JP2009244252A - Matrix having novel thiazole derivative immobilized thereon, and production method of same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a matrix having a lower-molecular-weight compound immobilized thereon for analysis or purification of a protein. <P>SOLUTION: The matrix having thiazole derivative immobilized thereon is useful for the analysis or purification of the protein, and is represented by general formula (1). In the formula, R<SP>1</SP>and R<SP>2</SP>represent a hydrogen atom or an optionally substituted alkyl group having the carbon number 1-6, Ar represents an optionally substituted aromatic group, n represents an integer of 1 to 12, and M represents a matrix. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a matrix to which a thiazole derivative that can be used for analysis or purification of proteins, particularly immunoglobulins, and a method for producing the same.

  In recent years, the demand for medical proteins has been expanding, and establishment of an industrial purification technique that can be carried out simply and on a large scale is eagerly desired. In general, protein purification techniques include chromatographic separation techniques such as gel permeation chromatography, ion exchange chromatography, hydrophobic interaction chromatography, reverse phase chromatography, and affinity chromatography. In the case of practical implementation, the use of a separating agent or apparatus used in a laboratory is not economical and is not practical. In addition, since many kinds of proteins are mixed in the process of industrially producing medical proteins, it is extremely difficult to obtain the target protein purely by using one of the above purification techniques. For this reason, a purification process in which several techniques are combined is generally employed (Patent Document 1).

  In recent years, many low molecular weight compounds for purifying immunoglobulin, which is one of medical proteins, and matrices to which they are bound have been disclosed, including sulfone derivatives (Patent Document 2), triazine derivatives (Patent Document 3), mercapto. A matrix in which a low molecular weight compound such as a heterocyclic compound (Patent Document 4) or a 4-pyridylethylthioalkyl derivative (Non-Patent Document 1) is bound is known. However, there is no report that the thiazole derivative (1a) used for the production of the matrix of the present invention is useful as a low molecular weight compound for purifying immunoglobulin. Moreover, although the thiazole derivative which has a chemical structure similar to the thiazole derivative (1a) used for manufacture of the matrix of this invention is reported in patent document 5 and nonpatent literature 2, the chemistry of the thiazole derivative described in these documents The structure is limited to the amino group or the alkyl group at the 2-position of the thiazole ring, and the description of the compound in which the benzoylamino group is substituted at the 2-position as in the thiazole derivative (1a) used for the production of the matrix of the present invention is as follows. Absent. In particular, the compound described in Non-Patent Document 2 is a thiazole derivative similar to the thiazole derivative (1a) used in the production of the matrix of the present invention, but their use is limited to use as a histamine H2 receptor agonist, There is no description about the use for purifying immunoglobulin.

  Patent Document 6 describes a thiazole derivative in which a benzoylamino group is substituted at the 2-position of the thiazole ring, but the substituent at the 4-position of the thiazole ring of these compounds is carbonyl, and the 5-position is unsubstituted. It is limited, and as its use, mention is made of an active ingredient of a medicament having an action for improving gastrointestinal motility. Furthermore, the compound of Patent Document 6 is not immobilized on a matrix, and there is no description about the use for purifying immunoglobulin.

WO2004 / 087661 US Pat. No. 4,696,980 US Pat. No. 6,117,996 Japanese Patent No. 384496 WO1991 / 10656 WO1998 / 17654 WO 1989/10360

Bioseparation, 9 (4), 211-221, 2000 Journal of Medicinal Chemistry, 35 (17), 3239-3246, 1992 First Ligand Coupling Handbook, published by Amersham Biosciences, 2005

  The problem to be solved by the present invention is to provide a matrix on which a thiazole derivative useful for analysis or purification of proteins, particularly immunoglobulins, is immobilized, and a method for producing them.

  As a result of intensive studies to solve the above-mentioned problems, the present inventor has found that the thiazole derivative-immobilized matrix represented by the following general formula (1) of the present invention can easily analyze or purify proteins, particularly immunoglobulins. The present invention has been completed.

  The present invention relates to a general formula (1)

（式中、Ｒ^１及びＲ^２は水素原子又は置換されていてもよい炭素数１から６のアルキル基を表し、Ａｒは置換されていてもよい芳香族基を表し、ｎは１から１２の整数を表し、Ｍはマトリックスを表す。）で表されるチアゾール誘導体固定化マトリックスに関するものである。

Wherein R ¹ and R ² represent a hydrogen atom or an optionally substituted alkyl group having 1 to 6 carbon atoms, Ar represents an optionally substituted aromatic group, and n represents 1 to 12 It represents an integer, and M represents a matrix.)

  The present invention also provides a general formula (1)

（式中、Ｒ^１、Ｒ^２、Ａｒ、ｎ及びＭは前記と同じ意味を表す。）においてＭのマトリックスがビニルポリマー、アガロース、キトサン、デキストラン、セルロース、シリカ、ポリスチレンのいずれかであることを特徴とする、チアゾール誘導体固定化マトリックスに関するものである。

(Wherein R ¹ , R ² , Ar, n and M represent the same meaning as described above), the matrix of M is any of vinyl polymer, agarose, chitosan, dextran, cellulose, silica and polystyrene. The present invention relates to a thiazole derivative-immobilized matrix.

  The present invention also provides a compound represented by the general formula (1a)

（式中、Ｒ^１、Ｒ^２、Ａｒ及びｎは前記と同じ意味を表し、Ｒ^３はアミノ基又はアンモニウム塩を表す。）で示されるチアゾール誘導体と、活性化基含有マトリックスとを反応させることを特徴とする、一般式（１）

(Wherein R ¹ , R ² , Ar and n represent the same meaning as described above, and R ³ represents an amino group or an ammonium salt) and an activated group-containing matrix is reacted. The general formula (1)

（式中、Ｒ^１、Ｒ^２、Ａｒ、ｎ及びＭは前記と同じ意味を表す。）で示されるチアゾール誘導体固定化マトリックスの製造方法に関するものである。

(Wherein R ¹ , R ² , Ar, n and M represent the same meanings as described above).

  The present invention also provides a general formula (1)

（式中、Ｒ^１、Ｒ^２、Ａｒ、ｎ及びＭは前記と同じ意味を表す。）で示されるチアゾール誘導体固定化マトリックスを使用し、タンパク質の分析または精製を行なう方法に関するものである。

(Wherein R ¹ , R ² , Ar, n and M represent the same meaning as described above), and relates to a method for analyzing or purifying proteins using a thiazole derivative-immobilized matrix.

  Hereinafter, the present invention will be described in more detail.

The thiazole derivative-immobilized matrix (1) of the present invention and the substituent represented by R ¹ , R ² and Ar, and the ammonium salt represented by R ³ in the thiazole derivative (1a) used for the production of the immobilized matrix The definition of (Z ⁻ H ₃ ⁺ N) is shown below.

The alkyl group having 1 to 6 carbon atoms represented by R ¹ and R ² may be linear or branched, and includes a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, Isobutyl group, sec-butyl group, tert-butyl group, pentyl group, isoamyl group, neopentyl group, 2-pentyl group, 3-pentyl group, tert-pentyl group, hexyl group, isohexyl group, 2-hexyl group, 3- A hexyl group can be exemplified. Further, these alkyl groups having 1 to 6 carbon atoms may be substituted with a halogen atom or the like, and more specifically, a difluoromethyl group, a trifluoromethyl group, a 2,2,2-trifluoroethyl group, a per Examples thereof include a fluorobutyl group, a hydroxymethyl group, a carboxymethyl group, a 4-morpholinocarbonylmethyl group, and the like. In particular, as a thiazole derivative used in the production of the immobilized matrix of the present invention, R ¹ is preferably a methyl group and R ² is preferably a hydrogen atom in view of good performance in immunoglobulin analysis or purification.

Ammonium salt represented by R ³ is _{^{Z - H 3 + N (Z}} - represents a conjugate base.) Is represented by, specifically _{^{Cl - H 3 + N, Br}} - H 3 + N, F - H _{^{3 + N, I - H 3}} + N, NO 3 - H 3 + N, 1/2 (SO 4 2-) H 3 + N, 1/3 (PO 4 3-) H 3 + N, CH 3 SO _{^{3 - H 3 + N, 4}} -CH 3 C 6 H 4 SO 3 - H 3 + N, CH 3 COO - H 3 + N, CF 3 COO - Although _H ^{3 +} N, etc. can be exemplified, matrix in terms of excellent reactivity and operability of immobilization to, ^Cl - _H ^{3 +} N or ^Br - _H ^{3 +} N are preferred.

  The aromatic group represented by Ar is not particularly limited, but furan-2-yl group, furyl group, thienyl group, thiophen-3-yl group, pyrrol-2-yl group, pyrrol-3-yl group, pyrazole -3-yl group, pyrazol-4-yl group, isothiazol-3-yl group, isothiazol-4-yl group, isoxazol-3-yl group, isoxazol-4-yl group, imidazol-2-yl group, Imidazol-4-yl group, imidazol-5-yl group, oxazol-2-yl group, oxazol-4-yl group, oxazol-5-yl group, thiazol-2-yl group, thiazol-4-yl group, thiazole -5-yl group, 1,2,4-triazol-3-yl group, phenyl group, 2-pyridyl group, 3-pyridyl group, 4-pyridyl group, 2-pyrimidyl group, 4-pyrimid Group, 5-pyrimidyl group, 1,2,4-triazin-3-yl group, 1,2,4-triazin-5-yl group, 1,2,4-triazin-6-yl group, benzodioxolane- 3-yl group, benzofuran-2-yl group, benzofuran-3-yl group, benzofuran-4-yl group, benzofuran-5-yl group, benzofuran-6-yl group, benzofuran-7-yl group, benzothiophene- 2-yl group, benzothiophen-3-yl group, benzothiophen-4-yl group, benzothiophen-5-yl group, benzothiophen-6-yl group, benzothiophen-7-yl group, indol-2-yl Group, indol-3-yl group, indol-4-yl group, indol-5-yl group, indol-6-yl group, indol-7-yl group, benzothiazol-2 Yl group, benzothiazol-4-yl group, benzothiazol-5-yl group, benzothiazol-6-yl group, benzothiazol-7-yl group, indazol-3-yl group, indazol-4-yl group, indazole -5-yl group, indazol-6-yl group, indazol-7-yl group, benzisoxazol-3-yl group, benzisoxazol-4-yl group, benzisoxazol-5-yl group, Benzisoxazol-6-yl group, benzisoxazol-7-yl group, naphthalen-1-yl group, naphthalen-2-yl group, quinolin-2-yl group, quinolin-3-yl group, quinolin- 4-yl group, quinolin-5-yl group, quinolin-6-yl group, quinolin-7-yl group, quinolin-8-yl group, quinoxalinyl group, quinoxaline-5- Yl, quinoxalin-6-yl, quinazolinyl, quinazolin-4-yl, quinazolin-5-yl, quinazolin-6-yl, quinazolin-7-yl, quinazolin-8-yl, benzoxazine -5-yl group, benzoxazin-6-yl group, benzoxazin-7-yl group, benzoxazin-8-yl group, benzothiazin-5-yl group, benzothiazin-6-yl group, benzothiazin-7-yl group Benzothiazin-8-yl group, 1,3-benzodioxol-4-yl group, 1,3-benzodioxol-5-yl group, 1,3-benzodioxol-6-yl group, Examples thereof include a 1,3-benzodioxol-7-yl group and an anthracen-1-yl group. In particular, Ar is preferably a phenyl group, a 2-pyridyl group, a 3-pyridyl group, or a 4-pyridyl group from the viewpoint of showing good performance in immunoglobulin analysis or purification.

  The substituent of the optionally substituted aromatic group represented by Ar is not particularly limited, but may be a halogen atom, an optionally substituted alkyl group having 1 to 6 carbon atoms, or optionally substituted. An alkoxy group having 1 to 12 carbon atoms, an optionally substituted cycloalkoxy group having 3 to 8 carbon atoms, an optionally substituted alkenyloxy group having 3 to 6 carbon atoms, and an optionally substituted carbon number 3 To alkynyloxy group having 1 to 6 carbon atoms, optionally substituted acyloxy group having 1 to 6 carbon atoms, optionally substituted alkylthio group having 1 to 6 carbon atoms, optionally substituted alkyl having 1 to 6 carbon atoms A sulfinyl group, an optionally substituted alkylsulfonyl group having 1 to 6 carbon atoms, an optionally substituted alkoxycarbonyl group having 1 to 12 carbon atoms, a carboxyl group, nitro , Optionally substituted amino group, a hydroxyl group or a cyano group is preferable.

  As a halogen atom, a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom can be illustrated.

  The optionally substituted alkyl group having 1 to 6 carbon atoms may be linear, branched or cyclic, and may be a methyl group, an ethyl group, a cyclopropyl group, a propyl group, an isopropyl group, Cyclopropyl group, butyl group, isobutyl group, sec-butyl group, tert-butyl group, pentyl group, isoamyl group, neopentyl group, 2-pentyl group, 3-pentyl group, 2-methylbutyl group, tert-pentyl group, hexyl Examples thereof include a group, an isohexyl group, a 2-hexyl group and a 3-hexyl group. These alkyl groups having 1 to 6 carbon atoms are halogen atoms, cycloalkyl groups having 3 to 8 carbon atoms, alkoxy groups having 1 to 6 carbon atoms, alkoxycarbonyl groups having 1 to 6 carbon atoms, carboxy groups, and acyl groups. , One or more aromatic groups which may be substituted may be substituted, and more specifically, 2-chloroethyl group, 3-chloropropyl group, difluoromethyl group, 3-fluoropropyl group, methoxymethyl group 2-ethoxyethyl group, cyclopropylmethyl group, cyclopentylmethyl group, cyclohexylmethyl group, 2-methylthioethyl group, 2-ethylthioethyl group, 2-allylthioethyl group, 2-propargylthioethyl group, 2-benzyl Thioethyl group, 2- (2-chlorobenzyl) thioethyl group, 2- (2,4-dichlorobenzyl) thioethyl Group, 2-methylsulfinylethyl group, 2-methylsulfonylethyl group, ethoxymethyl group, 2-methoxyethyl group, 2-chloroethoxymethyl group, methoxycarbonylmethyl group, ethoxycarbonylmethyl group, 1-methoxycarbonylethyl group, 1-ethoxycarbonylethyl group, 2-ethoxycarbonylethyl group, carboxymethyl group, acetonyl group, 1-acetylethyl group, 3-acetylpropyl group, phenacyl group, 4-chlorophenacyl group, 2,4-difluorophenacyl Group, 4-methylphenacyl group, 4-isopropylphenacyl group, 4-isobutylphenacyl group, 4-cyclohexylphenacyl group, 4-cyanophenacyl group, 4-nitrophenacyl group, thienyl group, 2- (thiophene- 2-yl) ethyl group, 2- (thiophene) 3-yl) ethyl group, furfuryl group, (5-methylfuran-2-yl) methyl group, (5-ethylfuran-2-yl) methyl group, (5-chlorofuran-2-yl) methyl group, 2 -(5-fluorofuran-2-yl) ethyl group, tetrahydrofurfuryl group, 2-pyridylmethyl group, 3-pyridylmethyl group, 4-pyridylmethyl group, 2- (pyridin-2-yl) ethyl group, 2 -(Pyridin-3-yl) ethyl group, 2- (pyridin-4-yl) ethyl group, 3- (pyridin-2-yl) propyl group, 3- (pyridin-3-yl) propyl group, 3- ( An example is pyridin-4-yl) propyl group.

  The optionally substituted alkoxy group having 1 to 12 carbon atoms may be linear or branched, and is a methoxy group, ethoxy group, propyloxy group, isopropyloxy group, butoxy group, isobutyl group. Oxy group, sec-butyloxy group, tert-butoxy group, pentyloxy group, isoamyloxy group, neopentyloxy group, 2-pentyloxy group, 3-pentyloxy group, tert-pentyloxy group, hexyloxy group, isohexyl Examples thereof include an oxy group, a 2-hexyloxy group, a 3-hexyloxy group, and a decenyloxy group. These alkoxy groups may be halogen atoms, cycloalkyl groups having 3 to 8 carbon atoms, alkoxy groups having 1 to 6 carbon atoms, alkoxycarbonyl groups having 1 to 6 carbon atoms, carboxy groups, acyl groups, One or more may be substituted with a good aromatic group, and more specifically, 2-chloroethoxy group, 3-chloropropyloxy group, difluoromethoxy group, 4-trifluoromethoxy group, 3-fluoropropyloxy Group, cyclopropylmethoxy group, cyclopentylmethoxy group, cyclohexylmethoxy group, 2-methylthioethoxy group, 2-ethylthioethoxy group, 2-allylthioethoxy group, 2-propargylthioethoxy group, 2-benzylthioethoxy group, 2 -(2-chlorobenzyl) thioethoxy group, 2- (2,4-dichlorobenzyl) Thioethoxy group, 2-methylsulfinylethoxy group, 2-methylsulfonylethoxy group, methoxymethoxy group, ethoxymethoxy group, 2-methoxyethoxy group, 2-chloroethoxymethoxy group, methoxycarbonylmethoxy group, ethoxycarbonylmethoxy group, 1- Methoxycarbonylethoxy group, 1-ethoxycarbonylethoxy group, 2-ethoxycarbonylethoxy group, carboxymethoxy group, 1-acetylethoxy group, 3-acetylpropoxy group, 2-pyridylmethoxy group, 3-pyridylmethoxy group, 4-pyridyl Methoxy group, 2- (pyridin-2-yl) ethoxy group, 2- (pyridin-3-yl) ethoxy group, 2- (pyridin-4-yl) ethoxy group, 3- (pyridin-2-yl) propoxy group 3- (Pyridin-3-yl) propyl Epoxy group can be exemplified benzyl group.

  Examples of the optionally substituted cycloalkoxy group having 3 to 8 carbon atoms include cyclopropyloxy group, cyclobutyloxy group, cyclopentyloxy group, 2-methylcyclobutyloxy group, cyclohexyloxy group, and 2-methylcyclopentyloxy group. , 3-methylcyclopentyloxy group, 4-methylcyclopentyloxy group, and cyclooctyloxy group.

  Examples of the optionally substituted alkenyloxy group having 3 to 6 carbon atoms include allyloxy group, 2-methyl-2-propenyloxy group, 2-butenyloxy group, 1-buten-3-yloxy group, 3-butenyloxy group, A 4-pentenyloxy group and a 5-hexenyloxy group can be exemplified. Further, these alkenyloxy groups having 3 to 6 carbon atoms may be substituted with a halogen atom or the like, such as 3,3-difluoroallyloxy group, 3,3-dichloroallyloxy group, 3,3-dibromoallyloxy group. Groups can be exemplified.

  Examples of the optionally substituted alkynyloxy group having 3 to 6 carbon atoms include propargyloxy group, 1-butyn-3-yloxy group, 2-butynyloxy group, 3-butynyloxy group, 2-pentynyloxy group, 3- Examples thereof include a pentynyloxy group, a 4-pentynyloxy group, a 2-hexynyloxy group, a 3-hexynyloxy group, a 4-hexynyloxy group, and a 5-hexynyloxy group. In addition, these optionally substituted alkynyloxy groups having 3 to 6 carbon atoms may be substituted with a halogen atom or the like, and examples thereof include a 4,4,4-trifluorobutyn-2-yl group and the like. Can do.

  Examples of the acyloxy group which may be substituted include an acetoxy group and a propionyloxy group. These optionally substituted acyloxy groups may be substituted with a halogen atom or the like, and examples thereof include a trifluoroacetoxy group and a 2,2,2-trifluoropropionyloxy group.

  Examples of the optionally substituted alkylthio group having 1 to 6 carbon atoms include methylthio group, ethylthio group, propylthio group, isopropylthio group, butylthio group, isobutylthio group, sec-butylthio group, pentylthio group, isoamylthio group, neo Examples thereof include a pentylthio group, a 2-pentylthio group, a 3-pentylthio group, a 2-methylbutylthio group, a hexylthio group, an isohexylthio group, a 3-methylpentylthio group, and a 2-methylpentylthio group. Further, one or more of these alkylthio groups may be substituted with a halogen atom, a cycloalkyl group having 3 to 8 carbon atoms, and more specifically, a 2-chloroethylthio group, a 3-chloropropylthio group, a difluoro. Examples thereof include a methylthio group, a 3-fluoropropylthio group, a cyclopropylmethylthio group, a cyclopentylmethylthio group, and a cyclohexylmethylthio group.

  Examples of the optionally substituted alkylsulfinyl group having 1 to 6 carbon atoms include methylsulfinyl group, ethylsulfinyl group, propylsulfinyl group, isopropylsulfinyl group, butylsulfinyl group, isobutylsulfinyl group, sec-butylsulfinyl group, pentylsulfinyl group Group, isoamylsulfinyl group, neopentylsulfinyl group, 2-pentylsulfinyl group, 3-pentylsulfinyl group, 2-methylbutylsulfinyl group, hexylsulfinyl group, isohexylsulfinyl group, 3-methylpentylsulfinyl group, 2-methylpentyl A sulfinyl group can be exemplified. One or more of these alkylsulfinyl groups may be substituted with a halogen atom, a cycloalkyl group having 3 to 8 carbon atoms, and more specifically, 2-chloroethylsulfinyl group, 3-chloropropylsulfinyl group, Examples thereof include a difluoromethylsulfinyl group, a 3-fluoropropylsulfinyl group, a cyclopropylmethylsulfinyl group, a cyclopentylmethylsulfinyl group, and a cyclohexylmethylsulfinyl group.

  Examples of the optionally substituted alkylsulfonyl group having 1 to 6 carbon atoms include methylsulfonyl group, ethylsulfonyl group, propylsulfonyl group, isopropylsulfonyl group, butylsulfonyl group, isobutylsulfonyl group, sec-butylsulfonyl group, and pentylsulfonyl. Group, isoamylsulfonyl group, neopentylsulfonyl group, 2-pentylsulfonyl group, 3-pentylsulfonyl group, 2-methylbutylsulfonyl group, hexylsulfonyl group, isohexylsulfonyl group, 3-methylpentylsulfonyl group, 2-methylpentyl A sulfonyl group can be illustrated. In addition, one or more of these alkylsulfonyl groups may be substituted with a halogen atom or a cycloalkyl group having 3 to 8 carbon atoms, and more specifically, 2-chloroethylsulfonyl group, 3-chloropropylsulfonyl group, difluoro Examples thereof include a methylsulfonyl group, a 3-fluoropropylsulfonyl group, a cyclopropylmethylsulfonyl group, a cyclopentylmethylsulfonyl group, and a cyclohexylmethylsulfonyl group.

  Examples of the optionally substituted alkoxycarbonyl group having 1 to 12 carbon atoms include methoxycarbonyl group, ethoxycarbonyl group, propoxycarbonyl group, butoxycarbonyl group, hexyloxycarbonyl group, dodecyloxycarbonyl group, benzyloxycarbonyl group, tert -A butoxycarbonyl group can be illustrated. In addition, one or more of these alkoxycarbonyl groups may be substituted with a halogen atom or the like, and more specifically, trifluoromethoxycarbonyl group, 2,2,2-trifluoroethoxycarbonyl group and the like can be exemplified. .

  Examples of the optionally substituted amino group include a methylamino group, a dimethylamino group, an ethylamino group, an N-ethyl-N-methylamino group, a diethylamino group, an N-methyl-N-propylamino group, and an N-ethyl- group. N-propylamino group, 2,2,2-trifluoroethylamino group, dipropylamino group, isopropylamino group, N-methyl-N-isopropylamino group, butylamino group, N-butyl-N-methylamino group , Isobutylamino group, sec-butylamino group, pentylamino group, isoamylamino group, neopentylamino group, 2-pentylamino group, 3-pentylamino group, 2-methylbutylamino group, hexylamino group, isohexylamino Group, 4-methylpentylamino group, benzenesulfonylamino group, N-benzenesulfonyl N-methylamino group, p-toluenesulfonylamino group, 4-chlorobenzenesulfonylamino group, 4-nitrobenzenesulfonylamino group, methylsulfonylamino group, chloromethylsulfonylamino group, trifluoromethylsulfonylamino group, N-methylsulfonyl- N-methylamino group, N-methylsulfonyl-N-ethylamino group, N-methylsulfonyl-N-propylamino group, ethylsulfonylamino group, N-ethylsulfonyl-N-methylamino group, N-ethylsulfonyl-N -An ethylamino group, a methoxycarbonylamino group, and an ethoxycarbonylamino group can be illustrated.

