Production method for compound

Abstract

Disclosed is a production method for a compound, the production method comprising: a step in which a covalent bond is formed between a support and a covalent bond-forming group of a compound that is represented by formula (I): CBFG–PG–MBG (in formula (I), CBFG represents a monovalent group having the covalent bond-forming group, PG represents a divalent group obtained by removing one hydrogen atom from a monovalent protective group, and MBG represents a monovalent group having a core group and a scaffold group that is protected by PG), and the compound represented by formula (I) is captured in the support; and a step in which PG is removed from the scaffold group, and a compound comprising the scaffold group and the core group or a group obtained by converting the core group by means of a chemical reaction having at least one stage on a solid phase is obtained.

Description

Compound manufacturing method

[0001] The present disclosure relates to methods for making compounds.

[0002] When artificially producing a compound, it is important to obtain a high-yield, high-purity product as easily as possible at each stage of the synthetic pathway from the viewpoint of overall yield and cost. In particular, obtaining a high-purity product by removing impurities such as reaction raw materials, reaction reagents, and by-products at each stage is extremely important in increasing the yield of the entire synthetic pathway. Generally, product purification is carried out by extraction, crystallization, chromatography, etc.

[0003] Solid-phase synthesis is one of the conversion methods that can be used to synthesize compounds. Solid-phase synthesis is a technique in which a compound (a raw compound) that serves as the raw material for a chemical reaction is captured on a support, and the captured molecule is transformed through a chemical reaction. In solid-phase synthesis, the target compound is obtained by liberating the converted compound from its elemental form.

[0004] The synthesis of compounds by solid-phase synthesis has also been applied to the construction of compound libraries. In solid-phase synthesis, compounds captured on a support are converted. Therefore, even if different compounds are captured on each support, these compounds can be reacted in one pod, and the individual compounds can be separated by separating the supports and then liberating the compounds from the support. Therefore, solid-phase synthesis allows libraries to be expanded in a manner that allows for easy separation of intermediate products and final products at each stage. A representative method for constructing a compound library using solid-phase synthesis is the One-Bead-One-Compound method (OBOC method). Regarding the synthesis of molecular compounds and library construction using solid-phase synthesis, Non-Patent Document 1 discloses that a peptide chain was extended from the aniline amino group of a rhodamine derivative captured on a support by solid-phase synthesis.

[0005] Furthermore, examples have been reported in which capture onto a solid phase has been applied to the purification of compounds. The methodology used in such examples is called synthesis-based on affinity separation (SAS). In SAS, a compound is selectively captured on a support through the interaction between a functional group of the compound and a group on a support that has a high affinity for that functional group. The support is then separated from the system, and the compound is then released from the support, thereby purifying the compound. For example, Non-Patent Document 2 discloses that the phosphono group of a coumarin derivative was captured on a support surface-modified with phos-tag (registered trademark), and the support was then separated from the system, and the coumarin derivative was then released from the support, thereby purifying the coumarin derivative.

[0006] Yugo Kuriki et al., "Development of a fluorescent probe library enabling efficient screening of tumor-imaging probes based on discovery of biomarker enzymatic activities", Chem Sci. 13(16):4474-4481 (2022).Shingo Sakamoto et al., "Identification of activity-based biomarkers for early-stage pancreatic tumors in blood using single-molecule enzyme activity screening", Cell Reports Methods 4, 100688 (2024).

[0007] In the methods described in Non-Patent Documents 1 and 2, when a compound is captured on a carrier, a functional group of the molecule is directly bound to or interacts with the carrier. In such methods, the efficiency of capturing the compound on the carrier and the efficiency of releasing the compound or a compound converted from the compound from the carrier depend greatly on the reactivity of the functional group, and therefore, depending on the molecular structure, these efficiencies may be low, which can result in a decrease in purification efficiency.

[0008] Furthermore, in the method described in Non-Patent Document 2, a raw material compound is captured on a carrier by a non-covalent interaction between a phosphono group and a phospho-tag. However, functional groups that can be used to capture a compound on a carrier by a non-covalent interaction are limited to a very small number of groups, such as a phosphono group. Therefore, the method described in Non-Patent Document 2 has the problem that it can only be used for purifying and producing compounds having such limited functional groups.

[0009] The present disclosure aims to provide a method for producing a compound. In view of the above-mentioned problems, the present disclosure also aims to provide a method for producing a compound that utilizes SAS purification and is less restricted by the structure of the compound.

[0010] The present inventors have found that the above-mentioned problems can be solved by a production method including capturing a compound protected by a protecting group modified with a covalent bond-forming group on a carrier by a covalent bond via the covalent bond-forming group, and then deprotecting the protecting group from the compound or a compound obtained by converting the compound on a solid phase.

[0011] The present disclosure relates to, for example, the following: [1] A method for producing a compound, comprising: a step of forming a covalent bond between a covalent bond-forming group of a compound represented by formula (I): CBFG-PG-MBG (I) [in formula (I), CBFG represents a monovalent group having a covalent bond-forming group, PG represents a divalent group obtained by removing one hydrogen atom from a monovalent protecting group, and MBG represents a monovalent group having a scaffold group protected by PG and a backbone group] and a support, thereby capturing the compound represented by formula (I) on the support; and a step of removing PG from the scaffold group to obtain a compound comprising the scaffold group and the backbone group or a group obtained by converting the scaffold group through at least one chemical reaction on a solid phase. [2] The method according to [1], further comprising a step of obtaining a compound represented by formula (I), the step comprising protecting the scaffold group with a compound represented by formula (II): CBFG-PG-LG (II) [in formula (II), CBFG and PG are the same as in formula (I), and LG represents a monovalent leaving group] or a compound in which the covalent bond-forming group of the compound represented by formula (I) has been protected. [3] The production method according to [1] or [2], further comprising a step of converting the backbone group of the compound represented by formula (I) captured on the support by at least one chemical reaction on a solid phase. [4] The production method according to any one of [1] to [3], wherein CBFG is a monovalent group having a click-reactive group. [5] CBFG is an ethynyl group, a 2-propynyl group, a 3-butynyl group, a 4-pentynyl group, a 5-hexynyl group, or a C group in which one hydrogen atom has been substituted with an azide group. １－５ [6] The method according to any one of [1] to [4], wherein PG is a group represented by formula (III-A) or formula (III-B): [In formula (III-A) and formula (III-B), R １ａ , R １ｂ and R １ｃ are each independently a hydrogen atom or a C １－５ [7] The method according to any one of [1] to [5], wherein PG is a divalent group obtained by removing one hydrogen atom from a group represented by the formula:１－５ Alkoxybenzyloxycarbonyl, para C １－５ The production method according to any one of [1] to [6], wherein the alkoxy group is a divalent group obtained by removing one hydrogen atom from alkoxybenzyl, trityl, tert-butoxycarbonyl, or 9-fluorenylmethyloxycarbonyl. [8] A method for screening a compound, comprising: forming a covalent bond between a covalent bond-forming group of a compound represented by formula (I): CBFG-PG-MBG (I) [in formula (I), CBFG represents a monovalent group having a covalent bond-forming group, PG represents a divalent group obtained by removing one hydrogen atom from a monovalent protecting group, and MBG represents a monovalent group having a scaffold group protected by PG and a backbone group] and a carrier, thereby capturing the compound represented by formula (I) on the carrier; removing PG from the scaffold group to obtain a compound consisting of the scaffold group and the backbone group or a group obtained by converting the scaffold group through at least one chemical reaction on a solid phase; and evaluating a compound consisting of the scaffold group and the backbone group or a group obtained by converting the backbone group, or a compound obtained by further converting the scaffold group. [9] A compound represented by formula (II-A): CRG-PG-LG (II-A) [In formula (II-A), CRG represents a monovalent group having a click-reactive group, with the proviso that the click-reactive group may be protected, PG represents a divalent group obtained by removing one hydrogen atom from a monovalent protecting group, and LG represents a monovalent leaving group].

[10] A kit comprising a compound represented by formula (IV): CRG-PG-OH (IV) [In formula (IV), CRG represents a monovalent group having a click-reactive group, with the proviso that the click-reactive group may be protected, and PG represents a divalent group obtained by removing one hydrogen atom from a monovalent protecting group] and a reagent for converting the hydroxyl group of the compound represented by formula (IV) to introduce a monovalent leaving group.

[0012] According to the present disclosure, a method for producing a compound is provided. The method for producing a compound according to the present disclosure uses SAS, which allows impurities such as raw materials, reaction reagents, and by-products other than the compound (target product) captured on the solid phase to be washed away, thereby making it possible to purify the compound extremely easily. This eliminates the need to optimize purification conditions for each individual compound, for example, when producing a library containing a large number of novel compounds, and also avoids the need to perform laborious and time-consuming purification procedures such as chromatography for each type of compound.

[0013] In the compound production method according to the present disclosure, the compound is captured on a support via a covalent bond-forming group obtained by modifying a protecting group that protects the functional group of the compound. As a result, the compound production method according to the present disclosure can eliminate the influence of the efficiency of capturing the functional group of the compound onto the support and the efficiency of releasing it from the support, and can highly efficiently purify the compound captured on the solid phase or a compound obtained by converting the compound.

[0014] In the compound production method according to the present disclosure, a protecting group can be selected according to the structure of the compound, and capture onto a carrier is performed via a covalent bond-forming group modified with the protecting group. As a result, in the compound production method according to the present disclosure, purification by SAS can be applied to the production of compounds having any protecting group that can be protected by a protecting group, not limited to compounds having limited functional groups such as phosphono groups.

[0015] A compound protected by a protecting group modified with a covalent bond-forming group is captured on a support by a covalent bond via the covalent bond-forming group, and then the protecting group is removed from the compound or a compound converted therefrom on a solid phase to release the compound or a compound converted therefrom on a solid phase. This can be easily applied to a synthetic route by replacing a protecting group used in a certain step in a conventional synthetic route with a protecting group modified with a covalent bond-forming group. Thus, the production method of the present disclosure can be a method that improves the yield and simplifies purification by improving existing schemes, by replacing, among the purification steps in conventional production methods, purification steps in which separation of the product and reagents or their reaction products is difficult, or purification after a step that requires the addition of an excess amount of reagent to increase the yield, with purification by SAS.

[0016] Furthermore, when the production method according to one embodiment of the present disclosure further includes a step of converting the backbone group of the compound represented by formula (I) captured on the carrier through at least one chemical reaction on a solid phase, the following effects are further achieved.

[0017] In a method for producing a compound according to one embodiment of the present disclosure, a compound is captured on a support via a protecting group that protects a starting compound of a chemical reaction. Therefore, in the method for producing a compound according to the present disclosure, a protecting group suitable for protecting the starting compound can be selected as the protecting group, and a protecting group suitable for capturing the starting compound on the support can be selected as the covalent bond-forming group suitable for capturing the compound on the support in terms of the efficiency of capturing the compound on the support, and these can be independently optimized.

[0018] In a compound production method according to one embodiment of the present disclosure, a starting compound is captured on a support by a covalent bond between the support and a covalent bond-forming group that modifies a protecting group. Therefore, since covalent bonds are more resistant to cleavage than non-covalent bonds, the production method according to one embodiment of the present disclosure can be applied to production using a wider variety of chemical reactions than when starting compounds are captured on a support by non-covalent interactions. Therefore, the production method according to one embodiment of the present disclosure is also suitable for, for example, converting starting compounds captured on a solid phase through multiple chemical reactions in multiple steps to obtain a target compound.

[0019] FIG. 1 shows the results of HPLC analysis of each compound of the fluorescent probe group synthesized in Example 1. FIG. 2 shows the results of HPLC analysis of each compound of the fluorescent probe group synthesized in Example 1. FIG. 3 shows the results of HPLC analysis of each compound of the fluorescent probe group synthesized in Example 1. FIG. 4 shows the results of HPLC analysis of each compound of the fluorescent probe group synthesized in Example 1. FIG. 5 shows the results of HPLC analysis of each compound of the fluorescent probe group synthesized in Example 1. FIG. 6 shows the results of HPLC analysis of each compound of the fluorescent probe group synthesized in Example 1. FIG. 7 shows the chemical structure and HPLC chromatogram of Suc-GDEVD-dsSiR, a type of fluorescent probe obtained in Example 2. FIG. 8 shows the results of HPLC-MS analysis of a model of an ADC molecule synthesized in Example 3. 1 shows the results of comparing the concentrations of untrapped sHMRG-Cpz remaining in the reaction solution when a compound is trapped on a carrier via a covalent bond-forming group modified with a protecting group that protects the functional group of the compound, and when a compound is trapped in a unit that is commonly used in conventional solid-phase synthesis, in Example 4. 1 shows the results of evaluating the release efficiency of sHMRG by deprotection of the functional group when a compound is trapped on a carrier via a covalent bond-forming group modified with a protecting group that protects the functional group of the compound, in Example 5. 1 shows liquid-phase chromatography charts before (Reaction-0 h) and after (Reaction-1 h) reacting sHMRG-Cpz with Fmoc-Tyr(tBu)-OH for 1 hour, and before (Flowthrough) capturing the resulting Fmoc-Tyr(tBu)-sHMRG-Cpz on Tentagel-azide, and after (Release) capturing and deprotecting Cpz from the amino group to recover Fmoc-Tyr(tBu)-sHMRG in Example 6. 1 shows the results of evaluating the product obtained when Fmoc-Lys(Cpz)-OH was exposed to conditions for forming an amide bond in solid-phase synthesis, conditions for deprotecting Fmoc, or conditions for deprotecting a protecting group bound to a solid phase in Example 7.

[0033] Figure 1 shows the results of evaluating the product when Fmoc-Cir(Ert)-OH was exposed to conditions for forming an amide bond in solid-phase synthesis, conditions for deprotecting Fmoc, or conditions for deprotecting a protecting group bound to a solid phase, in Example 7. Figure 2 shows the results of evaluating the enzyme activity of plasma samples prepared from thioacetamide (TAA) model mice or 4,4'-methylenedianiline (MDA) model mice, using various fluorescent probes, in Example 8. Figure 3 shows the results of evaluating the enzyme activity of plasma samples prepared from thioacetamide (TAA) model mice or 4,4'-methylenedianiline (MDA) model mice, using various fluorescent probes, in Example 8. Figure 4 shows the results of evaluating the enzyme activity of plasma samples prepared from thioacetamide (TAA) model mice or 4,4'-methylenedianiline (MDA) model mice, using various fluorescent probes, in Example 8. 18 shows the results of evaluating the enzyme activity of plasma samples prepared from thioacetamide (TAA) model mice or 4,4'-methylenedianiline (MDA) model mice using Suc-GLEVD-sHMRG in Example 8.

[0044] FIG. 19 shows the results of evaluating the enzyme activity of plasma samples prepared from thioacetamide (TAA) model mice or 4,4'-methylenedianiline (MDA) model mice using Suc-KHLY-sHMRG and Suc-AAVY-dsSiR in Example 8.

[0045] FIG. 19 shows the results of evaluating the enzyme activity of plasma samples prepared from thioacetamide (TAA) model mice or 4,4'-methylenedianiline (MDA) model mice using Suc-KHLY-sHMRG and Suc-AAVY-dsSiR in Example 8.

[0046] FIG. 19 shows the results of analyzing the linker molecules of the ADCs synthesized in Example 9 by HPLC-MS.

[0047] FIG. 19 shows the results of quantifying the amount of MMAE released by contacting the linker molecules of the ADCs with a plasma sample from a healthy donor (left) or an SKBR3 cell lysate (right) in Example 10.

[0020] Hereinafter, embodiments for carrying out the present disclosure will be described, but the present disclosure is not limited to the following embodiments.

[0021] In the present disclosure, "-" and "=" between two groups in a chemical formula indicate a single bond and a double bond, respectively.

[0022] The alkyl is a group obtained by removing one hydrogen atom from an alkane. The alkyl may be a group obtained by removing one hydrogen atom from a chain or cyclic alkane, and preferably a group obtained by removing one hydrogen atom from a chain alkane, and the chain alkane may be linear or branched. In one embodiment, the alkyl is １－５ It may be alkyl. １－５ Alkyl is an alkyl having from 1 to 5 carbon atoms. Alkyl may be, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, cyclopropyl, cyclobutyl, or cyclopentyl; １－５ It is alkyl.

