阅读说明：本技术 异型单分散聚乙二醇衍生物的制备方法 (Preparation method of heterotype monodisperse polyethylene glycol derivative ) 是由 小野泽昭英 市川贵志 于 2020-03-25 设计创作，主要内容包括：本发明是提供一种异型单分散聚乙二醇的制备方法,其包括：实施式(2)的化合物和式(3)的化合物之间的亲核取代反应使得满足式(F1)的要求以获得式(4)的化合物的步骤(A)；在10℃以下的温度条件下实施式(5)的化合物向式(4)的化合物的迈克尔加成反应以获得式(6)的化合物的步骤(B)；脱三苯甲基化或者脱苄基化式(6)的化合物以获得包含式(7)的化合物的反应产物的步骤(C)；从反应产物纯化式(7)的化合物的步骤(D)；式(7)的化合物与邻苯二甲酰亚胺反应并进行脱邻苯二甲酰亚胺化以获得式(8)的化合物的步骤(E)；式(8)的化合物经历酸水解处理以获得式(1)表示的化合物的步骤(F)。(The invention provides a preparation method of heterotype monodisperse polyethylene glycol, which comprises the following steps: a step (a) of carrying out a nucleophilic substitution reaction between the compound of formula (2) and the compound of formula (3) so as to satisfy the requirement of formula (F1) to obtain a compound of formula (4); a step (B) of carrying out a Michael addition reaction of the compound of formula (5) to the compound of formula (4) at a temperature of 10 ℃ or lower to obtain a compound of formula (6); a step (C) of detritylating or debenzylating the compound of formula (6) to obtain a reaction product comprising the compound of formula (7); a step (D) of purifying the compound of formula (7) from the reaction product; a step (E) of reacting the compound of formula (7) with phthalimide and performing dephthalimidization to obtain a compound of formula (8); a step (F) in which the compound of formula (8) is subjected to acid hydrolysis treatment to obtain a compound represented by formula (1).)

1. A process for producing a hetero-monodisperse polyethylene glycol represented by formula (1), which comprises the following steps (A), (B), (C), (D), (E) and (F):

wherein, in formula (1), a represents an integer of 6 to 12,

step (A): a step of carrying out a nucleophilic substitution reaction between the compound of formula (2) and the compound of formula (3) so as to satisfy the requirement of formula (F1) to obtain a compound of formula (4):

wherein, in formula (2), b represents an integer of 3 to 9,

wherein, in the formula (3), L represents a methanesulfonyl group or a toluenesulfonyl group, R¹Represents a trityl group or a benzyl group, and c represents an integer of 3 to 9,

6≤b+c≤12 (F1)

wherein, in the formula (4), R¹Represents a trityl group or a benzyl group and a represents an integer of 6 to 12;

step (B): a step of carrying out a Michael addition reaction of the compound of formula (5) to the compound of formula (4) obtained in the step (A) at a temperature of 10 ℃ or lower to obtain a compound of formula (6),

wherein, in formula (5), R²Represents a hydrocarbon group having 1 to 6 carbon atoms,

wherein, in formula (6), R¹Represents a trityl or benzyl group, R²Represents a hydrocarbon group having 1 to 6 carbon atoms, and a represents an integer of 6 to 12;

step (C): a step of detritylating or debenzylating the compound of formula (6) obtained in said step (B) to obtain a reaction product comprising the compound of formula (7),

wherein, in formula (7), R²Represents a hydrocarbon group having 1 to 6 carbon atoms and a represents an integer of 6 to 12;

step (D): a step of purifying a compound of formula (7) from the reaction product obtained in the step (C);

a step (E): a step of reacting the compound of formula (7) obtained in the step (D) with phthalimide and performing dephthalimidization to obtain a compound of formula (8),

wherein, in formula (8), R²Represents a hydrocarbon group having 1 to 6 carbon atoms and a represents an integer of 6 to 12; and

step (F): a step of subjecting the compound of formula (8) obtained in the step (E) to acid hydrolysis treatment to obtain a compound represented by formula (1).

2. The process of claim 1, wherein the compound of formula (7) is purified using dichloromethane or chloroform in the step (D).

3. The process according to claim 1 or 2, wherein in step (D), the compound of formula (7) is purified using water or an aqueous solution having an inorganic salt concentration of 10% by weight or less.

4. The method of any one of claims 1 to 3, wherein step (D) comprises a washing step at 1 to 25 ℃.

5. A process for producing a hetero-monodisperse polyethylene glycol represented by formula (1), which comprises the following steps (A), (B), (C), (E), (F) and (G):

wherein, in formula (1), a represents an integer of 13 to 40,

step (A): a step of carrying out a nucleophilic substitution reaction between the compound of formula (2) and the compound of formula (3) so as to satisfy the requirement of formula (F1) to obtain a compound of formula (4):

wherein, in formula (2), b represents an integer of 3 to 37,

wherein, in the formula (3), L represents a methanesulfonyl group or a toluenesulfonyl group, R¹Represents a trityl group or a benzyl group, and c represents an integer of 3 to 37,

13≤b+c≤40 (F1)

wherein, in the formula (4), R¹Represents a trityl group or a benzyl group and a represents an integer of 13 to 40;

step (B): a step of carrying out a Michael addition reaction of the compound of formula (5) to the compound of formula (4) obtained in the step (A) at a temperature of 10 ℃ or lower to obtain a compound of formula (6),

wherein, in formula (5), R²Is represented by having 1 to 6A hydrocarbon group of a carbon atom,

wherein, in formula (6), R¹Represents a trityl or benzyl group, R²Represents a hydrocarbon group having 1 to 6 carbon atoms, and a represents an integer of 13 to 40;

step (C): a step of detritylating or debenzylating the compound obtained in the step (B) to obtain a compound of formula (7),

wherein, in formula (7), R²Represents a hydrocarbon group having 1 to 6 carbon atoms and a represents an integer of 13 to 40;

a step (E): a step of reacting the compound of formula (7) obtained in the step (C) with phthalimide and performing dephthalimidization to obtain a compound of formula (8),

wherein, in formula (8), R²Represents a hydrocarbon group having 1 to 6 carbon atoms and a represents an integer of 13 to 40;

step (F): a step of subjecting the compound of formula (8) obtained in the step (E) to acid hydrolysis treatment to obtain a reaction product containing the compound represented by formula (1); and

a step (G): a step of purifying the compound of formula (1) from the reaction product obtained in the step (F).

6. The method according to claim 5, wherein the organic solvent to be used for purification in the step (G) is dichloromethane or chloroform.

7. The process according to claim 5 or 6, wherein the aqueous solution to be used for purification in step (G) is an alkaline aqueous solution having a pH of 8 or more.

8. The method of any one of claims 1 to 7, wherein R of the compound of formula (5) in step (B)²Is isopropyl or tert-butyl.

9. The method according to any one of claims 1 to 8, wherein flake potassium hydroxide or powder potassium hydroxide is used as the base in step (B).

Technical Field

The invention relates to a preparation method of heterotype monodisperse polyethylene glycol to be used for medicine application.

Background

In recent years, in the pharmaceutical field, Antibody-Drug conjugates (ADCs) in which a Drug is bound to an Antibody via a linker and which can actively carry the Drug to antigen-presenting cells (Toxins, March 2011, p.848-883 (non-patent document 1), j.med.chem.,2011,54, p.3606-3623 (non-patent document 2) have been put into practical use and are attracting high attention.

One material that has been used as a linker material for the ADC is heteromonodisperse polyethylene glycol. The hetero-monodisperse polyethylene glycol is a monodisperse polyethylene glycol having a prescribed molecular weight and having hetero polyethylene glycols having different functional groups at both ends as a main component.

In the above ADC, hetero-monodisperse polyethylene glycol is used as a linker, and an antibody and a drug are separately bound to each end thereof. Therefore, when a compound having the same functional groups at both ends (homo-polyethylene glycol or the like) is present as an impurity in hetero-monodisperse polyethylene glycol, a compound in which two antibodies are bound or a compound in which two drugs are bound is formed. The compound in which two antibodies bind does not act as an ADC because it does not bind to a drug, and the compound in which two drugs bind is carried to a site other than the antigen presenting cell because it does not bind to an antibody and causes side effects. Further, also in the case where another hetero compound having functional groups in a different combination from that in the case of hetero monodisperse polyethylene glycol having a target functional group is contained as an impurity, a compound lacking one of the target antibody and the drug is formed, so that the same problem as described above occurs. Therefore, from the viewpoint of the use and effect of the drug, it is important that the hetero monodisperse polyethylene glycol contains only one species of high purity hetero monodisperse polyethylene glycol having different functional groups from each other at both terminals.