The aromatic group which may be substituted represented by Ar is not particularly limited, but furan-2-yl group, 5-bromofuran-2-yl group, 5-methylfuran-2-yl group, furyl Group, thienyl group, 5-chlorothienyl group, 5-bromothienyl group, 5-methylthienyl group, 5-methoxythienyl group, 5-ethoxythienyl group, thiophen-3-yl group, 5-methylthiophen-3-yl Group, 5-methoxythiophen-3-yl group, 5-ethoxythiophen-3-yl group, 1-methylpyrrol-2-yl group, 1-methylpyrrol-3-yl group, 1-ethylpyrazol-3-yl Group, pyrazol-2-yl group, 3-ethoxy-1-methylpyrazol-2-yl group, 1-ethyl-4-methoxypyrazol-3-yl group, isothiazol-3-yl group, 5- Toxiisothiazol-3-yl group, isothiazol-4-yl group, isoxazol-3-yl group, isoxazol-4-yl group, 1-methylimidazol-2-yl group, 1-methylimidazol-4-yl group 1-methylimidazol-5-yl group, oxazol-2-yl group, oxazol-4-yl group, oxazol-5-yl group, thiazol-2-yl group, 3-methoxythiazol-2-yl group, 4 -Methoxythiazol-2-yl group, thiazol-4-yl group, thiazol-5-yl group, 1,2,4-triazol-3-yl group, 1,2,4-triazol-5-yl group, phenyl Group, 2-fluorophenyl group, 3-fluorophenyl group, 4-fluorophenyl group, 2,3-difluorophenyl group, 3,5-difluorophenyl 3,4,5-trifluorophenyl group, 2,3,4,5,6-pentafluorophenyl group, 3-fluoro-4-methylphenyl group, 4-trifluoromethoxyphenyl group, 2-chlorophenyl group, 3-chlorophenyl group, 4-chlorophenyl group, 4-chloro-3-methylphenyl group, 4-chloro-3-ethylphenyl group, 4-chloro-3-methoxyphenyl group, 4-chloro-3-ethoxyphenyl group, 2,3-dichlorophenyl group, 3,5-dichlorophenyl group, 3,4,5-trichlorophenyl group, 3-trifluoromethylphenyl group, 4-trifluoromethylphenyl group, 3-bromophenyl group, 4-bromophenyl Group, 4-bromo-3-methylphenyl group, 4-bromo-3-ethylphenyl group, 4-bromo-3-methoxyphenyl group 4-bromo-3-ethoxyphenyl group, 3,5-dibromo-4-hydroxyphenyl group, 3-iodophenyl group, 4-iodophenyl group, 4-iodo-3-methylphenyl group, 4-iodo-3 -Ethylphenyl group, 4-iodo-3-methoxyphenyl group, 4-iodo-3-ethoxyphenyl group, 2-methylphenyl group, 3-methylphenyl group, 4-methylphenyl group, 3,4-dimethylphenyl group 3,4,5-trimethylphenyl group, 3-hydroxy-4-methylphenyl group, 2-trifluorophenylmethylphenyl group, 3-trifluorophenylmethylphenyl group, 4-trifluorophenylmethylphenyl group, 4- Methoxymethylphenyl group, 3,4-diethoxyphenyl group, 3-ethyl-4-methylphenyl group, 4-ethyl-3 Methylphenyl group, 4-ethylphenyl group, 3,4-diethylphenyl group, 4-propylphenyl group, 4-isopropylphenyl group, 3-butylphenyl group, 4-butylphenyl group, 4-isobutylphenyl group, 4- (Sec-butyl) phenyl group, 4- (tert-butyl) phenyl group, 4-pentylphenyl group, 4-isoamylphenyl group, 4-neopentylphenyl group, 4- (2-pentyl) phenyl group, 4- ( 3-pentyl) phenyl group, 4- (2-methylbutyl) phenyl group, 4- (tert-pentyl) phenyl group, 4-hexylphenyl group, 4-isohexylphenyl group, 4- (2-chloroethyl) phenyl group, 4- (3-chloropropyl) phenyl group, 4- (difluoromethyl) phenyl group, 4- (3-fluoropropyl) Phenyl group, 4-methoxymethylphenyl group, 4-ethoxymethylphenyl group, 4-cyclopropylmethylphenyl group, 4-cyclopentylmethylphenyl group, cyclohexylmethylphenyl group, 4- (2-methylthioethyl) phenyl group, 4- (2-ethylthioethyl) phenyl group, 4- (2-allylthioethyl) phenyl group, 4- (2-propargylthioethyl) phenyl group, 4- (2-benzylthioethyl) phenyl group, 3-methoxyphenyl Group, 3-methoxy-4-methylphenyl group, 4-ethyl-3-methoxyphenyl group, 3-methoxy-4-propylphenyl group, 4-methoxyphenyl group, 4-methoxy-3-methoxyphenyl group, 3, 4-dimethoxyphenyl group, 3-ethoxy-4-methoxyphenyl group, 4-ethoxy-3- Methoxyphenyl group, 4-isopropyloxy-3-methoxyphenyl group, 3-isopropyloxy-4-methoxyphenyl group, 3-ethoxyphenyl group, 4-ethoxyphenyl group, 4-ethoxy-3-methylphenyl group, 4- Ethoxy-3-ethylphenyl group, 4-ethoxy-3-propylphenyl group, 4-ethoxy-3-isopropylphenyl group, 4-propyloxyphenyl group, 4-isopropyloxyphenyl group, 4-butoxyphenyl group, 4- Isobutyloxyphenyl group, 4- (sec-butyloxy) phenyl group, 3- (2-chloroethoxy) phenyl group, 3- (2-chloroethoxy) phenyl group, 4- (2-chloroethoxy) phenyl group, 3- (3-chloropropyloxy) phenyl group, 4- (3-chloropropyloxy) Enyl group, 3-difluoromethoxyphenyl group, 4-difluoromethoxyphenyl group, 4- (3-fluoropropyloxyphenyl group), 4-cyclopropylmethoxyphenyl group, 4-cyclopentylmethoxy group, 4-cyclohexylmethoxy group, 3 -Cyclopropyloxyphenyl group, 4-cyclopropyloxyphenyl group, 4-cyclopropyloxy-3-methoxyphenyl group, 4-cyclopropyloxy-3-methylphenyl group, 4-cyclobutyloxyphenyl group, 4-cyclopentyl Oxyphenyl group, 4- (2-methylcyclobutyloxy) phenyl group, 4-cyclohexyloxyphenyl, 4-allyloxyphenyl group, 4- (3,3-dichloroallyloxy) phenyl group, 4-propargyloxyphenyl group , 3-methyl 4-propargyloxyphenyl group, 3-ethyl-4-propargyloxyphenyl group, 3-methoxy-4-propargyloxyphenyl group, 3-ethoxy-4-propargyloxyphenyl group, 3-propoxy-4-propargyloxyphenyl group 4- (1-butyn-3-yloxy) phenyl group, 4- (2-butynyloxy) phenyl group, 3-methylthiophenyl group, 4-methylthiophenyl group, 4-methyl-3-methylthiophenyl group, 3-methyl -4-methylthiophenyl group, 4-methoxy-3-methylthiophenyl group, 3-ethoxy-4-methylthiophenyl group, 4-ethoxy-3-methylthiophenyl group, 3-propoxy-4-methylthiophenyl group, 4-ethyl Thiophenyl group, 3-propylthiophenyl group, 4-isopro Pyrthiophenyl group, 4-butylthiophenyl group, 4-isobutylthiophenyl group, 2- (sec-butylthio) phenyl group, 2-pentylthiophenyl group, 2-isoamylthiophenyl group, 4-neopentylthiophenyl group 4- (2-chloroethylthio) phenyl group, 3- (3-chloropropylthio) phenyl group, difluoromethylthiophenyl group, 3-acetoxyphenyl group, 4-acetoxyphenyl group, 3-methylsulfinylphenyl group, 4 -Methylsulfinylphenyl group, 4-methoxy-3-methylsulfinylphenyl group, 3-methoxy-4-methylsulfinylphenyl group, 4-ethoxy-3-methylsulfinylphenyl group, 3-ethoxy-4-methylsulfinylphenyl group, 4-Methyl-3-methylsulfinyl Nyl group, 3-ethyl-4-methylsulfinylphenyl group, 4- (2-chloroethylsulfinyl) phenyl group, 4- (3-chloropropylsulfinyl) phenyl group, 4- (difluoromethylsulfinyl) phenyl group, 3-methylsulfonylphenyl group, 4-methylsulfonylphenyl group, 4-methyl-3-methylsulfonylphenyl group, 4-ethyl-3-methylsulfonylphenyl group, 4-methoxy-3-methylsulfonylphenyl group, 4-ethoxy -3-methylsulfonylphenyl group, 3-methoxy-4-methylsulfonylphenyl group, 3-ethoxy-4-methylsulfonylphenyl group, ethylsulfonylphenyl group, propylsulfonylphenyl group, isopropylsulfonylphenyl group, butylsulfonylphenyl group, 2-black Ethylsulfonylphenyl group, 3-chloropropylsulfonylphenyl group, difluoromethylsulfonylphenyl group, 4-carboxyphenyl group, 4-methoxycarbonylphenyl group, 4-ethoxycarbonylphenyl group, 3-methylaminophenyl group, 4-methylamino Phenyl group, 4-methoxy-3-methylaminophenyl group, 3-methoxy-4-methylaminophenyl group, 4-ethoxy-3-methylaminophenyl group, 3-ethoxy-4-methylaminophenyl group, 4-methyl -3-methylaminophenyl group, 3-methyl-4-methylaminophenyl group, 4-ethyl-3-methylaminophenyl group, 3-dimethylaminophenyl group, 4-dimethylaminophenyl group, 4-methoxy-3- Dimethylaminophenyl group, 3-methoxy-4- Dimethylaminophenyl group, 4-ethoxy-3-dimethylaminophenyl group, 3-ethoxy-4-dimethylaminophenyl group, 4-methyl-3-dimethylaminophenyl group, 3-methyl-4-dimethylaminophenyl group, 4 -Ethyl-3-dimethylaminophenyl group, 3-methyl-4-dimethylaminophenyl group, 3- (N-ethyl-N-methylamino) phenyl group, 4- (N-ethyl-N-methylamino) phenyl group 4-methoxy-3- (N-ethyl-N-methylamino) phenyl group, 3-methoxy-4- (N-ethyl-N-methylamino) phenyl group, 4-ethoxy-3- (N-ethyl- N-methylamino) phenyl group, 3-ethoxy-4- (N-ethyl-N-methylamino) phenyl group, 4-methyl-3- (N-ethyl-N-methyl) Mino) phenyl group, 3-methyl-4- (N-ethyl-N-methylamino) phenyl group, 4-ethyl-3- (N-ethyl-N-methylamino) phenyl group, 3-methyl-4- ( N-ethyl-N-methylamino) phenyl group, 3-diethylaminophenyl group, 4-diethylaminophenyl group, 4-methoxy-3-diethylaminophenyl group, 3-methoxy-4-diethylaminophenyl group, 4-ethoxy-3- Diethylaminophenyl group, 3-ethoxy-4-diethylaminophenyl group, 4-methyl-3-diethylaminophenyl group, 3-methyl-4-diethylaminophenyl group, 4-ethyl-3-diethylaminophenyl group, 4-methylsulfonylaminophenyl Group, 4-trifluoromethylsulfonylaminophenyl group, 4-acetylamino Phenyl group, 2-hydroxyphenyl group, 3-hydroxyphenyl group, 4-hydroxyphenyl group, 3-hydroxy-4-methylphenyl group, 3-hydroxy-4-ethylphenyl group, 3-hydroxy-4-methoxyphenyl group 4-hydroxy-3-methylphenyl group, 4-hydroxy-3-ethylphenyl group, 4-hydroxy-3-methoxyphenyl group, 2-cyanophenyl group, 3-cyanophenyl group, 4-cyanophenyl group, 2 -Nitrophenyl group, 3-nitrophenyl group, 3-nitro-4-methylphenyl group, 4-nitrophenyl group, pyridin-2-yl group, 5-chloropyridin-2-yl group, 5-methylpyridin-2 -Yl group, 5-ethylpyridin-2-yl group, 5-methoxypyridin-2-yl group, 5-ethoxypyridin-2- Yl group, 2,3-dimethylpyridin-6-yl group, 2,3-dimethoxypyridin-6-yl group, pyridyl-3-yl group, 2-chloropyridin-5-yl group, 2-methylpyridin-5 -Yl group, 2-ethylpyridin-5-yl group, 2-methoxypyridin-5-yl group, 2
-Ethoxypyridin-5-yl group, 2,3-dimethylpyridin-5-yl group, pyridyl-4-yl group, 2-methylpyridin-4-yl group, 2-ethylpyridin-4-yl group, 2- Methoxypyridin-4-yl group, 2-ethoxypyridin-4-yl group, 2-pyrimidyl group, 5-methylpyrimidin-2-yl group, 5-ethylpyrimidin-2-yl group, 2-ethoxypyridin-5 Yl group, 2,3-dimethylpyridin-5-yl group, 2-methylpyridin-4-yl group, 2-ethylpyridin-4-yl group, 2-methoxypyridin-4-yl group, 2-ethoxypyridine- 4-yl group, 5-chloropyrimidin-2-yl group, 5-bromopyrimidin-2-yl group, 5-methoxypyrimidin-2-yl group, pyrimidyl-4-yl group, pyrimidyl-5-yl group, , 2,4-Triazin-3-yl group, 1,2,4-triazin-5-yl group, 1,2,4-triazin-6-yl group, benzodioxolan-3-yl group, benzofuran-2- Yl group, benzofuran-3-yl group, benzofuran-4-yl group, benzofuran-5-yl group, benzofuran-6-yl group, benzofuran-7-yl group, benzothiophen-2-yl group, benzothiophene-3 -Yl group, benzothiophen-4-yl group, benzothiophen-5-yl group, benzothiophen-6-yl group, benzothiophen-7-yl group, 1-methylindol-2-yl group, benzothiazol-2 -Yl group, benzothiazol-4-yl group, benzothiazol-5-yl group, benzothiazol-6-yl group, benzothiazol-7-yl group, benz Soxazol-3-yl group, benzisoxazol-4-yl group, benzisoxazol-5-yl group, benzisoxazol-6-yl group, benzisoxazol-7-yl group, naphthalene-1 -Yl group, naphthalen-2-yl group, quinolin-2-yl group, quinolin-3-yl group, quinolin-4-yl group, quinolin-5-yl group, quinolin-6-yl group, quinolin-7- Yl, quinolin-8-yl, quinoxalin-5-yl, quinoxalin-6-yl, quinazolin-4-yl, quinazolin-5-yl, quinazolin-6-yl, quinazolin-7-yl Group, quinazolin-8-yl group, benzoxazin-5-yl group, benzoxazin-6-yl group, benzoxazin-7-yl group, benzoxazin-8-yl group, benzothia Gin-5-yl group, benzothiazin-6-yl group, benzothiazin-7-yl group, benzothiazin-8-yl group, 1,3-benzodioxol-4-yl group, 1,3-benzodioxole Examples include a -5-yl group, a 1,3-benzodioxol-6-yl group, a 1,3-benzodioxol-7-yl group, and an anthracen-1-yl group.

  Ar is a 4-methylphenyl group, a 4-ethylphenyl group, a 3,4-dimethylphenyl group, a 3-ethyl-4-methylphenyl group, 4- Ethyl-3-methylphenyl group, 4-methoxyphenyl group, 4-ethoxyphenyl group, 3,4-dimethoxyphenyl group, 3,4-diethoxyphenyl group, 3-ethoxy-4-methoxyphenyl group, 4-ethoxy -3-methoxyphenyl group, 4-methoxy-3-methylphenyl group, 4-methyl-3-ethoxyphenyl group, 4-dimethylaminophenyl group, 2-methoxypyridin-5-yl group, 2-ethoxypyridine-5 -Yl group, 2,3-dimethylpyridin-5-yl group, 4-pyridyl group, 2-methylpyridin-4-yl group, 2-ethylpyridyl -4-yl group, 2-methoxypyridin-4-yl group, 2-ethoxypyridin-4-yl group, 2-chloropyridin-5-yl group, 3-methoxy-4-methylphenyl group, 3-hydroxy- Either a 4-methylphenyl group or a 4-methylthiophenyl group is preferable.

  The material of the matrix represented by M is not particularly limited. For example, a polypeptide or protein such as cross-linked albumin, agarose, alginate, carrageenan, chitin, cellulose, dextrin, dextran, starch or other polysaccharides, polyacrylamide, polystyrene , Synthetic polymers such as polyacrolein, polyvinyl alcohol, polymethacrylate, poly (2-hydroxyethyl methacrylate), polyurethane, inorganic compounds such as silica, glass, porous diatomaceous earth, alumina, zirconia, iron oxide or other metal oxides And a matrix such as a copolymer formed by arbitrarily combining two or more of the above substances. In addition, they contain compounds such as dextran, polyethylene glycol, polyvinyl alcohol or hydrolyzed polymers such as hydrolyzed starch used in liquid phase partitioning, or compounds such as perfluorodecalin used to form emulsions. An included matrix is also included. In particular, as a matrix material in the production of the thiazole derivative-immobilized matrix of the present invention, vinyl polymers such as Toyopearl (trade name) (manufactured by Tosoh Corporation), agarose, chitosan, dextran, cellulose, silica, and polystyrene are preferable.

  The vinyl polymer here refers to a polymer obtained by vinyl polymerization of a monomer having a vinyl group, and examples of the monomer include methyl methacrylate, 2-hydroxyethyl methacrylate, acrylamide, and vinyl ester. Furthermore, the vinyl polymer here includes a copolymer using two or more of the above-mentioned monomers, and a crosslinked one.

  The matrix may be granular or non-particulate, soluble or insoluble in an aqueous solvent, porous or non-porous.

  n can be appropriately selected from an integer of 1 to 12, but as a thiazole derivative used for producing the thiazole derivative-immobilized matrix of the present invention, n is preferably 3, 4 or 5.

  Next, the manufacturing method of the thiazole derivative used for manufacture of the thiazole derivative fixed matrix of this invention is demonstrated in detail.

  Manufacturing method 1

（式中、Ｒ^１、Ｒ^２、Ａｒ及びｎは前記と同じ意味を表し、Ｒ^３はアミノ基又はそのアンモニウム塩を表し、Ｙは脱離基を表す。）
工程１はフタルイミド誘導体（２）をアシルチオウレア誘導体（３）と反応させ、チアゾール誘導体（４）を合成する方法である。フタルイミド誘導体（２）は非特許文献２や特許文献７等を参考に合成することができる。

(Wherein R ¹ , R ² , Ar and n represent the same meaning as described above, R ³ represents an amino group or an ammonium salt thereof, and Y represents a leaving group.)
Step 1 is a method of synthesizing the thiazole derivative (4) by reacting the phthalimide derivative (2) with the acylthiourea derivative (3). The phthalimide derivative (2) can be synthesized with reference to Non-Patent Document 2, Patent Document 7, and the like.

  In the reaction of Step 1, the reaction is preferably carried out in a solvent, and any solvent that does not harm the reaction can be used. For example, diethyl ether, tetrahydrofuran (hereinafter THF), 1,2-dimethoxyethane ( DME), ether solvents such as 1,4-dioxane, ketone solvents such as acetone and methyl ethyl ketone, ester solvents such as ethyl acetate and butyl acetate, nitriles such as acetonitrile and propionitrile, benzene, toluene and xylene , Aromatic hydrocarbon solvents such as chlorobenzene, amide solvents such as N, N-dimethylformamide (hereinafter DMF), N-methylpyrrolidone, alcohol solvents such as methanol and ethanol, dimethyl sulfoxide (hereinafter DMSO), water or By using these mixed solvents , It is preferable to use DMF in terms of a good yield. Although there is no restriction | limiting in particular about reaction temperature, The target object can be obtained by making it react at the temperature chosen suitably from the range of 0 degreeC to 150 degreeC, and it is in the range of 60 degreeC to 150 degreeC at a point with a sufficient yield. It is preferable to react. This step can also be carried out in the presence of a base. Examples of the base include sodium hydride, sodium amide, sodium carbonate, potassium carbonate, sodium methoxide, sodium ethoxide, potassium tert-butoxide, sodium hydroxide, hydroxide Alkali metal bases such as potassium, organic amines such as triethylamine, tributylamine, N-methylmorpholine, pyridine and dimethylaniline can be used. The amount of the base used is not particularly limited, but the target product can be obtained with good yield by carrying out the reaction using an equal amount or more with respect to the reaction substrate. After completion of the reaction, the desired product can be obtained by ordinary post-treatment operations, but if necessary, it can be purified by a method such as column chromatography or recrystallization.

  Step 2 is a method for producing a thiazole derivative (1a) by treating the thiazole derivative (4) with a base. Bases include alkali metal bases such as sodium hydride, sodium amide, sodium carbonate, potassium carbonate, sodium methoxide, sodium ethoxide, potassium tert-butoxide, sodium hydroxide, potassium hydroxide, methylamine, ethylamine, dimethyl Organic amines such as amines and hydrazines can be used. Hydrazine or methylamine is preferred in terms of good yield. The target product can be obtained in good yield by carrying out the reaction with the base used in an amount of 2 equivalents or more with respect to the reaction substrate. In this reaction, the reaction is preferably carried out in a solvent, and any solvent that does not harm the reaction can be used. For example, ether solvents such as diethyl ether, THF, DME, dioxane, acetone, methyl ethyl ketone, etc. Ketone solvents, ester solvents such as ethyl acetate and butyl acetate, nitriles such as acetonitrile and propionitrile, aromatic hydrocarbon solvents such as benzene, toluene, xylene and chlorobenzene, DMF, N-methylpyrrolidone and the like An amide solvent, an alcohol solvent such as methanol, ethanol, isopropyl alcohol, DMSO, water, or a mixed solvent thereof can be used, and an alcohol such as methanol or ethanol can be used because of its good yield and simple post-treatment. It is preferable to use a system solvent. Although there is no restriction | limiting in particular about reaction temperature, A target object can be obtained by making it react at the temperature chosen suitably from the range of 0 to 150 degreeC. After completion of the reaction, the desired product can be obtained by ordinary post-treatment operations, but if necessary, it can be purified by a method such as column chromatography or recrystallization. Moreover, after completion | finish of reaction, it can process with an acid and can also be isolated as an ammonium salt. Any acid that does not harm the reaction can be used, for example, hydrochloric acid, hydrobromic acid, hydrofluoric acid, nitric acid, sulfuric acid, phosphoric acid and other inorganic acids, methanesulfonic acid, toluenesulfonic acid, trifluoroacetic acid However, it is preferable to use hydrochloric acid because the target product can be easily isolated.