[0023] Alkenyl is a group obtained by removing one hydrogen atom from an alkene. The alkenyl may be a group obtained by removing one hydrogen atom from a chain or cyclic alkene, and preferably a group obtained by removing one hydrogen atom from a chain alkene, and the chain alkene may be linear or branched. In one embodiment, the alkyl is C ２－４ It may be alkenyl. ２－４ Alkenyl is an alkenyl having from 2 to 4 carbon atoms. Alkenyl may be, for example, vinyl, allyl, 2-butenyl, or 3-butenyl, which are C ２－４ It is alkenyl.

[0024] An alkoxy is a monovalent group in which an alkyl is bonded to a root oxygen atom. In one embodiment, the alkoxy may be an alkoxy containing a linear alkyl. In one embodiment, the alkoxy may be C １－５ It may be alkoxy. １－５ Alkoxy is an alkoxy having from 1 to 5 carbon atoms. Alkoxy can be, for example, methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentoxy, isopentoxy or neopentoxy; １－５In one aspect, the alkoxy may be methoxy, ethoxy, propoxy, butoxy, or pentoxy, which include linear alkyls. １－５ It is an alkoxy.

[0025] Alkoxymethyl is a group in which one hydrogen atom of methyl is substituted with alkoxy. In one embodiment, the alkoxymethyl may be a group in which one hydrogen atom of methyl is substituted with alkoxy containing a linear alkyl. In one embodiment, the alkoxymethyl is a group having C １－５ It may be alkoxymethyl. １－５ Alkoxymethyl is a group in which one hydrogen atom of methyl is substituted with alkoxy having 1 to 5 carbon atoms. Alkoxymethyl may be, for example, methoxymethyl, ethoxymethyl, propoxymethyl, butoxymethyl or pentoxymethyl, which are straight-chain C １－５ It is an alkoxymethyl containing alkyl.

[0026] Halogen is an element belonging to Group 17 of the periodic table, and in one embodiment may be fluorine, chlorine, bromine, or iodine. Halogeno is a monovalent group corresponding to halogen, and in one embodiment may be fluoro, chloro, bromo, or iodo.

[0027] Amino is a group obtained by removing one hydrogen atom on the nitrogen atom from ammonia, a primary amine, or a secondary amine, and may be preferably a group obtained by removing one hydrogen atom on the nitrogen atom from ammonia or a primary amine, and more preferably a group obtained by removing one hydrogen atom on the nitrogen atom from ammonia. When amino is a group obtained by removing one hydrogen atom on the nitrogen atom from a primary amine or a secondary amine, the substituent on the nitrogen atom may be, for example, an alkyl which may have a substituent, an alkenyl which may have a substituent, or a phenyl which may have a substituent, and in one embodiment, it may be an alkyl.

[0028] A substituent is a monovalent group that replaces a hydrogen atom. In one embodiment, a substituent is a monovalent group that replaces a hydrogen atom on a carbon atom. An "optionally substituted" functional group may have no substituent, or may have one, two, three or more substituents, each of which is different. In one embodiment, an "optionally substituted" functional group may have no substituent or one substituent. In a preferred embodiment, an "optionally substituted" functional group may have no substituent. That is, in a preferred embodiment, an "optionally substituted" functional group may be the functional group itself.

[0029] The substituents may be, for example, but not limited to, alkyl, alkenyl, alkoxy, alkoxymethyl, halogeno, amino, phenyl, or carboxy, and these may further have substituents.

[0030] The electron-withdrawing group is a group that attracts electrons in a molecule, causing polarization. The electron-withdrawing group may be, for example, nitro, cyano, trifluoromethyl, halogeno, alkoxycarbonyl, acetyl, propionyl, or acetamido. Also, for example, the electron-withdrawing group may be a group having a Hammett constant at the para position of 0.10 or more, 0.20 or more, or 0.30 or more.

[0031] The present disclosure relates to a production method comprising capturing a compound protected by a protecting group modified with a covalent bond-forming group on a support by covalent bonding via the covalent bond-forming group, and then deprotecting the protecting group from the compound or a compound converted from the compound on a solid phase.

[0032] A first embodiment of the present disclosure is a method for producing a compound, the method comprising: a step of forming a covalent bond between a covalent bond-forming group of a compound represented by formula (I): CBFG-PG-MBG (I) and a support, thereby capturing the compound represented by formula (I) on the support (capturing step); and a step of removing PG from the scaffold group to obtain a compound comprising the scaffold group and a backbone group or a group obtained by converting the scaffold group through at least one chemical reaction on a solid phase (deprotecting step). In the above formula (I), CBFG represents a monovalent group having a covalent bond-forming group, PG represents a divalent group obtained by removing one hydrogen atom from a monovalent protecting group, and MBG represents a monovalent group having a scaffold group protected by PG and a backbone group. Hereinafter, this embodiment will also be referred to as the "production method of the first embodiment." Hereinafter, the compound produced by the production method of the first embodiment will also be referred to as the "target compound." Hereinafter, the compound represented by formula (I) will also be referred to as the "compound of formula (I)."

[0033] The unit represented by CBFG-PG in formula (I) is a unit corresponding to a protecting group modified with a covalent bond-forming group. In the production method of the first embodiment, the CBFG portion of the unit represented by CBFG-PG is bound to a carrier, and the PG portion is bound to MBG, which is a group corresponding to the target of purification or a raw material for a chemical reaction, thereby capturing the target of purification or the raw material for a chemical reaction (MBG) on the carrier. First, the unit represented by CBFG-PG and the carrier bound to the CBFG portion will be described in detail below.

[0034] PG represents a divalent group obtained by removing one hydrogen atom from a monovalent protecting group. In one embodiment, PG may represent a divalent group obtained by removing one hydrogen atom on a carbon atom from a monovalent protecting group. The monovalent protecting group in PG may be any protecting group that can form a covalent bond (protect) with the scaffolding group corresponding to the functional group to be protected in MBG, and that can cleave (deprotect) the covalent bond under conditions that do not dissociate the covalent bond between the CBFG moiety and the support. Such monovalent protecting groups can be a wide variety of protecting groups that are sometimes used to protect functional groups in the field of organic chemistry, and can be appropriately selected by a person skilled in the art based on common technical knowledge in accordance with the type of functional group to be protected.

[0035] The monovalent protecting group in PG may be, for example, a monovalent protecting group described in "GREEN'S PROTECTIVE GROUPS in Organic Synthesis" (5th edition, John Wiley & Sons, Inc., 2014).

[0036] When the functional group to be protected is hydroxy, the monovalent protecting group in PG may be, for example, an ether-based protecting group, a silyl ether-based protecting group, an ester-based protecting group, or a carbonate-based protecting group. The ether-based protecting group that protects hydroxy is a protecting group that forms an ether with the protected hydroxy, and may be, for example, methoxymethyl (MOM), 2-methoxyethoxymethyl (MEM), benzyloxymethyl, benzyl, p-methoxybenzyl (PMB), o-nitrobenzyl, or trityl, which may have a substituent, or in a preferred embodiment, any of these groups that do not have a substituent. These protecting groups can be removed by deprotection methods commonly used by those skilled in the art. The silyl ether-based protecting group for protecting a hydroxyl group is a protecting group that forms a silyl ether with the hydroxyl group to be protected. In one embodiment, the three methyl moieties of trimethylsilyl may be independently substituted with an alkyl group that may have a substituent, an alkenyl group that may have a substituent, an alkoxy group that may have a substituent, an alkoxymethyl group that may have a substituent, or a benzyl group that may have a substituent. In a preferred embodiment, the protecting group may be trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), tert-butyldimethylsilyl (TBDMS), or tert-butyldiphenylsilyl (TBDPS). These protecting groups can be easily deprotected by contacting them with fluoride ions. Fluoride ions can be supplied using a reagent such as tetrabutylammonium fluoride (TBAF). The ester-based protecting group for protecting hydroxy is a protecting group that forms an ester with the hydroxy to be protected, and in one embodiment may be a group in which the methyl moiety of acetyl is substituted with alkyl which may have a substituent, alkenyl which may have a substituent, alkoxymethyl which may have a substituent, benzyl which may have a substituent, or trityl which may have a substituent, and in a preferred embodiment may be acetyl, pivaloyl, benzoyl, p-methoxybenzoyl, phenylacetyl, or trifluoroacetyl. These protecting groups can be removed by deprotection methods commonly used by those skilled in the art.The carbonate-based protecting group that protects a hydroxyl group is a protecting group that forms a carbonate with the protected hydroxyl group, and in one embodiment may be a group in which the methyl moiety of a methoxycarbonyl group is substituted with an alkyl group which may have a substituent, an alkenyl group which may have a substituent, an alkoxymethyl group which may have a substituent, a benzyl group which may have a substituent, or a trityl group which may have a substituent, and in a preferred embodiment may be methoxycarbonyl, tert-butoxycarbonyl, or benzyloxycarbonyl. These protecting groups can be removed by deprotection methods commonly used by those skilled in the art.

[0037] When the functional group to be protected is carboxy, the monovalent protecting group in PG may be, for example, an ester-based protecting group. An ester-based protecting group that protects carboxy is a protecting group that forms an ester with the carboxy to be protected, and protects the carboxy by substituting a hydrogen atom of the carboxy. The ester-based protecting group that protects carboxy is, for example, a hydrocarbon group that may have a substituent, and in one aspect may be an alkyl that may have a substituent, an alkenyl that may have a substituent, an alkoxymethyl that may have a substituent, a benzyl that may have a substituent, or a trityl that may have a substituent, and in a preferred aspect, it may be methyl, ethyl, isopropyl, tert-butyl, allyl, methoxymethyl (MOM), benzyl, p-methoxybenzyl (PMB), or trityl. These protecting groups can be removed by deprotection methods commonly used by those skilled in the art.

[0038] When the functional group to be protected is amino, the monovalent protecting group in PG may be, for example, a carbamate-based protecting group, an amide-based protecting group, or an N-alkyl-based protecting group. A carbamate-based protecting group that protects an amino is a protecting group that forms a carbamate with the amino to be protected. The carbamate-based protecting group for protecting an amino group is, for example, a group in which the methyl moiety of a methoxycarbonyl group is substituted with a hydrocarbon group which may have a substituent. In one embodiment, the methyl moiety of a methoxycarbonyl group may be substituted with an alkyl group which may have a substituent, an alkenyl group which may have a substituent, an alkoxymethyl group which may have a substituent, a benzyl group which may have a substituent, or a 9-fluorenylmethyl group which may have a substituent. In a preferred embodiment, the protecting group may be tert-butoxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl (Fmoc), benzyloxycarbonyl (Cbz, Z), p-methoxybenzyloxycarbonyl (Cpz), 2-trimethylsilylethoxycarbonyl (Teoc), allyloxycarbonyl (Alloc), 2,2,2-trichloroethoxycarbonyl (Troc), or methoxycarbonyl. These protecting groups can be removed by deprotection methods commonly used by those skilled in the art. The amide-based protecting group for protecting an amino group is a protecting group that forms an amide with the protected amino group. In one embodiment, the amide-based protecting group for protecting an amino group may be a group in which the methyl moiety of acetyl is substituted with an alkyl group which may have a substituent, an alkenyl group which may have a substituent, an alkoxymethyl group which may have a substituent, or a benzyl group which may have a substituent, and in a preferred embodiment, the amide-based protecting group may be acetyl or trifluoroacetyl. These protecting groups can be removed by a deprotection method commonly used by those skilled in the art, such as base hydrolysis. The N-alkyl-based protecting group for protecting an amino group is a protecting group that protects the amino group by N-alkylating it, and is not limited to an alkyl group. The N-alkyl-based protecting group for protecting an amino group may be, for example, benzyl which may have a substituent, trityl or allyl which may have a substituent, and in a preferred embodiment, it may be benzyl, p-methoxybenzyl (PMB), trityl or allyl. These protecting groups can be removed by a deprotection method commonly used by those skilled in the art, such as hydrolysis or oxidation-reduction.

[0039] When the functional group to be protected is phosphono or phospho, the monovalent protecting group in PG may be, for example, a phosphate silyl ester protecting group or a phosphate ester protecting group. The phosphate silyl ester protecting group is a protecting group that forms a phosphate silyl ester together with the phosphono or phospho to be protected. As the phosphate silyl ester protecting group, the same groups as those described as the silyl ether protecting group that protects hydroxy may be used. As the phosphate ester protecting group, the same groups as those described as the ester protecting group that protects carboxy may be used.

[0040] When the functional group to be protected is sulfo, the monovalent protecting group in PG may be, for example, a sulfoester-based protecting group. A sulfoester-based protecting group is a protecting group that forms a sulfoester together with the sulfo to be protected. The sulfoester-based protecting group is, for example, a hydrocarbon group which may have a substituent, and in one embodiment may be alkyl which may have a substituent, alkenyl which may have a substituent, alkoxymethyl which may have a substituent, or phenyl which may have a substituent, and in a preferred embodiment may be isopropyl, isobutyl, or neopentyl.

[0041] In a preferred embodiment, the monovalent protecting group in PG is represented by the following formula (III-A) or formula (III-B): In other words, in a preferred embodiment, PG may be a divalent group obtained by removing one hydrogen atom from a group represented by formula (III-A) or formula (III-B). In formulas (III-A) and (III-B), R １ａ , R １ｂ and R １ｃ are each independently a hydrogen atom or a C １－５ It represents alkyl, phenyl which may have a substituent, or 9-fluorenylmethyl which may have a substituent.

[0042] In a more preferred embodiment, the monovalent protecting group in PG is para C１－５ Alkoxybenzyloxycarbonyl, para C １－５ It may be a divalent group obtained by removing one hydrogen atom from alkoxybenzyl, trityl, tert-butoxycarbonyl, or 9-fluorenylmethyloxycarbonyl. In a more preferred embodiment, the monovalent protecting group in PG may be a divalent group obtained by removing one hydrogen atom from paramethoxybenzyloxycarbonyl, paramethoxybenzyl, trityl, tert-butoxycarbonyl, or 9-fluorenylmethyloxycarbonyl. In a most preferred embodiment, the monovalent protecting group in PG may be a divalent group obtained by removing one hydrogen atom from the methoxy or benzene ring of paramethoxybenzyloxycarbonyl or paramethoxybenzyl, a divalent group obtained by removing one hydrogen atom from the benzene ring of trityl or 9-fluorenylmethyloxycarbonyl, or a divalent group obtained by removing one hydrogen atom from tert-butoxycarbonyl.

[0043] CBFG represents a monovalent group having a covalent bond-forming group. CBFG has at least a covalent bond-forming group and may further have a linker moiety. In one embodiment, CBFG may be a group consisting of a covalent bond-forming group, or a group consisting of a covalent bond-forming group and a linker moiety. When CBFG has a linker moiety, PG and the covalent bond-forming group form a conjugate molecule via the linker.

[0044] The covalent bond-forming group is a monovalent group that forms a covalent bond orthogonally with the carrier in the capture step. That is, the covalent bond-forming group is a monovalent group that forms a covalent bond with the carrier in the capture step but does not bond to other substances in the system. The covalent bond-forming group may be, for example, a click-reactive group, an activated ester (e.g., N-hydroxysuccinimide ester), an activated amide (e.g., acylimidazole), an acid halide, or an alkyl halide, and in one embodiment, may be a click-reactive group. When the covalent bond-forming group is a click-reactive group, the click reaction is highly orthogonal and the covalent bond formed is stable under a wide range of deprotection conditions for protecting groups, so a wide variety of protecting groups can be used as the monovalent protecting group in PG.

[0045] In the present disclosure, a click-reactive group refers to a monovalent group that undergoes a click reaction, which is a chemical reaction applicable to click chemistry. In one aspect, the click-reactive group may be a monovalent group that undergoes a cycloaddition reaction applicable to click chemistry. In a preferred aspect, the click-reactive group may be a monovalent group that undergoes a metal ion-catalyzed or steric strain-promoted cycloaddition reaction. In a more preferred aspect, the click-reactive group may be a monovalent group that undergoes an azide-alkyne cycloaddition reaction or a strain-promoted inverse electron demand Diels-Alder reaction. In an even more preferred aspect, the click-reactive group may be a monovalent group that undergoes an azide-alkyne cycloaddition reaction from the viewpoint of the stability of the covalent bond that is formed.

[0046] (1) Azide-Alkyne Cycloaddition Reaction Azide and ethynylene undergo an azide-alkyne cycloaddition reaction, which is a 1,3-dipolar cycloaddition reaction, to form a 1,2,3-triazole ring. In one embodiment, the covalent bond-forming group may be a group containing azide or ethynylene. The azide-alkyne cycloaddition reaction is known as a representative reaction in click chemistry, and the structure of the click-reactive group and the reaction conditions can be appropriately determined by those skilled in the art. It is known that the reaction conditions for the azide-alkyne cycloaddition reaction vary depending on the chemical structure of the ethynylene. The azide-alkyne cycloaddition reaction may be, for example, a Huisgen cyclization reaction or a strain-promoted alkyne-azide cycloaddition reaction (SPAAC).