In addition, in order to improve the effect of the above ADC, it is attempted to use an ADC in which a plurality of drugs are bound to an antibody. In producing the ADC, the amount of bound drug was confirmed using a mass spectrometer or HPLC. Therefore, when the hetero-monodisperse polyethylene glycol used as the linker material contains compounds having different glycol chain lengths, it is difficult to confirm the compounds in the production. Further, when compounds having different ethylene glycol chain lengths exist as impurities, there is a problem that since an equivalent amount of antibody or drug to be added at the time of manufacturing ADC becomes unclear, it is necessary to overuse expensive antibody or drug, and since compounds having different ethylene glycol chain lengths are regarded as compounds different from main drugs at the time of applying for medical products, there is a problem that identification of compounds, execution of various tests, evaluation of allowable amounts, and the like are further necessary. Therefore, as the hetero-monodisperse polyethylene glycol, it is important to contain only one kind of high purity polyethylene glycol having the same glycol chain length.

As described above, as the hetero-monodisperse polyethylene glycol used as a linker material for ADC, it has been desired to contain, particularly in high purity, a compound which is a main component of hetero-polyethylene glycol having different functional groups at both ends and in which the glycol chain lengths between the hetero-polyethylene glycols are the same.

The hetero-monodisperse polyethylene glycol having an amino group and a carboxyl group as functional groups at each end, respectively, can be used as it is as a linker of ADC, and further, the hetero-monodisperse polyethylene glycol obtained by conversion of the functional groups using such a compound as a raw material can also be used as a linker of ADC.

In the production of hetero-monodisperse polyethylene glycols, it is necessary to perform end functionalization efficiently. As the terminal functionalization step, patent documents 1 and 2 disclose a method of introducing a carboxyl group at the terminal of monomethoxypolyethylene glycol. In patent document 1, monomethoxypolyethylene glycol is subjected to michael addition reaction with acrylonitrile, nitrile is converted into amide under the condition of concentrated hydrochloric acid, and amide is hydrolyzed under the condition of aqueous potassium hydroxide solution, thereby effecting conversion into carboxyl group. However, under such strong acidic or strong basic conditions, a compound having a hydroxyl group instead of a carboxyl group is formed by the reverse reaction of the michael addition reaction and a compound having a short ethylene glycol chain length is formed by the cleavage of an ethylene glycol chain, so that purity and yield are decreased. In patent document 2, monomethoxypolyethylene glycol and t-butyl acrylate are subjected to michael addition reaction under trifluoroacetic acid conditions and converted into a carboxyl group. However, the method disclosed in the literature has a problem that the incorporation rate of t-butyl acrylate is as low as 70% or less and a compound having a hydroxyl group at the terminal remains.

Further, as a method of introducing an amino group, in patent document 3, in an octaethylene glycol derivative having a terminal vinyl group, the vinyl group is converted into an aldehyde group by ozone oxidation, and then converted into an amino group by a reductive amination reaction with sodium cyanoborohydride in an ammonium chloride solution. However, there are problems in industrial production in view of the toxicity of ozone oxidation to form explosive peroxides and sodium cyanoborohydride. In patent document 4, a hydroxyl group of an octaethyleneglycol derivative is reacted with tosyl chloride to introduce a tosyl group, and the resultant is reacted with potassium phthalimide to convert it into a phthalimide group, which is converted into an amino group by a deprotection reaction using hydrazine monohydrate. This Gabriel amine synthesis (Gabriel amine synthesis) is a general method for amino group conversion, but in order to introduce an amino group, a step of temporarily introducing a leaving group such as tosyl group into a hydroxyl group is required. In such a reaction step, the presence of residual unreacted raw materials and the formation of reaction by-products are inevitable, and generally the more the reaction steps, the lower the yield of the objective compound.

Further, non-patent document 3 discloses a method for synthesizing a compound having an ethylene glycol chain length of 8 and having a 4,4' -dimethoxytrityl group at one end and a hydroxyl group at the other end, wherein the two ends are different from each other in the compound. Part of the synthetic route is represented by the formula:

DmtrO-(CH₂CH₂O)₄-Ts+HO-(CH₂CH₂O)₄-H

→DmtrO-(CH₂CH₂O)₈-H+DmtrO-(CH₂CH₂O)₁₂-Dmtr

wherein, Dmtr represents 4,4' -dimethoxytrityl,

and describes the formation of the two terminal Dmtr bodies of the dodecamer by a 2:1 reaction between one terminal tosylate and tetraethylene glycol, when one terminal Dmtr body of the octamer is obtained by a 1:1 reaction between one terminal tosylate and tetraethylene glycol.

However, when hetero-polyethylene glycol having an amino group and a carboxyl group at both ends, respectively, is synthesized using a mixture containing a compound having Dmtr groups at both ends, a compound having an amino group at both ends or a compound having a carboxyl group at both ends is formed as an impurity. Therefore, when hetero-monodisperse polyethylene glycol containing such impurities is used to produce ADCs, compounds that bind to both drugs or both antibodies are formed, which results in reduced effectiveness as drugs.

As a method for producing hetero polyethylene glycol having different functional groups at both terminals, patent document 3 describes that a triethylene glycol derivative having an amino group-protecting group at one terminal and a ethylene glycol derivative having a carboxyl group-protecting group at the terminal are coupled and deprotected at each terminal to obtain hetero monodisperse polyethylene glycol having an amino group and a carboxyl group at each terminal. However, among the couplings between these polyethylene glycol derivatives to which respective protecting groups have been introduced, one of them is excessively used, and thus the cost is increased.

Further, patent document 4 describes that a hetero monodisperse polyethylene glycol having an amino group and a hydroxyl group is obtained at high purity using a hetero monodisperse polyethylene glycol having a leaving group at one end and a hydroxyl group at the other end as an intermediate, and the hetero monodisperse polyethylene glycol as an intermediate is prepared by continuous synthesis using readily commercially available tetraethylene glycol as a starting material.

However, in this preparation method, after repeating continuous synthesis until the target ethylene glycol chain length is obtained, in order to remove monodisperse polyethylene glycol impurities having protecting groups at both terminals from hetero monodisperse polyethylene glycol having a protecting group at one terminal and a hydroxyl group at the other terminal, the hydroxyl group is temporarily converted into a tosyl group. As described above, the more such reaction steps are added, the lower the yield of the target compound. Therefore, there is room for further improvement in industrial production.

Documents of the prior art

Patent document

Patent document 1: U.S. Pat. No.5672622

Patent document 2: JP-T-2007-538111

(the term "JP-T" as used herein refers to Japanese translation of a published PCT patent application)

Patent document 3: WO 9201474

Patent document 4: JP-A-2017-14371

Non-patent document

Non-patent document 1: toxins,2011, March, p.848-883

Non-patent document 2: med chem, 2011,54, p.3606-3623

Non-patent document 3: hem, 2014,5, p.694-697

Disclosure of Invention

Problems to be solved by the invention

The object of the present invention is to provide a process for the preparation of heteromonodisperse polyethylene glycol derivatives which can be used in pharmaceutical applications in high yields and industrially.

Means for solving the problems

As a result of intensive studies to achieve the above object, the present inventors have established a method for preparing hetero-monodisperse polyethylene glycol derivatives having the following constitution.

Thus, the present invention is as follows.

(1) A process for producing a hetero-monodisperse polyethylene glycol represented by formula (1), which comprises the following steps (A), (B), (C), (D), (E) and (F):

wherein, in formula (1), a represents an integer of 6 to 12,

step (A): carrying out the nucleophilic substitution reaction between the compound of formula (2) and the compound of formula (3) such that the requirements of formula (F1) are met to obtain the compound of formula (4):

wherein, in formula (2), b represents an integer of 3 to 9,

wherein, in the formula (3), L represents a methanesulfonyl group or a toluenesulfonyl group, R¹Represents a trityl group or a benzyl group, and c represents an integer of 3 to 9,

6≤b+c≤12 (F1)

wherein, in the formula (4), R¹Represents a trityl group or a benzyl group and a represents an integer of 6 to 12;

step (B): a step of carrying out a Michael addition reaction of the compound of formula (5) to the compound of formula (4) obtained in said step (A) at a temperature of 10 ℃ or lower to obtain a compound of formula (6),

wherein, in formula (5), R²Represents a hydrocarbon group having 1 to 6 carbon atoms,

wherein, in formula (6), R¹Represents a trityl or benzyl group, R²Represents a hydrocarbon group having 1 to 6 carbon atoms, and a represents an integer of 6 to 12;

step (C): a step of detritylating or debenzylating the compound of formula (6) obtained in said step (B) to obtain a reaction product comprising the compound of formula (7),

wherein, in formula (7), R²Represents a hydrocarbon group having 1 to 6 carbon atoms and a represents an integer of 6 to 12;

step (D): a step of purifying a compound of formula (7) from the reaction product obtained in the step (C);

a step (E): the compound of formula (7) obtained in said step (D) is reacted with phthalimide and dephthalimido

A step of amination to obtain a compound of formula (8),

wherein, in formula (8), R²Represents a hydrocarbon group having 1 to 6 carbon atoms and a represents an integer of 6 to 12; and

step (F): a step in which the compound of formula (8) obtained in said step (E) is subjected to acid hydrolysis treatment to obtain a compound represented by formula (1).