  Manufacturing method 2

（式中、Ｒ^１、Ｒ^２、Ａｒ及びｎは前記と同じ意味を表し、Ｗはハロゲン原子を表す。）
製造方法２は２−置換アミノチアゾール誘導体（５）と酸ハロゲン化物（６）を反応させ、チアゾール誘導体（４ａ）を製造する方法である。当該方法の反応では、反応は溶媒中で行なうことが好ましく、反応に害を及ぼさない溶媒であれば使用することができ、例えば、ジクロロメタン、クロロホルム、１，２−ジクロロエタンなどのハロゲン系溶媒、ジエチルエーテル、ＴＨＦ、ＤＭＥ、ジオキサンなどのエーテル系溶媒、アセトン、メチルエチルケトンなどのケトン系溶媒、酢酸エチル、酢酸ブチルなどのエステル系溶媒、アセトニトリル、プロピオニトリルなどのニトリル類、ベンゼン、トルエン、キシレン、クロロベンゼンなどの芳香族炭化水素系溶媒、ＤＭＦ、Ｎ−メチルピロリドンなどのアミド系溶媒、ＤＭＳＯ、水あるいはこれらの混合溶媒を用いることができ、収率が良い点でジクロロメタン、クロロホルム、１，２−ジクロロエタンなどのハロゲン系溶媒を用いるのが好ましい。反応温度については特に制限はないが、０℃から１５０℃の範囲から適宜選ばれた温度で反応させることにより目的物を得ることができ、収率が良い点で０℃から１００℃の範囲で反応させるのが好ましい。本工程では、塩基存在下に行なうこともでき、塩基としては、水素化ナトリウム、ナトリウムアミド、炭酸ナトリウム、炭酸カリウム、ナトリウムメトキシド、ナトリウムエトキシド、カリウム−ｔｅｒｔ−ブトキシド、水酸化ナトリウム、水酸化カリウムなどのアルカリ金属塩基、トリエチルアミン、トリブチルアミン、Ｎ−メチルモルホリン、ピリジン、ジメチルアニリンなどの有機アミン類を用いることができる。塩基の使用量は特に制限はないが、反応基質に対して等量以上用いて反応を実施することにより、収率良く目的物を得ることができる。反応終了後は、通常の後処理操作により目的物を得ることができるが、必要であればカラムクロマトグラフィーあるいは再結晶などにより精製することもできる。当該方法で合成されたチアゾール誘導体（４ａ）は、製造方法１の工程２を経由することによって、チアゾール誘導体（１ａ）へと導くことができる。

(In the formula, R ¹ , R ² , Ar and n represent the same meaning as described above, and W represents a halogen atom.)
Production method 2 is a method for producing a thiazole derivative (4a) by reacting a 2-substituted aminothiazole derivative (5) with an acid halide (6). In the reaction of this method, the reaction is preferably carried out in a solvent, and any solvent that does not harm the reaction can be used. For example, halogen solvents such as dichloromethane, chloroform, 1,2-dichloroethane, diethyl Ether solvents such as ether, THF, DME and dioxane, ketone solvents such as acetone and methyl ethyl ketone, ester solvents such as ethyl acetate and butyl acetate, nitriles such as acetonitrile and propionitrile, benzene, toluene, xylene and chlorobenzene Aromatic hydrocarbon solvents such as DMF, amide solvents such as N-methylpyrrolidone, DMSO, water or a mixed solvent thereof can be used, and dichloromethane, chloroform, 1,2-dichloroethane are preferable in terms of yield. Use halogenated solvents such as Preference is. Although there is no restriction | limiting in particular about reaction temperature, The target object can be obtained by making it react at the temperature chosen suitably from the range of 0 degreeC to 150 degreeC, and it is in the range of 0 degreeC to 100 degreeC at a point with a sufficient yield. It is preferable to react. This step can also be carried out in the presence of a base. Examples of the base include sodium hydride, sodium amide, sodium carbonate, potassium carbonate, sodium methoxide, sodium ethoxide, potassium tert-butoxide, sodium hydroxide, hydroxide Alkali metal bases such as potassium, organic amines such as triethylamine, tributylamine, N-methylmorpholine, pyridine and dimethylaniline can be used. The amount of the base used is not particularly limited, but the target product can be obtained with good yield by carrying out the reaction using an equal amount or more with respect to the reaction substrate. After completion of the reaction, the desired product can be obtained by ordinary post-treatment operations, but can be purified by column chromatography or recrystallization if necessary. The thiazole derivative (4a) synthesized by the method can be led to the thiazole derivative (1a) by passing through step 2 of production method 1.

  Next, the manufacturing method of the thiazole derivative fixed matrix of this invention is demonstrated in detail.

  Manufacturing method 3

（式中、Ｒ^１、Ｒ^２、Ｒ^３、Ｍ、Ａｒ及びｎは前記と同じ意味を表す。）
製造方法３はチアゾール誘導体（１ａ）と活性化基（活性化基とは、アミノ基あるいはアンモニウム塩と容易に反応できる官能基のことで、例えば、スクシンイミドオキシカルボニル基、ホルミル基、カルボキシル基、２，２，２−トリフルオロエチルスルホニル基（トレシル基）、塩化スルホニル基、トシル基、ビニルスルホニル基、エポキシ基をあげることができる。）含有マトリックスと反応させてチアゾール誘導体固定化マトリックス（１）を製造する方法である。なお、当該方法で用いる活性化基含有マトリックスの活性化基としては、スクシンイミドオキシカルボニル基、ホルミル基、２，２，２−トリフルオロエチルスルホニル基（トレシル基）、エポキシ基、カルボキシル基が特に好ましい。また、当該方法で用いる活性化基含有マトリックスは、活性化基を有しないマトリックスから周知の方法で活性化基を付加することで調製しても良く、ＨｉＴｒａｐ ＮＨＳ−ａｃｔｉｖａｔｅｄ ＨＰ（商品名）（ＧＥヘルスケアバイオサイエンス株式会社製）、エポキシトヨパール（商品名）、ホルミルトヨパール（商品名）、トレシルトヨパール（商品名）、カルボキシトヨパール（商品名）（以上東ソー株式会社製）、活性化キトパールＫ−６６（商品名）ゲル（富士紡ホールディングス株式会社製）、ＢＩＯＡＣＴ ＥＰＯ（商品名）ゲル（昭和電工株式会社製）、ＰＯＲＯＳ−ＥＰ（商品名）ゲル（アプライドバイオシステムズ株式会社製）、セルファインホルミル（商品名）ゲル（チッソ株式会社製）、Ｐｒｏｆｉｎｉｔｙ Ｅｐｏｘｉｄｅ（商品名）ゲル（バイオラッド株式会社製）、Ｍ．Ｓ．ＧＥＬ Ｅｐｏｘｙ−Ｄ−５０−１０００ＡＷ（商品名、ＡＧＣエスアイテック株式会社製）などのリガンド固定化用担体として市販されているものをそのまま用いても良い。

(Wherein R ¹ , R ² , R ³ , M, Ar and n have the same meaning as described above).
Production method 3 is a thiazole derivative (1a) and an activating group (an activating group is a functional group that can easily react with an amino group or an ammonium salt. For example, succinimideoxycarbonyl group, formyl group, carboxyl group, 2 , 2,2-trifluoroethylsulfonyl group (tresyl group), sulfonyl chloride group, tosyl group, vinylsulfonyl group, and epoxy group.) The thiazole derivative-immobilized matrix (1) is reacted with a matrix containing It is a manufacturing method. The activating group of the activating group-containing matrix used in the method is particularly preferably a succinimideoxycarbonyl group, formyl group, 2,2,2-trifluoroethylsulfonyl group (tresyl group), epoxy group, or carboxyl group. . In addition, the activation group-containing matrix used in the method may be prepared by adding an activation group from a matrix having no activation group by a known method. HiTrap NHS-activated HP (trade name) (GE) Healthcare Biosciences Inc.), Epoxy Toyopearl (trade name), Formyl Toyopearl (trade name), Tresyl Toyopearl (trade name), Carboxy Toyopearl (trade name) (above Tosoh Corporation), activity Chitopearl K-66 (trade name) gel (manufactured by Fujibo Holdings Co., Ltd.), BIOACT EPO (trade name) gel (manufactured by Showa Denko KK), POROS-EP (trade name) gel (manufactured by Applied Biosystems) , Cellufine Formyl (trade name) Gel (manufactured by Chisso Corporation), Profinity Epo xide (trade name) gel (manufactured by Bio-Rad Co., Ltd.), M.I. S. A commercially available carrier for immobilizing a ligand such as GEL Epoxy-D-50-1000AW (trade name, manufactured by AGC S-Tech Co., Ltd.) may be used as it is.

  In this reaction, the desired product can be easily obtained by reacting under neutral or basic conditions. In particular, by carrying out the reaction under basic conditions, a matrix with a high immobilization rate of thiazole derivatives can be obtained. Examples of the base include sodium hydride, sodium amide, sodium carbonate, potassium carbonate, sodium methoxide, sodium ethoxide, potassium tert-butoxide, sodium hydroxide, potassium hydroxide and other alkali metal bases, triethylamine, tributylamine, N -Organic amines such as methylmorpholine, pyridine, dimethylaniline can be used. The amount of the base used is not particularly limited, but by carrying out the reaction using equimolar amounts or more with respect to the reaction substrate, a matrix having a high immobilization ratio of the desired thiazole derivative can be obtained. Moreover, in order to maintain the pH of a solution, it can also react by adding a buffer solution. The reaction is preferably carried out in a solvent, and any solvent that does not harm the reaction can be used. For example, ether solvents such as diethyl ether, THF, DME, dioxane, and ketones such as acetone and ethyl methyl ketone. Solvents, ester solvents such as ethyl acetate and butyl acetate, nitriles such as acetonitrile and propionitrile, aromatic hydrocarbon solvents such as benzene, toluene, xylene and chlorobenzene, amides such as DMF and N-methylpyrrolidone A solvent, DMSO, water, or a mixed solvent thereof can be used. Although there is no restriction | limiting in particular about reaction temperature, The target thiazole derivative fixed matrix can be obtained by making it react at the temperature selected suitably from the range of 0 to 150 degreeC. The amount of the thiazole derivative immobilized on the matrix can be measured by using an analytical method such as absorbance analysis, high performance liquid chromatography, elemental analysis alone or in combination.

  Next, a method for analyzing, separating, isolating and purifying proteins using the thiazole derivative-immobilized matrix of the present invention will be described.

  The thiazole derivative-immobilized matrix obtained by the production method 3 is packed in a column tube, and a buffer solution, an aqueous solution of a metal salt, an amino acid solution, an alcohol solution, or the like is passed as an eluent, and the protein is analyzed or separated. Isolation and purification can be performed. Proteins that can be analyzed, separated, or isolated and purified by this method include plasma protein components and milk protein components, and naturally occurring proteins such as immunoglobulins in plasma, serum albumin, blood coagulation Examples include factors, lactalbumin, and lactoferrin. In addition to the above, the recombinant protein may be exemplified by peptide hormones, interferons, interleukins, growth factors, growth inhibitory factors, and vaccines that are naturally present in minute amounts or are artificially designed. In particular, the thiazole derivative-immobilized matrix of the present invention is preferred for immunoglobulin purification. Examples of the buffer solution to be passed include phosphate buffer solution, citrate buffer solution, Tris, PIPES, ACES, Cholamine, BES, MOPS, TES, HEPES, and metal salts include sodium sulfate, potassium sulfate, lithium sulfate, Examples include magnesium sulfate, ammonium sulfate, sodium citrate, potassium citrate, sodium chloride, and potassium chloride. Examples of amino acids include glycine, arginine, beta-alanine, and gamma aminobutyric acid. Examples of alcohol include ethanol, isopropyl alcohol, glycerol, and ethylene. A glycol can be illustrated and these can also be used as a mixed solvent. The target protein can be separated, isolated or purified within the pH range of 3 to 11, but the pH is preferably purified within the range of 5 to 9. In addition, the protein can be analyzed, separated, isolated, or purified using a chromatography apparatus such as AKTAprime plus (trade name) (manufactured by GE Healthcare Bioscience Co., Ltd.). When analyzing or purifying immunoglobulins or analogs, fragments or fusions thereof using the thiazole derivative-immobilized matrix obtained by production method 3, a phosphate buffer containing sodium sulfate or ammonium sulfate is passed through. The pH is preferably in the range of 5 to 9, and it is preferable to perform chromatography utilizing hydrophobic interaction. An immunoglobulin analog here refers to a natural or artificially produced protein or protein conjugate in which the structure and function of the immunoglobulin is at least partially retained, and an immunoglobulin fragment is an enzymatic treatment. Alternatively, it refers to a protein having an immunoglobulin partial structure produced by genetic engineering design. An immunoglobulin fusion is a functional part of a protein having biological activity such as various cytokines or cytokine receptors. It refers to those produced by fusing all or part of them with genetic engineering.

  The thiazole derivative-immobilized matrix obtained by the production method 3 includes alkaline aqueous solutions such as sodium hydroxide aqueous solution and potassium hydroxide aqueous solution, acidic aqueous solutions such as hydrochloric acid aqueous solution, sulfuric acid aqueous solution, nitric acid aqueous solution and phosphoric acid aqueous solution, guanidine hydrochloride aqueous solution, urea Protein denaturant aqueous solution such as aqueous solution, aqueous solution containing amino acids such as arginine and histidine, aqueous surfactant solution such as SDS, Tween, alcohol solution such as methanol, ethanol, isopropanol, glycerol, ethylene glycol, or these By washing with the mixed aqueous solution, it can be returned to the initial state and can be used repeatedly.

  The thiazole derivative-immobilized matrix of the present invention is a highly durable selective adsorption / desorption agent useful for the analysis and purification of proteins, particularly immunoglobulins. Therefore, the thiazole derivative-immobilized matrix is extremely useful for industrial purification of medical proteins, particularly immunoglobulins. Useful.

The chromatographic result of the immunoglobulin using the thiazole derivative fixed HiTrap column (result of compound 286). The chromatographic result of the immunoglobulin using the thiazole derivative fixed HiTrap column (result of the compound 304). The chromatographic result of the immunoglobulin using the thiazole derivative fixed HiTrap column (result of the compound 320). The chromatographic result of the immunoglobulin using the thiazole derivative fixed HiTrap column (result of compound 329). The chromatographic result of the immunoglobulin using the thiazole derivative fixed HiTrap column (result of compound 384). Chromatographic results of immunoglobulin using thiazole derivative-immobilized HiTrap column (results of compound 423) Chromatographic results of bovine serum albumin using thiazole derivative-immobilized HiTrap column (results of compound 286) Chromatographic results of bovine serum albumin using thiazole derivative-immobilized HiTrap column (results of compound 304) Chromatographic results of bovine serum albumin using a thiazole derivative-immobilized HiTrap column (results of compound 320) Chromatographic results of bovine serum albumin using a thiazole derivative-immobilized HiTrap column (results of compound 329) Chromatographic results of bovine serum albumin using thiazole derivative-immobilized HiTrap column (results of compound 384) Chromatographic results of bovine serum albumin using a thiazole derivative-immobilized HiTrap column (results of compound 423) Chromatographic results of a mixture of ribonuclease A, lysozyme, and α-chymotrypsinogen A using a compound 286-immobilized HiTrap column Chromatographic results of papain digest of human-mouse chimeric antibody using Compound 286-immobilized HiTrap column Results of SDS-PAGE analysis of fractions I, II, III and IV in FIG. Chromatographic results of immunoglobulins using Compound 286-immobilized Epoxy Toyopearl gel Chromatographic results of humanized monoclonal antibody using Compound 286-immobilized Epoxy Toyopearl gel Chromatographic results of bovine serum albumin using Compound 286-immobilized Epoxy Toyopearl gel Chromatographic results of humanized monoclonal antibody using Compound 329-immobilized Epoxy Toyopearl gel Chromatographic results of bovine serum albumin using Compound 329-immobilized Epoxy Toyopearl gel Chromatographic results of humanized monoclonal antibody using Compound 286 immobilized formyl toyopearl gel Chromatographic results of bovine serum albumin using Compound 286 immobilized formyl toyopearl gel BIACORE sensorgram (A: mouse IgM, B: mouse IgG, C: chicken IgY, D: bovine serum albumin) in which various antibodies were run as analytes on the compound 286-immobilized BIACORE sensor chip CM5

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited to these.

Example 1 Synthesis of Thiazole Derivative (Synthesis Example 1)

６−フタルイミド−１−ヘキサナール（４．９５ｇ，２０．２ｍｍｏｌ）を四塩化炭素（１００ｍＬ）に溶解し臭素（１．０３ｍＬ，２０．２ｍｍｏｌ）の四塩化炭素溶液（１００ｍＬ）を滴下し２時間反応した。反応液を水（１００ｍＬ）、飽和食塩水（１００ｍＬ）で洗浄し無水硫酸マグネシウムで乾燥した後、乾燥剤を濾別し溶媒を減圧留去し油状物を得た。得られた油状物をＤＭＦ（１００ｍＬ）に溶解し、４−メチルベンゾイルチオウレア（５．８７ｇ，２６．３ｍｍｏｌ）を加え、１０時間加熱還流した。反応終了後、反応混合物に１Ｍ塩酸（３００ｍＬ）を加え酢酸エチル（３００ｍＬ）で二回抽出し、有機層を合わせて飽和食塩水（３００ｍＬ）で二回洗浄した。有機層を無水硫酸マグネシウムで乾燥した後、乾燥剤を濾別し溶媒を減圧留去した。得られた混合物をシリカゲルカラムクロマトグラフィー（ヘキサン：酢酸エチル＝２：１）で精製することによって、４−メチル−Ｎ−［５−｛４−（フタルイミド）ブチル｝チアゾール−２−イル］ベンズアミドの白色固体（０．４２４ｇ，５％）を得た。ｍ．ｐ．２０３から２０５℃；^１Ｈ−ＮＭＲ（ＣＤＣｌ_３，ＴＭＳ，ｐｐｍ）：δ １．５８から１．８２（ｍ，４Ｈ），２．４４（ｓ，３Ｈ），２．８２（ｔ，Ｊ＝７．５Ｈｚ，２Ｈ），３．７２（ｔ，Ｊ＝７．５Ｈｚ，２Ｈ），６．９７（ｓ，１Ｈ），７．３２（ｄ，Ｊ＝７．５Ｈｚ，２Ｈ），７．６９から７．８６（ｍ，４Ｈ），７．９１（ｄ，Ｊ＝７．５Ｈｚ，２Ｈ）。アミドのプロトンは帰属できなかった。

6-phthalimido-1-hexanal (4.95 g, 20.2 mmol) is dissolved in carbon tetrachloride (100 mL), and a solution of bromine (1.03 mL, 20.2 mmol) in carbon tetrachloride (100 mL) is added dropwise and reacted for 2 hours. did. The reaction mixture was washed with water (100 mL) and saturated brine (100 mL) and dried over anhydrous magnesium sulfate, and then the desiccant was filtered off and the solvent was evaporated under reduced pressure to give an oil. The obtained oil was dissolved in DMF (100 mL), 4-methylbenzoylthiourea (5.87 g, 26.3 mmol) was added, and the mixture was heated to reflux for 10 hr. After completion of the reaction, 1M hydrochloric acid (300 mL) was added to the reaction mixture, and the mixture was extracted twice with ethyl acetate (300 mL). The organic layers were combined and washed twice with saturated brine (300 mL). The organic layer was dried over anhydrous magnesium sulfate, the desiccant was filtered off, and the solvent was distilled off under reduced pressure. The resulting mixture was purified by silica gel column chromatography (hexane: ethyl acetate = 2: 1) to give 4-methyl-N- [5- {4- (phthalimido) butyl} thiazol-2-yl] benzamide. A white solid (0.424 g, 5%) was obtained. m. p. ¹ ₃ H-NMR (CDCl ₃ , TMS, ppm): δ 1.58 to 1.82 (m, 4H), 2.44 (s, 3H), 2.82 (t, J = 7) .5 Hz, 2H), 3.72 (t, J = 7.5 Hz, 2H), 6.97 (s, 1H), 7.32 (d, J = 7.5 Hz, 2H), 7.69 to 7 .86 (m, 4H), 7.91 (d, J = 7.5 Hz, 2H). The proton of the amide could not be assigned.

  (Synthesis Example 2)

２−アミノ−４−メチル−５−｛４−（フタルイミド）ブチル｝チアゾール（０．５０ｇ，１．６６ｍｍｏｌ）のクロロホルム（１０ｍＬ）溶液に、４−メチルベンゾイルクロリド（０．３１ｇ，１．９９ｍｍｏｌ）とトリエチルアミン（１ｍＬ）を加え、２日間撹拌した。反応終了後、反応液を水（１０ｍＬ）で洗浄し、有機層を無水硫酸マグネシウムで乾燥した後、乾燥剤を濾別し溶媒を減圧留去した。得られた混合物をシリカゲルカラムクロマトグラフィー（酢酸エチル−ヘキサン＝１：２）で精製し、４−メチル−Ｎ−［４−メチル−５−｛４−（フタルイミド）ブチル｝チアゾール−２−イル］ベンズアミドの白色固体（０．４０ｇ，５６％）を得た。ｍ．ｐ．１７８から１８０℃；^１Ｈ−ＮＭＲ（ＣＤＣｌ_３，ＴＭＳ，ｐｐｍ）：δ １．５８から１．６６（ｍ，４Ｈ），２．２１（ｓ，３Ｈ）， ２．４１（ｓ，３Ｈ），２．７３（ｔ，Ｊ＝７．４Ｈｚ，２Ｈ），３．７２（ｔ，Ｊ＝７．５Ｈｚ，２Ｈ），７．２６（ｄ，Ｊ＝７．５Ｈｚ，２Ｈ），７．７６から７．８６（ｍ，６Ｈ）。アミドのプロトンは帰属できなかった。

To a solution of 2-amino-4-methyl-5- {4- (phthalimido) butyl} thiazole (0.50 g, 1.66 mmol) in chloroform (10 mL) was added 4-methylbenzoyl chloride (0.31 g, 1.99 mmol). And triethylamine (1 mL) were added and stirred for 2 days. After completion of the reaction, the reaction solution was washed with water (10 mL), the organic layer was dried over anhydrous magnesium sulfate, the desiccant was filtered off, and the solvent was distilled off under reduced pressure. The resulting mixture was purified by silica gel column chromatography (ethyl acetate-hexane = 1: 2) to give 4-methyl-N- [4-methyl-5- {4- (phthalimido) butyl} thiazol-2-yl]. A white solid of benzamide (0.40 g, 56%) was obtained. m. p. 178 to 180 ° C .; ¹ H-NMR (CDCl ₃ , TMS, ppm): δ 1.58 to 1.66 (m, 4H), 2.21 (s, 3H), 2.41 (s, 3H), 2.73 (t, J = 7.4 Hz, 2H), 3.72 (t, J = 7.5 Hz, 2H), 7.26 (d, J = 7.5 Hz, 2H), 7.76 to 7 .86 (m, 6H). The proton of the amide could not be assigned.

  (Synthesis Example 3)

Ｎ−（５−ブロモ−６−オキソヘプチル）フタルイミド（１．３０ｇ，３．８５ｍｍｏｌ）のＤＭＦ（２０ｍＬ）溶液に、Ｎ−（３−アセトキシ−４−メチルベンゾイル）チオウレアを加え、８０℃で７時間反応させた。反応終了後、反応混合物に１Ｍ塩酸（３０ｍＬ）を加え酢酸エチル（５０ｍＬ）で二回抽出し、有機層を合わせて飽和食塩水（３０ｍＬ）で二回洗浄した。有機層を無水硫酸マグネシウムで乾燥した後、乾燥剤を濾別し溶媒を減圧留去した。得られた混合物を自動設定中圧カラムクロマトグラフィーシステム（山善社製；Ｒｆ＝０．３２：ヘキサン／酢酸エチル＝１／１）で精製し、得られた褐色固体を酢酸エチル／ヘキサンの混合溶媒で洗浄することによって、白色固体の３−アセトキシ−４−メチル−Ｎ−［４−メチル−５−｛４−（フタルイミド）ブチル｝チアゾール−２−イル］ベンズアミド（０．８２３ｇ，４９％）を得た。：ｍ．ｐ．１７０から１７２℃；^１Ｈ−ＮＭＲ（ＤＭＳＯ−ｄ_６，ＤＭＳＯ，ｐｐｍ）：δ １．６０から１．６６（ｍ，４Ｈ），２．２０（ｓ，３Ｈ），２．２１（ｓ，３Ｈ），２．３５（ｓ，３Ｈ），２．７２から２．７４（ｍ，２Ｈ），３．５３から３．６４（ｍ，２Ｈ），７．４５（ｄ，Ｊ＝７．５Ｈｚ，１Ｈ），７．７９から７．９３（ｍ，６Ｈ），１２．４（ｂｒ ｓ，１Ｈ）。

N- (3-acetoxy-4-methylbenzoyl) thiourea was added to a solution of N- (5-bromo-6-oxoheptyl) phthalimide (1.30 g, 3.85 mmol) in DMF (20 mL), and the mixture was stirred at 80 ° C. for 7 minutes. Reacted for hours. After completion of the reaction, 1M hydrochloric acid (30 mL) was added to the reaction mixture, and the mixture was extracted twice with ethyl acetate (50 mL). The organic layers were combined and washed twice with saturated brine (30 mL). The organic layer was dried over anhydrous magnesium sulfate, the desiccant was filtered off, and the solvent was distilled off under reduced pressure. The resulting mixture was purified by an automatic medium pressure column chromatography system (manufactured by Yamazen; Rf = 0.32: hexane / ethyl acetate = 1/1), and the resulting brown solid was mixed with ethyl acetate / hexane. To give white solid 3-acetoxy-4-methyl-N- [4-methyl-5- {4- (phthalimido) butyl} thiazol-2-yl] benzamide (0.823 g, 49%). Obtained. : M. p. 170 to 172 ° C .; ¹ H-NMR (DMSO-d ₆ , DMSO, ppm): δ 1.60 to 1.66 (m, 4H), 2.20 (s, 3H), 2.21 (s, 3H) ), 2.35 (s, 3H), 2.72 to 2.74 (m, 2H), 3.53 to 3.64 (m, 2H), 7.45 (d, J = 7.5 Hz, 1H) ), 7.79 to 7.93 (m, 6H), 12.4 (br s, 1H).