[0047] (1-1) Huisgen Cyclization Reaction (CuAAC, Copper-Catalyzed Azide-Alkyne Cycloaddition) The Huisgen cyclization reaction is a cycloaddition reaction catalyzed by copper(I) ions, and is known to have a high tolerance for ethynylene structures. That is, in one embodiment, the covalent bond-forming group may be a group containing azide or ethynylene, and in this case, the covalent bond may be formed in the capture step by a 1,3-dipolar cycloaddition reaction catalyzed by copper(I) ions.

[0048] (1-2) Strain-Promoted Alkyne-Azide Cycloaddition (SPAAC) SPAAC is an azide-alkyne cycloaddition reaction that occurs between an azide and a strained ethynylene. In SPAAC, the ethynylene is sterically strained and in a high-energy state, allowing the azide-alkyne cycloaddition reaction to occur in the absence of a metal catalyst. SPAAC is known as a type of so-called copper-free click reaction. That is, in one embodiment, the covalent bond-forming group may be an azide or a group containing a strained (highly strained) ethynylene. In this case, a covalent bond may be formed in the capture step in the absence of a metal catalyst, and the covalent bond is formed by a 1,3-dipolar cycloaddition reaction.

[0049] The group containing strained ethynylene is not particularly limited as long as it is known as a group applicable to SPAAC, and may be, for example, a group in which one hydrogen atom has been removed from cyclooctyne, dibenzocyclooctyne (DBCO), or bicyclo[6.1.0]nonyne (BCN), which may have a substituent, and one or two carbon atoms forming the ring may be substituted with a nitrogen atom.

[0050] (2) Strain-Promoted Inverse Electron-Demand Diels-Alder Cycloaddition (SPIEDAC) SPIEDAC is an inverse electron-demand Diels-Alder reaction that occurs between an electron-deficient heterocycle and a sterically strained dienophile. SPIEDAC is known as a type of copper-free click reaction because it occurs without a metal ion catalyst such as copper ion and also occurs under physiological conditions such as in water and at room temperature. That is, in one embodiment, the covalent bond-forming group may be a group containing an electron-deficient heterocycle or a group containing a sterically strained (highly strained) dienophile. In this case, a covalent bond may be formed in the capture step in the absence of a metal catalyst, and the covalent bond is formed by an inverse electron-demand Diels-Alder reaction.

[0051] The group containing an electron-deficient heterocycle is not particularly limited as long as it is known as a group applicable to SPIEDAC, and may be, for example, a group described in the literature (BL Oliveira et al., "Inverse electron demand Diels-Alder reactions in chemical biology", Chem. Soc. Rev., 2017, 46, 4895-4950, Aysun Degirmenci et al., "Metal-Free Click-Chemistry: A Powerful Tool for Fabricating Hydrogels for Biomedical Applications", Bioconjugate Chemistry 2024, 35, 4, 433-452). The group containing an electron-deficient heterocycle may be, for example, a monovalent group containing a tetrazine ring or a triazine ring, and in one embodiment, it may be a monovalent group containing a 1,2,4,5-tetrazine ring. In a preferred embodiment, it may be a monovalent group obtained by removing a hydrogen atom from a compound in which at least one hydrogen atom of 1,2,4,5-tetrazine is substituted with a substituent. In a more preferred embodiment, it may be a group obtained by removing one hydrogen atom from a compound in which one hydrogen atom of 1,2,4,5-tetrazine is substituted with an aromatic ring compound (e.g., benzene, pyridine, or pyrimidine) which may have a substituent or an electron-withdrawing group (e.g., trifluoromethyl or methoxycarbonyl) and the other hydrogen atom is substituted with a substituent, or a group obtained by removing one hydrogen atom from an aromatic ring compound in which both hydrogen atoms of 1,2,4,5-tetrazine are each independently substituted with a substituent or an electron-withdrawing group.

[0052] The group containing a sterically strained dienophile is not particularly limited as long as it is known as a group applicable to SPIEDAC, and may be, for example, a group in which one hydrogen atom has been removed from trans-cyclooctene (TCO), bicyclo[6.1.0]nonyne (BCN), cyclooctyne, dibenzocyclooctyne (DBCO), or norbornene, or in one embodiment, a group in which one hydrogen atom has been removed from TCO or BCN, which may have a substituent, and one or two carbon atoms forming the ring may be substituted with nitrogen atoms.

[0053] In one embodiment, the covalent bond-forming group may be a monovalent group that forms a covalent bond with the carrier but does not bond to a functional group on the backbone of the compound of Formula (I). In this case, the compound of Formula (I) can be captured on the carrier without protecting the backbone, which is convenient. In a preferred embodiment, the covalent bond-forming group may be a monovalent group that forms a covalent bond with the carrier in the capture step but does not bond to amino, carboxy, hydroxy, phosphono, phospho, or sulfo. In this case, the compound of Formula (I) can be captured on the carrier without protecting the backbone, and even when an intermediate for preparing the compound of Formula (I) contains these functional groups, the compound of Formula (I) can be prepared without protecting the functional groups. An example of the covalent bond-forming group in this preferred embodiment is a click-reactive group. That is, in a preferred embodiment, CBFG may be a monovalent group having a click-reactive group.

[0054] As described above, the CBFG may or may not contain a linker moiety, but when the CBFG contains a linker moiety, the chemical structure of the linker is not particularly limited as long as it can be used to form a conjugate molecule. The linker may, for example, have one or more, two or more, three or more, four or more, five or more, seven or more, or ten or more atoms, or may have 200 or less, 50 or less, 30 or less, 12 or less, 8 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 or less, and these upper and lower limits may be freely combined. The linker may, for example, have a minimum number of chemical bonds connecting the covalent bond-forming group and PG of 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 7 or more, or 10 or more, or may have 200 or less, 50 or less, 30 or less, 12 or less, 8 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 or less, and these upper and lower limits may be freely combined. Furthermore, the linker may be, for example, a linker consisting of at least one atom selected from the group consisting of a hydrogen atom, a carbon atom, a nitrogen atom, an oxygen atom, a phosphorus atom, and a sulfur atom. Furthermore, the linker may be, for example, a linker consisting of an aliphatic hydrocarbon, a peptide linker, or a polyethylene glycol (PEG) linker, or a linker in which multiple linkers selected from the group consisting of these are bonded via an amide bond, an ether bond, an ester bond, or a triazole. The linker consisting of an aliphatic hydrocarbon may be, for example, an alkylene linker, and in one embodiment, C １－５ It may be alkylene, and in a preferred embodiment from the viewpoint of ease of handling in synthesis, it may be methylene, ethylene, linear propylene, linear butylene, or linear pentylene.

[0055] In one embodiment, CBFG may be a group consisting of a monovalent group that undergoes an azide-alkyne cycloaddition reaction or a strain-promoted inverse electron demand Diels-Alder reaction, and a linker moiety consisting of a linker made of an aliphatic hydrocarbon, a peptide linker, a PEG linker, or a linker in which a plurality of linkers selected from the group consisting of these are bonded via an amide bond, an ether bond, an ester bond, or a triazole bond. In a preferred embodiment, CBFG is an ethynyl group, a 2-propynyl group, a 3-butynyl group, a 4-pentynyl group, a 5-hexynyl group, or a C 1 -alkyl group in which one hydrogen atom is substituted with an azide group. １－５ It may be alkyl.

[0056] In one embodiment, CBFG is a group consisting of a monovalent group that undergoes an azide-alkyne cycloaddition reaction or a strain-promoted inverse electron demand Diels-Alder reaction, and a linker moiety consisting of a linker made of an aliphatic hydrocarbon, a peptide linker, a PEG linker, or a linker in which a plurality of linkers selected from the group consisting of these are bonded via an amide bond, an ether bond, an ester bond, or a triazole bond, and PG may be a divalent group obtained by removing one hydrogen atom from the group represented by formula (III-A) or formula (III-B). In a preferred embodiment, CBFG is an ethynyl group, a 2-propynyl group, a 3-butynyl group, a 4-pentynyl group, a 5-hexynyl group, or a C group in which one hydrogen atom is substituted with an azide group. １－５ alkyl, and PG is para C １－５ Alkoxybenzyloxycarbonyl, para C １－５ It may be a divalent group obtained by removing one hydrogen atom from alkoxybenzyl, trityl, tert-butoxycarbonyl, or 9-fluorenylmethyloxycarbonyl. In a more preferred embodiment, the monovalent group represented by CBFG-PG- may be a group selected from the following chemical structures: In the following structural formula, X represents ethynyl or azide, and R ２ represents a single bond, methylene, ethylene, linear propylene, linear butylene, or linear pentylene; R ３ represents a single bond or —C(═O)—O— in which a carbonyl is directly bonded to a bond.２ and / or R ３ represents a single bond, R ２ and / or R ３ This means that two groups bonded to two bonds extending from the aromatic ring directly form a single bond. In the structural formula below, the bond extending from the center of the aromatic ring means that one of the hydrogen atoms on the aromatic ring is substituted with a substituent at the end of the bond. Among the groups selected from the chemical structures below, R is selected so that when the group to be protected by PG is amino, it becomes a carbamate-based protecting group or an N-alkyl-based protecting group, when it is carboxy, it becomes an ester-based protecting group, and when it is hydroxy, it becomes an ether-based protecting group. ３ That is, when the group to be protected by PG is amino, among the groups selected from the following chemical structures, R ３ More preferably, R represents —C(═O)—O—, in which a carbonyl is directly bonded to the bond, or a single bond. When the group protected by PG is carboxy or hydroxy, R ３ Even more preferably, represents a single bond.

[0057] The support according to the present disclosure is a support for solid-phase synthesis, and has on its surface a group capable of covalently bonding with the covalent bond-forming group of CBFG. Hereinafter, the group on the surface of the support capable of covalently bonding with the covalent bond-forming group of CBFG is also referred to as a "surface-modifying group." Another embodiment of the present disclosure may be such a support itself.

[0058] The type of support according to the present disclosure may be stable under the reaction conditions of the capture step and the deprotection step and have affinity with the solvents used in those steps. Furthermore, when a method according to one embodiment of the present disclosure includes a conversion step described below, the type of support according to the present disclosure may be stable under the reaction conditions of the capture step and the deprotection step and have affinity with the solvents used in those steps. For example, when these steps include a step performed in an aqueous solvent, a support having high affinity with the aqueous solvent is preferred. Such a support having high affinity with the aqueous solvent may be, for example, a copolymer of a hydrophobic monomer and a hydrophilic monomer, or a hydrophobic gel particle whose surface is treated with a hydrophilic polymer. The hydrophobic monomer may be, for example, styrene, ethylene, propylene, or (meth)acrylate. The hydrophilic monomer may be, for example, ethylene glycol, (meth)acrylamide, or vinylpyrrolidone. The hydrophobic gel particle may be, for example, a polystyrene resin or silica gel. The hydrophilic polymer may be, for example, polyethylene glycol, polypropylene glycol, poly(meth)acrylamide, polyvinyl alcohol, or polyvinylpyrrolidone. In one embodiment, the carrier may be a polystyrene resin gel particle whose surface is treated with polyethylene glycol. Examples of such a carrier include the TentaGel® series, such as the TentaGel® S series. The carrier according to the present disclosure may be, for example, in the form of beads, resin, or film, but is not limited thereto.

[0059] The surface-modifying group can be one of those described as the covalent bond-forming group of CBFG. Among the combinations of groups forming a covalent bond described as the covalent bond-forming group of CBFG, one can be selected as the covalent bond-forming group of CBFG and the other as the surface-modifying group of the carrier. For example, when the covalent bond-forming group is azide, the surface-modifying group can be an ethynylene-containing group or a strained ethynylene-containing group. Also, when the covalent bond-forming group is an ethynylene-containing group or a strained ethynylene-containing group, the surface-modifying group can be azide. Alternatively, when the covalent bond-forming group is a group containing an electron-deficient heterocycle, the surface-modifying group can be a group containing a sterically strained dienophile. Also, when the covalent bond-forming group is a group containing a sterically strained dienophile, the surface-modifying group can be a group containing an electron-deficient heterocycle.

[0060] Such supports can be prepared by further modifying a commercially available carrier material having functional groups for surface modification with a surface-modifying group via a linker. Such modification can be carried out by contacting the carrier material with a molecule in which a group that forms a bond (e.g., an amide bond) with the functional group and the surface-modifying group are linked via a linker in the presence of a necessary additive. The linker is, for example, a polyethylene glycol linker or an alkylene linker. For example, the support can be prepared by contacting a carrier material having an amino group on its surface with a molecule in which an activated ester or activated amide and a surface-modifying group are linked via a linker in an organic solvent in the presence of a base. As another example, a carrier raw material having a carboxyl group on its surface can be contacted with a molecule having an amino group and a surface-modifying group bonded via a linker, in an organic solvent in the presence of a condensing agent such as HATU (1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate) or DMT-MM (2-chloro-4,6-dimethoxy-1,3,5-triazine + N-methyl morpholine), and a base. As another example, a carrier raw material having a bromine atom on its surface can be contacted with a molecule having a phenolic hydroxyl group and a surface-modifying group bonded via a linker, in an organic solvent in the presence of a base. Carrier raw materials having functional groups for surface modification are available from many suppliers.

[0061] Next, MBG, which is a group that is a target of purification or a raw material for a chemical reaction in the production method of the first embodiment, will be described. MBG is a monovalent group having a scaffold group protected by PG and a backbone group. In one aspect, MBG may be a monovalent group in which the scaffold group protected by PG and the backbone group are directly bonded, or may be a monovalent group consisting of a scaffold group protected by PG and a backbone group.

[0062] A scaffold group protected by PG is a group in which one hydrogen atom of the scaffold group has been removed to form a bond to PG. That is, the scaffold group protected by PG is a divalent group, and is bonded to PG and the backbone group, respectively. The scaffold group may be any group that can be protected by a protecting group, for example, amino, carboxy, hydroxy, phosphono, phospho, sulfo, mercapto, formyl, or boryl, and in one embodiment, it is amino, carboxy, hydroxy, phosphono, phospho, or sulfo, and in a preferred embodiment, it is amino, carboxy, or hydroxy.

[0063] The backbone group is a monovalent group other than the scaffold group portion of a group (MBG) that is the target of purification or a raw material for a chemical reaction in the production method of the first embodiment. The backbone group according to one aspect may be a monovalent group other than the scaffold group portion of a group that is the target of purification, and in this case, the method according to the present disclosure does not include the conversion step described below, and the structure of the compound obtained in the deprotection step is represented by MBG-H (H is a hydrogen atom). The backbone group according to one aspect may be a monovalent group other than the scaffold group portion of a group that is the raw material for a chemical reaction, and in this case, the method according to the present disclosure includes the conversion step described below.

[0064] The backbone group is, for example, a monovalent organic group, and in one embodiment, is a monovalent group consisting of at least one atom selected from the group consisting of a hydrogen atom, a carbon atom, a nitrogen atom, an oxygen atom, a phosphorus atom, and a sulfur atom. When the backbone group in one embodiment is a monovalent group other than a scaffold group portion among groups that serve as raw materials for a chemical reaction, the chemical structure of the backbone group can be converted by a chemical reaction in the conversion step.

[0065] When the backbone group according to one embodiment is a monovalent group other than the scaffold group portion among groups serving as raw materials for a chemical reaction, the backbone group may include a terminal group that can be modified with a monovalent group (modifying group). The terminal group is a group that acts as a scaffold for extending the modifying group by solid-phase synthesis in the conversion step. When the backbone group has a terminal group, the modifying group can be extended from the terminal group by a condensation reaction, a coupling reaction, a nucleophilic substitution reaction, or the like. The terminal group is, for example, amino, carboxy, hydroxy, phosphono, phospho, halogeno, or boryl, and in one embodiment, may be amino, carboxy, or hydroxy.