(2) The process of (1), wherein the compound of formula (7) is purified using dichloromethane or chloroform in the step (D).

(3) The method according to (1) or (2), wherein in the step (D), the compound of formula (7) is purified using water or an aqueous solution having an inorganic salt concentration of 10% by weight or less.

(4) The method according to any one of (1) to (3), wherein the step (D) comprises a washing step at 1 to 25 ℃.

(5) A process for producing a hetero-monodisperse polyethylene glycol represented by formula (1), which comprises the following steps (A), (B), (C), (E), (F) and (G):

wherein, in formula (1), a represents an integer of 13 to 40,

step (A): carrying out the nucleophilic substitution reaction between the compound of formula (2) and the compound of formula (3) such that the requirements of formula (F1) are met to obtain the compound of formula (4):

wherein, in formula (2), b represents an integer of 3 to 37,

wherein, in the formula (3), L represents a methanesulfonyl group or a toluenesulfonyl group, R¹Represents a trityl group or a benzyl group, and c represents an integer of 3 to 37,

13≤b+c≤40 (F1)

wherein, in the formula (4), R¹Represents a trityl group or a benzyl group and a represents an integer of 13 to 40;

step (B): a step of carrying out a Michael addition reaction of the compound of formula (5) to the compound of formula (4) obtained in said step (A) at a temperature of 10 ℃ or lower to obtain a compound of formula (6),

wherein, in formula (5), R²Represents a hydrocarbon group having 1 to 6 carbon atoms,

wherein, in formula (6), R¹Represents a trityl or benzyl group, R²Represents a hydrocarbon group having 1 to 6 carbon atoms, and a represents an integer of 13 to 40;

step (C): a step of detritylating or debenzylating the compound obtained in the step (B) to obtain a compound of formula (7),

wherein, in formula (7), R²Represents a hydrocarbon group having 1 to 6 carbon atoms and a represents an integer of 13 to 40;

a step (E): a step of reacting the compound of formula (7) obtained in said step (C) with phthalimide and performing dephthalimidization to obtain a compound of formula (8),

wherein, in formula (8), R²Represents a hydrocarbon group having 1 to 6 carbon atoms and a represents an integer of 13 to 40;

step (F): a step in which the compound of formula (8) obtained in the step (E) is subjected to acid hydrolysis treatment to contain a reaction product that obtains a compound represented by formula (1); and

a step (G): a step of purifying the compound of formula (1) from the reaction product obtained in the step (F).

(6) The process according to (5), wherein the organic solvent to be used for purification in the step (G) is dichloromethane or chloroform.

(7) The method according to (5) or (6), wherein the aqueous solution to be used for purification in the step (G) is an alkaline aqueous solution having a pH of 8 or more.

(8) The method according to any one of (1) to (7), wherein R of the compound of formula (5) in the step (B)²Is isopropyl or tert-butyl.

(9) The method according to any one of (1) to (8), wherein flake potassium hydroxide or powdery potassium hydroxide is used as the base in the step (B).

Effects of the invention

The invention relates to a novel preparation method of high-purity heterotype monodisperse polyethylene glycol with amino and carboxyl at two ends. In this preparation method, highly pure hetero-monodisperse polyethylene glycol can be produced by simple liquid phase separation extraction during the step without using a purification method such as column chromatography. In addition, in order to purify both-terminal protected impurities having a specific molecular weight and different chain lengths formed as by-products in the conventional chain extension step, the impurities can be removed without a step of once converting a hydroxyl group into a tosyl group. In this way, since the number of steps is smaller than ever, the presence of residual unreacted raw materials and the formation of reaction by-products, which lead to a reduction in yield, can be suppressed. Therefore, the method can be provided as an industrial method for preparing highly pure hetero-monodisperse polyethylene glycol having an amino group and a carboxyl group at both terminals, respectively.

Detailed Description

The hetero-monodisperse polyethylene glycol derivative according to the present invention is represented by the following formula (1). A "monodisperse polyethylene glycol" is a compound that contains more than 90% of components with a particular ethylene glycol chain length.

A in formula (1) of the present invention is an integer of 6 to 12 or 13 to 40, and represents a repeating unit of ethylene glycol. From the viewpoint of serving as a linker for ADC, a is preferably an integer of 6 to 12 or 13 to 24.

The hetero-monodisperse polyethylene glycol having an amino group and a carboxyl group at each of both ends can be produced at high purity as follows.

(step (A))

Step (a) according to the present invention is a step of carrying out a nucleophilic substitution reaction between compounds represented by the following formula (2):

HO-(CH₂CH₂O)_b-H...(2)

and a compound represented by the following formula (3):

R¹O-(CH₂CH₂O)_c-L...(3)

the following requirement is satisfied as represented by the following formula (F1):

6≤b+c≤12 or 13≤b+c≤40...(F1)

to obtain a compound represented by the following formula (4):

R¹O-(CH₂CH₂O)_a-H...(4)。

b in formula (2) represents an integer of 3 to 9 or 3 to 37, and preferably an integer of 3 to 9 or 10 to 21. R in the formula (3)¹Represents a trityl group or a benzyl group, L represents a methanesulfonyl group or a toluenesulfonyl group, and c represents an integer of 3 to 9 or 3 to 37, and preferably an integer of 3 to 9 or 10 to 21. In addition, the method can be used for producing a composite materialIn the formula (2), b and c in the formula (3) satisfy b + c of 6 to 12 or 13 to 40, and thus satisfy the requirement represented by the formula (F1), and preferably b + c of 6 to 12 or 13 to 24.

As the compound represented by formula (2) and the compound represented by formula (3), commercially available products can be used or the compounds can be obtained by known synthetic methods. Further, as the compound represented by formula (3), a compound having a long ethylene glycol chain length, that is, a large value of a in formula (4), can be synthesized using a compound obtained by methanesulfonic acid or toluenesulfonic acid the compound represented by formula (4) obtained in step (a).

By carrying out a nucleophilic substitution reaction between the compound represented by the formula (2) and the compound represented by the formula (3) in the presence of a base, a reaction mixture containing the compound represented by the formula (4) can be obtained. In the formula (4), R¹Represents a trityl group or a benzyl group, and a represents an integer of 6 to 12 or 13 to 40, and preferably an integer of 6 to 12 or 13 to 24. R in the formula (4)¹And R in the formula (3)¹Represent the same substituents. The reaction mixture contains a compound represented by the following formula (9):

R¹O-(CH₂CH₂O)_d-R1...(9)

as impurities.

In the formula (9), R¹Represents a trityl group or a benzyl group, and d represents an integer of 3 to 80. R in the formula (9)¹And R in the formula (3)¹Represent the same substituents.

Nucleophilic substitution reactions can be carried out in solvents. The solvent is not particularly limited as long as it is a solvent that does not react with the compound represented by formula (2) and the compound represented by formula (3). Specifically, aprotic polar solvents such as acetonitrile, dimethylformamide, tetrahydrofuran, dichloromethane, chloroform, and the like, and mixed solvents thereof can be used, and acetonitrile, dimethylformamide are preferred. The amount of the solvent used is generally 1 to 100 times, preferably 2 to 50 times, and more preferably 3 to 30 times the weight ratio of the compound represented by formula (3). When the amount of the solvent used is less than the lower limit, the amount of the compound represented by formula (9) in which the compound represented by formula (3) is bound to both terminals of the compound represented by formula (2) tends to increase, and when the amount exceeds the upper limit, the progress of the nucleophilic substitution reaction tends to slow down.

In the nucleophilic substitution reaction, the compound represented by formula (2) is used in an amount of usually 1.1 to 50 times, preferably 5.0 to 30 times, more preferably 5.0 to 20 times as much as the molar ratio of the compound represented by formula (3). When the amount of the compound represented by the formula (2) is less than the above, the amount of the compound represented by the formula (9) in which the compound represented by the formula (3) is bonded to both terminals of the compound represented by the formula (2) tends to increase, and when the amount exceeds the upper limit, the progress of the nucleophilic substitution reaction tends to slow down.

The base used in the nucleophilic substitution reaction is not particularly limited as long as the reaction proceeds, and specifically, sodium hydride, metallic sodium, or potassium tert-butoxide, and sodium hydride or metallic sodium is preferable. The molar ratio of the base to the compound represented by formula (2) is usually 1.1 to 10 times, preferably 1.2 to 5 times.