  (Synthesis Example 4)

モノテレフタル酸メチルクロリド（１．４１ｇ，７．１０ｍｍｏｌ）のクロロホルム（３０ｍＬ）溶液に、氷冷下にて２−アミノ−４−メチル−５−｛４−（フタルイミド）ブチル｝チアゾール（１．５６ｇ，４．７４ｍｍｏｌ）とトリエチルアミン（１．２０ｍＬ，７．９８ｍｍｏｌ）のクロロホルム（１０ｍＬ）溶液を加え、一晩反応させた。反応終了後、反応混合物を水（２０ｍＬ）、飽和食塩水（２０ｍＬ）で順次洗浄し、有機層を無水硫酸マグネシウムで乾燥した後、乾燥剤を濾別し溶媒を減圧留去した。得られた混合物を自動設定中圧カラムクロマトグラフィーシステム（山善社製；Ｒｆ＝０．１６：ヘキサン／酢酸エチル＝２／１）で精製し、４−メトキシカルボニル−Ｎ−［４−メチル−５−｛４−（フタルイミド）ブチル｝チアゾール−２−イル］ベンズアミドの白色固体（０．８１５ｇ，３３％）を得た。：ｍ．ｐ．１７３から１７４℃；^１Ｈ−ＮＭＲ（ＤＭＳＯ−ｄ_６，ＤＭＳＯ，ｐｐｍ）：δ １．６０から１．６７（ｍ，４Ｈ），２．２２（ｓ，３Ｈ），２．７２（ｔ，Ｊ＝７．５Ｈｚ，２Ｈ），３．６２（ｔ，Ｊ＝７．５Ｈｚ，２Ｈ），３．９０（ｄ，３Ｈ），７．８０から７．９０（ｍ，４Ｈ），８．０６から８．２０（ｍ，４Ｈ），１２．７（ｂｒ ｓ，１Ｈ）。

To a solution of methyl terephthalate methyl chloride (1.41 g, 7.10 mmol) in chloroform (30 mL) was added 2-amino-4-methyl-5- {4- (phthalimido) butyl} thiazole (1.56 g) under ice cooling. , 4.74 mmol) and triethylamine (1.20 mL, 7.98 mmol) in chloroform (10 mL) were added and allowed to react overnight. After completion of the reaction, the reaction mixture was washed successively with water (20 mL) and saturated brine (20 mL), the organic layer was dried over anhydrous magnesium sulfate, the desiccant was filtered off, and the solvent was evaporated under reduced pressure. The resulting mixture was purified by an automatic medium pressure column chromatography system (manufactured by Yamazen; Rf = 0.16: hexane / ethyl acetate = 2/1), and 4-methoxycarbonyl-N- [4-methyl-5. A white solid (0.815 g, 33%) of-{4- (phthalimido) butyl} thiazol-2-yl] benzamide was obtained. : M. p. ¹ H-NMR (DMSO-d ₆ , DMSO, ppm): δ 1.60 to 1.67 (m, 4H), 2.22 (s, 3H), 2.72 (t, J = 7.5 Hz, 2H), 3.62 (t, J = 7.5 Hz, 2H), 3.90 (d, 3H), 7.80 to 7.90 (m, 4H), 8.06 to 8 .20 (m, 4H), 12.7 (br s, 1H).

  (Synthesis Example 5)

２−アミノ−４−メチル−５−｛４−（フタルイミド）ブチル｝チアゾール（１．０ｇ，３．１７ｍｍｏｌ）のクロロホルム（１０ｍＬ）溶液に、３−シアノベンゾイルクロリド（０．７９ｇ，４．７５ｍｍｏｌ）を氷冷下にて加えた。次いでトリエチルアミン（０．７２ｍＬ，４．７９ｍｍｏｌ）のクロロホルム（１０ｍＬ）溶液を滴下し、一晩反応させた。反応終了後、反応混合物を水（２０ｍＬ）、飽和食塩水（２０ｍＬ）で順次洗浄し、有機層を無水硫酸マグネシウムで乾燥した後、乾燥剤を濾別し溶媒を減圧留去した。得られた混合物をシリカゲルカラムクロマトグラフィー（酢酸エチル／ヘキサン＝１／２）で精製し、３−シアノ−Ｎ−［４−メチル−５−｛４−（フタルイミド）ブチル｝チアゾール−２−イル］ベンズアミドの白色固体（０．５７ｇ，４０％）を得た。ｍ．ｐ．１７０から１７２℃；^１Ｈ−ＮＭＲ（ＣＤＣｌ_３，ＴＭＳ，ｐｐｍ）：δ １．６９から１．７０（ｍ，４Ｈ），１．９７（ｓ，３Ｈ），２．７４（ｔ，Ｊ＝６．５，２Ｈ），３．７３（ｔ，Ｊ＝６．８，２Ｈ），７．５６（ｄｄ，Ｊ＝８．０ ａｎｄ ８．０Ｈｚ，１Ｈ），７．７１から７．８６（ｍ，５Ｈ），８．０５（ｍ，１Ｈ），８．１６（ｓ，１Ｈ）。アミドのプロトンは帰属できなかった。

To a solution of 2-amino-4-methyl-5- {4- (phthalimido) butyl} thiazole (1.0 g, 3.17 mmol) in chloroform (10 mL) was added 3-cyanobenzoyl chloride (0.79 g, 4.75 mmol). Was added under ice cooling. Next, a solution of triethylamine (0.72 mL, 4.79 mmol) in chloroform (10 mL) was added dropwise and allowed to react overnight. After completion of the reaction, the reaction mixture was washed successively with water (20 mL) and saturated brine (20 mL), the organic layer was dried over anhydrous magnesium sulfate, the desiccant was filtered off, and the solvent was evaporated under reduced pressure. The resulting mixture was purified by silica gel column chromatography (ethyl acetate / hexane = 1/2), and 3-cyano-N- [4-methyl-5- {4- (phthalimido) butyl} thiazol-2-yl]. A white solid of benzamide (0.57 g, 40%) was obtained. m. p. 170 to 172 ° C .; ¹ H-NMR (CDCl ₃ , TMS, ppm): δ 1.69 to 1.70 (m, 4H), 1.97 (s, 3H), 2.74 (t, J = 6) .5, 2H), 3.73 (t, J = 6.8, 2H), 7.56 (dd, J = 8.0 and 8.0 Hz, 1H), 7.71 to 7.86 (m, 5H), 8.05 (m, 1H), 8.16 (s, 1H). The proton of the amide could not be assigned.

(Reference Example 1)
Under an argon atmosphere, 4-methyl-N- {5- (4-phthalimidobutyl) thiazol-2-yl} benzamide (0.105 g, 0.25 mmol) and ferrocene (0.023 g, 0.125 mmol) were added to DMSO (0.023 g, 0.125 mmol). 1.5 mL), 3M-trifluoromethyl iodide / DMSO solution (0.25 mL, 0.75 mmol), and 1M-sulfuric acid / DMSO solution (0.25 mL, 0.25 mmol) were added and stirred. To this solution, 31% hydrogen peroxide solution (75 μL, 0.75 mmol) was added dropwise at room temperature. After completion of the reaction, the reaction solution was poured into water (200 mL) and stirred for a while. After extracting with chloroform and drying the obtained organic layer with magnesium sulfate, the desiccant was filtered off and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography to give 4-methyl-N- {4-trifluoromethyl-5- (4-phthalimidobutyl) thiazol-2-yl} benzamide as a white solid (0.023 g, 19% ) ¹ H-NMR (CDCl ₃ , TMS, ppm): δ 1.79 to 1.82 (m, 4H), 2.47 (s, 3H), 3.01 (t, J = 7.5 Hz, 2H) , 3.76 (t, J = 7.5 Hz, 2H), 7.34 (d, J = 7.5 Hz, 1H), 7.72 to 7.90 (m, 6H), 9.37 (s, 1H); ¹⁹ F-NMR (CDCl ₃ , TMS, ppm): −60.87.

(Synthesis Examples 6 to 81)
In accordance with the method of Synthesis Example 1 or 2, thiazole derivatives described in Tables 1 to 12 were obtained.

（合成例８２）

(Synthesis Example 82)

４−メチル−Ｎ−｛５−（４−フタルイミドブチル）チアゾール−２−イル｝ベンズアミド（０．６０ｇ，１．４３ｍｍｏｌ）のエタノール（１０ｍＬ）溶液に、ヒドラジン一水和物（０．３５ｍＬ、７．１２ｍｍｏｌ）を室温にて加え、一晩撹拌した。反応終了後、反応溶液を減圧留去し、１Ｍ 塩酸（６ｍＬ）を加えた。析出した固体を濾別し、濾液を酢酸エチルで洗浄し、水層を減圧留去した。得られた固体にエタノールとトルエンを加え共沸し十分に乾燥し、固体を瀘取した。得られた固体を酢酸エチルにて洗浄することによって、４−｛２−（４−メチルベンゾイル）アミノチアゾール−５−イル｝ブチルアミン塩酸塩の白色固体（０．４４ｇ，収率：９５％）を得た。^１Ｈ−ＮＭＲ（ＤＭＳＯ−ｄ_６，ＤＭＳＯ，ｐｐｍ）：δ １．６４から１．６７（ｍ，４Ｈ），２．４０（ｓ，３Ｈ），２．７７から２．８０（ｍ，４Ｈ），７．３２（ｓ，１Ｈ），７．３５（ｄ，Ｊ＝７．５Ｈｚ，２Ｈ），８．０１（ｄ，Ｊ＝７．５Ｈｚ，２Ｈ），８．０７（ｂｒ ｓ，３Ｈ）。アミドのプロトンは帰属できなかった。

To a solution of 4-methyl-N- {5- (4-phthalimidobutyl) thiazol-2-yl} benzamide (0.60 g, 1.43 mmol) in ethanol (10 mL) was added hydrazine monohydrate (0.35 mL, 7 .12 mmol) was added at room temperature and stirred overnight. After completion of the reaction, the reaction solution was distilled off under reduced pressure, and 1M hydrochloric acid (6 mL) was added. The precipitated solid was separated by filtration, the filtrate was washed with ethyl acetate, and the aqueous layer was distilled off under reduced pressure. Ethanol and toluene were added to the obtained solid, azeotroped and dried sufficiently, and the solid was collected. The obtained solid was washed with ethyl acetate to obtain a white solid (0.44 g, yield: 95%) of 4- {2- (4-methylbenzoyl) aminothiazol-5-yl} butylamine hydrochloride. Obtained. ¹ H-NMR (DMSO-d ₆ , DMSO, ppm): δ 1.64 to 1.67 (m, 4H), 2.40 (s, 3H), 2.77 to 2.80 (m, 4H) 7.32 (s, 1H), 7.35 (d, J = 7.5 Hz, 2H), 8.01 (d, J = 7.5 Hz, 2H), 8.07 (brs, 3H). The proton of the amide could not be assigned.

  (Synthesis Example 83)

２−クロロ−Ｎ−［４−メチル−５−｛４−（フタルイミド）ブチル｝チアゾール−２−イル］ベンズアミド（０．２２ｇ，０．４８ｍｍｏｌ）のエタノール（５ｍＬ）溶液に、０．０５Ｍのヒドラジンエタノール溶液（３４ｍＬ）を室温にて加え、一晩撹拌した。反応終了後、反応溶液を減圧留去し、１Ｍ 塩酸（３ｍＬ）を加えた。析出した固体を濾別し、濾液を減圧留去した。得られた固体をエタノール並びに酢酸エチルを用いて固体を洗浄することにより、４−｛２−（２−クロロベンゾイルアミノ）−４−メチルチアゾール−５−イル｝ブチルアミン塩酸塩の褐色固体（０．０５０ｇ，収率：２１％）を得た。^１Ｈ−ＮＭＲ（ＤＭＳＯ−ｄ_６，ＤＭＳＯ，ｐｐｍ）：δ １．４２から１．７７（ｍ，４Ｈ），２．２１（ｓ，３Ｈ），２．６３から２．９２（ｍ，４Ｈ），７．３３から７．６７（ｍ，４Ｈ），７．６８から８．１６（ｍ，３Ｈ）。アミドのプロトンは帰属できなかった。

To a solution of 2-chloro-N- [4-methyl-5- {4- (phthalimido) butyl} thiazol-2-yl] benzamide (0.22 g, 0.48 mmol) in ethanol (5 mL), 0.05 M hydrazine Ethanol solution (34 mL) was added at room temperature and stirred overnight. After completion of the reaction, the reaction solution was distilled off under reduced pressure, and 1M hydrochloric acid (3 mL) was added. The precipitated solid was filtered off and the filtrate was distilled off under reduced pressure. The obtained solid was washed with ethanol and ethyl acetate to give a brown solid (0. 2- (2- (2-chlorobenzoylamino) -4-methylthiazol-5-yl} butylamine hydrochloride). 050 g, yield: 21%). ¹ H-NMR (DMSO-d ₆ , DMSO, ppm): δ 1.42 to 1.77 (m, 4H), 2.21 (s, 3H), 2.63 to 2.92 (m, 4H) , 7.33 to 7.67 (m, 4H), 7.68 to 8.16 (m, 3H). The proton of the amide could not be assigned.

  (Synthesis Example 84)

３，４−ジクロロ−Ｎ−［４−メチル−５−｛４−（フタルイミド）ブチル｝チアゾール−２−イル］ベンズアミド（０．２５ｇ，０．５１ｍｍｏｌ）のエタノール（５ｍＬ）溶液に、ヒドラジン一水和物（０．０７ｍＬ，１．５３ｍｍｏｌ）を室温にて加え、一晩撹拌した。反応終了後、反応溶液を留去し、１Ｍ 塩酸（３ｍＬ）を加え、室温で一晩撹拌した。析出した固体を濾別し、濾液を減圧留去した。得られた固体をエタノール並びに酢酸エチルを用いて洗浄することにより、４−｛２−（３，４−ジクロロベンゾイルアミノ）−４−メチルチアゾール−５−イル｝ブチルアミン塩酸塩の白色固体（０．１０ｇ， 収率：５０％）を得た。^１Ｈ−ＮＭＲ（ＤＭＳＯ−ｄ_６，ＤＭＳＯ，ｐｐｍ）：δ １．４６から１．７４（ｍ，４Ｈ），２．２４（ｓ，３Ｈ）， ２．６２から２．９１（ｍ，４Ｈ），７．８２（ｄ，Ｊ＝８．４Ｈｚ，１Ｈ），７．８５から７．９９（ｍ，３Ｈ），８．０３から８．１３（ｍ，１Ｈ），８．０４（ｄｄ，Ｊ＝２．０ ａｎｄ ８．４Ｈｚ，１Ｈ），８．３３（ｄ，Ｊ＝２．０Ｈｚ，１Ｈ）。

To a solution of 3,4-dichloro-N- [4-methyl-5- {4- (phthalimido) butyl} thiazol-2-yl] benzamide (0.25 g, 0.51 mmol) in ethanol (5 mL) was added hydrazine monohydrate. The Japanese product (0.07 mL, 1.53 mmol) was added at room temperature and stirred overnight. After completion of the reaction, the reaction solution was distilled off, 1M hydrochloric acid (3 mL) was added, and the mixture was stirred overnight at room temperature. The precipitated solid was filtered off and the filtrate was distilled off under reduced pressure. The obtained solid was washed with ethanol and ethyl acetate to give 4- {2- (3,4-dichlorobenzoylamino) -4-methylthiazol-5-yl} butylamine hydrochloride as a white solid (0. 10 g, yield: 50%). ¹ H-NMR (DMSO-d ₆ , DMSO, ppm): δ 1.46 to 1.74 (m, 4H), 2.24 (s, 3H), 2.62 to 2.91 (m, 4H) 7.82 (d, J = 8.4 Hz, 1H), 7.85 to 7.99 (m, 3H), 8.03 to 8.13 (m, 1H), 8.04 (dd, J = 2.0 and 8.4 Hz, 1H), 8.33 (d, J = 2.0 Hz, 1H).

  (Synthesis Example 85)

４−メチル−Ｎ−［４−メチル−５-｛４−（フタルイミド）ブチル｝チアゾール−２−イル］ベンズアミド（０．２３ｇ，０．５３ｍｍｏｌ）のエタノール（５ｍＬ）溶液に、ヒドラジン一水和物（０．２３ｍＬ，５．３０ｍｍｏｌ）を加え３時間還流した。反応終了後、反応溶液を留去し、残渣に１Ｍ 塩酸（５ｍＬ）を加え、撹拌した。析出した固体を濾別し、濾液を減圧留去した。得られた固体をエタノール並びに酢酸エチルを用いて洗浄することにより、４−｛４−メチル−２−（４−メチルベンゾイルアミノ）チアゾール−５−イル｝ブチルアミン塩酸塩の白色固体（０．０４３ｇ，収率：２４％）を得た。^１Ｈ−ＮＭＲ（ＤＭＳＯ−ｄ_６，ＤＭＳＯ，ｐｐｍ）：δ １．５５から１．８０（ｍ，４Ｈ），２．２４（ｓ，３Ｈ），２．３９（ｓ，３Ｈ），２．６４から２．９２（ｍ，４Ｈ），７．３４（ｄ，Ｊ＝８．２Ｈｚ，２Ｈ），７．７８から８．０５（ｍ，３Ｈ），７．９９（ｄ，Ｊ＝８．２Ｈｚ，２Ｈ）。アミドのプロトンは帰属できなかった。

Hydrazine monohydrate was added to a solution of 4-methyl-N- [4-methyl-5- {4- (phthalimido) butyl} thiazol-2-yl] benzamide (0.23 g, 0.53 mmol) in ethanol (5 mL). (0.23 mL, 5.30 mmol) was added and refluxed for 3 hours. After completion of the reaction, the reaction solution was distilled off, and 1M hydrochloric acid (5 mL) was added to the residue and stirred. The precipitated solid was filtered off and the filtrate was distilled off under reduced pressure. The obtained solid was washed with ethanol and ethyl acetate to give a white solid (0.043 g, 4- {4-methyl-2- (4-methylbenzoylamino) thiazol-5-yl} butylamine hydrochloride). Yield: 24%). ¹ H-NMR (DMSO-d ₆ , DMSO, ppm): δ 1.55 to 1.80 (m, 4H), 2.24 (s, 3H), 2.39 (s, 3H), 2.64 To 2.92 (m, 4H), 7.34 (d, J = 8.2 Hz, 2H), 7.78 to 8.05 (m, 3H), 7.99 (d, J = 8.2 Hz, 2H). The proton of the amide could not be assigned.

  (Synthesis Example 86)

４−トリフルオロメチル−Ｎ−［４−メチル−５−｛４−（フタルイミド）ブチル｝チアゾール−２−イル］ベンズアミド（０．３０ｇ，０．６２ｍｍｏｌ）のエタノール（５ｍＬ）溶液に、０．０５Ｍ ヒドラジンエタノール溶液（３０ｍＬ）を室温にて加え、２時間還流した。反応終了後、反応溶液を留去し、１Ｍ 塩酸（３ｍＬ）を加え、室温で２時間撹拌した。析出した固体を濾別し、濾液を減圧留去した。得られた固体をエタノール並びに酢酸エチルを用いて洗浄することにより、４−｛４−メチル−２−（４−トリフルオロメチルベンゾイルアミノ）チアゾール−５−イル｝ブチルアミン塩酸塩の白色固体（０．１０ｇ，収率：４１．７％）を得た。^１Ｈ−ＮＭＲ（ＤＭＳＯ−ｄ_６，ＤＭＳＯ，ｐｐｍ）：δ １．４９から１．７３（ｍ，４Ｈ），２．２４（ｓ，３Ｈ），２．６０から２．９０（ｍ，４Ｈ），７．９１（ｄ，Ｊ＝８．１Ｈｚ，２Ｈ），７．９７から８．１９（ｍ，３Ｈ），８．２６（ｄ，Ｊ＝８．１Ｈｚ，２Ｈ）。アミドのプロトンは帰属できなかった。

To a solution of 4-trifluoromethyl-N- [4-methyl-5- {4- (phthalimido) butyl} thiazol-2-yl] benzamide (0.30 g, 0.62 mmol) in ethanol (5 mL) was added 0.05M. A hydrazine ethanol solution (30 mL) was added at room temperature, and the mixture was refluxed for 2 hours. After completion of the reaction, the reaction solution was distilled off, 1M hydrochloric acid (3 mL) was added, and the mixture was stirred at room temperature for 2 hours. The precipitated solid was filtered off and the filtrate was distilled off under reduced pressure. The obtained solid was washed with ethanol and ethyl acetate to give a white solid (0. 4-methyl-4- (4-trifluoromethylbenzoylamino) thiazol-5-yl} butylamine hydrochloride). 10 g, yield: 41.7%). ¹ H-NMR (DMSO-d ₆ , DMSO, ppm): δ 1.49 to 1.73 (m, 4H), 2.24 (s, 3H), 2.60 to 2.90 (m, 4H) 7.91 (d, J = 8.1 Hz, 2H), 7.97 to 8.19 (m, 3H), 8.26 (d, J = 8.1 Hz, 2H). The proton of the amide could not be assigned.

  (Synthesis Example 87)

４−メトキシカルボニル−Ｎ−［４−メチル−５−｛４−（フタルイミド）ブチル｝チアゾール−２−イル］−ベンズアミド（０．５３５ｇ，１．１２ｍｍｏｌ）に０．５Ｍヒドラジンエタノール溶液（１３．５ｍＬ）を加え常温で一晩反応させた。反応終了後、溶液を減圧留去した。残渣に１Ｍ塩酸を加え超音波で懸濁し、セライトを敷いたガラスフィルターで固体を濾別した。濾液を減圧留去し固体を再度析出させ、少量の熱エタノールを加え洗浄することによって、４−［２−｛４−（メトキシカルボニル）ベンゾイルアミノ｝−４−メチルチアゾール−５−イル］ブチルアミン塩酸塩の白色固体（０．３６ｇ，収率：８４％）を得た。^１Ｈ−ＮＭＲ（ＤＭＳＯ−ｄ_６，ＤＭＳＯ，ｐｐｍ）：δ １．６０から１．７０（ｍ，４Ｈ），２．４２（ｓ，３Ｈ），２．７２から２．８０（ｍ，４Ｈ），３．９０（ｓ，３Ｈ），７．９７（ｂｒ ｓ，３Ｈ），８．０７から８．２１（ｍ，４Ｈ）。アミドのプロトンは帰属できなかった。

4-Methoxycarbonyl-N- [4-methyl-5- {4- (phthalimido) butyl} thiazol-2-yl] -benzamide (0.535 g, 1.12 mmol) in 0.5 M hydrazine ethanol solution (13.5 mL) ) And allowed to react overnight at room temperature. After completion of the reaction, the solution was distilled off under reduced pressure. 1M hydrochloric acid was added to the residue, and the mixture was suspended with ultrasound, and the solid was filtered off with a glass filter covered with celite. The filtrate was distilled off under reduced pressure and the solid was precipitated again, and washed with a small amount of hot ethanol to give 4- [2- {4- (methoxycarbonyl) benzoylamino} -4-methylthiazol-5-yl] butylamine hydrochloride. A white solid salt (0.36 g, yield: 84%) was obtained. ¹ H-NMR (DMSO-d ₆ , DMSO, ppm): δ 1.60 to 1.70 (m, 4H), 2.42 (s, 3H), 2.72 to 2.80 (m, 4H) 3.90 (s, 3H), 7.97 (br s, 3H), 8.07 to 8.21 (m, 4H). The proton of the amide could not be assigned.

