude product tripeptide. The crude product was purified by silica gel column chromatography (ethyl acetate / hexane = 30: 70 → 100: 0) to give a tripeptide (50 mg, 84%).
［合成実施例７］
可溶化剤の検討
Ｃｂｚ−Ｐｈｅ−Ｖａｌ−ＳＨとアラニンとの以下の縮合反応において、可溶化剤の種類を変更した。その結果を以下にまとめる。
Examination of solubilizer In the following condensation reaction of Cbz-Phe-Val-SH and alanine, the type of solubilizer was changed. The results are summarized below.
ジアルキルホスファイトが良好な結果をあたえ、特にジエチルホスファイトを用いた時最も高い反応加速効果が見られた。反応開始から６時間の時点での収率は非添加の５９％から８４％に向上した。エトキシ基からさらに嵩高い官能基を持つジアルキルホスファイトを用いた場合、収率の低減が見られたことから、ホスファイト上の立体が反応性に寄与していることが考えられる。また、アルキル基がリン上に直接導入されているホスファイトを用いた場合にも反応性が低下したことから、立体効果に加え、電子状態も反応性に寄与していることが示唆された。また、ジエチルチオホスファイトを用いた場合には、チオ酸とチオホスファイトが直接反応しているためか系が複雑化し、トリペプチドの収率も３４％まで低下した。 Dialkylphosphite gave good results, and the highest reaction acceleration effect was observed especially when diethylphosphite was used. The yield at 6 hours from the start of the reaction improved from 59% without addition to 84%. When dialkylphosphite having a bulkier functional group than the ethoxy group was used, the yield was reduced, suggesting that the steric structure on the phosphite contributes to the reactivity. In addition, the reactivity was also reduced when phosphite in which the alkyl group was directly introduced onto phosphorus was used, suggesting that the electronic state also contributes to the reactivity in addition to the stereoscopic effect. In addition, when diethylthiophosphite was used, the system was complicated probably because thioacid and thiophosphite reacted directly, and the yield of tripeptide was reduced to 34%.
［合成実施例８］
フラグメントカップリング反応の一般的方法
Ｎ-ヒドロキシピリドン添加剤１（１２μｍｏｌ）、亜りん酸ジエチル（１２μｍｏｌ），ＤＩＰＥＡ（２４μｍｏｌ）及びペプチドチオ酸（１２μミリモル）を、それぞれ原液として用いた。１．５ｍＬマイクロチュ-ブに、ペプチド断片（１４．４μｍｏｌ）を添加した。次いで，Ｎ-ヒドロキシピリドン添加剤１（ＤＭＳＯ中１２μｍｏｌ、１２μＬ）、亜リン酸ジエチル（ＤＭＳＯ中１２μｍｏｌ、１２μＬ）、及びＤＩＰＥＡ（ＤＭＳＯ中１２μｍｏｌ、１２μＬ）の原液をマイクロチュ-ブに添加した。最後に、ペプチドチオ酸（ＤＭＳＯ中１２μｍｏｌ、４８μＬ）の溶液を加え、指示された反応時間の間、３０℃で撹拌した。反応混合物をＤＭＳＯ（６８０μＬ）の１％ ＴＦＡ溶液で希釈し、ＨＰＬＣで分析した。検量線に基づいてＨＰＬＣにより収率を測定した。
[Synthesis Example 8]
General Method of Fragment Coupling Reaction N-hydroxypyridone additive 1 (12 μmol), diethyl phosphite (12 μmol), DIPEA (24 μmol) and peptide thioic acid (12 μmol) were used as stock solutions. A peptide fragment (14.4 μmol) was added to a 1.5 mL microtube. Then, a stock solution of N-hydroxypyridone additive 1 (12 μmol in DMSO, 12 μL), diethyl phosphite (12 μmol in DMSO, 12 μL), and DIPEA (12 μmol in DMSO, 12 μL) was added to the microtube. Finally, a solution of peptide thioic acid (12 μmol in DMSO, 48 μL) was added and stirred at 30 ° C. for the indicated reaction time. The reaction mixture was diluted with a 1% TFA solution of DMSO (680 μL) and analyzed by HPLC. The yield was measured by HPLC based on the calibration curve.