The reaction temperature varies depending on the solvent used, etc., but is usually 0 ℃ to 100 ℃, preferably 50 ℃ to 90 ℃. When the reaction temperature is lower than the lower limit, the reaction may be slowed, and when the reaction temperature exceeds the upper limit, side reactions may occur due to an excessively high temperature. The reaction time varies depending on the conditions such as the reaction temperature, and is usually 0.2 to 48 hours, preferably 2 to 24 hours. When the reaction time is short, the reaction will be insufficient.

In step (a), the reaction mixture containing the compound represented by formula (4) and the compound represented by formula (9) by such nucleophilic substitution reaction may be used as it is in step (B) without purification, or may be used after the compound represented by formula (4) above is purified by silica gel column chromatography, liquid phase separation extraction treatment, adsorption treatment, or the like. The compound represented by formula (9) can be used without purification because it has no reactivity in the reaction of the next step (B) and can be removed by purification in the step described later.

(step (B))

Step (B) according to the present invention is a step of carrying out michael addition of a compound represented by the following formula (5) to a compound represented by the formula (4) under a temperature condition of 10 ℃ or lower:

to obtain a compound represented by formula (6):

r in the formula (5)²Represents a hydrocarbon group having 1 to 6 carbon atoms. Specifically, as the hydrocarbon group having 1 to 6 carbon atoms, methyl group, ethyl group, propyl group, isopropyl group, butyl group, tert-butyl group, isobutyl group and sec-butyl group can be mentioned. From the viewpoint of stability under alkaline conditions, R²Preferably isopropyl or tert-butyl. From the viewpoint of improving the efficiency of the purification step described later, R²More preferably, it is a tert-butyl group.

Further, in formula (6), R¹Represents trityl or benzyl, R²Represents a hydrocarbon group having 1 to 6 carbon atoms, and a represents an integer of 6 to 12 or 13 to 40, and preferably an integer of 6 to 12 or 13 to 24. R in the formula (6)¹And R in the formula (4)¹Are the same substituent, and R in the formula (6)²And R in the formula (5)²Are the same substituents.

The michael addition reaction can be carried out in a solvent. The solvent is not particularly limited as long as it is a solvent that does not react with the compound represented by formula (4) and the compound represented by formula (5). Specifically, organic solvents such as tetrahydrofuran, acetonitrile, dichloromethane, chloroform, toluene, and the like, and mixed solvents thereof can be used, and dichloromethane and chloroform are preferable. The amount of the solvent used is generally 1 to 100 times, preferably 3 to 50 times, and more preferably 5 to 30 times the weight ratio of the compound represented by formula (4). When the amount of the solvent used is less than the lower limit, such an excess reaction may occur that a compound obtained by further Michael addition reaction of the compound represented by formula (5) with the compound represented by formula (6) is formed, whereas when the amount exceeds the upper limit, the progress of the Michael addition reaction tends to be slowed down.

In the Michael addition reaction, the compound represented by the formula (5) is used in an amount of usually 1 to 50 times, preferably 1.5 to 25 times, the molar ratio of the compound represented by the formula (4). When the amount of the compound represented by formula (5) is below the lower limit, the michael addition reaction may not be completed, and when the amount exceeds the upper limit, there is a risk of side reactions such as formation of a polymer of the compound represented by formula (5) occurring.

The base used in the michael addition reaction is not particularly limited as long as the reaction proceeds, but specifically, an inorganic catalyst such as sodium hydroxide or potassium hydroxide can be used, and potassium hydroxide is preferable. From the viewpoint of reactivity, potassium hydroxide in the form of flakes or powdery potassium hydroxide is more preferable. The amount of the base catalyst to be used is usually 0.1 to 10 times, preferably 0.1 to 5 times, the molar ratio to the compound represented by the formula (4).

The reaction temperature is usually 10 ℃ or lower, preferably 5 ℃ or lower. When the reaction temperature exceeds the upper limit, such an excessive reaction to form a compound resulting from a further Michael addition reaction of the compound represented by formula (5) with the compound represented by formula (6) may occur. The reaction time varies depending on conditions such as the reaction temperature, the base catalyst, etc., but is usually 0.2 to 12 hours, preferably 0.5 to 6 hours.

In step (B), the reaction mixture containing the compound represented by formula (6) obtained by such michael addition reaction and the compound represented by formula (9) formed in step (a) may be used as it is without purification in the next step (C), or may be used after purifying the compound represented by formula (6) by silica gel column chromatography, liquid phase separation extraction treatment, adsorption treatment, or the like. However, in the present invention, since purification can be performed in the step described later, the reaction mixture can be used without purification.

(step (C))

Step (C) of the present invention is a step of detritylating or debenzylating the compound represented by formula (6) to obtain the compound represented by formula (7):

when R in the formula (6)¹By detritylation when it is trityl or when R is¹The reaction product containing the compound represented by the formula (7) can be obtained by debenzylation at the time of the benzyl group incidentally, the reaction product contains a compound represented by the following formula (10):

HO-(CH₂CH₂O)_d-H...(10)

as an impurity, it is formed by detritylating or debenzylating the compound represented by formula (9) formed in step (a). In the above formula (10), d represents an integer of 3 to 80.

As the method of detritylation and debenzylation, known methods can be used, and for example, the method described in Protective Groups in Organic Synthesis (Protective Synthesis) by GRENE WUTS is effective. R in the formulae (6) and (9)¹In the case of trityl, detritylation can be achieved by a conversion reaction under acidic conditions, catalytic hydrogenation, or the like. The conversion reaction under acidic conditions is carried out under acidic conditions without decomposing the target compound (7) and has no problem. Specifically, there is a method of adding a catalytic amount of p-toluenesulfonic acid monohydrate to a methanol solvent to perform detritylation reaction.

Further, as a method of carrying out the catalytic hydrogenation, there is a method of carrying out the debenzylation reaction in a methanol solvent under a hydrogen atmosphere by adding a catalytic amount of palladium/carbon. On the other hand, when R in the formulae (6) and (9)²In the case of the benzyl group, debenzylation can be achieved by catalytic hydrogenation. Specifically, debenzylation can be carried out by adding a catalytic amount of palladium/carbon to a methanol solvent under a hydrogen atmosphere.

In step (C), by such detritylation or debenzylation, a reaction mixture containing the compound represented by formula (7) and the compound represented by formula (10) can be obtained. The reaction mixture may be used as it is in the next step (D) without purification, or may be used after the compound represented by formula (6) is purified by silica gel column chromatography, liquid phase separation extraction treatment, adsorption treatment or the like. However, in the present invention, since purification can be performed in the step described later, the mixture can be used without purification.

(step (D))

Step (D) of the present invention is a step of purifying a reaction product containing the compound represented by formula (7) obtained in step (C), wherein a is specifically an integer of 6 to 12. This purification step is a step of removing the compound represented by formula (10) formed as an impurity in step (C), and it is also preferable as an industrial production method of removing the impurity compound represented by formula (10) in this step (D) from the viewpoints of determining an appropriate amount of a reagent for the reaction and suppressing complication of the purification step.

Since both terminals of the compound represented by formula (10) formed as an impurity in step (C) are hydroxyl groups, a polarity difference is generated compared to the compound represented by formula (7) in which a is an integer of 6 to 12, so that the impurity of formula (10) can be removed only by simple liquid phase separation extraction without silica gel chromatography purification. On the other hand, in the case of the compound represented by formula (7) in which a is an integer of 13 to 40, the polarity difference from the impurity compound represented by formula (10) becomes small, so that the removal efficiency is lowered.

In the step (D), it is a step of dissolving the reaction product obtained in the step (C) in an organic solvent and then performing liquid phase separation washing with water or an aqueous solution at a temperature of 25 ℃ or lower. The organic solvent includes dichloromethane, chloroform and toluene, and dichloromethane and chloroform are preferable from the viewpoint of the solubility of the compound represented by formula (7). The amount of the organic solvent to be used is usually 2 to 30 times, preferably 3 to 20 times, the weight ratio of the reaction mixture containing the compound represented by formula (7) and the compound represented by formula (10). When the amount of the organic solvent is less than the lower limit, the compound represented by formula (7) may be dissolved in the aqueous layer, and when the amount exceeds the upper limit, the compound represented by formula (10) may be dissolved in the organic layer, and the liquid phase separation washing efficiency is lowered.