  (Synthesis Example 88)

３−アセトキシ−４−メチル−Ｎ−［４−メチル−５−｛４−（フタルイミド）ブチル｝チアゾール−２−イル］ベンズアミド（０．８０ｇ，１．６２ｍｍｏｌ）に０．５Ｍヒドラジンエタノール溶液（１０ｍＬ）を加え常温で一晩反応させた。反応終了後、溶液を減圧留去した後、残渣に１Ｍ塩酸を加え超音波で懸濁し、セライトを敷いたガラスフィルターで固体を濾別した。濾液を減圧留去し固体を再度析出させ、少量の熱エタノールを加え洗浄することによって、４−｛２−（３−ヒドロキシ４−メチルベンゾイルアミノ）−４−メチルチアゾール−５−イル｝ブチルアミン塩酸塩の白色固体（０．４８ｇ，収率：７５％）を得た。^１Ｈ−ＮＭＲ（ＤＭＳＯ−ｄ_６，ＤＭＳＯ，ｐｐｍ）：δ １．６０から１．７０（ｍ，４Ｈ），２．１９（ｓ，３Ｈ），２．２３（ｓ，３Ｈ），２．７２から２．８１（ｍ，４Ｈ），７．２０（ｄ，Ｊ＝７．５Ｈｚ，１Ｈ），７．４４から７．５０（ｍ，２Ｈ），７．９４（ｂｒ ｓ，３Ｈ）９．８０（ｂｒ ｓ，１Ｈ）。アミドのプロトンは帰属できなかった。

3-acetoxy-4-methyl-N- [4-methyl-5- {4- (phthalimido) butyl} thiazol-2-yl] benzamide (0.80 g, 1.62 mmol) in 0.5 M hydrazine ethanol solution (10 mL ) And allowed to react overnight at room temperature. After completion of the reaction, the solution was evaporated under reduced pressure, 1M hydrochloric acid was added to the residue, and the mixture was suspended with ultrasound, and the solid was filtered off with a glass filter covered with celite. The filtrate was distilled off under reduced pressure, the solid was precipitated again, and washed with a small amount of hot ethanol to give 4- {2- (3-hydroxy-4-methylbenzoylamino) -4-methylthiazol-5-yl} butylamine hydrochloride A white solid salt (0.48 g, yield: 75%) was obtained. ¹ H-NMR (DMSO-d ₆ , DMSO, ppm): δ 1.60 to 1.70 (m, 4H), 2.19 (s, 3H), 2.23 (s, 3H), 2.72 To 2.81 (m, 4H), 7.20 (d, J = 7.5 Hz, 1H), 7.44 to 7.50 (m, 2H), 7.94 (br s, 3H) 9.80 (Br s, 1H). The proton of the amide could not be assigned.

  (Synthesis Example 89)

３−シアノ−Ｎ−［４−メチル−５−｛４−（フタルイミド）ブチル｝チアゾール−２−イル］ベンズアミドに０．５Ｍヒドラジンエタノール溶液（７ｍＬ）を加え常温で一晩反応させた。反応終了後、溶媒を減圧留去し、残渣に１Ｍ塩酸を加え超音波で懸濁し、セライトを敷いたガラスフィルターで濾過した。溶媒を減圧留去し、少量のエタノールを加え溶解した後、酢酸エチルで再沈殿させて精製し、４−｛２−（３−シアノベンゾイルアミノ）−４−メチルチアゾール−５−イル｝ブチルアミン塩酸塩の白色固体（０．２５ｇ，収率：４６％）を得た。^１Ｈ−ＮＭＲ（ＤＭＳＯ−ｄ_６，ＤＭＳＯ，ｐｐｍ）：δ １．６２から１．６５（ｍ，４Ｈ），２．２４（ｓ，３Ｈ），３．４３から３．４６（ｍ，４Ｈ），７．５６（ｄｄ，Ｊ＝８．０ ａｎｄ ８．０Ｈｚ，１Ｈ），８．０５（ｄ，Ｊ＝８．０Ｈｚ，１Ｈ），８．１０から８．２２（ｍ，４Ｈ），８．４９（ｓ，１Ｈ）。アミドのプロトンは帰属できなかった。

A 0.5 M hydrazine ethanol solution (7 mL) was added to 3-cyano-N- [4-methyl-5- {4- (phthalimido) butyl} thiazol-2-yl] benzamide and reacted at room temperature overnight. After completion of the reaction, the solvent was distilled off under reduced pressure, 1M hydrochloric acid was added to the residue, the mixture was suspended with ultrasound, and filtered through a glass filter with celite. The solvent was distilled off under reduced pressure, a small amount of ethanol was added and dissolved, and then purified by reprecipitation with ethyl acetate, and 4- {2- (3-cyanobenzoylamino) -4-methylthiazol-5-yl} butylamine hydrochloride A white solid salt (0.25 g, yield: 46%) was obtained. ¹ H-NMR (DMSO-d ₆ , DMSO, ppm): δ 1.62 to 1.65 (m, 4H), 2.24 (s, 3H), 3.43 to 3.46 (m, 4H) 7.56 (dd, J = 8.0 and 8.0 Hz, 1H), 8.05 (d, J = 8.0 Hz, 1H), 8.10 to 8.22 (m, 4H), 8. 49 (s, 1H). The proton of the amide could not be assigned.

(Synthesis Examples 90 to 179)
The thiazole derivatives described in Tables 13 to 26 were obtained according to the method of Synthesis Example 89.

合成例１から８１に例示した方法によって合成した本発明に関わるチアゾール誘導体（４）を表２７から３２にまとめて例示した。

Examples of thiazole derivatives (4) according to the present invention synthesized by the methods exemplified in Synthesis Examples 1 to 81 are summarized in Tables 27 to 32.

また、合成例８２から１７９に例示した方法によって合成した本発明に関わるチアゾール誘導体（１ａ）を表３３から４１にまとめて例示した。

In addition, the thiazole derivatives (1a) according to the present invention synthesized by the methods exemplified in Synthesis Examples 82 to 179 are collectively shown in Tables 33 to 41.

（参考例２）

(Reference Example 2)

化合物２７６（２．５０ｇ，８．６４ｍｍｏｌ）のクロロホルム（５０ｍＬ）溶液にトリエチルアミン（２．５０ｍＬ，１８ｍｍｏｌ）を添加した。次に氷冷下でメタクリル酸クロリド（０．９３ｍＬ，９．５ｍｍｏｌ）を滴下したのち、氷冷下で３０分反応させた。反応終了後、反応混合物にクロロホルム（５０ｍＬ）を追加し、水（１００ｍＬ）、飽和炭酸ナトリウム水溶液（１００ｍＬ）、飽和食塩水（１００ｍＬ）で順次洗浄した。有機層を無水硫酸マグネシウムで乾燥した後、乾燥剤を濾別し溶媒を減圧留去した。得られた混合物をシリカゲルクロマトグラフィー（酢酸エチル／ヘキサン＝１／２（ｖ／ｖ））で精製することで、淡黄色粘性液体の４−メチル−Ｎ−［４−メチル−５−｛３−（２−プロペニルカルボニルアミノ）プロピル｝チアゾール−２−イル］ベンズアミド（１．０７ｇ，３．０ｍｍｏｌ，収率：３５％）を得た。^１Ｈ−ＮＭＲ（ＤＭＳＯ−ｄ_６，ＤＭＳＯ，ｐｐｍ）：δ １．７０から１．８２（ｍ，２Ｈ），１．８７（ｓ，３Ｈ），２．２１（ｓ，３Ｈ），２．３９（ｓ，３Ｈ），２．６８（ｔ，Ｊ＝７．５Ｈｚ，２Ｈ），３．１０から３．２５（ｍ，２Ｈ），５．３２（ｓ，１Ｈ），５．６４（ｓ，１Ｈ），７．３４（ｄ，Ｊ＝８．５Ｈｚ，２Ｈ），７．９０（ｓ，１Ｈ），７．９８（ｄ，Ｊ＝８．５Ｈｚ，２Ｈ）。

Triethylamine (2.50 mL, 18 mmol) was added to a solution of compound 276 (2.50 g, 8.64 mmol) in chloroform (50 mL). Next, methacrylic acid chloride (0.93 mL, 9.5 mmol) was added dropwise under ice cooling, followed by reaction for 30 minutes under ice cooling. After completion of the reaction, chloroform (50 mL) was added to the reaction mixture, and the mixture was washed successively with water (100 mL), saturated aqueous sodium carbonate solution (100 mL), and saturated brine (100 mL). The organic layer was dried over anhydrous magnesium sulfate, the desiccant was filtered off, and the solvent was distilled off under reduced pressure. The obtained mixture was purified by silica gel chromatography (ethyl acetate / hexane = 1/2 (v / v)) to give 4-methyl-N- [4-methyl-5- {3- (2-propenylcarbonylamino) propyl} thiazol-2-yl] benzamide (1.07 g, 3.0 mmol, yield: 35%) was obtained. ¹ H-NMR (DMSO-d ₆ , DMSO, ppm): δ 1.70 to 1.82 (m, 2H), 1.87 (s, 3H), 2.21 (s, 3H), 2.39 (S, 3H), 2.68 (t, J = 7.5 Hz, 2H), 3.10 to 3.25 (m, 2H), 5.32 (s, 1H), 5.64 (s, 1H) ), 7.34 (d, J = 8.5 Hz, 2H), 7.90 (s, 1H), 7.98 (d, J = 8.5 Hz, 2H).

Example 2 Immobilization of thiazole derivative on agarose gel (Part 1)
Dissolve thiazole derivative in DMSO to 40 μmol / mL, add 0.2 M sodium carbonate buffer (pH 8.3) containing an equal amount of 0.5 M sodium chloride to obtain a thiazole derivative solution with a concentration of 20 μmol / mL. Prepared. Non-patent to HiTrap NHS-activated HP 1 mL (trade name) (manufactured by GE Healthcare Biosciences; hereinafter abbreviated as HiTrap column), which is an agarose gel activated with N-hydroxysuccinimide (NHS). Immobilization was carried out by passing 2 mL of the thiazole derivative solution according to the method described in Document 3. Unreacted succinimideoxycarbonyl group was blocked according to the method described in Non-Patent Document 3. As for the amount of immobilization, according to “B. Acidic condition method” among the methods described in Non-Patent Document 3, the absorbance at the maximum absorption wavelength (around 300 nm) of the ligand (thiazole derivative) solution before and after passing through the HiTrap column was measured as U−. It was calculated by measuring with a 2900 spectrophotometer (manufactured by Hitachi, Ltd.). Table 42 shows the immobilized ligand density of each compound immobilized by this method. In addition, the compound numbers described in the examples and thereafter correspond to the compound numbers described in Tables 33 to 41.

実施例３ チアゾール誘導体のアガロースゲルへの固定化（その２）
チアゾール誘導体を２０μｍｏｌ／ｍＬ、トリエチルアミンを４０μｍｏｌ／ｍＬ含むＤＭＳＯ溶液を調製した。ＨｉＴｒａｐカラムをＤＭＳＯで置換後、これに前記チアゾール誘導体−トリエチルアミンのＤＭＳＯ溶液を２ｍＬ通液した。一時間放置した後、ＨｉＴｒａｐカラムに３ｍＬのＤＭＳＯを通液して、さらに５ｍＬの０．５Ｍ 塩化ナトリウムを含む０．２Ｍ 炭酸ナトリウム緩衝液（ｐＨ８．３）を通液した。未反応のスクシンイミドオキシカルボニル基のブロッキングは、非特許文献３に記載された方法に従って行なった。固定化量は非特許文献３に記載された方法のうち「Ｂ．酸性条件法」に従って、ＨｉＴｒａｐカラム通液前後のリガンド（チアゾール誘導体）溶液の紫外吸収極大波長（３００ｎｍ付近）における吸光度をＵ−２９００スペクトロフォトメーター（日立製作所製）で測定することにより算出した。当該方法で固定化した化合物３０４の固定化リガンド密度を表４３に示す。

Example 3 Immobilization of thiazole derivative on agarose gel (Part 2)
A DMSO solution containing a thiazole derivative at 20 μmol / mL and triethylamine at 40 μmol / mL was prepared. After replacing the HiTrap column with DMSO, 2 mL of the above-mentioned thiazole derivative-triethylamine in DMSO was passed through it. After standing for 1 hour, 3 mL of DMSO was passed through the HiTrap column, and then 0.2 mL of sodium carbonate buffer (pH 8.3) containing 5 mL of 0.5 M sodium chloride was passed. Unreacted succinimideoxycarbonyl group was blocked according to the method described in Non-Patent Document 3. As for the amount of immobilization, according to “B. Acidic condition method” among the methods described in Non-Patent Document 3, the absorbance at the maximum absorption wavelength (around 300 nm) of the ligand (thiazole derivative) solution before and after passing through the HiTrap column was measured as U−. It was calculated by measuring with a 2900 spectrophotometer (manufactured by Hitachi, Ltd.). Table 43 shows the immobilized ligand density of Compound 304 immobilized by this method.

実施例４ チアゾール誘導体固定化アガロースゲルを用いたタンパク質の吸脱着（その１）
実施例２あるいは３に記載の方法にて調製したチアゾール誘導体固定化ＨｉＴｒａｐカラムをＡＫＴＡｐｒｉｍｅ ｐｌｕｓ（商品名）（ＧＥヘルスケアバイオサイエンス株式会社製クロマトグラフィー装置）に取り付け、０．７Ｍ 硫酸ナトリウムを含む１０ｍＭ リン酸ナトリウム−１０ｍＭ クエン酸ナトリウム緩衝液（ｐＨ７．５）（以下平衡化緩衝液と呼ぶ）を２０ｍＬ通液し平衡化した。ＡＫＴＡｐｒｉｍｅ ｐｌｕｓを流速１ｍＬ／ｍｉｎで自動運転し、平衡化緩衝液を１０ｍＬ通液後、同緩衝液に溶解したヒト血漿由来免疫グロブリン製剤（化血研製、本製剤の免疫グロブリンは免疫グロブリンＧである）０．５ｍｇ（ＯＤ_２８０＝０．７）を添加してカラムに通液した。さらに平衡化緩衝液を１０ｍＬ通液後、平衡化緩衝液から１０ｍＭ リン酸ナトリウム−１０ｍＭ クエン酸ナトリウム緩衝液（ｐＨ７．５）への硫酸ナトリウムに関する直線濃度勾配クロマトグラフィーを行なった。免疫グロブリンのカラムからの溶出は２８０ｎｍにおける吸光度をモニターして検出した。溶出液は１ｍＬずつ分画し、各フラクションの２８０ｎｍにおける吸光度をＵ−２９００スペクトロフォトメーター（日立製作所製）で測定し回収率を算出した。チアゾール誘導体を固定化したＨｉＴｒａｐカラムを用いて行なったクロマトグラフィーの結果を図１から図６に示し、これらのクロマトグラフィーにおける免疫グロブリンの回収率を表４４に示す。いずれのチアゾール誘導体を用いたときも免疫グロブリンは５０％以上の回収率で回収され、チアゾール誘導体固定化ＨｉＴｒａｐカラムによる免疫グロブリンの吸脱着が確認された。特に化合物２８６、３０４、３２９を用いた場合の回収率は９０％以上と極めて高かった。

Example 4 Adsorption / desorption of protein using agarose gel immobilized with thiazole derivative (Part 1)
The thiazole derivative-immobilized HiTrap column prepared by the method described in Example 2 or 3 was attached to AKTAprime plus (trade name) (a chromatography device manufactured by GE Healthcare Biosciences), and 10 mM containing 0.7 M sodium sulfate. 20 mL of sodium phosphate-10 mM sodium citrate buffer (pH 7.5) (hereinafter referred to as equilibration buffer) was passed through to equilibrate. AKTAprime plus is automatically operated at a flow rate of 1 mL / min, equilibrated buffer solution is passed through 10 mL, and human plasma-derived immunoglobulin preparation dissolved in the same buffer solution (manufactured by Kakenken, the immunoglobulin of this preparation is immunoglobulin G) ) 0.5 mg (OD ₂₈₀ = 0.7) was added and passed through the column. Further, after 10 mL of the equilibration buffer was passed, linear concentration gradient chromatography on sodium sulfate from the equilibration buffer to 10 mM sodium phosphate-10 mM sodium citrate buffer (pH 7.5) was performed. Elution of the immunoglobulin from the column was detected by monitoring the absorbance at 280 nm. The eluate was fractionated by 1 mL, and the absorbance at 280 nm of each fraction was measured with a U-2900 spectrophotometer (manufactured by Hitachi, Ltd.) to calculate the recovery rate. The results of chromatography performed using a HiTrap column immobilized with a thiazole derivative are shown in FIG. 1 to FIG. 6, and the immunoglobulin recovery rate in these chromatography is shown in Table 44. When any thiazole derivative was used, the immunoglobulin was recovered at a recovery rate of 50% or more, and the adsorption / desorption of the immunoglobulin by the thiazole derivative-immobilized HiTrap column was confirmed. In particular, the recovery rate when using compounds 286, 304, and 329 was as extremely high as 90% or more.

それぞれの吸脱着操作後には１００ｍＭ 水酸化ナトリウム水溶液を１０ｍＬ通液してカラムの再生を行なった。前記のチアゾール誘導体固定化ＨｉＴｒａｐカラムはいずれもこの再生処理によって初期状態に復帰させることができ、繰り返し使用によってもその特性は変化しなかった。

After each adsorption / desorption operation, the column was regenerated by passing 10 mL of a 100 mM aqueous sodium hydroxide solution. Any of the above-mentioned thiazole derivative-immobilized HiTrap columns could be restored to the initial state by this regeneration treatment, and the characteristics were not changed by repeated use.

Example 5 Adsorption / desorption of protein using agarose gel immobilized with thiazole derivative (Part 2)
The Thiazole derivative-immobilized HiTrap column prepared by the method described in Example 2 or 3 was attached to AKTAprime plus (trade name) (a chromatography apparatus manufactured by GE Healthcare Biosciences), and 20 mL of the equilibration buffer was passed through. Liquid and equilibrate. AKTA prime plus is automatically operated at a flow rate of 1 mL / min, and after 10 mL of equilibration buffer is passed, 0.5 mg (OD ₂₈₀ = 0.3) of bovine serum albumin (manufactured by SIGMA) dissolved in the same buffer is added. And passed through the column. Further, after 10 mL of the equilibration buffer was passed, linear concentration gradient chromatography on sodium sulfate from the equilibration buffer to 10 mM sodium phosphate-10 mM sodium citrate buffer (pH 7.5) was performed. The elution of bovine serum albumin from the column was detected by monitoring the absorbance at 280 nm. The eluate was fractionated by 1 mL, and the absorbance at 280 nm of each fraction was measured with a U-2900 spectrophotometer (manufactured by Hitachi, Ltd.) to calculate the recovery rate. The results of chromatography performed using HiTrap columns with various thiazole derivatives immobilized are shown in FIGS. In addition, Table 45 shows the recovery rates of bovine serum albumin in these chromatography. When any thiazole derivative was used, bovine serum albumin was eluted by the 10th fraction, and almost no adsorption was observed on the column. Also, the results shown in FIGS. 1 to 6 were significantly different from the results obtained when the immunoglobulin was passed. Therefore, it was shown that the use of the thiazole derivative-immobilized matrix of the present invention enables separation of immunoglobulins and bovine serum albumin in a sample.

それぞれの吸脱着操作後には１００ｍＭ 水酸化ナトリウム水溶液を１０ｍＬ通液してカラムの再生を行なった。前記のチアゾール誘導体を固定化ＨｉＴｒａｐカラムはいずれもこの再生処理によって初期状態に復帰させることができ、繰り返し使用によってもその特性は変化しなかった。

After each adsorption / desorption operation, the column was regenerated by passing 10 mL of a 100 mM aqueous sodium hydroxide solution. Any HiTrap column immobilized with the thiazole derivative could be restored to its initial state by this regeneration treatment, and its characteristics did not change even after repeated use.

Example 6 Adsorption / desorption of protein using thiazole derivative-immobilized agarose gel (Part 3)
A HiTrap column on which compound 286 prepared by the method described in Example 2 was immobilized was attached to AKTAprime plus (trade name) (a chromatography device manufactured by GE Healthcare Biosciences), and 20 mL of the equilibration buffer was passed through. Liquid and equilibrate. AKTAprime plus was automatically operated at a flow rate of 1 mL / min, and 10 mL of equilibration buffer was passed through, followed by a mixture of ribonuclease A, lysozyme, α chymotrypsinogen A dissolved in the same buffer (ribonuclease A 0.15 mg, lysozyme 0.03 mg, α chymotrypsinogen A 0.04 mg) (OD ₂₈₀ = 0.24) was added and passed through the column. Further, after 10 mL of the equilibration buffer was passed, linear concentration gradient chromatography on sodium sulfate from the equilibration buffer to 10 mM sodium phosphate-10 mM sodium citrate buffer (pH 7.5) was performed. Protein elution from the column was detected by monitoring absorbance at 280 nm. The eluate was fractionated by 1 mL, and the absorbance at 280 nm of each fraction was measured with a U-2900 spectrophotometer (manufactured by Hitachi, Ltd.) to calculate the recovery rate. The results of chromatography performed using a HiTrap column with compound 286 immobilized thereon are shown in FIG. 13 and the protein recovery rate in this chromatography is shown in Table 46. All the proteins were eluted within the 10th fraction, which was a fraction different from that of immunoglobulin (eluted in the 25th to 45th fractions, see FIG. 1). From this, it was shown that the use of the thiazole derivative-immobilized matrix of the present invention makes it possible to separate the immunoglobulin in the sample from ribonuclease A, lysozyme, and α-chymotrypsinogen A.

吸脱着操作後には１００ｍＭ 水酸化ナトリウム水溶液を１０ｍＬ通液してカラムの再生を行なった。前記のチアゾール誘導体固定化ＨｉＴｒａｐカラムはこの再生処理によって初期状態に復帰させることができ、繰り返し使用によってもその特性は変化しなかった。

After the adsorption / desorption operation, the column was regenerated by passing 10 mL of a 100 mM sodium hydroxide aqueous solution. The above-mentioned thiazole derivative-immobilized HiTrap column could be restored to its initial state by this regeneration treatment, and its characteristics did not change even after repeated use.

Example 7 Adsorption / desorption of protein using agarose gel immobilized with thiazole derivative (Part 4)
Rituxan (trade name) (manufactured by Chugai Pharmaceutical Co., Ltd., 10 mg / mL), which is a human-mouse chimeric antibody, was immobilized at 37 ° C. using the immobilized papain (Immobilized Papain, manufactured by PIERCE) at 10 ° C. Limited hydrolyzate was obtained by time treatment.

A HiTrap column on which compound 286 prepared by the method described in Example 2 was immobilized was attached to AKTAprime plus (trade name) (a chromatography device manufactured by GE Healthcare Biosciences), and 20 mL of the equilibration buffer was passed through. Liquid and equilibrate. AKTAprime plus was automatically operated at a flow rate of 1 mL / min, 10 mL of equilibration buffer was passed, 1.2 mg (OD ₂₈₀ = 1.7) of the papain-treated antibody was added, and passed through the column. Further, after 10 mL of the equilibration buffer was passed, linear concentration gradient chromatography on sodium sulfate from the equilibration buffer to 10 mM sodium phosphate-10 mM sodium citrate buffer (pH 7.5) was performed. The elution of the protein from the column was detected by monitoring the absorbance at 280 nm, and the eluate was fractionated by 1 mL. The result of this chromatography is shown in FIG. In addition, 5 μL of each of the seventh, 29th, 39th, and 43rd fractions (represented as I, II, III, and IV, respectively) was subjected to SDS-PAGE containing 15% acrylamide under reducing conditions to obtain a silver staining reagent (silver staining It dye | stained using "1st" (brand name), the Dai-ichi Chemical Co., Ltd. product (FIG. 15). Thus, I is Fab (composed of 27 kDa protein derived from H chain and 27 kDa protein which is L chain), II is Fc (composed of dimer of 30 kDa protein derived from H chain), and III is partial digestion An antibody (consisting of a 60 kDa protein that is an H chain, a 30 kDa protein derived from the H chain, and a 27 kDa protein that is an L chain), and an IV is an undigested antibody (a dimer of an 60 kDa protein that is an H chain and an 27 kDa protein that is an L chain) It was shown that each fragment derived from an antibody can be separated.