［合成実施例９］
連続ペプチド合成
（１）Ｃｂｚ−Ｔｙｒ（ＯｔＢｕ）−ＯＨ −＞ Ｃｂｚ−Ｔｙｒ（ＯｔＢｕ）−ＳＨ
フレ-ム乾燥した試験管に、Ｃｂｚ−Ｔｙｒ（ＯｔＢｕ）−ＯＨ（７４ｍｇ，０．２ｍｍｏｌ）とＤＭＦ（２ｍＬ）をアルゴン雰囲気下で添加した。次いで、ＡｃＳＫ（１１４ｍｇ、１．０ｍｍｏｌ）とＡｃ２Ｓ（１２μＬ、０．１ミリモル）を溶液に添加した。反応混合物を０℃で５時間攪拌し、混合物を酢酸エチルで抽出し、合わせた有機層を１Ｍ ＨＣｌ水溶液及び食塩水で洗浄し、次いでＮａ２ＳＯ４上で乾燥した。乾燥剤を濾取した後、濾液を減圧下で濃縮した。残渣を凍結乾燥し、さらなる精製なしに次の反応に使用した。
[Synthesis Example 9]
Continuous Peptide Synthesis (1) Cbz-Tyr (O t Bu) -OH -> Cbz-Tyr (O t Bu) -SH
Frame - the arm dry test tube, Cbz-Tyr (O t Bu ) -OH (74mg, 0.2mmol) and DMF and (2 mL) was added under an argon atmosphere. It was then added AcSK (114 mg, 1.0 mmol) and Ac 2 S (12μL, 0.1 mmol) to the solution. The reaction mixture was stirred at 0 ° C. for 5 hours, the mixture was extracted with ethyl acetate and the combined organic layers were washed with 1M aqueous HCl and brine and then dried over Na 2 SO 4. After the desiccant was collected by filtration, the filtrate was concentrated under reduced pressure. The residue was lyophilized and used in the next reaction without further purification.
（２）Ｃｂｚ−Ｔｙｒ（ＯｔＢｕ）−ＳＨ −＞ Ｃｂｚ−Ｔｙｒ（ＯｔＢｕ）−Ｇｌｙ−ＯＨ
試験管に、Ｃｂｚ−Ｔｙｒ（ＯｔＢｕ）−Ｇｌｙ−ＳＨ及びＤＭＳＯ：トルエン＝１：１（２ｍＬ）の混合溶媒液を加えた。次いで、Ｎ-ヒドロキシピリドン添加剤１（３４ｍｇ、０．２ ｍｍｏｌ）、Ｇｌｙ（１８ｍｇ、０．２４ｍｍｏｌ）、亜リン酸ジエチル（２６μＬ）を加え、３０℃で６時間攪拌し、反応混合物を１．５ｍＭに希釈し、分析用ＨＰＬＣで分析したところ、ＨＰＬＣ収率は８９％であった。次いで、シリンジフィルタ-で不溶性物質を除去し、反応混合物を分取ＨＰＬＣで精製した。Ｃｂｚ−Ｔｙｒ（ＯｔＢｕ）−Ｇｌｙ−ＯＨを６６ｍｇ（７７％）得た。
Ｃｂｚ−Ｔｙｒ（ＯｔＢｕ）−Ｇｌｙ−ＯＨの特性を調べた結果は以下の通りである。
MS (ESI): m/z 451.15 (calcd [M+Na]+ = 451.47)
Purity: >95% (HPLC analysis at 230 nm) Retention time: 26.87 min
(2) Cbz-Tyr (O t Bu) -SH -> Cbz-Tyr (O t Bu) -Gly-OH
In a test tube, Cbz-Tyr (O t Bu ) -Gly-SH and DMSO: toluene = 1: plus 1 mixed solvent solution (2 mL). Next, N-hydroxypyridone additive 1 (34 mg, 0.2 mmol), Gly (18 mg, 0.24 mmol), and diethyl phosphite (26 μL) were added, and the mixture was stirred at 30 ° C. for 6 hours to prepare the reaction mixture. When diluted to 5 mM and analyzed by analytical HPLC, the HPLC yield was 89%. The insoluble material was then removed with a syringe filter and the reaction mixture was purified by preparative HPLC. Cbz-Tyr the (O t Bu) -Gly-OH 66mg (77%) was obtained.