The water or aqueous solution used for washing is not particularly limited as long as it can dissolve the compound represented by formula (10), but specifically includes water such as ion-exchanged water or distilled water and aqueous solutions of inorganic salts such as sodium chloride and potassium chloride. The concentration of the inorganic salt is not particularly limited as long as the inorganic salt is dissolved in the aqueous solution, but is preferably 10% by weight or less, and more preferably 5% by weight or less. When the concentration of the inorganic salt in the aqueous solution exceeds the upper limit, the washing efficiency of the compound represented by formula (10) is lowered. The amount of water or aqueous solution used to the weight ratio of the reaction mixture containing the compound represented by formula (7) and the compound represented by formula (10) is usually 2 to 30 times, preferably 3 to 20 times. When the amount of water or aqueous solution is less than the lower limit, the washing efficiency of the compound represented by formula (10) is decreased, whereas when the amount exceeds the upper limit, the compound represented by formula (7) may be dissolved in the aqueous layer, and the yield of the objective compound is decreased.

In step (D), the ratio of organic solvent to water or aqueous solution is generally such that the weight ratio organic solvent/water or aqueous solution is from 0.2 to 3.5, preferably from 1.0 to 3.5.

In step (D), the washing temperature is from 1 to 25 ℃, preferably from 5 to 20 ℃. When the temperature is lower than the lower limit, water or an aqueous solution is solidified, and thus purification becomes difficult, whereas when the temperature exceeds the upper limit, the compound represented by formula (10) is dissolved in the organic layer, and removal tends to be difficult. The number of times of performing the liquid phase separation washing is not particularly limited, and it is preferable to perform the liquid phase separation washing a plurality of times while confirming the compound represented by formula (10) contained in the organic solvent by TLC, MS measurement, or the like.

In step (D), since impurities formed in steps (a) to (C) can be removed by a simple liquid phase separation extraction treatment, purification by silica gel chromatography or the like in each step is not required. The obtained compound represented by formula (7) can be used as it is in step (E), or can be further purified by crystallization, adsorption treatment, silica gel chromatography or the like before use.

(step (E))

Step (E) of the present invention is a step of binding the hydroxyl group of the compound represented by the above formula (7) obtained in step (C) or step (D) to phthalimide by a mitsunobu reaction and deprotecting the resultant with a polyvalent amine to obtain a compound represented by the following formula (8):

when the purification step of step (D) is not performed (in the case where a in formula (7) is 13 to 40), the reaction product contains a compound represented by the following formula (11):

H₂N-(CH₂CH₂O)_d-1-CH₂CH₂-NH₂...(11)

which is formed as an impurity by combining the compound represented by the formula (10) formed in the step (C) with phthalimide and deprotecting the resultant. In formula (11), d represents an integer of 3 to 80.

The reaction with phthalimide in step (E) can be carried out in a solvent. The solvent is not particularly limited as long as it does not react with the compound represented by formula (7) and phthalimide. Specifically, organic solvents such as dichloromethane, chloroform, toluene, and the like, and mixed solvents thereof can be used, and dichloromethane, chloroform are preferred. The amount of the solvent used is usually 1 to 50 times, preferably 3 to 30 times, and more preferably 5 to 20 times the weight ratio of the compound represented by formula (7).

In the reaction with phthalimide in step (E), phthalimide is used in an amount of usually 1.0 to 10 times, preferably 1.3 to 5 times, the molar ratio to the compound represented by formula (7). When the amount of phthalimide is less than the lower limit, the reaction may not be completed, and when the amount exceeds the upper limit, unreacted phthalimide remains, and thus, a removal step is required, resulting in a reduction in yield.

As the azo-based reagent used in the reaction with phthalimide in step (E), 1' -azobis (N, N-dimethylformamide), 1' - (azodicarbonyl) dipiperidine, dibenzyl azocarboxylate, diethyl azodicarboxylate, diisopropyl azodicarboxylate, dimethyl azocarboxylate, 1' -azobis (N, N-diisopropylformamide), 1, 6-dimethyl-1, 5, 7-hexahydro-1, 4,6, 7-tetraazaoctan-2, 5-dione, and the like can be mentioned, and diethyl azodicarboxylate, diisopropyl azodicarboxylate, and diisopropyl azodicarboxylate are preferable, and diisopropyl azodicarboxylate is more preferable. The molar ratio of the azo-based reagent to the compound represented by formula (7) is usually 1.0 to 10 times, preferably 1.1 to 5 times. As the phosphine-based reagent to be used, dicyclohexylphenylphosphine, diethylphenylphosphine, 4- (dimethylamino) phenyldiphenylphosphine, diphenyl-2-pyridylphosphine, isopropyldiphenylphosphine, triisobutylphosphine, tri-n-butylphosphine, tri-tert-butylphosphine, tricyclohexylphosphine, tri-n-hexylphosphine, tri-n-octylphosphine, triphenylphosphine and the like may be mentioned, and triphenylphosphine is preferred. The molar ratio of the phosphine reagent to the compound represented by formula (7) is usually 1.0 to 10 times, preferably 1.3 to 5 times.

The reaction temperature is usually 0 ℃ to 100 ℃, preferably 10 ℃ to 50 ℃. The reaction time is usually 10 minutes to 12 hours, preferably 0.5 to 6 hours.

In the deprotection in step (E), the method is not particularly limited as long as it is described in Protective Groups in Organic Synthesis (Protective Groups in Organic Synthesis) written by GREENE WUTS, but polyvalent amines are preferably used.

In the deprotection in step (E), the solvent used includes dichloromethane, chloroform, methanol, ethanol, etc., and methanol is preferable. The amount of the solvent used is generally 1 to 50 times, preferably 2 to 30 times, and more preferably 3 to 20 times the weight ratio of the compound represented by formula (7).

In the deprotection of step (E), the polyvalent amines used include hydrazine monohydrate, ethylenediamine and ethylenediamine monohydrate. The polyvalent amine is used in an amount of usually 1 to 50 times, preferably 5 to 30 times, the molar ratio relative to the compound represented by formula (7). When the amount of the polyvalent amine is less than the lower limit, the deprotection reaction becomes insufficient. The reaction temperature is usually 10 ℃ to 80 ℃, preferably 20 ℃ to 60 ℃. The reaction time is usually 0.5 to 24 hours, preferably 1.0 to 12 hours.

In step (E), the reaction mixture containing the compound represented by formula (8) and the compound represented by formula (11) obtained by the mitsunobu reaction and deprotection may be used as it is in the next step (F) without purification, or may be used after the compound represented by formula (8) is purified by silica gel column chromatography, liquid phase separation extraction treatment, adsorption treatment, or the like. In the present invention, since purification can be performed in the step described later, the reaction mixture can be used without purification.

(step (F))

Step (F) according to the present invention is a step of subjecting the reaction product represented by formula (8) obtained in step (E) to acid hydrolysis treatment to obtain heterotypic monodisperse polyethylene glycol containing the compound represented by formula (1).

The hydrolysis treatment can be carried out in a solvent. The solvent is a solvent such as water, tetrahydrofuran, acetonitrile, chloroform, dichloromethane, etc., and a mixed solvent thereof. Water or dichloromethane is preferred. The amount of the solvent used is usually 0.5 to 50 times, preferably 0.8 to 40 times, and more preferably 1 to 30 times, the weight ratio relative to the compound represented by formula (8). When the amount of the solvent is less than the lower limit, the viscosity of the reaction solution becomes high, the stirring efficiency may be lowered, and the reaction may not be completed. On the other hand, when the amount exceeds the upper limit, the reaction progress tends to slow down.

An acid catalyst is used in the hydrolysis. The acid catalyst is not particularly limited as long as the reaction proceeds, and specifically, hydrochloric acid, phosphoric acid, trifluoroacetic acid, and the like. Hydrochloric acid is preferred from the viewpoint of suppressing side reactions. The amount of the acid catalyst to be used varies depending on the kind of the acid catalyst to be used, but specifically, in the case of using 1M hydrochloric acid, the weight ratio to the compound represented by the formula (8) is usually 0.5 to 10 times.

The reaction temperature for acid hydrolysis varies depending on the acid catalyst used, but is usually 10 to 100 ℃. The reaction time of the acid hydrolysis varies depending on conditions such as the reaction temperature, and is usually about 0.5 to 12 hours.

In step (F), by such acid hydrolysis treatment, a reaction mixture containing the compound represented by formula (1) and the compound represented by formula (11) can be obtained. This reaction mixture can be used without purification because it can be purified in the step described later.

(step (G))

Step (G) according to the present invention is a step of purifying the reaction mixture containing the compound represented by formula (1) obtained in step (F). This step (G) is not necessarily performed, but may be performed as long as the compound in which a in formula (1) is an integer of 6 to 12 is purified in step (D). However, step (G) is a purification step necessary for the compound in which a in formula (1) is an integer of 13 to 40.