Example 8 Immobilization of thiazole derivative on vinyl polymer gel (Part 1)
Compound 286 (102 mg, 0.3 mmol) was weighed into a centrifuge tube, and 0.3 mL of acetonitrile solution (1 mmol / mL) of 1,2-dimethoxybenzene was added. Next, acetonitrile (1.7 mL), buffer (composition: 0.2 M NaHCO ₃ , 0.5 M NaCl, pH 8.3; 8 mL), triethylamine (90 μL, 0.6 mmol) were added to the centrifuge tube to obtain compound 286. After confirming dissolution, 0.3 g of Epoxy Toyopearl (trade name) gel (manufactured by Tosoh Corporation, epoxy group content: 804 μmol / g (gel)) was added. The centrifuge tube containing the mixture obtained by the above operation was set on a desktop shaker set at an internal temperature of 40 ° C., and stirred and shaken at 165 rpm for 16 hours. After completion of the reaction, acetonitrile (5 mL) was added to the centrifuge tube, and Toyopearl gel and the reaction solution were separated with a glass filter. Next, the Toyopearl gel on the glass filter was washed twice with a washing solution (composition: CH ₃ CN / MeOH / 50 mM NH ₄ H ₂ PO ₄ = 4/1/5; 10 mL). The combined reaction solution and washing solution, high performance liquid chromatography (Column: manufactured by Tosoh Corporation TSK-gel ODS-80T _M (trade name) (inner diameter 4.6 mm × length 15cm), _eluent: CH 3 CN / MeOH / 50mM NH ₄ H ₂ PO ₄ = 4/1/5, flow rate: 1 mL / min, temperature: 25 ° C., detection: UV 254 nm), by quantifying the remaining compound 286, the compound 286 was 127 μmol / It was confirmed that mL (gel) was immobilized. Furthermore, in order to block unreacted epoxy groups in the Toyopearl gel obtained by the above operation, the obtained Toyopearl gel was placed in a centrifuge tube and a blocking solution (composition: 0.5 M monoethanolamine, 0.5 M sodium chloride, pH 8). .3 (solvent is water); 5 mL) was added. The centrifuge tube containing the reaction solution was set on a desktop shaker set to an internal temperature of 40 ° C., and the remaining epoxy groups in the gel were blocked with monoethanolamine by stirring and shaking at 165 rpm for 16 hours. After completion of the reaction, acetonitrile (5 mL) was added to the centrifuge tube, and Toyopearl gel and the blocking solution were separated with a glass filter. Subsequently, the gel was washed twice with ethanol (5 mL) and water (5 mL), respectively, to obtain an epoxy toyopearl gel in which compound 286 was immobilized. Further, when the compound 286 remaining in the solution obtained by combining the blocking solution and the washing solution was measured by high performance liquid chromatography, it was confirmed that the compound 286 did not remain in the solution obtained by combining the blocking solution and the washing solution.

Example 9 Immobilization of thiazole derivative on vinyl polymer gel (Part 2)
Compound 329 (107 mg, 0.3 mmol) was weighed into a centrifuge tube, and 0.3 mL of acetonitrile solution (1 mmol / mL) of 1,2-dimethoxybenzene was added. Next, acetonitrile (1.7 mL), 0.5 M aqueous sodium hydroxide solution (2.4 mL), and water (5.6 mL) were added to the centrifuge tube to confirm that compound 329 was dissolved, and then 0.3 g of Epoxy Toyopearl (trade name) gel (manufactured by Tosoh Corporation, epoxy group content: 804 μmol / g (gel)) was added. The centrifuge tube containing the mixture obtained by the above operation was set on a desktop shaker set at an internal temperature of 40 ° C., and stirred and shaken at 165 rpm for 24 hours. After completion of the reaction, acetonitrile (5 mL) was added to the centrifuge tube, and Toyopearl gel and the reaction solution were separated with a glass filter. Next, the Toyopearl gel on the glass filter was washed twice with a washing solution (composition: CH ₃ CN / MeOH / 50 mM NH ₄ H ₂ PO ₄ = 4/1/5; 10 mL). The combined reaction solution and washing solution, high performance liquid chromatography (Column: manufactured by Tosoh Corporation TSK-gel ODS-80T _M (internal diameter 4.6 mm × length 15cm), _{eluent: CH 3 CN / MeOH / 50mM} NH 4 H 2 By quantifying the remaining compound 329 at PO ₄ = 4/1/5, flow rate: 1 mL / min, temperature: 25 ° C., detection: UV 254 nm), compound 329 was found to be 81 μmol / mL (gel) in epoxy toyopearl. It was confirmed that it was immobilized. Furthermore, in order to block unreacted epoxy groups in the Toyopearl gel obtained by the above operation, the Toyopearl gel obtained was placed in a centrifuge tube and a blocking solution (composition: 0.5 M monoethanolamine, 0.5 M sodium chloride aqueous solution, pH 8.3 (solvent is water); 5 mL) was added. The centrifuge tube containing the reaction solution was set on a desktop shaker set at an internal temperature of 40 ° C., and the remaining epoxy groups in the gel were blocked with monoethanolamine by stirring and shaking at 165 rpm for 20 hours. After completion of the reaction, acetonitrile (5 mL) was added to the centrifuge tube, and Toyopearl gel and the blocking solution were separated with a glass filter. Subsequently, the gel was washed twice with ethanol (5 mL) and water (5 mL), respectively, to obtain an epoxy toyopearl gel in which compound 329 was immobilized. Moreover, when the compound 329 which remains in the solution which combined the blocking solution and the washing | cleaning liquid was measured by the high performance liquid chromatography, it was confirmed that the compound 329 does not remain in the solution which combined the blocking solution and the washing | cleaning liquid.

Example 10 Immobilization of thiazole derivative on vinyl polymer gel (Part 3)
Compound 286 (30 mg, 88 μmol) was weighed into a centrifuge tube, and 0.1 mL of acetonitrile solution (1 mmol / mL) of 1,2-dimethoxybenzene was added. Next, acetonitrile (2.4 mL), buffer (composition: 0.1 M Na ₂ HPO ₄ —NaH ₂ PO ₄ , pH 7.0; 2.5 mL), triethylamine (30 μL, 0.2 mmol) were added to the centrifuge tube. After confirming that compound 286 was dissolved, 3 mL of formyl toyopearl (trade name) gel (manufactured by Tosoh Corporation, formyl group content: 65 μmol / g (gel)) was added. The centrifuge tube containing the mixture obtained by the above operation was set on a desktop shaker set at an internal temperature of 25 ° C., and stirred and shaken at 165 rpm. 30 minutes after the start of stirring and shaking, borane-pyridine complex (250 μL, 2.5 mmol) was added to the centrifuge tube, and the mixture was further stirred and shaken at 165 rpm for 2.5 hours. After completion of the reaction, acetonitrile (5 mL) was added to the centrifuge tube, and Toyopearl gel and the reaction solution were separated with a glass filter. Next, the Toyopearl gel on the glass filter was washed twice with a washing solution (composition: CH ₃ CN / MeOH / 50 mM NH ₄ H ₂ PO ₄ = 4/1/5; 10 mL). The combined reaction solution and washing solution, high performance liquid chromatography (Column: manufactured by Tosoh Corporation TSK-gel ODS-80T _M (trade name) (inner diameter 4.6 mm × length 15cm), _eluent: CH 3 CN / MeOH / 50mM NH ₄ H ₂ PO ₄ = 4/1/5, flow rate: 1 mL / min, temperature: 25 ° C., detection: UV 254 nm), by quantifying the remaining compound 286, compound 286 was 19 μmol / mL (gel) An immobilized formyl toyopearl gel was obtained.

Example 11 Immobilization of thiazole derivative on vinyl polymer gel (Part 4)
Compound 320 (111 mg, 0.3 mmol) was weighed into a centrifuge tube, and 0.3 mL of acetonitrile solution (1 mmol / mL) of 1,2-dimethoxybenzene was added. Next, acetonitrile (1.7 mL), 0.5 M aqueous sodium hydroxide solution (0.6 mL), and water (7.4 mL) were added to the centrifuge tube to confirm that compound 320 was dissolved, and then 0.3 g of Epoxy Toyopearl (trade name) gel (manufactured by Tosoh Corporation, epoxy group content: 804 μmol / g (gel)) was added. The centrifuge tube containing the mixture obtained by the above operation was set on a desktop shaker set at an internal temperature of 40 ° C., and stirred and shaken at 165 rpm for 24 hours. After completion of the reaction, acetonitrile (5 mL) was added to the centrifuge tube, and Toyopearl gel and the reaction solution were separated with a glass filter. Next, the Toyopearl gel on the glass filter was washed twice with a washing solution (composition: CH ₃ CN / MeOH / 50 mM NH ₄ H ₂ PO ₄ = 4/1/5; 10 mL). The combined reaction solution and washing solution, high performance liquid chromatography (Column: manufactured by Tosoh Corporation TSK-gel ODS-80T _M (trade name) (inner diameter 4.6 mm × length 15cm), _eluent: CH 3 CN / MeOH / 50mM NH ₄ H ₂ PO ₄ = 4/1/5, flow rate: 1 mL / min, temperature: 25 ° C., detection: UV254 nm), by quantifying the remaining compound 320, the compound 320 was 176 μmol / mL in Epoxy Toyopearl. (Gel) It was confirmed to be immobilized. Furthermore, in order to block unreacted epoxy groups in the Toyopearl gel obtained by the above operation, the Toyopearl gel obtained was placed in a centrifuge tube and a blocking solution (composition: 0.5M monoethanolamine, 0.5M aqueous sodium chloride solution ( The solvent was water), pH 8.3; 5 mL). The centrifuge tube containing the reaction solution was set on a desktop shaker set at an internal temperature of 40 ° C., and the remaining epoxy groups in the gel were blocked with monoethanolamine by stirring and shaking at 165 rpm for 20 hours. After completion of the reaction, acetonitrile (5 mL) was added to the centrifuge tube, and Toyopearl gel and the blocking solution were separated with a glass filter. Subsequently, the gel was washed twice with ethanol (5 mL) and water (5 mL), respectively, to obtain an epoxy toyopearl gel in which compound 320 was immobilized. Moreover, when the compound 320 which remains in the solution which combined the blocking solution and the washing | cleaning liquid was measured by the high performance liquid chromatography, it was confirmed that the compound 320 does not remain in the solution which combined the blocking solution and the washing | cleaning liquid.

Example 12 Immobilization of thiazole derivative on vinyl polymer gel (Part 5)
Compound 384 (112 mg, 0.3 mmol) was weighed into a centrifuge tube, and 0.3 mL of acetonitrile solution (1 mmol / mL) of 1,2-dimethoxybenzene was added. Next, acetonitrile (1.7 mL), 0.5 M aqueous sodium hydroxide solution (0.9 mL), and water (7.1 mL) were added to the centrifuge tube to confirm that compound 384 was dissolved, and then 0.3 g of Epoxy Toyopearl (trade name) gel (manufactured by Tosoh Corporation, epoxy group content: 804 μmol / g (gel)) was added. The centrifuge tube containing the mixture obtained by the above operation was set on a desktop shaker set at an internal temperature of 40 ° C., and stirred and shaken at 165 rpm for 24 hours. After completion of the reaction, acetonitrile (5 mL) was added to the centrifuge tube, and Toyopearl gel and the reaction solution were separated with a glass filter. Next, the Toyopearl gel on the glass filter was washed twice with a washing solution (composition: CH ₃ CN / MeOH / 50 mM NH ₄ H ₂ PO ₄ = 4/1/5; 10 mL). The combined reaction solution and washing solution, high performance liquid chromatography (Column: manufactured by Tosoh Corporation TSK-gel ODS-80T _M (trade name) (inner diameter 4.6 mm × length 15cm), _eluent: CH 3 CN / MeOH / 50mM NH ₄ H ₂ PO ₄ = 4/1/5, flow rate: 1 mL / min, temperature: 25 ° C., detection: UV 254 nm), by quantifying the remaining compound 384, the compound 384 was 126 μmol / It was confirmed that mL (gel) was immobilized. Furthermore, in order to block unreacted epoxy groups in the Toyopearl gel obtained by the above operation, the Toyopearl gel obtained was placed in a centrifuge tube and a blocking solution (composition: 0.5 M monoethanolamine, 0.5 M sodium chloride aqueous solution, pH 8.3 (solvent is water); 5 mL) was added. The centrifuge tube containing the reaction solution was set on a desktop shaker set at an internal temperature of 40 ° C., and the remaining epoxy groups in the gel were blocked with monoethanolamine by stirring and shaking at 165 rpm for 20 hours. After completion of the reaction, acetonitrile (5 mL) was added to the centrifuge tube, and Toyopearl gel and the blocking solution were separated with a glass filter. Subsequently, the gel was washed twice with ethanol (5 mL) and water (5 mL), respectively, to obtain an epoxy toyopearl gel in which compound 384 was immobilized. Moreover, when the compound 384 which remains in the solution which combined the blocking solution and the washing | cleaning liquid was measured by the high performance liquid chromatography, it was confirmed that the compound 384 does not remain in the solution which combined the blocking solution and the washing | cleaning liquid.

Example 13 Immobilization of thiazole derivative on vinyl polymer gel (Part 6)
Compound 423 (111 mg, 0.3 mmol) was weighed into a centrifuge tube, and 0.3 mL of acetonitrile solution (1 mmol / mL) of 1,2-dimethoxybenzene was added. Next, acetonitrile (1.7 mL), 0.5 M aqueous sodium hydroxide solution (0.9 mL), and water (7.1 mL) were added to the centrifuge tube to confirm that compound 423 was dissolved, and then 0.3 g of Epoxy Toyopearl (trade name) gel (manufactured by Tosoh Corporation, epoxy group content: 804 μmol / g (gel)) was added. The centrifuge tube containing the mixture obtained by the above operation was set on a desktop shaker set at an internal temperature of 40 ° C., and stirred and shaken at 165 rpm for 24 hours. After completion of the reaction, acetonitrile (5 mL) was added to the centrifuge tube, and Toyopearl gel and the reaction solution were separated with a glass filter. Next, the Toyopearl gel on the glass filter was washed twice with a washing solution (composition: CH ₃ CN / MeOH / 50 mM NH ₄ H ₂ PO ₄ = 4/1/5; 10 mL). The combined reaction solution and washing solution, high performance liquid chromatography (Column: manufactured by Tosoh Corporation TSK-gel ODS-80T _M (trade name) (inner diameter 4.6 mm × length 15cm), _eluent: CH 3 CN / MeOH / 50mM NH ₄ H ₂ PO ₄ = 4/1/5, flow rate: 1 mL / min, temperature: 25 ° C., detection: UV 254 nm), by quantifying remaining compound 423, compound 423 was found to be 114 μmol / epoxy toyopearl. It was confirmed that mL (gel) was immobilized. Furthermore, in order to block unreacted epoxy groups in the Toyopearl gel obtained by the above operation, the Toyopearl gel obtained was placed in a centrifuge tube and a blocking solution (composition: 0.5 M monoethanolamine, 0.5 M sodium chloride aqueous solution, pH 8.3 (solvent is water); 5 mL) was added. The centrifuge tube containing the reaction solution was set on a desktop shaker set at an internal temperature of 40 ° C., and the remaining epoxy groups in the gel were blocked with monoethanolamine by stirring and shaking at 165 rpm for 20 hours. After completion of the reaction, acetonitrile (5 mL) was added to the centrifuge tube, and Toyopearl gel and the blocking solution were separated with a glass filter. Subsequently, the gel was washed twice each with ethanol (5 mL) and water (5 mL) to obtain an epoxy toyopearl gel in which compound 423 was immobilized. Moreover, when the compound 423 which remains in the solution which combined the blocking solution and the washing | cleaning liquid was measured by the high performance liquid chromatography, it was confirmed that the compound 423 does not remain in the solution which combined the blocking solution and the washing | cleaning liquid.

Example 14 Immobilization of thiazole derivative on vinyl polymer gel (Part 7)
Compound 283 (94 mg, 0.3 mmol) was measured in a centrifuge tube, and 0.3 mL of 1,2-dimethoxybenzene in acetonitrile (1 mmol / mL) was added. Next, acetonitrile (1.7 mL), buffer (composition: 0.2 M NaHCO ₃ , 0.5 M NaCl, pH 8.3; 8 mL), triethylamine (90 μL, 0.6 mmol) were added to the centrifuge tube to add compound 283. After confirming dissolution, 0.3 g of Epoxy Toyopearl (trade name) gel (manufactured by Tosoh Corporation, epoxy group content: 804 μmol / g (gel)) was added. The centrifuge tube containing the mixture obtained by the above operation was set on a desktop shaker set at an internal temperature of 40 ° C., and stirred and shaken at 165 rpm for 16 hours. After completion of the reaction, acetonitrile (5 mL) was added to the centrifuge tube, and Toyopearl gel and the reaction solution were separated with a glass filter. Next, the Toyopearl gel on the glass filter was washed twice with a washing solution (composition: CH ₃ CN / MeOH / 50 mM NH ₄ H ₂ PO ₄ = 4/1/5; 10 mL). The combined reaction solution and washing solution, high performance liquid chromatography (Column: manufactured by Tosoh Corporation TSK-gel ODS-80T _M (trade name) (inner diameter 4.6 mm × length 15cm), _eluent: CH 3 CN / MeOH / 50mM NH ₄ H ₂ PO ₄ = 4/1/5, flow rate: 1 mL / min, temperature: 25 ° C., detection: UV 254 nm), by quantifying remaining compound 283, compound 283 was found to be 102 μmol / epoxy toyopearl. It was confirmed that mL (gel) was immobilized. Furthermore, in order to block unreacted epoxy groups in the Toyopearl gel obtained by the above operation, the Toyopearl gel obtained was placed in a centrifuge tube and a blocking solution (composition: 0.5 M monoethanolamine, 0.5 M sodium chloride aqueous solution, pH 8.3 (solvent is water); 5 mL) was added. The centrifuge tube containing the reaction solution was set on a desktop shaker set to an internal temperature of 40 ° C., and the remaining epoxy groups in the gel were blocked with monoethanolamine by stirring and shaking at 165 rpm for 16 hours. After completion of the reaction, acetonitrile (5 mL) was added to the centrifuge tube, and Toyopearl gel and the blocking solution were separated with a glass filter. Subsequently, the gel was washed twice with ethanol (5 mL) and water (5 mL), respectively, to obtain an epoxy toyopearl gel in which compound 283 was immobilized. Moreover, when the compound 283 which remains in the solution which combined the blocking solution and the washing | cleaning liquid was measured by the high performance liquid chromatography, it was confirmed that the compound 283 does not remain in the solution which combined the blocking solution and the washing | cleaning liquid.

Example 15 Immobilization of thiazole derivative on vinyl polymer gel (Part 8)
Compound 284 (98 mg, 0.3 mmol) was weighed into a centrifuge tube, and 0.3 mL of an acetonitrile solution (1 mmol / mL) of 1,2-dimethoxybenzene was added. Next, acetonitrile (1.7 mL), buffer (composition: 0.2 M NaHCO ₃ , 0.5 M NaCl, pH 8.3; 8 mL), triethylamine (90 μL, 0.6 mmol) were added to the centrifuge tube to obtain compound 284. After confirming dissolution, 0.3 g of Epoxy Toyopearl (trade name) gel (manufactured by Tosoh Corporation, epoxy group content: 804 μmol / g (gel)) was added. The centrifuge tube containing the mixture obtained by the above operation was set on a desktop shaker set at an internal temperature of 40 ° C., and stirred and shaken at 165 rpm for 16 hours. After completion of the reaction, acetonitrile (5 mL) was added to the centrifuge tube, and Toyopearl gel and the reaction solution were separated with a glass filter. Next, the Toyopearl gel on the glass filter was washed twice with a washing solution (composition: CH ₃ CN / MeOH / 50 mM NH ₄ H ₂ PO ₄ = 4/1/5; 10 mL). The combined reaction solution and washing solution, high performance liquid chromatography (Column: manufactured by Tosoh Corporation TSK-gel ODS-80T _M (trade name) (inner diameter 4.6 mm × length 15cm), _eluent: CH 3 CN / MeOH / 50mM NH ₄ H ₂ PO ₄ = 4/1/5, flow rate: 1 mL / min, temperature: 25 ° C., detection: UV 254 nm), by quantifying the remaining compound 284, the compound 284 was added to the epoxy toyopearl at 117 μmol / It was confirmed that mL (gel) was immobilized. Furthermore, in order to block unreacted epoxy groups in the Toyopearl gel obtained by the above operation, the Toyopearl gel obtained was placed in a centrifuge tube and a blocking solution (composition: 0.5 M monoethanolamine, 0.5 M sodium chloride aqueous solution, pH 8.3 (solvent is water); 5 mL) was added. The centrifuge tube containing the reaction solution was set on a desktop shaker set to an internal temperature of 40 ° C., and the remaining epoxy groups in the gel were blocked with monoethanolamine by stirring and shaking at 165 rpm for 16 hours. After completion of the reaction, acetonitrile (5 mL) was added to the centrifuge tube, and Toyopearl gel and the blocking solution were separated with a glass filter. Subsequently, the gel was washed twice with ethanol (5 mL) and water (5 mL), respectively, to obtain an epoxy toyopearl gel in which compound 284 was immobilized. Moreover, when the compound 284 which remains in the solution which combined the blocking solution and the washing | cleaning liquid was measured by the high performance liquid chromatography, it was confirmed that the compound 284 does not remain in the solution which combined the blocking solution and the washing | cleaning liquid.

Example 16 Immobilization of thiazole derivative on vinyl polymer gel (No. 9)
Compound 292 (191 mg, 0.5 mmol) was weighed into a centrifuge tube, and 0.5 mL of acetonitrile solution (1 mmol / mL) of 1,2-dimethoxybenzene was added. Next, acetonitrile (1.5 mL), buffer solution (composition: 0.2 M NaHCO ₃ , 0.5 M NaCl, pH 8.3; 4 mL), triethylamine (140 μL, 1.0 mmol) were added to the centrifuge tube, and compound 292 was added. After confirming dissolution, 0.3 g of Epoxy Toyopearl (trade name) gel (manufactured by Tosoh Corporation; epoxy group content: 804 μmol / g (gel)) was added. The centrifuge tube containing the mixture obtained by the above operation was set on a desktop shaker set at an internal temperature of 40 ° C., and stirred and shaken at 165 rpm for 16 hours. After completion of the reaction, acetonitrile (5 mL) was added to the centrifuge tube, and Toyopearl gel and the reaction solution were separated with a glass filter. Next, the Toyopearl gel on the glass filter was washed twice with a washing solution (composition: CH ₃ CN / MeOH / 50 mM NH ₄ H ₂ PO ₄ = 4/1/5; 10 mL). The combined reaction solution and washing solution, high performance liquid chromatography (Column: manufactured by Tosoh Corporation TSK-gel ODS-80T _M (trade name) (inner diameter 4.6 mm × length 15cm), _eluent: CH 3 CN / MeOH / 50mM NH ₄ H ₂ PO ₄ = 4/1/5, flow rate: 1 mL / min, temperature: 25 ° C., detection: UV 254 nm) by quantifying the remaining compound 292, the compound 292 was found to be 56 μmol / mol in the epoxy toyopearl. It was confirmed that mL (gel) was immobilized. Furthermore, in order to block unreacted epoxy groups in the Toyopearl gel obtained by the above operation, the Toyopearl gel obtained was placed in a centrifuge tube and a blocking solution (composition: 0.5 M monoethanolamine, 0.5 M sodium chloride aqueous solution, pH 8.3 (solvent is water); 5 mL) was added. The centrifuge tube containing the reaction solution was set on a desktop shaker set to an internal temperature of 40 ° C., and the remaining epoxy groups in the gel were blocked with monoethanolamine by stirring and shaking at 165 rpm for 16 hours. After completion of the reaction, acetonitrile (5 mL) was added to the centrifuge tube, and Toyopearl gel and the blocking solution were separated with a glass filter. Subsequently, the gel was washed twice with ethanol (5 mL) and water (5 mL), respectively, to obtain an epoxy toyopearl gel in which compound 292 was immobilized. Moreover, when the compound 292 which remains in the solution which combined the blocking solution and the washing | cleaning liquid was measured by the high performance liquid chromatography, it was confirmed that the compound 292 does not remain in the solution which combined the blocking solution and the washing | cleaning liquid.