Cbz-Tyr (O t Bu) -Gly-OH results properties were examined in are as follows.
MS (ESI): m / z 451.15 (calcd [M + Na] + = 451.47)
Purity:> 95% (HPLC analysis at 230 nm) Retention time: 26.87 min
（３）Ｃｂｚ-Ｔｙｒ（ＯｔＢｕ）-Ｇｌｙ-ＯＨ -＞ Ｃｂｚ-Ｔｙｒ（ＯｔＢｕ）-Ｇｌｙ-ＳＨ
フレ-ム乾燥した試験管に、Ｃｂｚ−Ｔｙｒ（ＯｔＢｕ）−Ｇｌｙ−ＯＨ（８５ｍｇ、０．２ｍｍｏｌ）及びＤＭＦ（２ｍＬ）をアルゴン雰囲気下で添加した。次いで、ＡｃＳＫ（１１４ｍｇ、１．０ｍｍｏｌ）とＡｃ２Ｓ（１２μＬ、０．１ミリモル）を溶液に添加した。反応混合物を０℃で５時間攪拌し、混合物を酢酸エチルで抽出し、合わせた有機層を１Ｍ ＨＣｌ水溶液及び食塩水で洗浄し、次いでＮａ２ＳＯ４上で乾燥した。乾燥剤を濾取した後、濾液を還元圧力下で濃縮した。残渣を凍結乾燥し、さらなる精製なしに次の反応に使用した。
(3) Cbz-Tyr (O t Bu) -Gly-OH -> Cbz-Tyr (O t Bu) -Gly-SH
Frame - the arm dry test tube, Cbz-Tyr (O t Bu ) -Gly-OH (85mg, 0.2mmol) and DMF (2 mL) was added under an argon atmosphere. It was then added AcSK (114 mg, 1.0 mmol) and Ac 2 S (12μL, 0.1 mmol) to the solution. The reaction mixture was stirred at 0 ° C. for 5 hours, the mixture was extracted with ethyl acetate and the combined organic layers were washed with 1M aqueous HCl and brine and then dried over Na 2 SO 4. After the desiccant was collected by filtration, the filtrate was concentrated under reducing pressure. The residue was lyophilized and used in the next reaction without further purification.
（４）Ｃｂｚ−Ｔｙｒ（ＯｔＢｕ）−Ｇｌｙ−ＳＨ −＞ Ｃｂｚ−Ｔｙｒ（ＯｔＢｕ）−Ｇｌｙ−Ｇｌｙ−ＯＨ
試験管に、Ｃｂｚ−Ｔｙｒ（ＯｔＢｕ）−Ｇｌｙ−ＳＨ及びＤＭＳＯ：トルエン＝１：１（２ｍＬ）の混合溶媒液を加えた。次いで、Ｎ-ヒドロキシピリドン添加剤１（３４ｍｇ、０．２ ｍｍｏｌ）、Ｇｌｙ（１８ｍｇ、０．２４ｍｍｏｌ）、亜リン酸ジエチル（２６μＬ）を加え、３０℃で６時間攪拌し、反応混合物を１．５ｍＭに希釈し、分析用ＨＰＬＣで分析したところ、ＨＰＬＣ収率は９４％であった。次いで、シリンジフィルタ-で不溶性物質を除去し、反応混合物を分取ＨＰＬＣで精製した。Ｃｂｚ−Ｔｙｒ（ＯｔＢｕ）−Ｇｌｙ−Ｇｌｙ−ＯＨを７１ｍｇ（７３％）得た。
Ｃｂｚ−Ｔｙｒ（ＯｔＢｕ）−Ｇｌｙ−Ｇｌｙ−ＯＨの特性を調べた結果は以下の通りである。
MS (ESI): m/z 508.15 (calcd [M+Na]+ = 508.21)
Purity: >95% (HPLC analysis at 230 nm) Retention time: 26.87 min
(4) Cbz-Tyr (O t Bu) -Gly-SH -> Cbz-Tyr (O t Bu) -Gly-Gly-OH
In a test tube, Cbz-Tyr (O t Bu ) -Gly-SH and DMSO: toluene = 1: plus 1 mixed solvent solution (2 mL). Next, N-hydroxypyridone additive 1 (34 mg, 0.2 mmol), Gly (18 mg, 0.24 mmol), and diethyl phosphite (26 μL) were added, and the mixture was stirred at 30 ° C. for 6 hours to prepare the reaction mixture. When diluted to 5 mM and analyzed by analytical HPLC, the HPLC yield was 94%. The insoluble material was then removed with a syringe filter and the reaction mixture was purified by preparative HPLC. Cbz-Tyr the (O t Bu) -Gly-Gly -OH 71mg (73%) was obtained.