[0066] In one aspect, the backbone group may have a functional moiety. The functional moiety is a moiety that directly exerts a function in the compound produced by the production method of the first embodiment. When the backbone group according to one aspect is a monovalent group other than a scaffold group moiety among groups that serve as raw materials for a chemical reaction, if the backbone group contains a functional moiety, the production method of the first embodiment can improve the function, such as enhancing its function or enabling its function to be exerted under specific conditions. In this case, the functional moiety may be, for example, a divalent group obtained by removing two hydrogen atoms from a functional molecule, in which case each hydrogen atom may be substituted with a scaffold group and a terminal group. Furthermore, for example, the functional moiety may be, in a functional molecule having a functional group corresponding to a scaffold group or a terminal group, a divalent group obtained by removing one hydrogen atom each from the scaffold group or terminal group and another portion, in which case each hydrogen atom may be substituted with a scaffold group and a terminal group. In one aspect, the backbone group may be a monovalent group consisting of a terminal group and a functional moiety, or a monovalent group consisting of a functional moiety.

[0067] The function of the functional moiety may be, for example, a pharmacological effect, a visualization effect, or radioactivity. When the function of the functional moiety is a pharmacological effect, the functional moiety may be, for example, a moiety obtained by removing one, two, or more hydrogen atoms from a molecule having a pharmacological effect. When the function of the functional moiety is a pharmacological effect, for example, a product having a high purity sufficient for clinical administration can be obtained by simple purification. Furthermore, for example, when the method according to one embodiment includes a conversion step, if the function of the functional moiety is a pharmacological effect, a prodrug that exhibits its medicinal effect only in a disease environment can be produced by adding a modifying group that is cleaved in a disease environment to the terminal group. Furthermore, when the method according to one embodiment includes a conversion step, if the function of the functional moiety is a pharmacological effect, a medicinal substance with improved drug delivery and / or pharmacokinetics can be produced by adding a modifying group that is excellent in localization (targeting) to a disease site and / or improved retention in the body. When the function of the functional moiety is a visualization effect, the functional moiety may be, for example, a moiety obtained by removing one, two, or more hydrogen atoms from a molecule having a visualization effect. A molecule having a visualization function is a molecule that emits a detectable optical signal, such as a molecule that emits fluorescence (fluorescent molecule), a molecule that emits phosphorescence (phosphorescent molecule), a molecule that generates Raman scattering, or a molecule that generates chemiluminescence (luminescent molecule), with fluorescent molecules being preferred. When the function of the functional moiety is a visualization function, for example, a product having a high purity sufficient for clinical diagnosis or administration can be obtained by simple purification. Furthermore, for example, when the method according to one embodiment includes a conversion step, when the function of the functional moiety is a visualization function, a diagnostic probe (imaging probe) that emits an optical signal only in a disease environment can be produced by, for example, adding a modifying group that is detached in a disease environment to the terminal group. Radioactivity is the property of emitting radiation. A radioactive functional moiety (radioactive moiety) can be, for example, fluorine-18 ( １８ F), Iodine-131 ( １３１The moiety may be a moiety containing fluorine-1, fluorine-1, iodine-2, iodine-3, iodine-4, iodine-5, iodine-6, iodine-7, iodine-8, iodine-9, iodine-10, iodine-11, iodine-12, iodine-13, iodine-14, iodine-15, iodine-16, iodine-17, iodine-18, iodine-19, iodine-20, iodine-21, iodine-22, iodine-23, iodine-24, iodine-25, iodine-26, iodine-27, iodine-28, iodine-29, iodine-29, iodine-29, iodine-29, iodine-29, iodine-29, iodine-29, iodine-30, iodine-31, iodine-32, iodine-33, iodine-34, iodine-35, iodine-36, iodine-37, iodine-38, iodine-39 ...

[0068] In one embodiment, the backbone group may have at least one functional group protected with a protecting group, for example, at least one functional group including a terminal group may be protected with a protecting group, or the terminal group may be protected with a protecting group, as long as the protecting group can protect the backbone group and can be deprotected under conditions that do not deprotect PG from the scaffold group.

[0069] The production method according to the first embodiment includes a capture step and a deprotection step in this order. In one aspect, the production method according to the first embodiment may include a step (conversion step) of converting the backbone group of the compound represented by formula (I) captured on the support into a resultant group by at least one chemical reaction on the solid phase after the capture step and before the deprotection step. The resultant group is a group obtained by converting the backbone group by at least one chemical reaction on the solid phase. In one aspect, the production method according to the first embodiment may also include a step (pretreatment step) of obtaining the compound represented by formula (I), and the pretreatment step may be included before the capture step. In other words, the production method according to one aspect of the first embodiment may include a capture step, a conversion step, and a deprotection step in this order, a pretreatment step, a capture step, and a deprotection step in this order, or a pretreatment step, a capture step, a conversion step, and a deprotection step in this order.

[0070] In the pretreatment step, a compound of formula (I) is obtained, which can be obtained by chemical synthesis and / or biological synthesis (e.g., enzymatic synthesis), in one aspect by organic chemical synthesis, according to methods commonly used by those skilled in the art.

[0071] For example, the compound of formula (I) can be obtained by protecting the scaffold group in MBG with PG in CBFG-PG (scaffold group protection treatment). The scaffold group protection treatment can be carried out according to conditions that are sometimes used for protection with a protecting group from which PG is derived, and those skilled in the art can set the conditions appropriately. In one embodiment, the compound of formula (I) may be obtained by a method comprising protecting the scaffold group using a compound represented by the following formula (II): CBFG-PG-LG (II) In other words, in one embodiment, the scaffold group protection treatment may comprise protecting the scaffold group using a compound represented by formula (II) above. In formula (II), CBFG and PG are the same as in formula (I), and LG represents a monovalent leaving group. Protection of the scaffold group using a compound represented by formula (II) can be carried out according to conditions that are commonly used by those skilled in the art, for example, by contacting the compound represented by formula (II) with a compound having a scaffold group in an organic solvent, and the organic solvent may further contain necessary additives (e.g., a base, a catalyst). In this case, the leaving group in LG may be introduced into a reagent added to the reaction system from the beginning (i.e., the compound of formula (II) itself may be added to the reaction system), or may be introduced by converting a functional group such as a hydroxy group in the system (i.e., in situ).

[0072] The leaving group in LG is a group that, when protecting a functional group with a protecting group, is released (liberated) before or after the protecting group is bonded, thereby inducing the formation of a bond between the functional group and the protecting group or the generation of a reactive intermediate for protection with the protecting group. LG is not particularly limited as long as it is a leaving group used when protecting a functional group with a protecting group, and examples thereof include chloro, bromo, iodo, tosyl, In this case, the root of LG may be carbonyl or methylene and the scaffold group may be amino, hydroxy or carboxy; in one aspect, the root of LG may be carbonyl or methylene and the scaffold group may be amino, or the root of LG may be carbonyl and the scaffold group may be hydroxy; in a preferred aspect, the root of LG may be carbonyl and the scaffold group may be amino.

[0073] In the scaffold group protection treatment, the covalent bond-forming group of CBFG may be protected with a protecting group. In this case, the protecting group is not particularly limited as long as it can protect the covalent bond-forming group and can be deprotected under conditions that do not deprotect PG from the scaffold group. For example, when the covalent bond-forming group is an ethynyl group, trimethylsilyl or the like can be used as the protecting group. When the covalent bond-forming group is protected with a protecting group in the scaffold group protection treatment, the pretreatment step further includes a treatment of removing the protecting group from the covalent bond-forming group after the scaffold group protection treatment.

[0074] In one embodiment, the scaffold group protected in the scaffold group protection treatment may be a scaffold group contained in a compound represented by MBG-H (H represents a hydrogen atom), and in another embodiment, it may be a scaffold group contained in a compound that is a precursor to the compound represented by MBG-H. In other words, in one embodiment, in the pretreatment step, the compound of formula (I) may be obtained by carrying out the scaffold group protection treatment as the final treatment, or in another embodiment, the compound of formula (I) may be obtained by carrying out a conversion treatment by at least one further chemical reaction step after the scaffold group protection treatment.

[0075] The conversion treatment performed after the scaffold group protection treatment may be any treatment that does not include a chemical reaction that deprotects PG from the scaffold group. The conversion treatment may consist of a liquid-phase reaction and / or a solid-phase reaction, and in one embodiment, may consist of a liquid-phase reaction. The conversion treatment performed after the scaffold group protection treatment may include, for example, a reaction to protect the backbone group with a protecting group, and in one embodiment, a reaction to protect a terminal group in the backbone group with a protecting group. Furthermore, the conversion treatment performed after the scaffold group protection treatment may include, for example, a reaction to introduce a group containing a covalent bond-forming group in the compound of formula (I) or a group in which the covalent bond-forming group has been protected with a protecting group, or may include a reaction to elongate the covalent bond-forming group or a group in which the covalent bond-forming group has been protected with a protecting group by, for example, a condensation reaction, a nucleophilic substitution reaction, or a coupling reaction. For example, when the covalent bond-forming group is an azide, it may include a reaction to azido a terminal haloalkyl of a compound that is a precursor to the compound represented by MBG-H with sodium azide. Furthermore, for example, when the covalent bond-forming group is ethynyl, the reaction may include a reaction of introducing an ethynyl group or a group containing an ethynyl group protected by trimethylsilyl into a compound that is a precursor of the compound represented by MBG-H by a condensation reaction, a nucleophilic substitution reaction, or a coupling reaction.

[0076] In the capture step, a covalent bond is formed between the covalent bond-forming group of the compound of formula (I) and the carrier, thereby capturing the compound represented by formula (I) on the carrier. The conditions for the reaction to form a covalent bond between the covalent bond-forming group and the carrier can be appropriately set by a person skilled in the art based on common technical knowledge depending on the combination of the covalent bond-forming group and the surface-modifying group on the carrier. For example, the solvent, reaction concentration, reaction time, type of further addition, and concentration of each component may be appropriately set by a person skilled in the art.

[0077] In the conversion step, the backbone group of the compound of formula (I) captured on the support is converted into a resultant group by at least one chemical reaction on the solid phase. In this step, the backbone group is converted into a resultant group by solid-phase synthesis. The type of chemical reaction in the conversion step may be any reaction that does not impair the covalent capture between the support and the scaffold group, for example, any reaction that does not deprotect PG from the scaffold group, and a wide variety of chemical reactions can be used. The chemical reaction in each step of the conversion step may be, for example, a substitution reaction, addition reaction, elimination reaction, oxidation-reduction reaction, rearrangement reaction, cyclization reaction, or ring-opening reaction that does not deprotect PG from the scaffold group. In addition, the chemical reaction in each step of the conversion step may be, for example, a dehydration-condensation reaction, hydrolysis reaction, decarboxylation reaction, nucleophilic substitution reaction, electrophilic substitution reaction, oxidation reaction, reduction reaction, coupling reaction, or functional group conversion reaction that does not deprotect PG from the scaffold group. The conversion step may include at least one selected from the group consisting of, for example, extension of a modifying group, protection of a functional group, removal of a protecting group from a functional group, oxidation, reduction, and functional group conversion. The functional group conversion may be a reaction known as a reaction that can be used to convert a functional group in a molecule, such as conversion of hydroxy to halogeno.

[0078] In one embodiment, the at least one chemical reaction step may include a reaction to extend (add) the modifying group. In other words, in one embodiment, the conversion step may include a reaction to extend the modifying group from the terminal group. For example, the resulting group may be a group in which a modifying group is extended from the terminal group of the backbone group or a converted form thereof, or a converted form thereof. Furthermore, for example, the resulting group may be a group in which a modifying group is extended from the terminal group of the backbone group. The type of reaction to extend the modifying group can be appropriately selected by those skilled in the art depending on the structures of the terminal group and the modifying group. For example, it may be a dehydration condensation reaction, a nucleophilic substitution reaction, an electrophilic substitution reaction, or a coupling reaction. In one embodiment, it may be a dehydration condensation reaction or a nucleophilic substitution reaction. Note that if the terminal group is protected in the capture step, the conversion step may include a reaction to remove the protecting group protecting the terminal group. The type and conditions of the reaction may be appropriately selected by those skilled in the art as long as it can deprotect the terminal group and does not deprotect PG from the scaffold group.

[0079] In one embodiment, the modifying group may include an oligomer chain, and in a preferred embodiment, the oligomer chain or its terminal may be further modified with a group. The oligomer chain may be, for example, an oligopeptide chain, an oligonucleotide chain, or an oligosaccharide chain. The number of monomers constituting the oligomer chain may be, for example, 2 or more, 3 or more, 4 or more, or 5 or more, or 100 or less, 20 or less, 10 or less, 7 or less, 5 or less, 4 or less, or 3 or less, and these upper and lower limits may be freely combined. In one embodiment, the monomer constituting the oligomer chain may be a natural or unnatural amino acid, nucleotide, or monosaccharide. In a preferred embodiment, it may be a natural or unnatural amino acid or nucleotide, and in a more preferred embodiment, it may be a natural or unnatural amino acid.

[0080] Each amino acid constituting the oligopeptide chain may be, for example, an L- or D-amino acid, and in one embodiment, may be an L-amino acid. Each amino acid constituting the oligopeptide chain may be, for example, an α-amino acid, a β-amino acid, or a γ-amino acid, and in one embodiment, may be an α-amino acid. In the present disclosure, proline is treated as an α-amino acid. Each amino acid constituting the oligopeptide chain may be, for example, a primary or secondary amino acid bonded to the α-carbon, and in one embodiment, a primary amino or N-C １－５It may be an alkylamino, and in a preferred embodiment, it may be a primary amino. In a more preferred embodiment, each amino acid constituting the oligopeptide chain may be alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine. The reaction for extending the modifying group, which is the oligopeptide chain, may be, for example, amide condensation or a nucleophilic substitution reaction between an activated ester or an activated amide and an amino, and in one embodiment, the extension of the modifying group may be carried out by Fmoc solid-phase synthesis or Boc solid-phase synthesis. Fmoc solid-phase synthesis is a method for extending a peptide chain from the C-terminus toward the N-terminus using Fmoc as an amino-protecting group. The Boc solid-phase synthesis method is a method in which a peptide chain is elongated from the N-terminus toward the C-terminus using Boc as a carboxy protecting group.

[0081] Each nucleotide constituting the oligonucleotide chain may be, for example, a ribonucleotide or a deoxynucleotide. The nucleic acid base in each nucleotide constituting the oligonucleotide chain may be, for example, adenine, guanine, cytosine, thymine or uracil. The method for extending the modified group of the oligonucleotide chain may be any method that may be used when extending an oligonucleotide chain, such as the phosphoramidite method.

[0082] Each sugar constituting the oligosaccharide chain may be, for example, a pentose or a hexose, and in one embodiment, may be a hexose. Each sugar constituting the oligosaccharide chain may be, for example, an aldose or a ketose, or an aldose or a ketose of a hexose. Each sugar constituting the oligosaccharide chain may be, for example, glucose, mannose, galactose, or fructose. The method for elongating the modifying group, which is an oligosaccharide chain, may be any method that is sometimes used for elongating an oligosaccharide chain, such as a method using an oligosaccharide chain synthesized in liquid phase or enzymatically and having a halogeno- or phenylmercapto-sugar leaving group at the terminal, and elongating the terminal group by glycosylation with the cation generated by elimination of the leaving group.

[0083] In the deprotection step, PG is deprotected from the scaffold group. When the method according to one embodiment of the present disclosure does not include a conversion step, deprotecting PG from the scaffold group releases a compound comprising the scaffold group and the backbone group, i.e., a compound represented by MBG-H (H is a hydrogen atom), from the support. In this way, the compound comprising the scaffold group and the backbone group can be purified by the method according to one embodiment of the present disclosure. On the other hand, when the method according to one embodiment of the present disclosure includes a conversion step, deprotecting PG from the scaffold group releases a compound comprising the scaffold group and the resultant group from the support, thereby producing a compound in which the backbone group has been converted to the resultant group by at least one chemical reaction step on a solid phase.

[0084] The conditions for deprotecting PG from the scaffold group may be any conditions that do not cause unintended changes in the chemical structure of the backbone group or the resulting group, and can be appropriately selected by those skilled in the art within the range of conditions commonly used for removing the protecting group from which PG is derived. Examples of reactions for deprotecting PG from the scaffold group include acid hydrolysis, base hydrolysis, acid-catalyzed decarboxylation, oxidation, and reduction (e.g., hydrogenation). Regarding the deprotection method, for example, when the protecting group from which PG is derived is one that can be deprotected by acid hydrolysis, the deprotection step may be a step of deprotecting PG from the scaffold group by acid hydrolysis in water, and the acid may be, for example, trifluoroacetic acid or hydrochloric acid. Furthermore, for example, when the protecting group from which PG is derived is one that can be deprotected by acid-catalyzed decarboxylation, the deprotection step may be a step of deprotecting PG from the scaffold group by contacting with an acid in an organic solvent, and the acid may be, for example, trifluoroacetic acid or hydrochloric acid. Also, for example, if the protecting group from which PG is derived is one that can be deprotected by base hydrolysis, the deprotection step can be a step of deprotecting PG from the scaffold group by base hydrolysis in water, and the base can be, for example, sodium hydroxide or potassium carbonate.