Example 17 Immobilization of thiazole derivative on vinyl polymer gel (Part 10)
Compound 364 (193 mg, 0.5 mmol) was weighed into a centrifuge tube, and 0.5 mL of acetonitrile solution (1 mmol / mL) of 1,2-dimethoxybenzene was added. Next, acetonitrile (1.5 mL), buffer solution (composition: 0.2 M NaHCO ₃ , 0.5 M NaCl, pH 8.3; 8 mL), triethylamine (140 μL, 1.0 mmol) were added to the centrifuge tube to obtain compound 364. After confirmation of dissolution, 0.3 g of Epoxy Toyopearl (trade name) gel (manufactured by Tosoh Corporation, epoxy group content: 804 μmol / g (gel)) was added. The centrifuge tube containing the mixture obtained by the above operation was set on a desktop shaker set at an internal temperature of 40 ° C., and stirred and shaken at 165 rpm for 16 hours. After completion of the reaction, acetonitrile (5 mL) was added to the centrifuge tube, and Toyopearl gel and the reaction solution were separated with a glass filter. Next, the Toyopearl gel on the glass filter was washed twice with a washing solution (composition: CH ₃ CN / MeOH / 50 mM NH ₄ H ₂ PO ₄ = 4/1/5; 10 mL). The combined reaction solution and washing solution, high performance liquid chromatography (Column: manufactured by Tosoh Corporation TSK-gel ODS-80T _M (trade name) (inner diameter 4.6 mm × length 15cm), _eluent: CH 3 CN / MeOH / 50mM NH ₄ H ₂ PO ₄ = 4/1/5, flow rate: 1 mL / min, temperature: 25 ° C., detection: UV 254 nm), by quantifying the remaining compound 364, epoxide Toyopearl was compounded with 257 μmol / It was confirmed that mL (gel) was immobilized. Furthermore, in order to block unreacted epoxy groups in the Toyopearl gel obtained by the above operation, the Toyopearl gel obtained was placed in a centrifuge tube and a blocking solution (composition: 0.5 M monoethanolamine, 0.5 M sodium chloride aqueous solution, pH 8.3 (solvent is water); 5 mL) was added. The centrifuge tube containing the reaction solution was set on a desktop shaker set to an internal temperature of 40 ° C., and the remaining epoxy groups in the gel were blocked with monoethanolamine by stirring and shaking at 165 rpm for 16 hours. After completion of the reaction, acetonitrile (5 mL) was added to the centrifuge tube, and Toyopearl gel and the blocking solution were separated with a glass filter. Subsequently, the gel was washed twice with ethanol (5 mL) and water (5 mL), respectively, to obtain an epoxy toyopearl gel in which compound 364 was immobilized. Further, when the compound 364 remaining in the solution obtained by combining the blocking solution and the washing solution was measured by high performance liquid chromatography, it was confirmed that the compound 364 did not remain in the solution obtained by combining the blocking solution and the washing solution.

Example 18 Immobilization of thiazole derivative on vinyl polymer gel (Part 11)
Compound 286 (102 mg, 0.3 mmol) was weighed into a centrifuge tube, and 0.3 mL of acetonitrile solution (1 mmol / mL) of 1,2-dimethoxybenzene was added. Next, acetonitrile (1.7 mL), buffer (composition: 0.2 M NaHCO ₃ , 0.5 M NaCl, pH 8.3; 4 mL), triethylamine (90 μL, 0.6 mmol) were added to the centrifuge tube to obtain compound 286. After confirming dissolution, 0.3 g of Tresyl Toyopearl (trade name) gel (manufactured by Tosoh Corporation) was added. The centrifuge tube containing the mixture obtained by the above operation was set on a desktop shaker set at an internal temperature of 40 ° C., and stirred and shaken at 165 rpm for 16 hours. After completion of the reaction, acetonitrile (5 mL) was added to the centrifuge tube, and Toyopearl gel and the reaction solution were separated with a glass filter. Next, the Toyopearl gel on the glass filter was washed twice with a washing solution (composition: CH ₃ CN / MeOH / 50 mM NH ₄ H ₂ PO ₄ = 4/1/5; 10 mL). The combined reaction solution and washing solution, high performance liquid chromatography (Column: manufactured by Tosoh Corporation TSK-gel ODS-80T _M (trade name) (inner diameter 4.6 mm × length 15cm), _eluent: CH 3 CN / MeOH / 50mM NH ₄ H ₂ PO ₄ = 4/1/5, flow rate: 1 mL / min, temperature: 25 ° C., detection: UV 254 nm), by quantifying the remaining compound 286, 94 μmol of compound 286 was added to tresyl toyopearl. / ML (gel) was confirmed to be immobilized. Furthermore, in order to block the unreacted tresyl group in the Toyopearl gel obtained by the above operation, the Toyopearl gel obtained was placed in a centrifuge tube and a blocking solution (composition: 0.5M trishydroxymethylaminomethane hydrochloride, 0.5M Sodium chloride aqueous solution, pH 8.3; 5 mL) was added. The centrifuge tube containing the reaction solution was set on a desktop shaker set to an internal temperature of 40 ° C., and the remaining tresyl group in the gel was blocked with trishydroxymethylaminomethane by stirring and shaking at 165 rpm for 16 hours. After completion of the reaction, acetonitrile (5 mL) was added to the centrifuge tube, and Toyopearl gel and the blocking solution were separated with a glass filter. Subsequently, the gel was washed twice with ethanol (5 mL) and water (5 mL), respectively, to obtain a tresyl toyopearl gel in which compound 286 was immobilized. Further, when the compound 286 remaining in the solution obtained by combining the blocking solution and the washing solution was measured by high performance liquid chromatography, it was confirmed that the compound 286 did not remain in the solution obtained by combining the blocking solution and the washing solution.

Example 19 Immobilization of thiazole derivative on vinyl polymer gel (Part 12)
Compound 329 (30 mg, 84 μmol) was weighed into a centrifuge tube, and 0.1 mL of acetonitrile solution (1 mmol / mL) of 1,2-dimethoxybenzene was added. Next, acetonitrile (2.4 mL), buffer (composition: 0.1 M Na ₂ HPO ₄ —NaH ₂ PO ₄ , pH 7.0; 2.5 mL), triethylamine (30 μL, 0.2 mmol) were added to the centrifuge tube. After confirming that compound 329 was dissolved, 3 mL of formyl toyopearl (trade name) gel (manufactured by Tosoh Corporation, formyl group content: 65 μmol / mL (gel)) was added. The centrifuge tube containing the mixture obtained by the above operation was set on a desktop shaker set at an internal temperature of 25 ° C., and stirred and shaken at 165 rpm. 30 minutes after the start of stirring and shaking, borane-pyridine complex (250 μL, 2.5 mmol) was added to the centrifuge tube, and the mixture was further stirred and shaken at 165 rpm for 2.5 hours. After completion of the reaction, acetonitrile (5 mL) was added to the centrifuge tube, and Toyopearl gel and the reaction solution were separated with a glass filter. Next, the Toyopearl gel on the glass filter was washed twice with a washing solution (composition: CH ₃ CN / MeOH / 50 mM NH ₄ H ₂ PO ₄ = 4/1/5; 10 mL). The combined reaction solution and washing solution, high performance liquid chromatography (Column: manufactured by Tosoh Corporation TSK-gel ODS-80T _M (trade name) (inner diameter 4.6 mm × length 15cm), _eluent: CH 3 CN / MeOH / 50mM NH ₄ H ₂ PO ₄ = 4/1/5, flow rate: 1 mL / min, temperature: 25 ° C., detection: UV 254 nm), the remaining compound 329 was quantified, and compound 329 was immobilized at 14 μmol / mL (gel) The obtained formyl toyopearl gel was obtained.

Example 20 Immobilization of thiazole derivative on vinyl polymer gel (Part 13)
Compound 286 (170 mg, 0.5 mmol) was weighed into a centrifuge tube, and 0.5 mL of acetonitrile solution (1 mmol / mL) of 1,2-dimethoxybenzene was added. Next, acetonitrile (1.5 mL), buffer solution (composition: 0.2 M NaHCO ₃ , 0.5 M NaCl, pH 8.3; 8 mL), triethylamine (140 μL, 1.0 mmol) were added to the centrifuge tube, and compound 286 was added. After confirming dissolution, 0.3 g of Epoxy Toyopearl (trade name) gel (manufactured by Tosoh Corporation, epoxy group content: 804 μmol / g (gel)) was added. The centrifuge tube containing the mixture obtained by the above operation was set on a desktop shaker set at an internal temperature of 40 ° C., and stirred and shaken at 165 rpm for 63 hours. After completion of the reaction, acetonitrile (5 mL) was added to the centrifuge tube, and Toyopearl gel and the reaction solution were separated with a glass filter. Next, the Toyopearl gel on the glass filter was washed twice with a washing solution (composition: CH ₃ CN / MeOH / 50 mM NH ₄ H ₂ PO ₄ = 4/1/5; 10 mL). The combined reaction solution and washing solution, high performance liquid chromatography (Column: manufactured by Tosoh Corporation TSK-gel ODS-80T _M (trade name) (inner diameter 4.6 mm × length 15cm), _eluent: CH 3 CN / MeOH / 50mM NH ₄ H ₂ PO ₄ = 4/1/5, flow rate: 1 mL / min, temperature: 25 ° C., detection: UV 254 nm), by quantifying the remaining compound 286, epoxide Toyopearl was compounded with 167 μmol / It was confirmed that mL (gel) was immobilized. When the nitrogen atom content and sulfur atom content in this compound 286-immobilized epoxy toyopearl gel were measured, it was confirmed that 1.2% by weight of nitrogen atoms and 0.89% by weight of sulfur atoms were contained.

Example 21 Immobilization of Thiazole Derivative on Vinyl Polymer Gel (Part 14)
Compound 358 (134 mg, 0.4 mmol) was weighed in a centrifuge tube, and 0.4 mL of acetonitrile solution (1 mmol / mL) of 1,2-dimethoxybenzene was added. Next, 1,4-dioxane (10 mL) and triethylamine (120 μL, 0.9 mmol) were added to the centrifuge tube to confirm that compound 358 was dissolved, and then 2.5 mL of Toyopearl CM-650S (trade name) gel (Tosoh Corporation; ion exchange capacity: 0.1 mol / L (gel)) and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (154 mg, 0.8 mmol) were added. The centrifuge tube containing the mixture obtained by the above operation was set on a desktop shaker set at an internal temperature of 25 ° C., and stirred and shaken at 165 rpm for 14 hours. After completion of the reaction, Toyopearl gel and the reaction solution were separated with a glass filter. Next, the Toyopearl gel on the glass filter was washed twice with 1,4-dioxane (10 mL), and then the washing liquid (composition: 0.05% trifluoroacetic acid-containing CH ₃ CN / 0.05% trifluoroacetic acid aqueous solution = 45 / 55; 10 mL). The combined reaction solution and washing solution, high performance liquid chromatography (Column: manufactured by Tosoh Corporation TSK-gel ODS-80T _M (trade name) (inner diameter 4.6 mm × length 15cm), eluent: 0.05% trifluoroacetic acid The remaining compound 358 was quantified with CH ₃ CN / 0.05% trifluoroacetic acid aqueous solution, flow rate: 1 mL / min, temperature: 25 ° C., detection: UV254 nm), and compound 358 was immobilized at 16 μmol / mL (gel). Toyopearl CM-650S gel was obtained.

Example 22 Protein Adsorption / Desorption Using Thiazole Derivative-immobilized Vinyl Polymer Gel (Part 1)
1 mL of Compound 286-immobilized epoxy toyopearl gel prepared by the method described in Example 8 was packed into a TRICORN column (trade name) (manufactured by GE Healthcare Biosciences), and AKTAprime plus (trade name) (GE Healthcare). Attached to a chromatography device manufactured by Care Bioscience Co., Ltd.) and equilibrated by passing 20 mL of the equilibration buffer. AKTAprime plus was automatically operated at a flow rate of 1 mL / min, 10 mL of equilibration buffer was passed through, and 0.5 mg (OD ₂₈₀ = 0.7) of a human plasma-derived immunoglobulin preparation (manufactured by Kakenken) dissolved in the same buffer Was added and passed through the column. Further, after 10 mL of the equilibration buffer was passed, linear concentration gradient chromatography on sodium sulfate from the equilibration buffer to 10 mM sodium phosphate-10 mM sodium citrate buffer (pH 7.5) was performed. Elution of the immunoglobulin from the column was detected by monitoring the absorbance at 280 nm. The eluate was fractionated by 1 mL, and the absorbance at 280 nm of each fraction was measured with a U-2900 spectrophotometer (manufactured by Hitachi, Ltd.) to calculate the recovery rate. The results of chromatography carried out using Compound 286-immobilized epoxy toyopearl gel are shown in FIG. The immunoglobulin eluted in the 20th to 50th fractions somewhat wider than when the HiTrap column was used (see FIG. 1).

吸脱着操作後には１００ｍＭ 水酸化ナトリウムを１０ｍＬ通液してカラムの再生を行なった。化合物２８６固定化エポキシトヨパールゲルはこの再生処理によって初期状態に復帰させることができ、繰り返し使用によってもその特性は変化しなかった。

After the adsorption / desorption operation, 10 mL of 100 mM sodium hydroxide was passed through to regenerate the column. Compound 286-immobilized epoxy toyopearl gel could be restored to its initial state by this regeneration treatment, and its properties did not change even after repeated use.

Example 23 Adsorption / desorption of protein using thiazole derivative-immobilized vinyl polymer gel (Part 2)
1 mL of Compound 286-immobilized epoxy toyopearl gel prepared by the method described in Example 8 was packed into a TRICORN column (trade name) (manufactured by GE Healthcare Biosciences), and AKTAprime plus (trade name) (GE Healthcare). (Chromatography apparatus manufactured by Care Bioscience Co., Ltd.), and 20 mL of 0.7 M sodium sulfate, 10 mM sodium phosphate-10 mM sodium citrate buffer (pH 7.5) was passed and equilibrated. AKTAprime plus was automatically operated at a flow rate of 1 mL / min. After passing 10 mL of the equilibration buffer, 0.5 mg of a humanized monoclonal antibody (OD ₂₈₀ = 0.7) dissolved in the same buffer was added. Further, 10 mL of the equilibration buffer was passed through, followed by concentration gradient chromatography of sodium sulfate using 10 mM sodium phosphate-10 mM sodium citrate buffer (pH 7.5). Elution of the humanized monoclonal antibody from the column was detected by monitoring the absorbance at 280 nm. The eluate was fractionated by 1 mL, and the absorbance at 280 nm of each fraction was measured with a U-2900 spectrophotometer (manufactured by Hitachi, Ltd.) to calculate the recovery rate. FIG. 17 shows the results of chromatography performed using Compound 286-immobilized epoxy toyopearl gel, and Table 48 shows the recovery rates of humanized monoclonal antibodies in this chromatography. The humanized monoclonal antibody eluted in the 30th to 45th fractions, and was eluted at a position somewhat behind the immunoglobulin (see FIG. 16).

吸脱着操作後には１００ｍＭ 水酸化ナトリウムを１０ｍＬ通液してカラムの再生を行なった。化合物２８６固定化エポキシトヨパールゲルはこの再生処理によって初期状態に復帰させることができ、繰り返し使用によってもその特性は変化しなかった。

After the adsorption / desorption operation, 10 mL of 100 mM sodium hydroxide was passed through to regenerate the column. Compound 286-immobilized epoxy toyopearl gel could be restored to its initial state by this regeneration treatment, and its properties did not change even after repeated use.

Example 24 Adsorption / desorption of protein using thiazole derivative-immobilized vinyl polymer gel (Part 3)
1 mL of Compound 286-immobilized epoxy toyopearl gel prepared by the method described in Example 8 was packed into a TRICORN column (trade name) (manufactured by GE Healthcare Biosciences), and AKTAprime plus (trade name) (GE Healthcare). (Chromatography apparatus manufactured by Care Bioscience Co., Ltd.), and 20 mL of 0.7 M sodium sulfate, 10 mM sodium phosphate-10 mM sodium citrate buffer (pH 7.5) was passed and equilibrated. AKTAprime plus was automatically operated at a flow rate of 1 mL / min, and after 10 mL of the equilibration buffer was passed, 0.5 mg of bovine serum albumin (OD ₂₈₀ = 0.3) dissolved in the same buffer was added. Further, 10 mL of the equilibration buffer was passed through, followed by concentration gradient chromatography of sodium sulfate using 10 mM sodium phosphate-10 mM sodium citrate buffer (pH 7.5). The elution of bovine serum albumin from the column was detected by monitoring the absorbance at 280 nm. The eluate was fractionated by 1 mL, and the absorbance at 280 nm of each fraction was measured with a U-2900 spectrophotometer (manufactured by Hitachi, Ltd.) to calculate the recovery rate. The results of chromatography performed using Compound 286-immobilized Epoxy Toyopearl gel are shown in FIG. 18, and the recovery rate of bovine serum albumin in this chromatography is shown in Table 49. Bovine serum albumin elutes up to the 10th fraction, which is a fraction different from that of immunoglobulin (see FIG. 16), and results similar to those when using HiTrap columns (see FIG. 1 and FIG. 7). became. From this, it was shown that even when the matrix to be immobilized with the thiazole derivative was changed from agarose to Toyopearl, the same immunoglobulin separation performance was obtained.

吸脱着操作後には１００ｍＭ 水酸化ナトリウムを１０ｍＬ通液してカラムの再生を行なった。化合物２８６固定化エポキシトヨパールゲルはこの再生処理によって初期状態に復帰させることができ、繰り返し使用によってもその特性は変化しなかった。

After the adsorption / desorption operation, 10 mL of 100 mM sodium hydroxide was passed through to regenerate the column. Compound 286-immobilized epoxy toyopearl gel could be restored to its initial state by this regeneration treatment, and its properties did not change even after repeated use.

Example 25 Protein Adsorption / Desorption Using Thiazole Derivative-immobilized Vinyl Polymer Gel (Part 4)
Compound 329-immobilized epoxy toyopearl gel prepared by the method described in Example 9 was packed into a TRICORN column (trade name) (manufactured by GE Healthcare Biosciences), and AKTAprime plus (trade name) (GE Healthcare). A chromatography device manufactured by Bioscience Co., Ltd.) and 20 mL of 0.7 M sodium sulfate, 10 mM sodium phosphate-10 mM sodium citrate buffer solution (pH 7.5) was passed through and equilibrated. AKTAprime plus was automatically operated at a flow rate of 1 mL / min. After passing 10 mL of the equilibration buffer, 0.5 mg of a humanized monoclonal antibody (OD ₂₈₀ = 0.7) dissolved in the same buffer was added. Further, 10 mL of the equilibration buffer was passed through, followed by concentration gradient chromatography of sodium sulfate using 10 mM sodium phosphate-10 mM sodium citrate buffer (pH 7.5). Elution of the humanized monoclonal antibody from the column was detected by monitoring the absorbance at 280 nm. The eluate was fractionated by 1 mL, and the absorbance at 280 nm of each fraction was measured with a U-2900 spectrophotometer (manufactured by Hitachi, Ltd.) to calculate the recovery rate. FIG. 19 shows the results of chromatography performed using Compound 329-immobilized epoxy toyopearl gel, and Table 50 shows the recovery rates of humanized monoclonal antibodies in this chromatography. The humanized monoclonal antibody eluted in the 30th to 45th fractions and eluted in the same fractional position as when compound 286 was used as the thiazole derivative (see FIG. 17).

吸脱着操作後には１００ｍＭ 水酸化ナトリウムを１０ｍＬ通液してカラムの再生を行なった。化合物３２９固定化エポキシトヨパールゲルはこの再生処理によって初期状態に復帰させることができ、繰り返し使用によってもその特性は変化しなかった。

After the adsorption / desorption operation, 10 mL of 100 mM sodium hydroxide was passed through to regenerate the column. Compound 329-immobilized epoxy toyopearl gel could be restored to its initial state by this regeneration treatment, and its properties did not change even after repeated use.

Example 26 Protein Adsorption / Desorption Using Thiazole Derivative-immobilized Vinyl Polymer Gel (Part 5)
Compound 329-immobilized epoxy toyopearl gel prepared by the method described in Example 9 was packed into a TRICORN column (trade name) (manufactured by GE Healthcare Biosciences), and AKTAprime plus (trade name) (GE Healthcare). A chromatography device manufactured by Bioscience Co., Ltd.) and 20 mL of 0.7 M sodium sulfate, 10 mM sodium phosphate-10 mM sodium citrate buffer solution (pH 7.5) was passed through and equilibrated. AKTAprime plus was automatically operated at a flow rate of 1 mL / min, and after 10 mL of the equilibration buffer was passed, 0.5 mg of bovine serum albumin (OD ₂₈₀ = 0.3) dissolved in the same buffer was added. Further, 10 mL of the equilibration buffer was passed through, followed by concentration gradient chromatography of sodium sulfate using 10 mM sodium phosphate-10 mM sodium citrate buffer (pH 7.5). The elution of bovine serum albumin from the column was detected by monitoring the absorbance at 280 nm. The eluate was fractionated by 1 mL, and the absorbance at 280 nm of each fraction was measured with a U-2900 spectrophotometer (manufactured by Hitachi, Ltd.) to calculate the recovery rate. FIG. 20 shows the results of chromatography performed using Compound 329-immobilized epoxy toyopearl gel, and Table 51 shows the recovery rate of bovine serum albumin by this chromatography. Bovine serum albumin elutes by the 10th fraction, which is a fraction different from the humanized monoclonal antibody (see FIG. 19) and is the same as when compound 286 was used as the thiazole derivative (see FIG. 18). The result was. From this, it was shown that even when the thiazole derivative was changed from compound 286 to compound 329, the protein separation performance was similar.

吸脱着操作後には１００ｍＭ 水酸化ナトリウムを１０ｍＬ通液してカラムの再生を行なった。化合物３２９固定化エポキシトヨパールゲルはこの再生処理によって初期状態に復帰させることができ、繰り返し使用によってもその特性は変化しなかった。

After the adsorption / desorption operation, 10 mL of 100 mM sodium hydroxide was passed through to regenerate the column. Compound 329-immobilized epoxy toyopearl gel could be restored to its initial state by this regeneration treatment, and its properties did not change even after repeated use.

Example 27 Protein Adsorption / Desorption Using Thiazole Derivative-immobilized Vinyl Polymer Gel (Part 6)
Compound 286-immobilized formyltoyopearl gel prepared by the method described in Example 10 was packed into a 1 mL TRICORN column (trade name) (manufactured by GE Healthcare Biosciences), and AKTAprime plus (trade name) (GE Healthcare). A chromatography device manufactured by Care Bioscience Co., Ltd.) and equilibrated with 20 mL of 0.7 M sodium sulfate, 10 mM sodium phosphate-10 mM sodium citrate buffer (pH 7.5). AKTAprime plus was automatically operated at a flow rate of 1 mL / min. After passing 10 mL of the equilibration buffer, 0.5 mg of humanized monoclonal antibody (OD ₂₈₀ = 0.8) dissolved in the same buffer was added. Further, 10 mL of the equilibration buffer was passed through, followed by concentration gradient chromatography of sodium sulfate using 10 mM sodium phosphate-10 mM sodium citrate buffer (pH 7.5). Elution of the humanized monoclonal antibody from the column was detected by monitoring the absorbance at 280 nm. The eluate was fractionated by 1 mL, and the absorbance at 280 nm of each fraction was measured with a U-2900 spectrophotometer (manufactured by Hitachi, Ltd.) to calculate the recovery rate. The results of chromatography performed using Compound 286-immobilized formyl toyopearl gel are shown in FIG. 21, and the recovery rate of the humanized monoclonal antibody of this chromatography is shown in Table 52. The humanized monoclonal antibody eluted in the 30th to 45th fractions, and eluted in the same fractional position as when epoxytoyopearl was used for immobilization of thiazole derivatives (see FIG. 17).

吸脱着操作後には１００ｍＭ 水酸化ナトリウムを１０ｍＬ通液してカラムの再生を行なった。化合物２８６固定化ホルミルトヨパールゲルはこの再生処理によって初期状態に復帰させることができ、繰り返し使用によってもその特性は変化しなかった。

After the adsorption / desorption operation, 10 mL of 100 mM sodium hydroxide was passed through to regenerate the column. Compound 286-immobilized formyl toyopearl gel could be restored to its initial state by this regeneration treatment, and its properties were not changed by repeated use.

Example 28 Protein Adsorption / Desorption Using Thiazole Derivative-immobilized Vinyl Polymer Gel (Part 7)
Compound 286-immobilized formyltoyopearl gel prepared by the method described in Example 10 was packed into a TRICORN column (trade name) (manufactured by GE Healthcare Biosciences), and AKTAprime plus (trade name) (GE Healthcare). A chromatography device manufactured by Bioscience Co., Ltd.) and 20 mL of 0.7 M sodium sulfate, 10 mM sodium phosphate-10 mM sodium citrate buffer solution (pH 7.5) was passed through and equilibrated. AKTAprime plus was automatically operated at a flow rate of 1 mL / min, and after 10 mL of the equilibration buffer was passed, 0.5 mg of bovine serum albumin (OD ₂₈₀ = 0.3) dissolved in the same buffer was added. Further, 10 mL of the equilibration buffer was passed through, followed by concentration gradient chromatography of sodium sulfate using 10 mM sodium phosphate-10 mM sodium citrate buffer (pH 7.5). The elution of bovine serum albumin from the column was detected by monitoring the absorbance at 280 nm. The eluate was fractionated by 1 mL, and the absorbance at 280 nm of each fraction was measured with a U-2900 spectrophotometer (manufactured by Hitachi, Ltd.) to calculate the recovery rate. The results of chromatography performed using Compound 286-immobilized formyl toyopearl gel are shown in FIG. 22, and the recovery rate of bovine serum albumin by this chromatography is shown in Table 53. Bovine serum albumin elutes by the 10th fraction, which is a fraction different from the humanized monoclonal antibody (see FIG. 21), and when epoxytoyopearl was used for immobilization of thiazole derivatives (see FIG. 18). ) And similar results. From this, it was shown that even if the activation group of the matrix used for immobilizing the thiazole derivative was changed from an epoxy group to a formyl group, the same protein separation performance was obtained.