In the step (G), it is a step of dissolving the reaction product obtained in the step (F) in an aqueous solution and then performing liquid phase separation washing with an organic solvent at 30 ℃ or lower. The aqueous solution is an alkaline aqueous solution having a pH of 8 or more, and specifically, an aqueous sodium hydroxide solution or an aqueous potassium hydroxide solution adjusted to a pH of 9 to 11 is preferable. The amount of the basic or aqueous solution to be used is usually 2 to 30 times, preferably 3 to 20 times, the weight ratio of the reaction mixture containing the compound represented by formula (1) and the compound represented by formula (11). When the amount of the basic aqueous solution is less than the lower limit, the compound represented by formula (1) may be dissolved in the organic solvent, and when the amount exceeds the upper limit, the liquid phase separation washing efficiency may be decreased.

The organic solvent used in the washing is not particularly limited as long as it can dissolve the impurity compound represented by formula (11), but specifically, ethyl acetate, toluene, chloroform and dichloromethane are included, and chloroform and dichloromethane are preferable from the viewpoint of solubility of the impurities. The amount of the organic solvent to be used is usually 2 to 30 times, preferably 3 to 20 times, the weight ratio of the reaction mixture containing the compound represented by formula (8) and the compound represented by formula (11). When the amount of the organic solvent is less than the lower limit, the removal efficiency of the compound represented by formula (11) is lowered, whereas when the amount exceeds the upper limit, the compound represented by formula (1) may be dissolved in the organic layer and the yield of the objective compound is lowered.

In step (G), the ratio of organic solvent to aqueous alkaline solution is generally such that the weight ratio of organic solvent/aqueous alkaline solution has a value of 0.2 to 3.0, preferably 0.5 to 2.0. The number of liquid phase separation washes is not particularly limited, and it is preferable to perform a plurality of liquid phase separation washes while confirming the compound represented by formula (11) contained in the aqueous solution by TLC, MS measurement, or the like.

According to the present invention, it is possible to manufacture highly pure hetero-monodisperse polyethylene glycol by simple liquid phase separation extraction during the step without using a purification method such as column chromatography. In addition, in order to purify both-terminal protected impurities having a specific molecular weight and different chain lengths formed as by-products in the conventional chain extension step, the impurities can be removed without a step of once converting a hydroxyl group into a tosyl group. In this way, since the number of steps is smaller than ever, the presence of residual unreacted raw materials and the formation of reaction by-products, which lead to a reduction in yield, can be suppressed. Therefore, the method can provide an industrial production method of highly pure hetero-monodisperse polyethylene glycol having amino groups and carboxyl groups at both terminals, respectively, with good yield.

Examples

Hereinafter, the present invention will be described in more detail with reference to specific examples, however, the present invention is not limited to these examples.

For the measurement of the monodisperse polyethylene glycol obtained in the present invention, in¹JNM-ECP400 or JNM-ECP600 manufactured by JEOL Datum Co., Ltd. was used in H-NMR analysis. 5mm phi tubes for measurement, CDCl₃Or CDOD₃As a deuterated solvent, Tetramethylsilane (TMS) was used as an internal standard.

A compound (R) obtained by the production process of the step (A) and represented by the above formula (4)¹Trityl group) contains a monodisperse polyethylene glycol impurity having both terminal trityl groups as a compound represented by formula (9) wherein a is 8 or 12. Although it is difficult to precisely quantify the content, the hydroxyl group of monodisperse polyethylene glycol having a trityl group at one end reacts with methanesulfonyl chloride, and the content of the resulting compound is determined¹H-NMR measurement results, and the content of a monodisperse polyethylene glycol impurity having both terminal trityl groups contained in each step was estimated.

In the reactions of the examples, reagent equivalents were calculated under the assumption that all amounts were derived from monodisperse polyethylene glycols with a trityl group at one end, since the exact number of moles of monodisperse polyethylene glycol with a trityl group at one end was not known.

(example 1)

Synthesis of Compound 10 in which a is 8 in formula (1)

(example 1-1)

In the formula (3), a is 4 and R¹Synthesis of Compound 4 which is trityl and L is methylsulfonyl

Tetraethylene glycol 1(3416g, 17.6mol) and toluene (3.7L) were charged into a reaction vessel equipped with a thermometer, a nitrogen inlet tube, and a stirrer, all dissolved under a nitrogen atmosphere, and then azeotropically dehydrated at 110 ℃ to 120 ℃. After azeotropic dehydration, the mixture was cooled, triethylamine (736ml, 5.3mol), DMAP (54g, 0.44mol) and trityl chloride (TrtCl, 1226g, 4.4mol) were added, and the mixture was stirred at room temperature for 3 hours. After 3 hours, the disappearance of TrtCl was confirmed by Thin Layer Chromatography (TLC), and 5% sodium dihydrogenphosphate aqueous solution (6.1L) was added for liquid phase separation. The organic layer was washed once with 5% aqueous sodium dihydrogenphosphate (6.1L), once with saturated aqueous sodium hydrogencarbonate (6.1L) and once with saturated aqueous sodium chloride (6.1L). Sodium sulfate was added to the organic layer for dehydration, followed by filtration. The filtrate was concentrated under reduced pressure to obtain a reaction product containing compound 2 as a pale yellow transparent liquid. Furthermore, by TLC analysis and¹the H-NMR measurement confirmed that the obtained reaction product contained the above-mentioned compound 3.

Compound 2

¹H-NMR(CDCl₃TMS, internal standard); δ (ppm):

2.4(1H，t，-C-(OCH₂CH₂)₄-OH)，

3.23(2H，t，(C₆H₅)₃C-OCH₂CH₂-, including one derived from Compound 3)

3.45-3.85(14H，m，-OCH₂CH₂-(OCH₂CH₂)₃-OH, including the one derived from Compound 3),

7.21-7.47(15H，m，(C₆H₅)₃C-OCH₂CH₂comprising a compound derived from Compound 3)

Yield: 1687g

The reaction product containing compound 2 (compound 2: 1672g, less than 3.83mol) and toluene (8.4L) were charged into a reaction vessel equipped with a thermometer, a nitrogen inlet pipe, and an agitator and the product was dissolved under a nitrogen atmosphere, followed by addition of triethylamine (643ml, 4.62 mol). Methanesulfonyl chloride (326mL, 4.22mol) was added dropwise at 10 ℃, and the mixture was stirred at room temperature for 2 hours. After 2 hours, disappearance of compound 2 was confirmed by TLC analysis, 5% sodium dihydrogenphosphate aqueous solution (8.4L) was added, and liquid phase separation was performed. The organic layer was washed once with 5% aqueous sodium dihydrogenphosphate (8.4L), once with saturated aqueous sodium bicarbonate (8.4L), and once with saturated aqueous sodium chloride (8.4L). Sodium sulfate was added to the organic layer for dehydration, followed by filtration. The filtrate was concentrated under reduced pressure to obtain a reaction product containing compound 4 as a pale yellow transparent liquid. Furthermore, by TLC analysis and¹the H-NMR measurement confirmed that the obtained reaction product contained the above-mentioned compound 3.

Compound 4

¹H-NMR(CDCl₃TMS, internal standard); δ (ppm):

2.98(3H，s，-OCH₂CH₂-O-SO₂CH₃)，

3.23(2H，t，(C₆H₅)₃C-OCH₂CH₂-, including one derived from Compound 3)

3.45-3.85(12H，m，-OCH₂CH₂-(OCH₂CH₂)₂-OCH₂CH₂-, including the one derived from Compound 3),

4.33(2H，t，-OCH₂CH₂-O-SO₂CH₃)，

7.21-7.47(15H，m，(C₆H₅)₃C-OCH₂CH₂-, including one derived from Compound 3)

Yield: 1942g

Preparation of Compound 4 according to example 1-1¹As a result of H-NMR measurement, it was confirmed that Compound 3 was contained in an amount of about 6.2 mol%.

The calculation formula of the content of compound 3 based on the δ 3.23 peak is represented by the following formula.

(((2-[δ4.32])/4H)/([δ4.32]/2H))×100(mol％)

Further, the reaction product 2 obtained in example 1-1 contained the compound 3 in an amount of about 8.8 wt%.