[0085] In one aspect, the target compound (the compound produced by the production method of the first embodiment) may be a compound comprising a scaffold group and a backbone group or a resultant group obtained in the deprotection step. In another aspect, the target compound may be a compound obtained by further converting the chemical structure of the compound comprising a scaffold group and a backbone group or a resultant group obtained in the deprotection step. In other words, in another aspect, the production method of the first embodiment may include a step (post-treatment step) of further converting the compound comprising a scaffold group and a backbone group or a resultant group after the deprotection step. The conversion in the post-treatment step can be appropriately designed, selected, and performed by a person skilled in the art, and may be a single-step or multi-step conversion, for example, a conversion by chemical synthesis and / or biological synthesis (e.g., enzymatic synthesis), or in one aspect, a conversion by organic chemical synthesis.

[0086] The target compound produced by the production method of the first embodiment may be, for example, a molecular compound. In this case, the target compound is not limited to the form of the molecule itself of the molecular compound, and may be, for example, the molecular compound, a salt of the molecular compound, or a solvate thereof, or a metal complex containing them.

[0087] In these cases, the molecular weight of the molecular compound may be, for example, 100 or more, 200 or more, 300 or more, 500 or more, 700 or more, or 1000 or more, or 10000 or less, 7000 or less, 5000 or less, 3000 or less, 2000 or less, or 1500 or less, and these upper and lower limits may be freely combined.

[0088] The target compound produced by the production method of the first embodiment may be composed of, for example, at least one atom selected from the group consisting of atoms of elements with atomic numbers 1 to 20, or may be a molecular compound composed of at least one atom selected from that group, a salt of the molecular compound, or a solvate thereof. The target compound produced by the production method of the first embodiment may be composed of, for example, at least one atom selected from the group consisting of hydrogen atoms, lithium atoms, boron atoms, carbon atoms, nitrogen atoms, oxygen atoms, fluorine atoms, sodium atoms, magnesium atoms, silicon atoms, phosphorus atoms, sulfur atoms, chlorine atoms, potassium atoms, and calcium atoms. The target compound produced by the production method of the first embodiment may be, for example, a molecular compound composed of at least one atom selected from the group consisting of hydrogen atoms, boron atoms, carbon atoms, nitrogen atoms, oxygen atoms, fluorine atoms, silicon atoms, phosphorus atoms, sulfur atoms, and chlorine atoms, or a salt of the molecular compound, or a solvate thereof.

[0089] Specific examples of target compounds produced by the production method of the first embodiment include functional substances themselves, functional substances modified with modifying groups (e.g., prodrugs, targeting drugs, or fluorescent probes), and linkers of small molecule-small molecule conjugates or antibody-small molecule conjugates (ADCs).

[0090] A second embodiment of the present disclosure is a compound screening method comprising a capture step, a conversion step, a deprotection step, and a step (evaluation step) of evaluating a compound comprising a scaffold group and a backbone group or a resultant group, or a compound obtained by further converting the compound. Hereinafter, this embodiment will also be referred to as the "screening method of the second embodiment." In the screening method of the second embodiment, a compound comprising a scaffold group and a backbone group or a resultant group, or a compound obtained by further converting the compound, is used as a candidate compound, and the compound is evaluated in the evaluation step to select a compound having a desired function. The screening method of the second embodiment includes a capture step, a conversion step, a deprotection step, and an evaluation step, in this order. The capture step, conversion step, and deprotection step of the screening method of the second embodiment are performed in the same manner as described for the capture step, conversion step, and deprotection step of the production method of the first embodiment. Hereinafter, a compound consisting of a scaffold group and a backbone group or a resultant group, or a compound obtained by further converting the same (i.e., a compound consisting of a scaffold group and a backbone group, a compound obtained by further converting a compound consisting of a scaffold group and a backbone group, a compound consisting of a scaffold group and a resultant group, or a compound obtained by further converting a compound consisting of a scaffold group and a resultant group) will also be referred to as a "candidate compound".

[0091] In one aspect, the screening method of the second embodiment may include a pretreatment step prior to the capture step. The pretreatment step in the screening method of the second embodiment is carried out in the same manner as described for the pretreatment step in the production method of the first embodiment.

[0092] In one aspect, the screening method of the second embodiment may include a post-treatment step after the deprotection step and before the evaluation step. The post-treatment step in the screening method of the second embodiment is carried out in the same manner as described for the post-treatment step in the production method of the first embodiment.

[0093] In the evaluation step, a candidate compound is evaluated. More specifically, when the screening method of the second embodiment does not include a post-treatment step, the evaluation step evaluates a compound comprising the scaffold group and the resultant group obtained in the deprotection step, and when the screening method of the second embodiment includes a post-treatment step, the evaluation step evaluates a compound obtained by further converting the compound comprising the scaffold group and the resultant group obtained in the post-treatment step.

[0094] The evaluation items and evaluation methods for candidate compounds in the evaluation step can be appropriately selected by those skilled in the art from commonly used evaluation items and evaluation methods depending on the purpose of screening. The evaluation method in the evaluation step may be, for example, an in vitro method, a method using cells, or a method using experimental animals. The experimental animal may be an experimental animal other than a human, such as a mouse or a rat. The experimental animal may also be a model animal for the disease targeted by the compound, such as a cancer model animal.

[0095] For example, if the compound selected by the screening method of the second embodiment is a ligand for a certain target, the in vitro method in the evaluation step may be, for example, surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), or ELISA. In the same case, the method using cells in the evaluation step may be, for example, an affinity evaluation using the phenotype of cells expressing the target as an indicator when the compound is contacted with the cells. Such affinity measurement can be performed according to techniques commonly used by those skilled in the art.

[0096] For example, if the compound selected by the screening method of the second embodiment is a fluorescent probe, the in vitro method in the evaluation step may involve, for example, measuring a change in a fluorescence parameter due to the interaction between the candidate compound and the target molecule (e.g., a structural change caused by metabolism by the target enzyme or binding to the target molecule) using an optical instrument such as a microwell plate reader or a fluorometer. In the same case, the method using cells or experimental animals in the evaluation step may involve, for example, contacting a candidate compound with cells or experimental animals that contain or may contain the target molecule or environment, and then measuring the fluorescence parameter in the cells or experimental animals using an optical instrument such as a fluorescence microscope, an imaging device, or a microwell plate reader. In these cases, the fluorescence parameter may be, for example, fluorescence intensity, the ratio of fluorescence intensities at multiple wavelengths (ratio value), or fluorescence lifetime; in one aspect, it may be fluorescence intensity. Measurement of these fluorescence parameters can be performed according to techniques commonly used by those skilled in the art. Furthermore, when the compound selected by the screening method of the second embodiment is a radioactive substance for visualization, the method using cells or experimental animals in the evaluation step may involve evaluating the radiation intensity (e.g., dose) in cells or experimental animals similarly treated with a radioactive substance instead of a fluorescent probe by measuring the radiation intensity (e.g., dose) using a dosimeter or an imaging device capable of measuring radiation intensity, such as a PET device or a SPECT device.

[0097] For example, when the compound selected by the screening method of the second embodiment is a compound used as a substance having a therapeutic effect or as a part thereof (e.g., a linker for an ADC), the in vitro method in the evaluation step may be performed, for example, according to the same method as described for the case where the compound to be selected is a ligand. Furthermore, in the same case, the method using cells in the evaluation step may involve, for example, contacting cells derived from a patient with the disease (e.g., an established cancer cell line) with the candidate compound and evaluating the viability and / or proliferation rate of the cells. The method for evaluating the viability and proliferation rate of cells may be any method commonly used by those skilled in the art, and may include, for example, a method using the intensity of the absorbance signal or fluorescence signal in an MTT assay, a CCK-8 assay, or live-dead staining (calcein AM and ethidium homodimer) as an index, or a method in which the number of cells or viable cells is counted before and after a predetermined time (e.g., 1 day, 2 days, 3 days, or 7 days) has elapsed since contact with the candidate compound. In the same case, the cell-based method in the evaluation step may involve, for example, contacting cells derived from a patient with the disease (e.g., an established cancer cell line) with a candidate compound and assessing the expression level of a disease biomarker (e.g., a specific protein or the mRNA encoding it) or the enzymatic activity of a disease marker enzyme in the cells. The expression level of a disease biomarker may be assessed according to methods commonly performed by those skilled in the art. For example, the protein expression level may be assessed by ELISA or Western blotting, the mRNA expression level may be assessed by PCR or a method using a next-generation sequencer, and the enzymatic activity may be assessed by a method using a fluorescent probe or a standard substrate. In the same case, the experimental animal method in the evaluation step may involve, for example, administering the candidate compound to a disease model animal (e.g., a cancer model animal) and assessing parameters commonly obtained in clinically diagnosing the disease. Such parameters may be, for example, the animal's survival time, the size of the diseased area, and / or a biomarker for the disease.In addition, when the candidate compound is not a compound that has a therapeutic effect by itself but a compound that exerts a therapeutic effect by sensitizing an external stimulus, the cells or experimental animal may be subjected to a stimulus (e.g., light irradiation, radiation irradiation, neutron beam irradiation, etc.) after contacting or administering the candidate compound.

[0098] In one aspect, the screening method of the second embodiment may further include, after the evaluation step, a step (selection step) of selecting a compound having a desired function from among the candidate compounds based on the evaluation results of the evaluation step. In the selection step, for example, a candidate compound that shows a statistically significant or remarkable difference in the magnitude of a parameter corresponding to the function compared to a similar evaluation performed using a compound not having the desired function or a similar evaluation performed without exposure to the candidate compound may be selected as a compound having the desired function. In this case, showing a statistically significant or remarkable difference may mean, for example, that the p-value in a statistical test is less than 0.05 or less than 0.01, respectively. Furthermore, for example, a compound whose value of the parameter is a predetermined factor or more (e.g., 2-fold or more, 3-fold or more, 5-fold or more, or 10-fold or more) higher than the average value of the parameter corresponding to the function measured for all candidate compounds tested may be selected as a compound having the desired function. In these cases, the parameter corresponding to the desired function may be, for example, the affinity with the target compound (e.g., K ｄ , I.C. ５０ ), fluorescence parameters (fluorescence intensity, ratio value, fluorescence lifetime), survival rate, size of diseased area, expression level of disease biomarker, or enzymatic activity of disease biomarker enzyme.

[0099] The number of candidate compounds evaluated in the evaluation step may be, for example, 2 or more, 5 or more, 20 or more, 50 or more, 100 or more, 200 or more, or 500 or more. In the screening method according to the second embodiment of the present disclosure, a large number of compounds can be easily purified by SAS. Therefore, by applying a combinatorial chemistry technique such as the split-and-pool method, a large number of highly pure candidate compounds can be easily produced and evaluated. In combinatorial chemistry, a single compound is subjected to different transformations to obtain multiple compounds, and then these multiple compounds are further subjected to different transformations to obtain multiple compounds. This process allows a large number of candidate compounds to be easily produced combinatorially. Therefore, in the screening method according to the second embodiment, such a large number of candidate compounds can be easily purified by SAS, allowing for high-precision screening to be easily performed without optimizing purification conditions or tedious purification procedures. In addition, when the screening method of the second embodiment includes a conversion step, it becomes possible to obtain highly pure compounds that have been converted by solid-phase synthesis. Furthermore, from the many types of candidate compounds produced in this manner, candidate compounds having desired functions can be selected by comprehensively evaluating the compounds in an unknown structure using, for example, a microwell plate reader. Furthermore, the selected candidate compounds can be analyzed by mass spectrometry or nuclear magnetic resonance analysis to identify their chemical structures. As a result, the screening method of the second embodiment can achieve both improved screening size and accuracy, and can effectively find hit compounds.

[0100] A third embodiment of the present disclosure is a compound represented by formula (II-A): CRG-PG-LG (II-A), where in formula (II-A), CRG represents a monovalent group having a click-reactive group, with the proviso that the click-reactive group may be protected, PG represents a divalent group obtained by removing one hydrogen atom from a monovalent protecting group, and LG represents a monovalent leaving group. CRG in formula (II-A) is a group in which a monovalent group having a click-reactive group, which is one aspect of CBFG according to the production method of the first embodiment, is optionally protected, and more specific examples of the click-reactive group and its protecting group may be those described in the production method of the first embodiment. Furthermore, PG and LG in formula (II-A) may be those described in the production method of the first embodiment. In one aspect, the click-reactive group may be unprotected or may be protected. Hereinafter, the compound of this embodiment will also be referred to as a "compound of the third embodiment."

[0101] The compound of the third embodiment can be suitably used to first protect a compound with PG, then capture the compound on a support by covalent bonding via a covalent bond-forming group, and then deprotect the protecting group from the compound. Thus, in one aspect, the compound of the third embodiment can be used to purify a compound by SAS. In other words, one aspect of the third embodiment can be the use of the compound of the third embodiment in purifying a compound by SAS. The compound of the third embodiment can also be used to convert a raw material compound by solid-phase synthesis, by first protecting the raw material compound with PG, then capturing the raw material compound on a support by covalent bonding via a covalent bond-forming group, converting the raw material compound by a chemical reaction, and then removing the protecting group from the converted compound to liberate the converted compound. Thus, in one aspect, the compound of the third embodiment can be used to capture a compound on a support in solid-phase synthesis, and then releasing the converted compound by deprotection. In other words, one aspect of the third embodiment may be use of the compound of the third embodiment in capturing a compound on a support in a solid-phase synthesis method and releasing the converted compound by deprotection. Also, in a preferred aspect, the compound of the third embodiment may be used for the production method of the first embodiment or the screening method of the second embodiment. In other words, a preferred aspect of the third embodiment may be use of the compound of the third embodiment in the production method of the first embodiment or the screening method of the second embodiment.

[0102] A fourth embodiment of the present disclosure is a kit comprising a compound represented by formula (IV): CRG-PG-OH (IV) and a reagent that converts the hydroxyl group of the compound represented by formula (IV) to introduce a monovalent leaving group. In formula (IV), CRG represents a monovalent group having a click-reactive group, with the proviso that the click-reactive group may be protected, and PG represents a divalent group formed by removing one hydrogen atom from a monovalent protecting group. In addition, in formula (IV), OH represents a hydroxyl group. The click-reactive group and its protecting group, as well as PG, are the same as those described for the compound of the third embodiment. Hereinafter, the compound of this embodiment will also be referred to as the "kit of the fourth embodiment." Hereinafter, the compound represented by formula (IV) will also be referred to as the "formula (IV) compound." Hereinafter, the reagent that converts the hydroxyl group of the compound represented by formula (IV) to introduce a monovalent leaving group will also be referred to as the "leaving group introducing reagent."

[0103] In the kit of the fourth embodiment, contacting a compound of formula (IV) with a leaving group introducing reagent converts the hydroxyl group of the compound of formula (IV) to introduce a monovalent leaving group, thereby obtaining a compound according to one aspect of the third embodiment. The leaving group introducing reagent may be any reagent capable of converting the hydroxyl group in the compound of formula (IV) to introduce a leaving group, and a reagent that may be used to convert a hydroxyl group bonded to a carbon atom to introduce a leaving group can be appropriately selected by a person skilled in the art depending on the type of leaving group, the structure of the compound of formula (IV), and common general technical knowledge. Contacting these leaving group introducing reagents with a compound of formula (IV) under conditions that can be appropriately determined by a person skilled in the art based on common general technical knowledge can provide a compound according to one aspect of the third embodiment. In a preferred aspect, the introduction of the leaving group may involve substituting the hydroxyl group with the leaving group, or substituting the hydrogen atom of the hydroxyl group with a carbonyl to which the leaving group is directly bonded. In one aspect, the leaving group introducing reagent may be a reagent that substitutes a hydroxyl group for a monovalent leaving group by contacting the compound of Formula (IV) with another reagent (e.g., triphenylphosphine) in the presence of the other reagent. In this case, the kit of the fourth embodiment may further include the other reagent, which can be appropriately selected by a person skilled in the art based on common technical knowledge. For example, when the leaving group is chloro, the leaving group introducing reagent may be, for example, triphosgene, thionyl chloride, phosphoryl chloride, phosgene, hydrogen chloride, or carbon tetrachloride. In particular, when the leaving group introducing reagent is carbon tetrachloride, the kit of the fourth embodiment may further include triphenylphosphine. For example, when the leaving group is bromo, the leaving group introducing reagent may be phosphorus tribromide, phosphorus pentabromide, hydrogen bromide, hydrogen tetrabromide, or elemental bromine. In particular, when the leaving group introducing reagent is hydrogen tetrabromide or elemental bromine, the kit of the fourth embodiment may further include triphenylphosphine. For example, when the leaving group is iodine, the leaving group introducing reagent may be simple iodine, lithium iodide, sodium iodide, or potassium iodide. In particular, when the leaving group introducing reagent is simple iodine, the kit of the fourth embodiment may further include triphenylphosphine. For example, when the leaving group is tosyl (p-toluenesulfonyl), the leaving group introducing reagent may be tosyl chloride. For example, when the leaving group is When the substituent is When the substituent is Thus, the leaving group introducing reagent in one embodiment may be triphosgene, thionyl chloride, phosphoryl chloride, phosgene, hydrogen chloride, carbon tetrachloride, phosphorus tribromide, phosphorus pentabromide, hydrogen bromide, hydrogen tetrabromide, elemental bromine, elemental iodine, lithium iodide, sodium iodide, potassium iodide, tosyl chloride, CDI, NHS, or paranitrophenol.