Cbz-Tyr (O t Bu) -Gly-Gly-OH results properties were examined in are as follows.
MS (ESI): m / z 508.15 (calcd [M + Na] + = 508.21)
Purity:> 95% (HPLC analysis at 230 nm) Retention time: 26.87 min
（５）Ｃｂｚ−Ｔｙｒ（ＯｔＢｕ）−Ｇｌｙ−Ｇｌｙ−ＯＨ −＞ Ｃｂｚ−Ｔｙｒ（ＯｔＢｕ）−Ｇｌｙ−Ｇｌｙ−ＳＨ
フレ−ム乾燥試験管に、Ｃｂｚ−Ｔｙｒ（ＯｔＢｕ）−Ｇｌｙ−Ｇｌｙ−ＯＨ（４９ｍｇ、０．１ｍｍｏｌ）及びＤＭＦ（１ｍＬ）をアルゴン雰囲気下で添加した。次いで、ＡｃＳＫ（３４ｍｇ、０．３ｍｍｏｌ）とＡｃ２Ｓ（２．１μＬ、０．０２ミリモル）を溶液に添加した。反応混合物を０℃で３時間攪拌し、混合物を酢酸エチルで抽出し、合わせた有機層を１Ｍ ＨＣｌ水溶液及び食塩水で洗浄し、次いでＮａ２ＳＯ４上で乾燥した。乾燥剤を濾取した後、濾液を減圧下で濃縮した。残渣を凍結乾燥し、さらなる精製なしに次の反応に使用した。
(5) Cbz-Tyr (O t Bu) -Gly-Gly-OH -> Cbz-Tyr (O t Bu) -Gly-Gly-SH
Frame - the arm dry test tube, Cbz-Tyr (O t Bu ) -Gly-Gly-OH (49mg, 0.1mmol) and DMF (1 mL) was added under an argon atmosphere. AcSK (34 mg, 0.3 mmol) and Ac 2 S (2.1 μL, 0.02 mmol) were then added to the solution. The reaction mixture was stirred at 0 ° C. for 3 hours, the mixture was extracted with ethyl acetate and the combined organic layers were washed with 1M aqueous HCl and brine and then dried over Na 2 SO 4. After the desiccant was collected by filtration, the filtrate was concentrated under reduced pressure. The residue was lyophilized and used in the next reaction without further purification.
（６）Ｃｂｚ−Ｔｙｒ（ＯｔＢｕ）−Ｇｌｙ−Ｇｌｙ−ＳＨ −＞ Ｃｂｚ−Ｔｙｒ（ＯｔＢｕ）−Ｇｌｙ−Ｇｌｙ−Ｐｈｅ−ＯＨ
試験管に、Ｃｂｚ−Ｔｙｒ（ＯｔＢｕ）−Ｇｌｙ−Ｇｌｙ−ＳＨ及びＤＭＳＯ：トルエン＝１：１（１ｍＬ）の混合溶媒液を加えた。次いで、Ｎ-ヒドロキシピリドン添加剤１（１７ｍｇ、０．１ ｍｍｏｌ）、Ｐｈｅ（２０ｍｇ、０．１２ ｍｍｏｌ）、亜リン酸ジエチル（１３μＬ）を加え、３０℃で６時間攪拌し、反応混合物を１．５ ｍＭに希釈し、分析用ＨＰＬＣで分析したところ、ＨＰＬＣ収率は８７％であった。次いで、シリンジフィルタ-で不溶性物質を除去し、反応混合物を分取ＨＰＬＣで精製した。Ｃｂｚ−Ｔｙｒ（ＯｔＢｕ）−Ｇｌｙ−Ｇｌｙ−Ｐｈｅ−ＯＨを４０ｍｇ（６３％）た。
Ｃｂｚ−Ｔｙｒ（ＯｔＢｕ）−Ｇｌｙ−Ｇｌｙ−Ｐｈｅ−ＯＨの特性を調べた結果は以下の通りである。
MS (ESI): m/z 655.25 (calcd [M+Na]+ = 655.27)
Purity: >95% (HPLC analysis at 230 nm) Retention time: 28.39 min
(6) Cbz-Tyr (O t Bu) -Gly-Gly-SH -> Cbz-Tyr (O t Bu) -Gly-Gly-Phe-OH
In a test tube, Cbz-Tyr (O t Bu ) -Gly-Gly-SH and DMSO: toluene = 1: plus 1 mixed solvent solution (1 mL). Next, N-hydroxypyridone additive 1 (17 mg, 0.1 mmol), Ph (20 mg, 0.12 mmol), and diethyl phosphite (13 μL) were added, and the mixture was stirred at 30 ° C. for 6 hours to add 1 reaction mixture. When diluted to 5.5 mM and analyzed by analytical HPLC, the HPLC yield was 87%. The insoluble material was then removed with a syringe filter and the reaction mixture was purified by preparative HPLC. Cbz-Tyr the (O t Bu) -Gly-Gly -Phe-OH was 40mg (63%).