Example 1-2, step (A)

In the formula (4), a is 8 and R¹Synthesis of Compound 5 which is a Tribenzyl group

Sodium hydride (215g) was placed in a reaction vessel equipped with a thermometer, a nitrogen inlet pipe, and an agitator, and after nitrogen substitution, MeCN (3.9L) was added and the mixture was cooled to 0 ℃. MeCN (2.1L) was mixed with tetraethylene glycol 1(3660g, 18.8mol) azeotropically dehydrated with toluene (1.8L), and the mixed solution was added dropwise over 2 hours. After completion of the dropwise addition, MeCN (2.1L) was mixed with the reaction product containing compound 4 (compound 4: 1942g, less than 3.77mol), and the mixed solution was added dropwise over 20 minutes. After completion of the dropwise addition, the reaction mixture was heated to 75 ℃ and stirred for 3 hours. After 3 hours, disappearance of compound 4 was confirmed by TLC, and the mixture was cooled until the temperature became 40 ℃ or less. Saturated aqueous ammonium chloride (3.9L) and hexane (3L) were added to the reaction mixture solution and subjected to liquid phase separation. Concentrating under reduced pressure to removeThe lower layer except the hexane layer (upper layer), and toluene (9.7L) was added to the residue. The toluene solution was washed once with saturated aqueous ammonium chloride (5.8L) and three times with saturated aqueous sodium chloride (9.7L). Sodium sulfate was added to the organic layer for dehydration, followed by filtration. The filtrate was concentrated under reduced pressure to obtain a reaction product containing compound 5 as a pale yellow transparent liquid. Furthermore, by TLC analysis and¹H-NMR measurement confirmed that the obtained reaction product contained the above-mentioned compounds 3 and 6.

Compound 4

¹H-NMR(CDCl₃TMS, internal standard); δ (ppm):

2.52(1H，t，-C-(OCH₂CH₂)₈-OH)，

3.23(2H，t，(C₆H₅)₃C-OCH₂CH₂-, including those derived from compounds 3 and 6)

3.45-3.85(30H，m，-OCH₂CH₂-(OCH₂CH₂)₇-OH, including those derived from compounds 3 and 6),

7.21-7.47(15H，m，(C₆H₅)₃C-OCH₂CH₂-, including those derived from compounds 3 and 6)

Yield: 2195g

Examples 1 to 3, step (B)

In the formula (6), a is 8, R¹Is trityl, and R²Synthesis of Compound 7 which is t-butyl

The reaction product containing compound 5 (compound 5: 2190g, less than 3.58mol) and methylene chloride (11L) were charged into a reaction vessel equipped with a thermometer, a nitrogen inlet pipe, and a stirrer, and all were dissolved under a nitrogen atmosphere, followed by addition of powdery potassium hydroxide (21g, 0.37 mol). After cooling to 5 ℃ tert-butyl acrylate (R in formula (5)) was added dropwise²Compound as tert-butyl 780mL5.38mol) and the mixture was reacted at 5 ℃ for 1 hour. After the reaction, a saturated aqueous ammonium chloride solution (4.0L) was added and liquid phase separation was performed. The organic layer was washed once with saturated aqueous sodium chloride (5.5L). The organic layer was concentrated under reduced pressure to obtain a reaction product containing compound 4 as a pale yellow transparent liquid. Furthermore, by TLC analysis and¹H-NMR measurement confirmed that the obtained reaction product contained the above-mentioned compounds 3 and 6.

Compound 7

¹H-NMR(CDCl₃TMS, internal standard); δ (ppm):

1.44(9H，s，-CH₂CH₂-COO-C(CH₃)₃)，

2.49(2H，t，-CH₂CH₂-COO-C(CH₃)₃)，

3.23(2H，t，(C₆H₅)₃C-OCH₂CH₂-, including those derived from compounds 3 and 6),

3.45-3.85(32H，m，-OCH₂CH₂-(OCH₂CH₂)₇-OCH₂CH₂-, including those derived from compounds 3 and 6),

7.21-7.47(15H，m，(C₆H₅)₃C-OCH₂CH₂-, including those derived from compounds 3 and 6)

Yield: 2588g (yield: 98%)

Examples 1 to 4, Steps (C) and (D)

In the formula (7), a is 8 and R²Synthesis of Compound 8 which is t-butyl

The reaction product containing compound 7 (compound 7: 2588g, less than 3.49mol) and methanol (12.9L) were charged into a reaction vessel equipped with a thermometer, a nitrogen inlet pipe, and an agitator, and all were dissolved under a nitrogen atmosphere. Thereafter, p-toluenesulfonic acid monohydrate (338g, 1.78mol) and hexane (10) were addedL). After stirring at room temperature for 30 minutes, the hexane layer was removed, hexane (6.5L) was added again, and the mixture was stirred for 30 minutes. After 5 times of the same operation, by¹As a result of H-NMR measurement, disappearance of compounds 7, 3 and 6 was confirmed, and a saturated aqueous sodium hydrogencarbonate solution (5.2L) was added. The mixed solution was washed once with hexane (6.5L) to remove trityl methyl ether. The methanol solution was concentrated under reduced pressure to obtain a crude product containing compound 8. TLC analysis and¹H-NMR measurement confirmed that the obtained crude product contained the above-mentioned compounds 1 and 9.

Next, dichloromethane (12.9L) was added to the crude product, and the mixture was washed three times with ion-exchanged water (12.9L) and 1 time with a saturated aqueous sodium chloride solution (12.9L) under 10 ℃. According to TLC analysis, disappearance of compounds 1 and 9 was confirmed. Magnesium sulfate was added to the organic layer to dehydrate, followed by filtration. The filtrate was concentrated under reduced pressure to obtain a purified product containing compound 8 as a pale yellow transparent liquid.

Compound 8

¹H-NMR(CDCl₃TMS, internal standard); δ (ppm):

1.44(9H，s，-CH₂CH₂-COO-C(CH₃)₃)，

2.50(2H，t，-CH₂CH₂-COO-C(CH₃)₃)，

2.6(1H，t，H-(OCH₂CH₂)₈-OCH₂CH₂-)，

3.45-3.85(34H，m，H-(OCH₂CH₂)₈-OCH₂CH₂-)

yield: 1229g (yield: 71%)

Examples 1 to 5, step (E)

In the formula (8), a is 8 and R²Synthesis of Compound 9 which is t-butyl

Compound 8(1229g, 2.46mol) and methylene chloride (4.9L) were charged into a reaction vessel equipped with a thermometer, a nitrogen inlet pipe, and a stirrer, and all were dissolved under a nitrogen atmosphere. Then, phthalimide (508g, 3.45mol), triphenylphosphine (906g, 3.45mol) were added. After stirring at room temperature for 30 minutes, diisopropyl azodicarboxylate (599g, 2.96mol) diluted with dichloromethane (1.2L) was added dropwise, and the mixture was stirred for 1 hour. After 1 hour, TLC confirmed the disappearance of compound 8, the solvent was distilled off under reduced pressure, methanol (5.4L) and ethylenediamine monohydrate (2L, 24.7mol) were added, and the reaction was carried out at 40 ℃ for 2 hours. A6N aqueous hydrochloric acid solution was additionally added for neutralization, the solvent was distilled off under reduced pressure, sodium chloride and chloroform (6.1L) were added for extraction, and a saturated aqueous sodium chloride solution (6.2L) was added to the extract, followed by washing twice. To the organic layer was added 1% sodium dihydrogen phosphate aqueous solution (6.2L), and the mixture was extracted three times. To the extracted aqueous solution was added a mixed solvent of chloroform (2.2L) and toluene (4L), and the mixture was washed 7 times. After adding a further 5N aqueous sodium hydroxide solution for neutralization, sodium chloride was added and extraction was carried out four times with dichloromethane (6.2L). Sodium sulfate was added to the organic layer for dehydration, followed by filtration. The filtrate was concentrated under reduced pressure to obtain compound 9 as a pale yellow transparent liquid.

Compound 9

¹H-NMR(CDCl₃TMS, internal standard); δ (ppm):

1.44(9H，s，-CH₂CH₂-COO-C(CH₃)₃)，

2.50(2H，t，-CH₂CH₂-COO-C(CH₃)₃)，

3.1(2H，t，H₂N-CH₂CH₂O-(CH₂CH₂O)₇-)，

3.45-3.85(32H，m，H₂N-CH₂CH₂O-(CH₂CH₂O)₇-CH₂CH₂-)

yield: 1149g (yield: 94%)

Examples 1 to 6, step (F)

Synthesis of Compound 10 in which a is 8 in formula (1)

Compound 9(850g, 1.71mol) and 1M hydrochloric acid (4.2L) were added to a reaction vessel equipped with a thermometer, a nitrogen inlet pipe, and a stirrer and the compound was dissolved, followed by stirring at 50 ℃ to 55 ℃ for 1 hour. After cooling to 15 ℃, the mixture was neutralized with 10M aqueous sodium hydroxide solution, and sodium chloride and dichloromethane (4.3L) were added, followed by washing. Further, the aqueous layer was washed twice with dichloromethane (4.3L), adjusted to pH 2 with 6M hydrochloric acid, and concentrated under reduced pressure. Dichloromethane (2.6L) and ethanol (2.2L) were added and the mixture was stirred and filtered, then concentrated under reduced pressure. Then, after repeating the operation of adding dichloromethane (2.6L) and concentrating under reduced pressure three times, dichloromethane (2.6L) was added to dissolve the resultant, and the solution was filtered. The filtrate was concentrated under reduced pressure to obtain compound 10 as a pale yellow transparent liquid.