[0104] The kit of the fourth embodiment can be used in the same manner as the compound of the third embodiment. That is, in one aspect, the kit of the fourth embodiment may be used for purifying a compound by SAS. In other words, one aspect of the fourth embodiment may be the use of the kit of the fourth embodiment in purifying a compound by SAS. Furthermore, the kit of the fourth embodiment may be used for capturing a compound on a support in a solid-phase synthesis method and releasing the converted compound by deprotection. In other words, one aspect of the fourth embodiment may be the use of the kit of the fourth embodiment in capturing a compound on a support in a solid-phase synthesis method and releasing the converted compound by deprotection. Furthermore, in a preferred aspect, the kit of the fourth embodiment may be used for the production method of the first embodiment or the screening method of the second embodiment. In other words, a preferred aspect of the fourth embodiment may be the use of the kit of the fourth embodiment in the production method of the first embodiment or the screening method of the second embodiment. In one aspect of these cases, the kit of the fourth embodiment may further comprise instructions containing a description of the steps corresponding to the manufacturing method of the first embodiment or the screening method of the second embodiment, and the instructions may be electronic.

[0105] The present disclosure will be described in more detail below using examples, but the present disclosure should not be limited to the following examples.

[0106] <Preparation Example 1: Preparation of Tentagel-azide> All reactions were carried out using a fully automated peptide synthesizer, SyroI (Biotage). ２ 600 mg (156 μmol) of (Sigma-Aldrich, 86364, 90 μm, 0.26 mmol / g) was dissolved in 5 mL of DIEA-NMP (N-methyl-2-pyrrolidone) (1:2), and 25 mg (64 μmol) of Azide-PEG4-SE (TCI, A2388) was added and reacted at room temperature for 18 hours. The beads were then washed three times with DMF, dissolved in 5 mL of DIEA-NMP (1:2), and 156 μL of acetic anhydride was added and reacted at room temperature for 3 hours. The beads were then washed three times with DMF and then three times with DCM to obtain Tentagel-azide. The washed beads were dried under reduced pressure and stored in a refrigerator at 4°C. In the following examples, Tentagel-azide will also be simply referred to as "beads."

[0107] Example 1 In Example 1, a group of fluorescent probes was prepared in which sHMRG (WO 2023 / 219136), a highly water-soluble fluorescent base, was modified with a polypeptide. The sHMRG used in this example was prepared according to the method described in WO 2023 / 219136.

[0108] [Synthesis of p-Propargyloxy benzyl alcohol (Pzl-OH)] 2500 mg (15.6 mmol, 1 eq.) of p-Propargyloxy benzyaldehyde (APOLLO SCIENTIFIC, OR15265) was dissolved in 10 mL of methanol (MeOH). Sodium borohydride (NaBH ４ After adding 1180 mg (31 mmol, 2 eq.) of HCl in small portions, the solution was stirred at room temperature for 30 minutes. ２After adding 20 mL of HCl, extraction was performed three times using dichloromethane (DCM) through a liquid separation operation. The collected DCM layer was washed once using saturated brine through a liquid separation operation. The washed DCM layer was washed with sodium sulfate (Na ２ SO ４ ), and then the solvent was removed by distillation under reduced pressure to obtain 1760 mg of a colorless liquid as a residue (yield: 70%). Pzl-OH is an example of the compound of formula (IV) according to the kit of the fourth embodiment.

[0109] Pzl-OH １ H-NMR (400MHz, CD ３ CL): δ 2.49 (s, 1H), 4.61 (d, 2H, J = 6.0Hz), 4.68 (s, 2H), 6.91 (d, 2H, J = 8.0Hz), 7.28 (d, 2H, J = 8.0Hz). LRMS(ESI ＋ ): m / z=145 (M-OH) ＋

[0110] [Synthesis of sHMRG-Cpz] 150 mg (0.92 mmol) of Pzl-OH was dissolved in 5 mL of tetrahydrofuran (THF), and 805 μL (4.62 mmol, 5 eq.) of N,N-diisopropylethylamine (DIEA) and 69 mg (0.23 mmol, 0.25 eq.) of triphosgene dissolved in 1 mL of THF were added in that order on an ice bath under an argon atmosphere. The mixture was returned to room temperature and stirred for 30 minutes to obtain Solution A. 20 mg (0.05 mmol) of sHMRG was dissolved in a mixed solvent of 1 mL of dimethyl sulfoxide (DMSO) and 300 μL of N,N-diisopropylethylamine (DIEA). 500 μL of Solution A was added to the solution. The reaction mixture was analyzed by HPLC every 10 minutes, and when about 50% of sHMRG was converted to a Cpz(p-propargyloxy)benzyoxycarbonyl) protected product, the reaction mixture was added with H ２ 1 mL of O was added. After the addition, the solution was concentrated under reduced pressure and then ２ The mixture was diluted with 0.05% TFA (trifluoroacetic acid) in H O and purified by preparative MPLC (mobile phase: A = 0.1% TFA (trifluoroacetic acid) in H O). ２(O, B = 0.1% TFA in AcCN (acetonitrile), A:B = 95:5 to 5:95 over 15 min). Fractions containing the target substance were combined and lyophilized to obtain 7 mg of an orange solid (y. 24%).

[0111] sHMRG-Cpz １ H-NMR (400MHz, CD ３ OD): δ 3.45 (s, 1H), 4.33 (s, 2H), 4.71 (s, 2H), 5.18 (s, 2H), 6.7-7.1 (m, 6H), 7.2-7.4 (m, 5H), 7.95 (d, 1H, J=8.2Hz), 8.16 (s, 1H). LRMS(ESI ＋ ): m / z=585 (M+H) ＋

[0112] [Synthesis of Fluorescent Probes with sHMRG as the Core Nucleus] A group of fluorescent probes with various modification groups was synthesized using sHMRG as the core nucleus. The amino acids in the peptide sequence bound to the probe are expressed as P1, P2, P3, etc., from the amino acid directly linked to the fluorophore (C-terminal side) toward the N-terminal side. For modification of the P1 position, an appropriately protected building block (Fmoc-AA-OH) listed in Table 1 was used, and the reaction was carried out as described in Step 1 below. For modification of the P2 position and beyond, the reaction was carried out as described in Step 3 below using the building blocks listed in Table 1. In the following description, these amino acids will be represented by the single-letter or three-letter symbols listed in Table 1. Furthermore, the reaction of binding an organic acid such as succinic acid (Suc) to the N-terminal side was also carried out as described in Step 3 below, similar to the case of amino acid modification.

[0113]

[0114] More specifically, the probe was synthesized according to the following scheme.

[0115] [Step 1: Liquid-Phase Pretreatment] 1 μmol of sHMRG-Cpz was dissolved in 10 μL of NMP. To this solution, 10 μmol of an appropriately protected amino acid (shown as Fmoc-AA-OH in the reaction scheme) serving as a building block for peptide synthesis was added as an extension of the first amino acid residue, and 2.8 mg (10 μmol) of DMT-MM were added, followed by dissolution in 500 μL of DCM. The solution after addition was stirred in an open system at 55°C for 2 hours.

[0116] [Step 2: Capture on solid phase] 50 mg of beads were placed in a reactor for solid phase synthesis (a reaction vessel with a filter on the bottom that can aspirate the solvent), and 150 μL of Tris-HCl buffer (pH 7.4, 100 mM) was added. The solution after the reaction in step 1 was dissolved in 50 μL of tertiary butanol (t-BuOH) and added to the reactor. The vessel containing the solution after the reaction in step 1 was washed twice with 50 μL of t-BuOH, and these were also added to the same Tris-HCl buffer. A total of 300 μL of solution was added containing CuSO ４ (1 mM), BTTP (3-(4-((Bis((1-tert-butyl-1H-1,2,3-triazol-4-yl)methyl)amino)methyl)-1H-1,2,3-triazol-1-yl)propan-1-o, 3 mM), and sodium ascorbate (3 mM) (all concentrations are final concentrations) were added, and the mixture was stirred at room temperature for 2 hours, whereby the intermediate obtained in step 1 was captured on the beads by a click reaction.

[0117] [Step 3: Elongation Reaction] Using a fully automated peptide synthesizer, SyroI, amino acid peptide elongation from the second residue onward was carried out according to the standard protocol for Fmoc solid-phase synthesis. Specifically, the peptide was elongated one amino acid at a time from the C-terminus by repeating the following steps until the desired peptide sequence was obtained: (1) condensation, (2) washing, (3) deprotection of the Fmoc group, and (4) washing. (1) Condensation was carried out for 40 minutes in 2 mL of a DMF solution containing 0.16 M of the amino acid building block (Fmoc-AA-OH), 0.16 M HATU, and 0.64 M DIEA. (2) Washing was carried out by adding 2 mL of DMF, reacting for 1 minute, and then removing the solvent, a procedure repeated three times. (3) For deprotection of the Fmoc group, 2 mL of a 40% piperidine solution (DMF) was added and reacted for 3 minutes, after which the solution was removed and 2 mL of a 20% piperidine solution (DMF) was added and reacted for 3 minutes. (4) For washing, the same procedure as in (2) was repeated six times.

[0118] [Step 4: Deprotection] After step 3, the beads were washed three times with DMF and then three times with DCM. After washing, the beads were washed with 2 mL of TFA-H ２ A 9:1 mixture of acetone and HCl was added and stirred at room temperature for 3 hours to release the compound from the beads. For compounds with a terminal succinic acid moiety, the tertiary butyl group was deprotected. The solution components after the reaction were recovered. The remaining beads were washed twice with 500 μL of acetone, which was also added to the recovered solution components. ２ The solution was analyzed by HPLC, and those compounds that did not exceed 90% purity in the absorption chromatogram (absorbance at 500 nm) were subjected to simple purification using preparative MPLC. ２ Dissolved in 0 and loaded onto a preparative MPLC, mobile phase: A = 0.1% TFA (trifluoroacetic acid) in H ２ Purification was carried out under the conditions of O, B = 0.1% TFA in AcCN (acetonitrile), A:B = 95:5 to 5:95 over 15 min, and the fractions containing the target substance were mixed and lyophilized. The resulting solid was dissolved in DMSO at an appropriate concentration.

[0119] Figures 1, 2, 3, 4, 5, and 6 show the results of HPLC analysis of each compound (solid dissolved in DMSO) in the fluorescent probe group synthesized in Example 1. In the figures, the amino acid sequence of each compound is shown in the upper right corner of the chromatogram. The vertical axis of the chromatograms in Figures 1 to 6 represents absorbance at 500 nm. As shown in Figures 1 to 6, 55 compounds with different retention times were obtained in high purity. This demonstrates that the production method of the first embodiment can synthesize fluorescent probes with diverse structures by solid-phase synthesis. This production method is suitable for use in combinatorial chemistry. It was also revealed that the production method of the first embodiment can produce target compounds with high purity. This production method, which produces such highly pure target compounds, does not require or is easy to purify the target compounds, and they could be quickly used in screening experiments using enzyme activity measurements, as shown in Example 8. SEQ ID NO: 1: VDQQD SEQ ID NO: 2: DYEVD SEQ ID NO: 3: GDEVD SEQ ID NO: 4: GYVAD SEQ ID NO: 5: GLEVD SEQ ID NO: 6: VDVAD SEQ ID NO: 7: VVEID SEQ ID NO: 8: GPLGP SEQ ID NO: 9: AAPPD SEQ ID NO: 10: GVQVD SEQ ID NO: 11: LLEHD SEQ ID NO: 12: EGITD SEQ ID NO: 13: DYEVE SEQ ID NO: 14: GIETE SEQ ID NO: 15: GDEVE

[0120] Example 2 In Example 2, a fluorescent probe was prepared in which deSiR (WO 2023 / 219136), a highly water-soluble fluorescent base, was modified with a polypeptide. The Leuco deSiR used in this example was prepared according to the method described in WO 2023 / 219136.

[0121] [Synthesis of Ethynyl-trityl alcohol (Ert-OH)] 500 mg (1.97 mmol) of 4-bromophenylethylenyl)-trimethylsilane (TCI, B6028) was dissolved in 50 mL of THF and cooled to -78°C under an argon atmosphere. 1.5 mL (1.97 mmol, 1 eq.) of sec-BuLi in hexane (1.3 M) was added dropwise thereto, and the mixture was returned to room temperature and stirred for 30 minutes. The reaction solution was cooled again to -78°C, and a solution of 360 mg (1.97 mmol, 1 eq.) of benzophenone in 2 mL of THF was added dropwise thereto. After stirring at room temperature for 2 hours, H ２ After the addition, the solution was extracted three times with DCM by a separation operation, and the DCM layer was recovered. The recovered DCM layer was washed once with saturated saline, and then washed with Na ２ SO ４ The residue was purified by silica gel column chromatography (Hexane-DCM 100:0 to 0:100 over 15 min) to give 320 mg (y. 57%) of Ert-OH as a colorless liquid.

[0122] Ethynyl-trityl alcohol (Ert-OH) １ H-NMR (400MHz, CDCl ３ ): δ 2.77 (s, 1H), 3.05 (s, 1H), 7.3 (m, 12H), 7.45 (d, 2H, J=8.2Hz).

[0123] [Synthesis of leuco-dsSiR-Ert]

[0124] [Process 1: Synthesis of Ethynyl-trityl chloride (Ert-Cl)] 50 mg (0.18 mmol, 1 eq) of Ert-OH was dissolved in 1 mL of toluene, and the resulting solution was dissolved in SOCl. ２ 76 μL (1.1 mmol, 6 eq.) of toluene was added. After the addition, the solution was heated and stirred under reflux conditions for 18 hours. After the reaction, the solution was diluted with toluene, and the solvent was distilled off under reduced pressure to obtain Ert-Cl. The obtained Ert-Cl was used as it was in the next reaction.

[0125] Ethynyl-trityl chloride (Ert-Cl)１ H-NMR (400MHz, CDCl ３ ): δ 3.08 (s, 1H), 7.2 (m, 6H), 7.3 (m, 6H), 7.42 (d, 2H, J=8.2Hz).

[0126] [Treatment 2: Synthesis of leuco-dsSiR-Ert] 20 mg (0.04 mmol) of Leuco dsSiR was dissolved in 20 μL of NMP. A solution of 6.2 mg (0.02 mmol, 0.5 eq.) of Ert-Cl obtained in Treatment 1 and 36 μL (0.2 mmol, 5 eq.) of DIEA in 100 μL of DCM was added dropwise thereto, and the reaction was carried out at room temperature for 30 minutes while distilling off the DCM under reduced pressure. The concentrated solution was then heated in a 500-kJ / 2000 rpm flask. ２ The mixture was mixed with 0 and purified by preparative MPLC (mobile phase: A = 0.1% triethylamine in H ２ O, B = MeOH, A:B = 95:5 to 5:95 over 15 min). The fractions containing the target substance were mixed, triethylamine was added until the mixture was sufficiently basic, and the mixture was lyophilized to obtain 4.5 mg of an orange solid (y. 15%). LRMS (ESI － ): m / z=755 (MH) －

[0127] [Synthesis of Fluorescent Probes with dsSiR as a Core] Fluorescent probes with various modifying groups were synthesized using dsSiR as a core according to the following scheme: The amino acid building blocks used were those shown in Table 1 in Example 1.