吸脱着操作後には１００ｍＭ 水酸化ナトリウムを１０ｍＬ通液してカラムの再生を行なった。化合物２８６固定化ホルミルトヨパールゲルはこの再生処理によって初期状態に復帰させることができ、繰り返し使用によってもその特性は変化しなかった。

After the adsorption / desorption operation, 10 mL of 100 mM sodium hydroxide was passed through to regenerate the column. Compound 286-immobilized formyl toyopearl gel could be restored to its initial state by this regeneration treatment, and its properties were not changed by repeated use.

Example 29 Immobilization of thiazole derivatives on chitosan gel 5 mL of activated chitopearl K-66 (trade name) gel washed with water (Fujibo Holdings Co., Ltd .; succinimideoxycarbonyl group content: 15 μmol / mL (gel)) Was measured into a centrifuge tube, and 10 mL of DMSO was added thereto. Next, 900 μL of a DMSO solution of compound 286 (0.25M), 225 μL of 1M acetonitrile solution of 1,2-dimethoxybenzene, and 68 μL of triethylamine were sequentially added. The centrifuge tube containing the mixture obtained by the above operation was set on a desktop shaker set at an internal temperature of 40 ° C., and stirred and shaken at 165 rpm for 3 hours. After completion of the reaction, the chitopearl gel and the reaction solution were separated with a glass filter, and the chitopearl gel on the glass filter was washed with dimethyl sulfoxide (5 mL) and a washing solution (composition: CH ₃ CN / MeOH / 50 mM NH ₄ H ₂ PO ₄ = 4/1/5). ; 15 mL). The combined reaction solution and washing solution, high performance liquid chromatography (Column: manufactured by Tosoh Corporation TSK-gel ODS-80T _M (trade name) (inner diameter 4.6 mm × length 15cm), _eluent: CH 3 CN / MeOH / 50mM NH ₄ H ₂ PO ₄ = 4/1/5, flow rate: 1 mL / min, temperature: 25 ° C., detection: UV 254 nm), the remaining compound 286 was quantified, and compound 286 was 5.0 μmol / mL (gel) An immobilized chitopearl gel was obtained.

Example 30 Immobilization of thiazole derivative on vinyl polymer gel (Part 15)
Compound 286 (102 mg, 0.3 mmol) was weighed into a centrifuge tube, and 0.3 mL of acetonitrile solution (1 mmol / mL) of 1,2-dimethoxybenzene was added. Next, acetonitrile (1.7 mL), 1M aqueous sodium hydroxide solution (1.2 mL), and water (6.8 mL) were added to the centrifuge tube to confirm that compound 286 was dissolved, and then 0.5 g of BIOACT- EPO (trade name) gel (manufactured by Showa Denko KK, epoxy group content: 200 μmol / g (gel)) was added. The centrifuge tube containing the mixture obtained by the above operation was set on a desktop shaker set at an internal temperature of 40 ° C., and stirred and shaken at 165 rpm for 43 hours. After completion of the reaction, acetonitrile (5 mL) was added to the centrifuge tube, and the BIOACT-EPO gel and the reaction solution were separated with a glass filter. Next, the BIOACT-EPO gel on the glass filter was washed twice with a washing liquid (composition: 0.05% trifluoroacetic acid-containing CH ₃ CN / 0.05% trifluoroacetic acid aqueous solution = 1/1; 10 mL). The combined reaction solution and washing solution, high performance liquid chromatography (Column: manufactured by Tosoh Corporation TSK-gel ODS-80T _M (internal diameter 4.6 mm × length 15cm), eluent: 0.05% trifluoroacetic acid-containing _CH 3 CN /0.05% aqueous trifluoroacetic acid = 1/1, flow rate: 1 mL / min, temperature: 25 ° C., detection: UV 254 nm), by quantifying the remaining compound 286, the compound 286 was found on the BIOACT-EPO gel. It was confirmed that 46 μmol / mL (gel) was immobilized. Furthermore, in order to block unreacted epoxy groups in the BIOACT-EPO gel obtained by the above operation, the obtained BIOACT-EPO gel was placed in a centrifuge tube (blocking solution (composition: acetonitrile; 2 mL, 0.5 M mono). Ethanolamine, 0.5M aqueous sodium chloride solution, pH 8.3 (solvent is water); 4 mL) was added. The centrifuge tube containing the reaction solution was set on a desktop shaker set to an internal temperature of 40 ° C., and the remaining epoxy groups in the gel were blocked with monoethanolamine by stirring and shaking at 165 rpm for 23 hours. After completion of the reaction, acetonitrile (5 mL) was added to the centrifuge tube, and the BIOACT-EPO gel and the blocking solution were separated with a glass filter. Subsequently, the gel was washed twice with ethanol (5 mL) and water (5 mL), respectively, to obtain a BIOACT-EPO gel in which compound 286 was immobilized. Further, when the compound 286 remaining in the solution obtained by combining the blocking solution and the washing solution was measured by high performance liquid chromatography, it was confirmed that the compound 286 did not remain in the solution obtained by combining the blocking solution and the washing solution.

Example 31 Immobilization of thiazole derivative on styrene-divinylbenzene copolymer gel Compound 286 (102 mg, 0.3 mmol) was weighed into a centrifuge tube and 1,2-dimethoxybenzene in acetonitrile (1 mmol / mL) was added to 0. 3 mL was added. Next, acetonitrile (1.7 mL), 1M aqueous sodium hydroxide solution (1.2 mL), and water (6.8 mL) were added to the centrifuge tube to confirm that compound 286 was dissolved, and then 0.3 g of POROS- EP (trade name) gel (Applied Biosystems Japan Co., Ltd., epoxy group content: not disclosed) was added. The centrifuge tube containing the mixture obtained by the above operation was set on a desktop shaker set at an internal temperature of 40 ° C., and stirred and shaken at 165 rpm for 36 hours. After completion of the reaction, acetonitrile (5 mL) was added to the centrifuge tube, and the POROS-EP gel and the reaction solution were separated with a glass filter. Next, the POROS-EP gel on the glass filter was washed twice with a washing liquid (composition: 0.05% trifluoroacetic acid-containing CH ₃ CN / 0.05% trifluoroacetic acid aqueous solution = 1/1; 10 mL). The combined reaction solution and washing solution, high performance liquid chromatography (Column: manufactured by Tosoh Corporation TSK-gel ODS-80T _M (internal diameter 4.6 mm × length 15cm), eluent: 0.05% trifluoroacetic acid-containing _CH 3 CN /0.05% trifluoroacetic acid aqueous solution = 1/1, flow rate: 1 mL / min, temperature: 25 ° C., detection: UV 254 nm), by quantifying the remaining compound 286, compound 286 was found on the POROS-EP gel. It was confirmed that 91 μmol / mL (gel) was immobilized. Furthermore, in order to block the unreacted epoxy group in the POROS-EP gel obtained by the above operation, the obtained POROS-EP gel was placed in a centrifuge tube and a blocking solution (composition: acetonitrile; 2 mL, 0.5M mono). Ethanolamine, 0.5M aqueous sodium chloride solution, pH 8.3 (solvent is water); 4 mL) was added. The centrifuge tube containing the reaction solution was set on a desktop shaker set at an internal temperature of 40 ° C., and the remaining epoxy groups in the gel were blocked with monoethanolamine by stirring and shaking at 165 rpm for 6 hours. After completion of the reaction, acetonitrile (5 mL) was added to the centrifuge tube, and the POROS-EP gel and the blocking solution were separated with a glass filter. Subsequently, the gel was washed twice with ethanol (5 mL) and water (5 mL), respectively, to obtain a POROS-EP gel in which compound 286 was immobilized. Further, when the compound 286 remaining in the solution obtained by combining the blocking solution and the washing solution was measured by high performance liquid chromatography, it was confirmed that the compound 286 did not remain in the solution obtained by combining the blocking solution and the washing solution.

Example 32 Immobilization of thiazole derivative on sensor chip Compound 286 was dissolved in DMSO to 4 mg / mL, and this was diluted 8-fold with 10 mM boric acid-1 M NaCl buffer (pH 8.5) to obtain 0 A compound solution having a concentration of 5 mg / mL was prepared. BIACORE sensor chip CM5 (trade name, manufactured by GE Healthcare Biosciences Co., Ltd.) is mounted on BIACORE 2000 (trade name, manufactured by GE Healthcare Biosciences Co., Ltd.), and BIACORE amine coupling kit (trade name, GE Healthcare Biosciences, Inc.) Was used to activate carboxymethyldextran on sensor chip CM5 with N-hydroxysuccinimide (NHS). Immobilization was performed by contacting the compound 286 solution having a concentration of 0.5 mg / mL at 25 ° C. for 5 minutes. Thereafter, unreacted succinimideoxycarbonyl groups were blocked using a 1M monoethanolamine aqueous solution. A series of immobilization processes was monitored by increasing SPR resonance units (RU) on the surface of the sensor chip CM5.

Example 33 Affinity analysis using BIACORE (trade name) The HBS-EP buffer (composition: 10 mM HEPES) was applied to the ligand-matrix complex (sensor chip CM5 on which compound 286 was immobilized) prepared in Example 32. (PH 7.4), 0.15 M NaCl, 3 mM EDTA, 0.005% SP20) diluted mouse IgM (10 μg / mL, sample A), mouse IgG (320 μg / mL, sample B), chicken IgY (400 μg / mL, sample C) and bovine serum albumin (320 μg / mL, sample D) were each passed through for 120 seconds (binding phase), and then replaced with HBS-EP buffer (dissociation phase) for 280 seconds to analyze the affinity. . At the end of each analysis, 10 mM sodium hydroxide was passed for 80 seconds to regenerate the sensor chip CM5. FIG. 23 shows a sensorgram of the sensor chip CM5 on which the compound 286 is immobilized. Table 54 shows the residual RU after 240 seconds of dissociation time.

図２３及び表５４より、マウスＩｇＭ（試料Ａ）が他の試料（試料Ｂ、Ｃ、Ｄ）と比較し、化合物２８６を固定化したセンサーチップＣＭ５に対する親和性が高いことが示された。よって、化合物２８６を固定化したデキストランゲルが、マウスＩｇＭを特異的に吸着することが判明した。

FIG. 23 and Table 54 show that mouse IgM (sample A) has a higher affinity for the sensor chip CM5 on which compound 286 is immobilized, as compared with other samples (samples B, C, and D). Therefore, it was found that dextran gel immobilized with compound 286 specifically adsorbs mouse IgM.

Example 34 Immobilization of thiazole derivative on cellulose gel A mixture of 0.1 M N-cyclohexyl-2-aminoethanesulfonic acid buffer (pH 9.0) / acetonitrile = 1/1 was prepared, and this was used as a solvent. Compound 486 and sodium hydroxide were added to 41 mM each. Take 3.0 mL of Celfine Formyl (trade name) gel (manufactured by Chisso Corp., containing 10-15 μmol / mL formyl group) in a centrifuge tube, add 5 mL of the solution containing the compound 486 to it, and shake on the table. After shaking for 30 minutes at 25 ° C. and 160 rpm, borane-pyridine complex (0.17 mL, 1.7 mmol) was added and shaken for 16 hours under the same conditions. The reaction mixture was filtered through a glass filter, washed once with 5 mL of acetonitrile, and then 4 times with 5 mL of a washing solution (acetonitrile / water / trifluoroacetic acid = 45/55 / 0.05). Together these filtrate and washings high performance liquid chromatography (Column: manufactured by Tosoh Corporation TSK-gel ODS-80T _M (internal diameter 4.6 mm × length 15cm), eluent: same composition as the cleaning solution, flow rate: 1 mL / Min, temperature: 25 ° C., detection: UV 254 nm) was quantified to confirm that compound 486 was immobilized on Serfine Formyl gel at 5 μmol / mL (gel).

Example 35 Immobilization of Thiazole Derivative on Acrylamide-Vinyl Copolymer Gel Compound 486 in a mixed solvent of 0.1M N-cyclohexyl-2-aminoethanesulfonic acid buffer (pH 9.0) / acetonitrile = 1/1 And sodium hydroxide were added to 41 mM each. Take 0.3 g of Profitity Epoxide (trade name) gel (manufactured by Bio-Rad Co., Ltd., containing 50 to 132 μmol / g epoxy group) in a centrifuge tube, add 5 mL of the solution containing the compound 486 to it, and shake on the table The mixture was shaken for 15 hours at 40 ° C. and 160 rpm. The reaction mixture was filtered through a glass filter, washed once with 5 mL of acetonitrile, and then 4 times with 5 mL of a washing solution (acetonitrile / water / trifluoroacetic acid = 45/55 / 0.05). Together these filtrate and washings high performance liquid chromatography (Column: manufactured by Tosoh Corporation TSK-gel ODS-80T _M (internal diameter 4.6 mm × length 15cm), eluent: same composition as the cleaning solution, flow rate: 1 mL / Min, temperature: 25 ° C., detection: UV 254 nm) was quantified to confirm that compound 486 was immobilized on Profinity Epoxide gel at 4 μmol / mL (gel).

Example 36 Immobilization of thiazole derivative on silica gel 2-hydroxy-3- [4- (2-hydroxyethyl) -1-piperazinyl] propanesulfonic acid buffer (0.1 M, pH 8.0) / acetonitrile = 1 / Compound 486 and sodium hydroxide were each added to 1 mixed solvent so that it might become 41 mM. 1.3 g of M.p. S. GEL Epoxy-D-50-1000AW (trade name, manufactured by AGC S-Tech Co., Ltd., containing 73 μmol / g epoxy group), 6.1 mL of the above solution is added thereto, and using a desktop shaker, The mixture was shaken at 40 ° C. and 160 rpm for 4 days. The reaction mixture was filtered through a glass filter, washed once with 5 mL of acetonitrile, and then 4 times with 5 mL of a washing solution (acetonitrile / water / trifluoroacetic acid = 45/55 / 0.05). Together these filtrate and washings high performance liquid chromatography (Column: manufactured by Tosoh Corporation TSK-gel ODS-80T _M (internal diameter 4.6 mm × length 15cm), eluent: same composition as the cleaning solution, flow rate: 1 mL / min, temperature: 25 ° C., detection: UV 254 nm). S. It was confirmed that 22 μmol / mL (gel) of Compound 486 was immobilized on GEL Epoxy-D-50-1000AW.

Example 37 Immobilization of 4-methyl-N- [4-methyl-5- {3- (2-propenylcarbonylamino) propyl} thiazol-2-yl] benzamide on polystyrene beads (Part 1)
In a 200 mL eggplant-shaped flask, copper (I) chloride (159 mg, 1.61 mmol), pentamethyldiethylenetriamine (419 mg, 2.42 mmol), methanol (19.5 mL), and 2-hydroxyethyl methacrylate (19.5 mL, 161 mmol) Were sequentially added and stirred at room temperature to confirm that the reaction mixture was uniformly dissolved. Next, 1.4 g of chloromethylated polystyrene beads (Watanabe Chemical Co., Ltd., chloromethyl group content: 1.15 meq / g) was added to the reaction solution, and dissolved oxygen in the reaction solution was removed by freeze degassing. After that, the reaction vessel was stirred at 40 ° C. for 12 hours. After completion of the reaction, the polystyrene beads and the reaction solution were separated, and the polystyrene beads were washed twice with methanol (200 mL) and then washed three times with ethanol / 5% aqueous ammonia = 9/1 (v / v) (1 L). . After completion of the washing, the polystyrene beads and the washing liquid were separated, and then vacuum-dried to obtain pale blue poly (2-hydroxyethyl methacrylate) -modified polystyrene beads (18.4 g, graft ratio: 1200%). Here, the graft ratio is
Graft rate = (weight of polystyrene beads after reaction−weight of polystyrene beads before reaction) / (weight of polystyrene beads before reaction)
It is defined as

Next, copper (I) chloride (22 mg, 0.22 mmol), 1,4,8,11-tetramethyl-1,4,8,11-tetraazatetradecane (56 mg, 0.22 mmol) was placed in a 50 mL eggplant-shaped flask. 4-methyl-N- [4-methyl-5- {3- (2-propenylcarbonylamino) propyl} thiazol-2-yl] benzamide (525 mg, 1.47 mmol) synthesized in Reference Example 2, and 2- A mixed solvent (3 mL) of butanone / 2-propanol = 7/3 (v / v) was sequentially added, and the mixture was stirred at room temperature to confirm that the reaction mixture was uniformly dissolved. Next, 2.5 g of poly (2-hydroxyethyl methacrylate) modified polystyrene beads were added to the reaction solution, dissolved oxygen in the reaction solution was removed by freeze degassing, and the reaction vessel was stirred at 60 ° C. for 63 hours. After completion of the reaction, the polystyrene beads and the reaction solution were separated, twice with ethanol (300 mL), twice with ethanol / 5% aqueous ammonia = 9/1 (v / v) (300 mL), and once with DMF (200 mL). Washed. After completion of the washing, the polystyrene beads and the washing liquid were separated, followed by vacuum drying to obtain light brown poly (2-hydroxyethyl methacrylate) modified polystyrene beads (2.61 g) to which a thiazole derivative was bound. As a result of calculating from the weight increase of the obtained polystyrene beads, it was estimated that an average of 2 molecules of thiazole derivatives were bonded to the end of the graft chain, and the equivalent was 0.12 meq / g. FT-IR, ν (cm ⁻¹ , reflect): 3440 (br, O—H), 2949 (w), 1720 (s, C═O), 1637 (w, C═O), 1450 (m), 1243 (m), 1149 (s), 1074 (s).

Example 38 Immobilization of 4-methyl-N- [4-methyl-5- {3- (2-propenylcarbonylamino) propyl} thiazol-2-yl] benzamide on polystyrene beads (Part 2)
In a 200 mL eggplant-shaped flask, copper (I) chloride (500 mg, 5.0 mmol), pentamethyldiethylenetriamine (1.30 g, 7.5 mmol), 2-propanol (22 mL), and an aqueous acrylamide solution (composition: 17.8 g / 22 mL). 250 mmol) was sequentially added and stirred at room temperature to confirm that the reaction mixture was uniformly dissolved. Next, 5.0 g of chloromethylated polystyrene beads (Peptide Laboratories, Inc., chloromethyl group content: 0.97 meq / g) was added to the reaction solution, and dissolved oxygen in the reaction solution was removed by freeze degassing. After that, the reaction vessel was stirred at 80 ° C. for 18 hours. After completion of the reaction, the polystyrene beads and the reaction solution were separated, and the polystyrene beads were washed with ethanol (150 mL), and then washed twice with ethanol / 5% aqueous ammonia = 9/1 (v / v) (200 mL). After completion of the washing, the polystyrene beads and the washing liquid were separated, and vacuum-dried to obtain a light blue polyacrylamide-modified polystyrene beads (12.3 g, graft ratio: 146%). Here, the graft ratio is graft ratio = (weight of polystyrene beads after reaction−weight of polystyrene beads before reaction) / (weight of polystyrene beads before reaction).
It is defined as

Next, copper (I) chloride (60 mg, 0.60 mmol), tris (2- (dimethylamino) ethyl) amine (208 mg, 0.9 mmol), 4-methyl-synthesized in Reference Example 2 was placed in a 50 mL eggplant-shaped flask. N- [4-Methyl-5- {3- (2-propenylcarbonylamino) propyl} thiazol-2-yl] benzamide (1.07 g, 3.0 mmol), and 2-propanol / water = 2/1 (v / V) mixed solvent (3 mL) was sequentially added and stirred at room temperature to confirm that the reaction mixture was uniformly dissolved. Next, 1.5 g of polyacrylamide-modified polystyrene beads were added to the reaction solution, dissolved oxygen in the reaction solution was removed by freeze degassing, and the reaction vessel was stirred at 80 ° C. for 87 hours. After completion of the reaction, ethanol (30 mL) and 5% aqueous ammonia (5 mL) were added to the reaction solution, and the mixture was stirred at room temperature for 1 hour. Next, the polystyrene beads and the reaction solution were separated, and washed twice with ethanol / 5% aqueous ammonia = 9/1 (v / v) (50 mL) and once with DMF (250 mL). After completion of the washing, the polystyrene beads and the washing liquid were separated, and then vacuum-dried to obtain light green polyacrylamide-modified polystyrene beads (1.84 g) to which a thiazole derivative was bound. As a result of calculating from the weight increase of the obtained polystyrene beads, it was estimated that an average of 2 molecules of thiazole derivatives were bonded to the graft chain terminal and the equivalent was 0.52 meq / g. FT-IR, ν (cm ⁻¹ , reflect): 3336 (br), 2922 (m), 1654 (s, C═O), 1600 (m), 1492 (m), 1450 (m), 1028 (w ), 756 (m), 698 (s).

Claims (9)

General formula (1)

Wherein R ¹ and R ² represent a hydrogen atom or an optionally substituted alkyl group having 1 to 6 carbon atoms, Ar represents an optionally substituted aromatic group, and n represents 1 to 12 An integer, and M represents a matrix.) Ar is a halogen atom, an optionally substituted alkyl group having 1 to 6 carbon atoms, an optionally substituted alkoxy group having 1 to 12 carbon atoms, or an optionally substituted carbon group having 3 to 8 carbon atoms. A cycloalkoxy group, an optionally substituted alkenyloxy group having 3 to 6 carbon atoms, an optionally substituted alkynyloxy group having 3 to 6 carbon atoms, an optionally substituted acyloxy group having 1 to 6 carbon atoms Group, optionally substituted alkylthio group having 1 to 6 carbon atoms, optionally substituted alkylsulfinyl group having 1 to 6 carbon atoms, optionally substituted alkylsulfonyl group having 1 to 6 carbon atoms, substituted An optionally substituted alkoxycarbonyl group having 1 to 12 carbon atoms, a carboxyl group, a nitro group, an optionally substituted amino group, a hydroxyl group or a cyano group Thiazole derivatives immobilization matrix according to 1. Ar is a halogen atom, an optionally substituted alkyl group having 1 to 6 carbon atoms, an optionally substituted alkoxy group having 1 to 12 carbon atoms, an optionally substituted cycloalkoxy group having 3 to 8 carbon atoms Group, optionally substituted alkenyloxy group having 3 to 6 carbon atoms, optionally substituted alkynyloxy group having 3 to 6 carbon atoms, optionally substituted acyloxy group having 1 to 6 carbon atoms, substituted An optionally substituted alkylthio group having 1 to 6 carbon atoms, an optionally substituted alkylsulfinyl group having 1 to 6 carbon atoms, an optionally substituted alkylsulfonyl group having 1 to 6 carbon atoms, One or more selected from an alkoxycarbonyl group having 1 to 12 carbon atoms, a carboxyl group, a nitro group, an optionally substituted amino group, a hydroxyl group or a cyano group. Thiazole derivatives immobilization matrix according to claim 1 substituted with a substituent which is also an aromatic group. General formula (1)

(Wherein R ¹ , R ² , Ar, n and M represent the same meaning as described above), the matrix of M is any one of vinyl polymer, agarose, chitosan, dextran, cellulose, silica and polystyrene. The thiazole derivative-immobilized matrix according to claim 1, characterized in that it is characterized in that General formula (1a)

(Wherein R ¹ , R ² , Ar and n represent the same meaning as described above, and R ³ represents an amino group or an ammonium salt) and an activated group-containing matrix is reacted. The general formula (1)

(Wherein R ¹ , R ² , Ar, n, and M represent the same meaning as described above), a method for producing a thiazole derivative-immobilized matrix. 6. The activation group of the activation group-containing matrix is any one of succinimideoxycarbonyl group, epoxy group, formyl group, 2,2,2-trifluoroethylsulfonyl group, and carboxyl group. A method for producing a thiazole derivative-immobilized matrix. General formula (1)

(Wherein R ¹ , R ² , Ar, n and M represent the same meaning as described above), and a method for analyzing or purifying proteins using a thiazole derivative-immobilized matrix. 8. A method according to claim 7, characterized in that the protein is an immunoglobulin or an analogue, fragment or fusion thereof. 9. A method according to claim 8, characterized in that the immunoglobulin is immunoglobulin G (IgG).

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