Cbz-Tyr (O t Bu) -Gly-Gly-Phe-OH results properties were examined in are as follows.
MS (ESI): m / z 655.25 (calcd [M + Na] + = 655.27)
Purity:> 95% (HPLC analysis at 230 nm) Retention time: 28.39 min
（７）Ｃｂｚ−Ｔｙｒ（ＯｔＢｕ）−Ｇｌｙ−Ｇｌｙ−Ｐｈｅ−ＯＨ −＞ Ｃｂｚ−Ｔｙｒ（ＯｔＢｕ）−Ｇｌｙ−Ｇｌｙ−Ｐｈｅ−ＳＨ
フレ−ム乾燥試験管に、Ｃｂｚ−Ｔｙｒ（ＯｔＢｕ）−Ｇｌｙ−Ｇｌｙ−Ｐｈｅ−ＯＨ（１３ｍｇ、２０μｍｏｌ）及びＤＭＦ（２００μＬ）をアルゴン雰囲気下で添加した。次いで，ＡｃＳＫ（１１ｍｇ、１００μｍｏｌ）とＡｃ２Ｓ（１μＬ、１０μｍｏｌ）を溶液に添加した。反応混合物を０℃で５時間攪拌し、混合物を酢酸エチルで抽出し、合わせた有機層を１Ｍ ＨＣｌ水溶液及び食塩水で洗浄し、次いでＮａ２ＳＯ４上で乾燥した。乾燥剤を濾取した後、濾液を減圧下で濃縮した。残渣を凍結乾燥し、さらなる精製なしに次の反応に使用した。
(7) Cbz-Tyr (O t Bu) -Gly-Gly-Phe-OH -> Cbz-Tyr (O t Bu) -Gly-Gly-Phe-SH
Frame - the arm drying tube and Cbz-Tyr (O t Bu) -Gly-Gly-Phe-OH (13mg, 20μmol) and DMF (200 [mu] L) was added under an argon atmosphere. Then, AcSK (11 mg, 100 μmol) and Ac 2 S (1 μL, 10 μmol) were added to the solution. The reaction mixture was stirred at 0 ° C. for 5 hours, the mixture was extracted with ethyl acetate and the combined organic layers were washed with 1M aqueous HCl and brine and then dried over Na 2 SO 4. After the desiccant was collected by filtration, the filtrate was concentrated under reduced pressure. The residue was lyophilized and used in the next reaction without further purification.