[0128] Step 1: Liquid-Phase Pretreatment: 1 μmol of Leuco-dsSiR Ert was dissolved in 10 μL of NMP and 5 μL of DIEA. To this solution, 10 μmol of an appropriately protected amino acid (Fmoc-AA-OH) serving as a building block for peptide synthesis and 4.3 mg (10 μmol) of COMU ({{[(1-Cyano-2-ethoxy-2-oxoethylidene)amino]oxy}-4-morpholinomethylene}dimethylammonium Hexafluorophosphate) were added as an extension of the first amino acid residue, followed by the addition of 500 μL of DCM. The resulting solution was stirred at 55°C in an open system for 2 hours.

[0129] [Step 2: Capture on Solid Phase] In the same manner as in step 2 of Example 1, the intermediate obtained in step 1 was captured on beads.

[0130] [Step 3: Elongation Reaction] The peptide was elongated one amino acid at a time from the C-terminus according to the same method as in Step 3 of Example 1. [Step 4: Oxidation Reaction] The beads after Step 3 were washed three times with DMF. A solution of 10 mg of 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) in 1 mL of acetone was added to the washed beads, and the mixture was stirred at room temperature for 30 minutes to allow the reaction. This oxidized the dsSiR core moiety.

[0131] [Step 5: Deprotection] After step 4, the beads were washed five times with acetone. After washing, the beads were washed with 2 mL of TFA-H ２ A 9:1 mixture of acetone and HCl was added and stirred at room temperature for 3 hours to release the compound from the beads. For compounds with a terminal succinic acid moiety, the tertiary butyl group was deprotected. The solution components after the reaction were recovered. The remaining beads were washed twice with 500 μL of acetone, which was also added to the recovered solution components. ２ The solution was lyophilized and then purified using preparative MPLC. ２ Dissolved in 0 and loaded onto a preparative MPLC, mobile phase: A = 0.1% TFA (trifluoroacetic acid) in H２ Purification was performed under the following conditions: O, B = 0.1% TFA in AcCN (acetonitrile), A:B = 95:5 to 5:95 over 15 min, and the fractions containing the target substance were mixed and lyophilized. The resulting solid was dissolved in DMSO at an appropriate concentration. In this manner, a fluorescent probe with dsSiR as the parent nucleus was obtained. Figure 7 shows the chemical structure and HPLC chromatogram of Suc-GDEVD-dsSiR, one of the fluorescent probes obtained in this manner. The vertical axis of the chromatogram in Figure 7 represents absorbance at 500 nm. Example 2 demonstrated that the production method of the first embodiment is applicable not only to condensation reactions and mild deprotection reactions as performed in Example 1, but also to the production of compounds involving various types of reactions, including oxidation-reduction reactions.

[0132] Example 3 In Example 3, a compound was prepared in which a protecting group for SAS was bound to a reactive site (here, an amino acid site) different from the site exhibiting a function such as fluorescence, and a structure mimicking a linker molecule used in antibody-drug conjugates (ADCs) was synthesized. An ADC is a conjugate in which a drug (payload) is bound to a polymer such as an antibody via a linker site having a structure that can be cleaved by a hydrolysis reaction or the like. The linker structure used in ADCs consists of four sites: a modification site on the antibody, a linker site, an adapter site that connects the reaction in which the linker is cleaved to the release of the payload, and the payload. In ADCs, various peptides are used as linker sites with a structure that can be subjected to hydrolysis. For example, in a previous publication (Yasuaki Anami et al., "Glutamic acid-valine-citrulline linkers ensure stability and efficacy of antibody-drug conjugates in mice," Nat Commun 9, 2512 (2018)), a group of molecules with diverse peptide sequences was synthesized and the optimal molecule was selected. However, the synthesis in this publication required multiple purification steps, which limited the exploratory research that could be carried out. Therefore, in Example 3, we demonstrated that it is possible to synthesize a model molecule having the basic structure of an ADC linker using the concept of this patent. As shown below, for this model molecule, succinic acid was used as the antibody modification site instead of the commonly used click-reactive site, and O-Benzyl glycine was used as the payload instead of a drug.

[0133] [Synthesis of Fmoc-Lys(Cpz)-OH] 375 mg (0.92 mmol) of Fmoc-Lys-OH hydrochloride (Watanabe, L01166) was dissolved in 1 mL of NMP, and 120 μL (0.69 mmol) of DIEA was added. 5 mL of the same solution A used in the synthesis of sHMRG-Cpz in Example 1 was added thereto, and the mixture was allowed to react at room temperature for 2 hours. The concentrated solution was then dissolved in H ２The mixture was mixed with 0 and purified by preparative MPLC (mobile phase: A = 0.1% TFA in H ２ (O, B = MeOH, A:B = 95:5 to 5:95 over 15 min) Fractions containing the target substance were combined, triethylamine was added until the mixture was sufficiently basic, and the mixture was lyophilized to give 4.5 mg of an orange solid (y. 15%).

[0134] Fmoc-Lys(Cpz)-OH １ H-NMR (400MHz, CDCl ３ ):δ 1.2-2.0 (m, 6H), 3.22 (m, 2H), 4.18 (t, 1H, J=6.0Hz), 4.3 (m, 3H), 4.71 (s, 2H), 4.95 (s, 2H), 6 92 (d, 2H, J=8.0Hz), 7.24 (m, 4H), 7.35 (t, 2H, J=8.0Hz), 7.56 (m, 2H), 7.72 (d, 2H, J=8.0Hz). LRMS(ESI ＋ ): m / z=557 (M+H) ＋

[0135] [Synthesis of model molecules (models of ADC molecules) having the basic structure of an ADC linker]

[0136] [Step 1: Liquid Phase Pretreatment] 5 μmol of Fmoc-Lys(Cpz)-OH was dissolved in 20 μL of NMP. 30 μmol of p-aminobenzyl alcohol and 30 μmol of DMT-MM were added, followed by the addition of 500 μL of DCM to dissolve them. The resulting solution was stirred in an open system at 55°C for 2 hours.

[0137] [Step 2: Capture on Solid Phase] In the same manner as in step 2 of Example 1, the intermediate obtained in step 1 was captured on beads.

[0138] [Step 3: Conversion to p-Nitrophenyl carbonate] A solution of 20 mg of p-Nitrophenyl chloroformate in 1 mL of DMF and 200 μL of DIEA was added to the beads obtained after step 2, and the mixture was allowed to react at room temperature for 18 hours. The beads were then washed three times with DMF and three times with DCM.

[0139] [Step 4: Payload Binding] A solution of 20 mg of glycine benzyl Easter p-TsOH salt in 1 mL of DMF and 200 μL of DIEA was added to the beads after Step 3, and the reaction was allowed to proceed at room temperature for 3 hours. The beads were then washed three times with DMF and three times with DCM.

[0140] [Step 5: Elongation Reaction] According to the same method as in step 3 of Example 1, the peptide was elongated one amino acid at a time from the C-terminus.

[0141] [Step 6: Deprotection] After step 5, the beads were washed five times with DCM. After washing, the beads were washed with 2 mL of TFA-H ２ A 9:1 mixture of acetone and HO was added and stirred at room temperature for 3 hours to release the compound from the beads, and the solution components after the reaction were recovered. The remaining beads were washed twice with 500 μL of acetone, and this was also added to the recovered solution components. ２ The solution was lyophilized and then subjected to simple purification using preparative MPLC. ２ Dissolved in 0 and loaded onto a preparative MPLC, mobile phase: A = 0.1% TFA (trifluoroacetic acid) in H ２ Purification was carried out under the conditions of O, B = 0.1% TFA in AcCN (acetonitrile), A:B = 95:5 to 5:95 over 15 min, and the fractions containing the target substance were mixed and lyophilized. The resulting solid was dissolved in DMSO at an appropriate concentration.

[0142] Figure 8 shows the results of HPLC-MS analysis of the model ADC molecule synthesized in Example 3. The vertical axis of the HPLC chromatogram in Figure 8 represents absorbance at 254 nm. The MS spectrum in Figure 8 is the MS spectrum obtained at the time of elution of the main peak in the 254 nm chromatogram. In this way, it was possible to synthesize a compound having the same basic structure as the ADC linker using only a simple purification method. Example 3 demonstrated that the antibody binding site, peptide linker, and payload structures can be selected arbitrarily, making this method applicable to the synthesis of a wide range of functional molecules. Furthermore, molecules without an antibody binding site can also be used as prodrugs that release drugs by peptide hydrolysis around target cells. This demonstrates the applicability of this method to the synthesis of various functional molecules other than the fluorescent probes used in Examples 1 and 2.

[0143] Example 4 The compound capture efficiency was compared between a case where a compound was captured on a support via a covalent bond-forming group modified with a protecting group that protects the functional group of the compound, and a case where a compound was captured on a support commonly used in conventional solid-phase synthesis.

[0144] In the case of capturing a compound on a carrier via a covalent bond-forming group modified with a protecting group that protects the functional group of the compound, sHMRG-Cpz (0.5 μmol) obtained in Example 1 was dissolved in t-BuOH (40%), DMSO (10%), CuSO ４The compound was dissolved in 1 mL of Tris-HCl buffer (100 mM, pH 7.4) containing 1 mM benzotriazole, BTTP (3 mM), and sodium ascorbate (3 mM), and incubated with Tentagel-azide (0.26 mmol / g, 50 mg) obtained in Preparation Example 1 at 25°C for the time indicated in the figure. In the case of capturing a compound in a unit commonly used in conventional solid-phase synthesis, sHMRG-Cpz (0.5 μmol) was dissolved in 400 μL of NMP and 100 μL of DIEA, and incubated with CTC resin (1.55 mmol / g, 30 mg) at 25°C for the time indicated in the figure. During these incubations, the concentration of uncaptured sHMRG-Cpz remaining in the reaction solution was quantified using LC-MS.

[0145] FIG. 9 shows the results of comparing the concentration of untrapped sHMRG-Cpz remaining in the reaction solution when a compound is captured on a support via a covalent bond-forming group modified with a protecting group that protects the compound's functional groups, and when a compound is captured on a monomer commonly used in conventional solid-phase synthesis. The results in FIG. 9 are shown as mean ± standard deviation (n = 3). As shown in FIG. 9, in the former case, the target dye disappeared from the reaction solution after 30 minutes, whereas in the latter case, the rate of disappearance of the target dye from the reaction solution was slower, and the target dye still remained even after 18 hours. These results demonstrate that the method of capturing a compound on a support via a covalent bond-forming group modified with a protecting group that protects the compound's functional groups enables highly efficient capture of compounds.

[0146] Example 5 When a compound was captured on a carrier via a covalent bond-forming group modified with a protecting group that protects the functional group of the compound, the efficiency of compound release by deprotection of the functional group was evaluated.

[0147] Two hours after the start of incubation in Example 4, Tentagel was washed three times with DMF and three times with DCM. ２ 0 (500 μL) was added, and the mixture was shaken at 25°C for the time indicated in the figure, after which the released sHMRG was quantified by LC-MS.２ The amount was calculated by normalizing it with the amount released when treated with 500 μL of 0.

[0148] Figure 10 shows the results of evaluating the release efficiency of sHMRG by deprotection of functional groups when a compound was captured on a carrier via a covalent bond-forming group modified with a protecting group that protects the functional group of the compound. The vertical axis of Figure 10 represents the recovery rate of sHMRG. The results in Figure 10 are shown as the mean value ± standard deviation (n = 3). Figure 10 shows that the recovery rate exceeded 90% 90 minutes after the start of deprotection. This demonstrates that the compound captured on the carrier can remain stable on the carrier and can be recovered almost quantitatively by deprotection.

[0149] Example 6 In the same manner as in Example 1, sHMRG-Cpz was reacted with Fmoc-Tyr(tBu)-OH to form an amide bond, and the resulting Fmoc-Tyr(tBu)-sHMRG-Cpz was then covalently captured on Tentagel azide, followed by deprotection of Cpz from the amino group to recover Fmoc-Tyr(tBu)-sHMRG. The reaction was monitored by liquid-phase chromatography. The results of liquid-phase chromatography were monitored by absorbance at 254 nm.

[0150] 11 shows liquid-phase chromatography charts before (Reaction-0 h) and after (Reaction-1 h) reacting sHMRG-Cpz with Fmoc-Tyr(tBu)-OH for 1 hour, before (Flowthrough) capturing the resulting Fmoc-Tyr(tBu)-sHMRG-Cpz on Tentagel-azide, and after (Release) capturing and deprotecting Cpz from the amino group to recover Fmoc-Tyr(tBu)-sHMRG. Figure 11 demonstrates that the method according to the present disclosure can synthesize the target compound with a purity of 95% or higher without any special purification procedures and in a manner that allows for automation of all steps using a synthesis apparatus.

[0151] Preparation Example 2: Fmoc-Cir(Ert)-OH Fmoc-Cir(Ert)-OH, shown in the structural formula below, was prepared as a building block that can be used in the production method according to the present disclosure. Fmoc-Cir(Ert)-OH can form a covalent bond with a solid phase via an alkynyl group and has a carboxy group, making it suitable for use in producing compounds having citrulline or a peptide chain extended therefrom.

[0152] Fmoc-Cit-OH (Watanabe Chemical Co., Ltd., M00443, 640 mg, 1.61 mmol) was dissolved in 1 mL of glacial acetic acid. Ert-OH (307 mg, 1.08 mmol) obtained in Example 2 and acetic anhydride (457 μL, 4.83 mmol) were added thereto, and the mixture was stirred at 60°C for 30 minutes. Toluene was added to the reaction mixture, and the mixture was concentrated under reduced pressure. The residue was diluted with acetonitrile and directly purified by MPLC (C18 column; H ２ The resulting fraction was lyophilized to give Fmoc-Cit(Ert)-OH (48 mg, yield 6.7%) as a white solid.

[0153] Fmoc-Cit(Ert)-OH １ H-NMR (400MHz, AcCN-d3): δ 7.80 (d, 2H, J = 8.4Hz), 7.63 (d, 2H, J = 8.4Hz), 7.3-7.4 (m, 4H), 7.1-7.3 (m, 14H), 6.18 (s, 1H), 6.03 (2H, J = 8.0Hz), 5.29 (br , 1H), 4.28 (d, 2H, J = 8.4Hz), 4.21 (t, 1H, J = 8.4Hz), 4.15 (m, 1H), 3.32 (s, 1H), 2.9-3.0 (m, 2H), 1.4-1.7 (m, 2H), 1.37 (m, 2H) LRMS (ESI ＋ ): m / z=664 (M+H) ＋

[0154] Example 7: Fmoc-Lys(Cpz)-OH prepared in Example 3 and Fmoc-Cir(Ert)-OH prepared in Preparation Example 2 are suitable for the production method according to the present disclosure because the protecting group attached to the solid phase is not removed when forming an amide bond in solid-phase synthesis or when deprotecting the N-terminal protecting group (Fmoc), and the protecting group protecting the N-terminus is not removed when deprotecting the protecting group attached to the solid phase. Therefore, the products obtained by exposing Fmoc-Lys(Cpz)-OH and Fmoc-Cir(Ert)-OH to these conditions were evaluated using liquid chromatography with mass spectrometry (LC-MS). Stability under amide bond formation conditions was evaluated by LC-MS after stirring for 40 minutes at room temperature in NMP containing 40% by volume of DIEPA (N,N-diisopropylethylamine) based on the total amount. The stability under the conditions for deprotecting Fmoc was evaluated by LC-MS after stirring for 3 minutes in DMF containing 40% by volume of piperidine relative to the total volume. ２ After stirring in a 9:1 mixture of HCl and HCl for 3 hours, the product was evaluated by LC-MS.

[0155] Figure 12 shows the results of evaluating the products obtained when Fmoc-Lys(Cpz)-OH was exposed to conditions for forming an amide bond in solid-phase synthesis, conditions for deprotecting Fmoc, or conditions for deprotecting a protecting group bound to a solid phase. Figure 13 shows the results of evaluating the products obtained when Fmoc-Cir(Ert)-OH was exposed to conditions for forming an amide bond in solid-phase synthesis, conditions for deprotecting Fmoc, or conditions for deprotecting a protecting group bound to a solid phase. Figures 12 and 13 demonstrate that the protecting groups protecting the N-terminus and the protecting groups bound to a solid phase in the building blocks used were removed under the corresponding deprotection conditions but not under other conditions.