（８）Ｃｂｚ−Ｔｙｒ（ＯｔＢｕ）−Ｇｌｙ−Ｇｌｙ−Ｐｈｅ−ＳＨ −＞ Ｃｂｚ−Ｔｙｒ（ＯｔＢｕ）−Ｇｌｙ−Ｇｌｙ−Ｐｈｅ−Ｌｅｕ−ＯＨ
試験管に、Ｃｂｚ−Ｔｙｒ（ＯｔＢｕ）−Ｇｌｙ−Ｇｌｙ−Ｐｈｅ−ＳＨ及びＤＭＳＯ：トルエン＝１：１（２００μＬ）の混合溶媒液を加えた。次いで、Ｎ−ヒドロキシピリドン添加剤１（４ｍｇ、２０μｍｏｌ）、Ｌｅｕ（４ｍｇ、２４μｍｏｌ）、亜リン酸ジエチル（１３μＬ）を加え、３０℃で６時間攪拌し、反応混合物を１．５ ｍＭに希釈し、分析用ＨＰＬＣで分析したところ、ＨＰＬＣ収率は５４％であった。次いで、シリンジフィルタ−で不溶性物質を除去し、反応混合物を分取ＨＰＬＣで精製した。Ｃｂｚ−Ｔｙｒ（ＯｔＢｕ）−Ｇｌｙ−Ｇｌｙ−Ｐｈｅ−Ｌｅｕ−ＯＨを７ｍｇ（４７％）得た。
Ｃｂｚ-Ｔｙｒ（ＯｔＢｕ）-Ｇｌｙ-Ｇｌｙ-Ｐｈｅ-ＬｅｕＯＨの特性を調べた結果は以下の通りである。
MS (ESI): m/z 768.25 (calcd [M+Na]+ = 768.36)
Purity: >95% (HPLC analysis at 230 nm) Retention time: 30.00 min
(8) Cbz-Tyr (O t Bu) -Gly-Gly-Phe-SH -> Cbz-Tyr (O t Bu) -Gly-Gly-Phe-Leu-OH
In a test tube, Cbz-Tyr (O t Bu ) -Gly-Gly-Phe-SH and DMSO: toluene = 1: plus 1 mixed solvent solution (200 [mu] L). Next, N-hydroxypyridone additive 1 (4 mg, 20 μmol), Leu (4 mg, 24 μmol), and diethyl phosphite (13 μL) were added, and the mixture was stirred at 30 ° C. for 6 hours to dilute the reaction mixture to 1.5 mM. As a result of analysis by HPLC for analysis, the HPLC yield was 54%. The insoluble material was then removed with a syringe filter and the reaction mixture was purified by preparative HPLC. Cbz-Tyr the (O t Bu) -Gly-Gly -Phe-Leu-OH was obtained 7mg (47%).
Cbz-Tyr (O t Bu) -Gly-Gly-Phe-LeuOH result characteristics were examined in are as follows.
MS (ESI): m / z 768.25 (calcd [M + Na] + = 768.36)
Purity:> 95% (HPLC analysis at 230 nm) Retention time: 30.00 min
Claims (6)
保護基を表すか、Ｎ末端が保護基で保護されたアミノ酸又はペプチドを表し、
Ｒ１は、α−アミノ酸の側鎖であり、当該側鎖は保護基で保護されていてもよい。）
Ｌは、エステル基（−ＣＯ２Ｒ；Ｒは炭素数１〜３０のアルキル基）、置換又は無置換のアリ−ル基、置換又は無置換のベンゾイル基、アミノ基で置換されていてもよいアミド基（−ＣＯＮＲ’Ｒ’’；Ｒ’、Ｒ’’は、各々独立に、水素原子又は炭素数１〜４の置換又は無置換のアルキル基であり、Ｒ’及びＲ’’の少なくとも一方はアミノ基で置換されているアルキル基である）、及びアシル基（−ＣＯＲａ’； Ｒａ’は炭素数１〜４のアルキル基）からなる群から選択され、但し、Ｒ３及びＲ４の少なくとも１つがエステル基（−ＣＯ２Ｒａ；Ｒａは炭素数１〜４のアルキル基）である場合は、Ｌは水素（Ｈ）であってもよく、
Ｒ３及びＲ４は、各々独立に、水素、炭素数１〜４のアルキル基、ハロゲン又はエステル基（−ＣＯ２Ｒａ；Ｒａは炭素数１〜４のアルキル基）を表す。）
Ｒ５及びＲ６は一緒になってＲ５及びＲ６が結合しているリン原子を含む４〜７員の置換又は無置換のヘテロシクリルを形成してもよい。）
Represents a protecting group or represents an amino acid or peptide with the N-terminus protected by a protecting group.
R 1 is a side chain of α-amino acid, and the side chain may be protected by a protecting group. )
L may be substituted with an ester group (-CO 2 R; R is an alkyl group having 1 to 30 carbon atoms), a substituted or unsubstituted allyl group, a substituted or unsubstituted benzoyl group, or an amino group. The amide group (-CONR'R ";R',R" is an independently substituted or unsubstituted alkyl group having a hydrogen atom or 1 to 4 carbon atoms, and at least one of R'and R ". Is an alkyl group substituted with an amino group), and selected from the group consisting of an acyl group (-COR a '; Ra ' is an alkyl group having 1 to 4 carbon atoms), provided that R 3 and R 4 are used. When at least one of is an ester group (-CO 2 Ra ; Ra is an alkyl group having 1 to 4 carbon atoms), L may be hydrogen (H).
R 3 and R 4 independently represent hydrogen, an alkyl group having 1 to 4 carbon atoms, a halogen or an ester group (-CO 2 Ra ; Ra is an alkyl group having 1 to 4 carbon atoms). )
R 5 and R 6 may be combined to form a 4- to 7-membered substituted or unsubstituted heterocyclyl containing a phosphorus atom to which R 5 and R 6 are attached. )
（ＰＧは、Ｎ末端の保護基を表し、
ＡＡは、任意のアミノ酸残基を表し、各出現において同一又は異なっていてもよく、アミノ酸残基の一部又は全ての側鎖は保護基で保護されていてもよく、
ｎは、１〜４の整数である。）
で表される化合物と、
Ｈ−（ＡＡ’）ｍ−ＯＨ：
（ＡＡ’は、任意のアミノ酸残基を表し、各出現において同一又は異なっていてもよく、アミノ酸残基の一部又は全ての側鎖は保護基で保護されていてもよく、
ｍは、１〜４の整数である。）
で表されるアミノ酸又はペプチドを、
以下の式（III）で表される化合物及び以下の式（IV）で表される化合物の存在下で反応させることにより、ＰＧ−（ＡＡ）ｎ（ＡＡ’）ｍ−ＯＨで表される化合物を調製する方法。
Ｌは、エステル基（−ＣＯ２Ｒ；Ｒは炭素数１〜３０のアルキル基）、置換又は無置換のアリ−ル基、置換又は無置換のベンゾイル基、アミノ基で置換されていてもよいアミド基（−ＣＯＮＲ’Ｒ’’；Ｒ’、Ｒ’’は、各々独立に、水素原子又は炭素数１〜４の置換又は無置換のアルキル基であり、Ｒ’及びＲ’’の少なくとも一方はアミノ基で置換されているアルキル基である）、及びアシル基（−ＣＯＲａ’； Ｒａ’は炭素数１〜４のアルキル基）からなる群から選択され、但し、Ｒ３及びＲ４の少なくとも１つがエステル基（−ＣＯ２Ｒａ；Ｒａは炭素数１〜４のアルキル基）である場合は、Ｌは水素（Ｈ）であってもよく、
Ｒ３及びＲ４は、各々独立に、炭素数１〜４のアルキル基、又はハロゲン又はエステル基（−ＣＯ２Ｒａ；Ｒａは炭素数１〜４のアルキル基）を表す。）
Ｒ５及びＲ６は一緒になってＲ５及びＲ６が結合しているリン原子を含む４〜７員の置換又は無置換のヘテロシクリルを形成してもよい。） PG- (AA) n- SH:
(PG represents an N-terminal protecting group,
AA represents any amino acid residue, which may be the same or different at each appearance, and some or all side chains of the amino acid residue may be protected by a protecting group.
n is an integer of 1 to 4. )
And the compound represented by
H- (AA') m- OH:
(AA'represents any amino acid residue, which may be the same or different at each appearance, and some or all side chains of the amino acid residue may be protected by a protecting group.
m is an integer of 1 to 4. )
Amino acids or peptides represented by
A compound represented by PG- (AA) n (AA') m- OH by reacting in the presence of a compound represented by the following formula (III) and a compound represented by the following formula (IV). How to prepare.