[0156] Example 8: Using a library of fluorescent probes obtained using a method similar to that of Example 1, we searched for fluorescent probes capable of specifically detecting liver damage, thereby exploring the activity of specific biomarkers for liver damage. Two liver damage models with different molecular mechanisms were created. The thioacetamide (TAA) model was for hepatocellular damage, and the 4,4'-methylenedianiline (MDA) model was for cholestasis. While the currently used biomarkers AST / ALT cannot distinguish between these two types of liver damage, we hoped that using the library of fluorescent probes obtained using a method similar to that of Example 1 would reveal unique changes in enzyme activity patterns that reflect the differences in pathology. Enzyme activity was evaluated using a digital enzyme assay using a microdevice. Note that sHMRG modified with a peptide chain is originally weakly fluorescent, but when the peptide chain is cleaved from its base by an enzymatic reaction, it yields highly fluorescent sHMRG as a metabolite.

[0157] [Creation of liver injury model and preparation of plasma samples] Animal studies were performed with the approval of the University of Tokyo Animal Experiment Committee (P4-21, P31-9). Six-week-old male C57BL / 6JJcl mice were purchased from CLEA Japan (Tokyo, Japan) and allowed to acclimate for 5 days. To induce liver damage, mice were administered thioacetamide (TAA, T0817, Tokyo Chemical Industry Co., Ltd., Japan, 350 mg / L) or 4,4'-methylenedianiline (MDA, M0220, Tokyo Chemical Industry Co., Ltd., Japan, 750 mg / L) dissolved in drinking water, while the control group was given tap water only. After 4 days of treatment, the mice were euthanized, and blood was collected from the inferior vena cava into 1.5 mL tubes containing 1.5 μL heparin (Yoshida Pharmaceutical Co., Ltd., Japan). The collected blood was centrifuged at 1,700 g at 4°C for 15 minutes, and plasma was separated.

[0158] [Preparation of buffer solution containing fluorescent probes and plasma samples] Various fluorescent probes (30 μM) obtained in the same manner as in Example 1 were dissolved in CaCl ２ (1mM), MgCl ２The cells were incubated at 25°C for 18 hours with plasma samples diluted 100-fold in Tris-HCl buffer (100 mM, pH 7.4) containing 1 mM triphosphate (DTT), 1 mM triphosphate (DTT), and 250 μM Triton X-100.

[0159] Digital Enzyme Assay Using a Microdevice Digital enzyme assay was performed using a commercially available microdevice (Simoa disk; Quanterix). 40 μL of a buffer solution containing a fluorescent probe and plasma sample was manually pipetted into the microdevice. Subsequently, 80 μL of FC-70 (Sigma-Aldrich) was injected into the device to wash away excess reaction mixture. Fluorescence images of the chamber were acquired using an epifluorescence microscope (Ti2, Nikon) equipped with a 20x dry objective (Plan Apo 20x), an sCMOS camera (ORCA-Fusion C14440, Hamamatsu Photonics), a white LED illumination unit (X-Cite Xylis, Optoscience), and a motorized stage. The assay was performed using a solution containing 10 μM of the fluorescent dye sTM (described in S. Sakamoto et al., Sci. Adv. 2020, 6, eaay0888.) as an internal standard, and focus was adjusted using its fluorescence. Images were acquired using the tile scan mode and perfect focus function. The excitation and emission filters used were FITC (mirror = 510 nm, Ex. = 460-500 nm, Em. = 510-560 nm) and mCherry (mirror = 600 nm, Ex. = 550-590 nm, Em. = 608-683 nm).

[0160] Image Processing: In the following examples, when creating histograms and scatter plots, acquired fluorescent images were processed using the GA3 module of NIS Elements software (Nikon). First, background correction using rolling ball correction (3 mm) was performed on all fluorescent images. Next, ROIs were selected by bright spot detection (diameter = 3 μm, contrast = 500) using an mCherry filter. Irregular fluorescent spots resulting from fluorescent debris or air bubbles were excluded by dilating the ROIs and removing overlapping ROIs. The fluorescent signal detected by the FITC filter, which reflects enzyme activity, was obtained as the average signal obtained from the center of each ROI. Data processing was performed using Excel or Kaleidagraph software, and histograms and scatter plots were created.

[0161] [Results] Figures 14, 15, and 16 show the results of evaluating the enzyme activity of plasma samples prepared from thioacetamide (TAA) model mice or 4,4'-methylenedianiline (MDA) model mice using various fluorescent probes. By preparing a library containing a large number of fluorescent probes using the production method of the present disclosure, it was possible to easily search for fluorescent probes that can specifically detect diseases. This also made it possible to identify activities that could serve as specific biomarkers for liver damage.

[0162] As an example of the discovered activity, Figure 17 shows the results of evaluating the enzyme activity of plasma samples prepared from thioacetamide (TAA) model mice or 4,4'-methylenedianiline (MDA) model mice using Suc-GLEVD-sHMRG. As shown in Figure 17, Suc-GLEVD-sHMRG was able to selectively detect enzyme activity that was specifically enhanced in mice with hepatocellular injury, which is liver injury induced by TAA, among other liver damage. The discovered Suc-GLEVD-sHMRG had a sequence that was cleaved by Caspase-1&6.

[0163] As an example of other activities discovered, Figure 18 shows the results of evaluating the enzyme activity of plasma samples prepared from thioacetamide (TAA) model mice or 4,4'-methylenedianiline (MDA) model mice using Suc-KHLY-sHMRG and Suc-AAVY-dsSiR. The Suc-AAVY-dsSiR used was prepared by the method described in Example 2. Figure 19 shows the number of wells (i.e., the number of bright spots) located within the area designated as Spot III in Figure 18. The results in Figure 19 are shown as mean ± standard deviation for four mice (n = 4) in the control group and six mice (n = 6) in the TAA and MDA groups. As shown in Figures 18 and 19, the combination of Suc-KHLY-sHMRG and Suc-AAVY-dsSiR was able to selectively detect enzyme activities that were specifically elevated in mice with cholestasis, a liver injury caused by MDA, among other liver disorders. SEQ ID NO: 16: GWEHD SEQ ID NO: 17: VVEID SEQ ID NO: 18: GDEVD SEQ ID NO: 19: DYEVD SEQ ID NO: 20: VDQQD SEQ ID NO: 21: GYVAD SEQ ID NO: 22: GIETE SEQ ID NO: 23: GLEVD SEQ ID NO: 24: GPLGP

[0164] Thus, based on the results of Figures 17, 18, and 19, it was demonstrated that the screening method made possible by the production method of the present disclosure can discover activities that can serve as disease-specific biomarkers.

[0165] Example 9 ADC linker molecules were prepared in the same manner as in Example 3. The prepared ADC linker molecules had a payload moiety, a release module moiety, a substrate moiety, a colorimetric reporter moiety, a linker moiety, and an antibody labeling moiety, as shown in the structural example below. As the alkyne-containing building block, Fmoc-Lys(Cpz)-OH prepared in Example 3 or Fmoc-Cir(Ert)-OH prepared in Preparation Example 2 was used.

[0166] The linker molecule of the ADC was prepared by the method shown in the following reaction scheme: All operations were carried out using an automated peptide synthesizer Syro I (Biotage) equipped with a heating block kit.

[0167] Step 1: Amidation Fmoc-Cit(Ert)-OH prepared in Preparation Example 2 or Fmoc-Lys(Cpz)-OH (5 μmol) prepared in Example 3 was dissolved in NMP (10 μL) and DCM (500 μL) together with p-aminobenzyl alcohol (6.5 mg, 50 μmol), DMT-MM (14.6 mg, 50 μmol), and DIEA (2.8 μL, 15 μmol), and the solution was stirred at 65° C. for 2 hours.

[0168] Step 2: Capture and Washing Tentagel-azide beads (100 mg) were dissolved in Tris-HCl buffer (300 mM, pH 7.4; 300 μL) and introduced into the reactor of an automated peptide synthesizer. The reaction mixture obtained in step 1 was diluted with t-BuOH (400 μL) and added to the reactor. Furthermore, CuSO ４ (10 mM aqueous solution, 100 μL), TBTA (tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine, 30 mM DMSO solution, 100 μL), and sodium ascorbate (30 mM aqueous solution, 100 μL) were added and stirred at 25°C for 2 hours. Subsequently, benzyl propargyl ether (7.7 μL) dissolved in t-BuOH (100 μL) was added and stirred at 25°C for 1 hour. After the reaction, the beads were washed six times with DMF.

[0169] Step 3: Peptide Elongation The beads obtained in Step 2 were subjected to (a) Fmoc deprotection treatment and (b) amino acid coupling treatment alternately to carry out synthesis until the target peptide sequence was obtained. The Fmoc deprotection treatment in (a) was as follows: Piperidine (40% DMF solution, 1200 μL) was added to the beads and stirred at 25°C for 3 minutes. After removing the solvent, piperidine (40% DMF solution, 600 μL) and DMF (600 μL) were added to the beads and stirred for 12 minutes. After the reaction, the beads were washed six times with DMF. The amino acid coupling treatment in (b) was as follows: Fmoc-amino acid (0.4 M DMF solution, 800 μL), DMT-MM (0.4 M MeOH solution, 840 μL), and DIEA (1.6 M NMP solution, 400 μL) were added to the beads and stirred at 30°C for 40 minutes. After the reaction, the beads were washed three times with DMF. Hexanoic acid was used as the building block for the mAb modification site, and Fmoc-NH-PEG was used as the building block for the PEG linker. ２ The following compounds were used: -DGA-OH (Watanabe Chemical Industry Co., Ltd.), and Fmoc-Apd-OH (described in the literature (Norimichi Nagano et al., "Development of fluorogenic substrates for colorectal tumor-related neuropeptidases for activity-based diagnosis", Chem. Sci., 2023,14, 4495-4499)) as a building block for the colorimetric reporter. In the compound containing the GfFK sequence, "f" represents D-phenylalanine, and the corresponding building block was Fmoc-D-Phe-OH (Watanabe Chemical Industry Co., Ltd. K00453).

[0170] Step 4: 4-Nitrophenyl Carbonation The beads obtained in Step 3 were washed three times with DCM, and THF (500 μL) was added. 4-Nitrophenyl chloroformate (10.6 mg, 53 μmol) dissolved in THF (250 μL) and pyridine (43 μL, 530 μmol) dissolved in THF (250 μL) were added sequentially, and the mixture was stirred at 25° C. for 1 hour. After the reaction, the beads were washed six times with DCM.

[0171] Step 5: Introduction of Payload The beads obtained in Step 4 were dissolved in NMP (50 μL), and MMAE (Selleck, S7721, monomethyl auristatin E) or sarcosine benzyl ester (Santa Cruz, SC-286766, Sar-OBzl, 7.9 μmol) dissolved in NMP (50 μL) was added. DIEA (1.6 M NMP solution, 50 μL) and a solution of HOAt (2.2 mg, 16 μmol) in NMP (50 μL) were further added, followed by stirring at 25° C. for 2 hours. After the reaction, the beads were washed six times with DMF and six times with DCM.

[0172] Step 6: Cleavage and purification A 90% TFA aqueous solution (200 μL) was added to the beads obtained in step 5 and stirred at 25° C. for 15 minutes. After collecting the reaction solution, the beads were washed three times with acetone (200 μL) and added to the collected reaction solution. The collected reaction solution was diluted with water and directly subjected to purification MPLC (C18 column; H ２ The resulting fraction was freeze-dried to obtain the target product.

[0173] By the steps described above, the six ADC linker molecules shown in the table below were obtained. Figure 20 shows the results of HPLC-MS analysis of the ADC linker molecules synthesized in Example 9. The vertical axis of the HPLC chromatogram in Figure 20 represents absorbance at 254 nm. In this way, it was possible to synthesize ADC linker molecules with various chemical structures using only a simple purification method.

[0174] Example 10: It was examined whether the linker molecule of the ADC obtained in Example 9 was metabolized by an enzyme to release the active substance. The linker molecule (10 μM) of the ADC indicated as 1, 2, 3, or 5 in Table 2 was mixed with a plasma sample from a healthy donor (4-fold dilution) or an SKBR3 cell lysate (0.2 mg / mL) in a phosphate buffer solution (100 mM, pH 7.4 for the plasma sample, pH 5.0 for the cell lysate) containing CHAPS (0.1%) for 3 hours to allow the reaction to proceed. An equal amount of acetonitrile containing 10% by volume of formic acid was added to the reaction solution, and the mixture was centrifuged (14,000 rpm, 10 minutes, 4°C), after which the supernatant was collected. Five μL of the supernatant was analyzed using LC-MS / MS (Waters H-Class / Xevo TQD, H ２ O-0.1% formic acid / AcCN-H ２ The mixture was injected into a 0.1% formic acid (8:2) mixture (95 / 5 to 0 / 100, over 3.5 min). MMAE was quantified in multiple reaction monitoring (MRM) mode (m / z = 718.5 > 134.0, 152.0, 686.4, cone voltage = 40 V, collision energy = 30 V).

[0175] 21 shows the results of quantifying the amount of MMAE released by contacting the linker molecule of an ADC with a plasma sample from a healthy donor (left) or an SKBR3 cell lysate (right). Figure 21 demonstrates that the production method according to the present disclosure can be used to easily produce multiple candidate molecules through a fully automated process, making it easier to find molecules that exhibit desired properties (in this case, high MMAE release efficiency).

Claims

1. A method for producing a compound, comprising: forming a covalent bond between a covalent bond-forming group of a compound represented by formula (I): CBFG-PG-MBG (I) [in formula (I), CBFG represents a monovalent group having a covalent bond-forming group, PG represents a divalent group obtained by removing one hydrogen atom from a monovalent protecting group, and MBG represents a monovalent group having a scaffold group protected by PG and a backbone group] and a support, thereby capturing the compound represented by formula (I) on the support; and removing PG from the scaffold group to obtain a compound comprising the scaffold group and the backbone group or a group obtained by converting the scaffold group through at least one chemical reaction on a solid phase.

2. The method according to claim 1, further comprising a step of obtaining the compound represented by formula (I), the step comprising protecting the scaffold group with a compound represented by formula (II): CBFG-PG-LG (II) [in formula (II), CBFG and PG are the same as in formula (I), and LG represents a monovalent leaving group] or a compound in which the covalent bond-forming group of the compound has been protected.

3. The method according to claim 1, further comprising a step of converting the backbone of the compound represented by formula (I) captured on the carrier by at least one chemical reaction on a solid phase.

4. The method according to any one of claims 1 to 3, wherein CBFG is a monovalent group having a click-reactive group.

5. CBFG is an ethynyl, 2-propynyl, 3-butynyl, 4-pentynyl, or 5-hexynyl group, or a C group in which one hydrogen atom is replaced by an azide group. １－５ The method according to any one of claims 1 to 3, wherein the alkyl is alkyl.

6. PG is represented by formula (III-A) or formula (III-B): [In formula (III-A) and formula (III-B), R １ａ , R １ｂ and R １ｃ are each independently a hydrogen atom or a C １－５ wherein the alkyl group is a divalent group obtained by removing one hydrogen atom from a group represented by the formula (I):

7. PG is Para C １－５ Alkoxybenzyloxycarbonyl, para C １－５ The method according to any one of claims 1 to 3, wherein the alkoxy group is a divalent group obtained by removing one hydrogen atom from alkoxybenzyl, trityl, tert-butoxycarbonyl, or 9-fluorenylmethyloxycarbonyl.

8. A method for screening a compound, comprising: forming a covalent bond between a support and a covalent bond-forming group of a compound represented by formula (I): CBFG-PG-MBG (I) [in formula (I), CBFG represents a monovalent group having a covalent bond-forming group, PG represents a divalent group obtained by removing one hydrogen atom from a monovalent protecting group, and MBG represents a monovalent group having a scaffold group protected by PG and a backbone group], thereby capturing the compound represented by formula (I) on the support; removing PG from the scaffold group to obtain a compound consisting of the scaffold group and the backbone group or a group obtained by converting the scaffold group through at least one chemical reaction on a solid phase; and evaluating a compound consisting of the scaffold group and the backbone group or a group obtained by converting the scaffold group, or a compound obtained by further converting the scaffold group.

9. A compound represented by formula (II-A): CRG-PG-LG (II-A) [in formula (II-A), CRG represents a monovalent group having a click reactive group, with the proviso that the click reactive group may be protected, PG represents a divalent group obtained by removing one hydrogen atom from a monovalent protecting group, and LG represents a monovalent leaving group].

10. A kit comprising a compound represented by formula (IV): CRG-PG-OH (IV) [in formula (IV), CRG represents a monovalent group having a click-reactive group, which may be protected, and PG represents a divalent group formed by removing one hydrogen atom from a monovalent protecting group], and a reagent for converting a hydroxyl group in the compound represented by formula (IV) to introduce a monovalent leaving group.

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