L may be substituted with an ester group (-CO 2 R; R is an alkyl group having 1 to 30 carbon atoms), a substituted or unsubstituted allyl group, a substituted or unsubstituted benzoyl group, or an amino group. The amide group (-CONR'R ";R',R" is an independently substituted or unsubstituted alkyl group having a hydrogen atom or 1 to 4 carbon atoms, and at least one of R'and R ". Is an alkyl group substituted with an amino group), and selected from the group consisting of an acyl group (-COR a '; Ra ' is an alkyl group having 1 to 4 carbon atoms), provided that R 3 and R 4 are used. When at least one of is an ester group (-CO 2 Ra ; Ra is an alkyl group having 1 to 4 carbon atoms), L may be hydrogen (H).
R 3 and R 4 each independently represent an alkyl group having 1 to 4 carbon atoms, or a halogen or ester group (-CO 2 Ra ; Ra is an alkyl group having 1 to 4 carbon atoms). )
R 5 and R 6 may be combined to form a 4- to 7-membered substituted or unsubstituted heterocyclyl containing a phosphorus atom to which R 5 and R 6 are attached. )
（ｉ）以下の式（V）で表される化合物を、以下の式（１）の化合物と反応させることにより、式（VII）で表される化合物を調製する工程：
を含む、式（VIIII）の化合物を調製する方法。
Ｌは、エステル基（−ＣＯ２Ｒ；Ｒは炭素数１〜３０のアルキル基）、置換又は無置換のアリ−ル基、置換又は無置換のベンゾイル基、アミノ基で置換されていてもよいアミド基（−ＣＯＮＲ’Ｒ’’；Ｒ’、Ｒ’’は、各々独立に、水素原子又は炭素数１〜４の置換又は無置換のアルキル基であり、Ｒ’及びＲ’’の少なくとも一方はアミノ基で置換されているアルキル基である）、及びアシル基（−ＣＯＲａ’； Ｒａ’は炭素数１〜４のアルキル基）からなる群から選択され、但し、Ｒ３及びＲ４の少なくとも１つがエステル基（−ＣＯ２Ｒａ；Ｒａは炭素数１〜４のアルキル基）である場合は、Ｌは水素（Ｈ）であってもよく、
Ｒ３及びＲ４は、各々独立に、炭素数１〜４のアルキル基、ハロゲン又はエステル基（−ＣＯ２Ｒａ；Ｒａは炭素数１〜４のアルキル基）を表す。）
Ｒ５及びＲ６は一緒になってＲ５及びＲ６が結合しているリン原子を含む４〜７員の置換又は無置換のヘテロシクリルを形成してもよい。）
(I) A step of preparing a compound represented by the formula (VII) by reacting the compound represented by the following formula (V) with the compound represented by the following formula (1):
A method for preparing a compound of formula (VIIII), comprising:
L may be substituted with an ester group (-CO 2 R; R is an alkyl group having 1 to 30 carbon atoms), a substituted or unsubstituted allyl group, a substituted or unsubstituted benzoyl group, or an amino group. The amide group (-CONR'R ";R',R" is an independently substituted or unsubstituted alkyl group having a hydrogen atom or 1 to 4 carbon atoms, and at least one of R'and R ". Is an alkyl group substituted with an amino group), and selected from the group consisting of an acyl group (-COR a '; Ra ' is an alkyl group having 1 to 4 carbon atoms), provided that R 3 and R 4 are used. When at least one of is an ester group (-CO 2 Ra ; Ra is an alkyl group having 1 to 4 carbon atoms), L may be hydrogen (H).
R 3 and R 4 independently represent an alkyl group having 1 to 4 carbon atoms, a halogen or an ester group (-CO 2 Ra ; Ra is an alkyl group having 1 to 4 carbon atoms). )
R 5 and R 6 may be combined to form a 4- to 7-membered substituted or unsubstituted heterocyclyl containing a phosphorus atom to which R 5 and R 6 are attached. )
Ｒ５及びＲ６は一緒になってＲ５及びＲ６が結合しているリン原子を含む４〜７員の置換又は無置換のヘテロシクリルを形成してもよい。） A method of using a compound represented by the following formula (IV) as a solubilizer in peptide synthesis.
R 5 and R 6 may be combined to form a 4- to 7-membered substituted or unsubstituted heterocyclyl containing a phosphorus atom to which R 5 and R 6 are attached. )
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| CN115140763B (en) * | 2022-08-04 | 2023-05-30 | 先导薄膜材料有限公司 | Method for removing impurities in ITO (indium tin oxide) tower cleaning powder |
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