Compound 10

¹H-NMR(CD₃OD, internal TMS); δ (ppm):

2.56(2H，t，-CH₂CH₂-COOH)，

3.17(2H，t，H₂N-CH₂CH₂O-)，

3.6-3.9(32H，m，-CH₂CH₂O-(CH₂CH₂O)₇-CH₂CH₂-)

yield: 710g (yield: 87%)

Purity: 99.8% (HPLC-RI)

The HPLC measurement conditions for purity measurement are as follows.

The device comprises the following steps: alias Corporation

Column: inertsil ODS-3 (column size: 4.6 mm. times.25 cm, particle size 5 μm), manufactured by GL Science Inc

A detector: RI (Ri)

Developing solvent: methanol solution/5 mM ammonium acetate solution 15/85

Flow rate: 1.0 mL/min

Column temperature: 40 deg.C

Sample concentration: 1mg/mL

Injection amount: 50 μ L

(example 2)

Synthesis of Compound 18 in which a is 12 in formula (1)

(example 2-1)

In the formula (3), a is 8 and R¹Synthesis of Compound 11 which is trityl and L is methylsulfonyl

The reaction product containing compound 5 (compound 5: 841g, less than 1.37mol) and toluene (4.2L) were charged into a reaction vessel equipped with a thermometer, a nitrogen inlet pipe, and a stirrer and the product was dissolved under a nitrogen atmosphere, followed by addition of triethylamine (267ml, 1.92 mol). Methanesulfonyl chloride (128mL, 1.65mol) was added dropwise at 10 ℃, and the mixture was stirred at room temperature for 2 hours. After 2 hours, disappearance of compound 5 was confirmed by TLC analysis, 5% sodium dihydrogenphosphate aqueous solution (4.2L) was added, and liquid phase separation was performed. The organic layer was washed once with 5% aqueous sodium dihydrogenphosphate (4.2L), once with saturated aqueous sodium bicarbonate (4.2L) and once with saturated aqueous sodium chloride (4.2L). Sodium sulfate was added to the organic layer for dehydration, followed by filtration. The filtrate was concentrated under reduced pressure to obtain a reaction product containing compound 11 as a pale yellow transparent liquid. Furthermore, by TLC analysis and¹H-NMR measurement confirmed that the obtained reaction product contained the above-mentioned compounds 3 and 6.

Compound 11

¹H-NMR(CDCl₃TMS, internal standard); δ (ppm):

3.07(3H，s，-OCH₂CH₂-O-SO₂CH₃)，

3.23(2H，t，(C₆H₅)₃C-OCH₂CH₂-, including those derived from compounds 3 and 6)

3.45-3.85(28H，m，-OCH₂CH₂-(OCH₂CH₂)₆-OCH₂CH₂-, including those derived from compounds 3 and 6),

4.37(2H，t，-OCH₂CH₂-O-SO₂CH₃)，

7.21-7.47(15H，m，(C₆H₅)₃C-OCH₂CH₂-, including those derived from compounds 3 and 6)

Yield: 945g

Preparation of Compound 11 according to example 2-1¹As a result of H-NMR measurement, it was confirmed that the contents of the compounds 3 and 6 were about 11 mol% (compound 3: 6 mol%, compound 6: 5 mol%, rough estimate).

The calculated formula for the content of compounds 3 and 6 based on the delta 3.23 peak is represented by the following formula.

(((2-[δ4.32])/4H)/([δ4.32]/2H))×100(mol％)

As for the content of compound 3, the value calculated in example 1-1 was applied.

Further, the reaction product 5 used in example 2-1 contained the compounds 3 and 6 in an amount of about 12.6% by weight.

Example 2-2, step (A)

In the formula (4), a is 12 and R¹Synthesis of Compound 12 as a Tribenzyl group

Sodium hydride (78g) was placed in a reaction vessel equipped with a thermometer, a nitrogen inlet pipe, and an agitator, and after nitrogen substitution, MeCN (1.9L) was added and the mixture was cooled to 0 ℃. MeCN (940mL) was mixed with tetraethylene glycol 1(1327g, 6.83mol) azeotropically dehydrated with toluene (660mL) and added dropwise over 2 hoursThe mixed solution was added. After the dropwise addition was complete, MeCN (940mL) was mixed with the reaction product containing Compound 11 (Compound 11: 944g, less than 1.37mol), and the mixed solution was added dropwise over 20 minutes. After completion of the dropwise addition, the reaction mixture was heated to 75 ℃ and stirred for 3 hours. After 3 hours, use¹H-NMR confirmed the disappearance of compound 11, and the mixture was cooled until the temperature became 40 ℃ or less. Saturated aqueous ammonium chloride (1.9L) and hexane (1.4L) were added to the reaction mixture solution and subjected to liquid phase separation. The lower layer from which the hexane layer (upper layer) had been removed was concentrated under reduced pressure, and toluene (4.7L) was added to the residue. The toluene solution was washed once with saturated aqueous ammonium chloride (2.8L) and three times with saturated aqueous sodium chloride (4.7L). Sodium sulfate was added to the organic layer for dehydration, followed by filtration. The filtrate was concentrated under reduced pressure to obtain a reaction product containing compound 12 as a pale yellow transparent liquid. Furthermore, by TLC analysis and¹the results of H-NMR measurement confirmed that the obtained reaction product contained the above-mentioned compounds 3, 6 and 13.

Compound 12

¹H-NMR(CDCl₃TMS, internal standard); δ (ppm):

2.56(1H，t，-C-(OCH₂CH₂)₁₂-OH)，

3.23(2H，t，(C₆H₅)₃C-OCH₂CH₂-, including those derived from compounds 3, 6 and 13)

3.45-3.85(46H，m，-OCH₂CH₂-(OCH₂CH₂)₁₁-OH, including those derived from compounds 3, 6 and 13),

7.21-7.47(15H，m，(C₆H₅)₃C-OCH₂CH₂-, including those derived from compounds 3, 6 and 13)

Yield: 1027g

Examples 2 to 3, step (B)

In the formula (6), a is 12, R¹Is trityl, and R²Synthesis of Compound 14 which is t-butyl

The reaction product containing compound 12 (compound 12: 200g, less than 253mmol) and dichloromethane (1000mL) were charged to a reaction vessel equipped with a thermometer, nitrogen inlet tube and stirrer, and the product was dissolved under nitrogen atmosphere, followed by addition of powdered potassium hydroxide (1.4g, 25 mmol). After cooling to 5 ℃ tert-butyl acrylate (R in formula (5)) was added dropwise²Compound which is tert-butyl, 55mL, 380mmol), and the mixture was reacted at 5 ℃ for 2 hours. After the reaction, a saturated aqueous ammonium chloride solution (400mL) was added and liquid phase separation was performed. The organic layer was washed once with saturated aqueous sodium chloride (600 mL). The organic layer was concentrated under reduced pressure to obtain a reaction product containing compound 14 as a pale yellow transparent liquid. Furthermore, by TLC analysis and¹the results of H-NMR measurement confirmed that the obtained reaction product contained the above-mentioned compounds 3, 6 and 13.

Compound 14

¹H-NMR(CDCl₃TMS, internal standard); δ (ppm):

1.44(9H，s，-CH₂CH₂-COO-C(CH₃)₃)，

2.49(2H，t，-CH₂CH₂-COO-C(CH₃)₃)，

3.23(2H，t，(C₆H₅)₃C-OCH₂CH₂-, including those derived from compounds 3, 6 and 13),

3.45-3.85(48H，m，-OCH₂CH₂-(OCH₂CH₂)₁₁-OCH₂CH₂-, including those derived from compounds 3, 6 and 13),

7.21-7.47(15H，m，(C₆H₅)₃C-OCH₂CH₂-, including those derived from compounds 3, 6 and 13)

Yield: 226g (yield: 97%)

Examples 2 to 4, Steps (C) and (D)

In the formula (7), a is 12 and R²Synthesis of Compound 15 which is t-butyl

The reaction product containing compound 14 (compound 14: 226g, less than 246mmol) and methanol (1.1L) were charged into a reaction vessel equipped with a thermometer, nitrogen inlet pipe and stirrer, and the product was dissolved under a nitrogen atmosphere. Thereafter, p-toluenesulfonic acid monohydrate (23g, 123mmol) and hexane (900mL) were added. After stirring at room temperature for 30 minutes, the hexane layer was removed, hexane (565mL) was added again, and the mixture was stirred for 30 minutes. After four times of the same operation, by¹As a result of H-NMR measurement, disappearance of compounds 14, 3, 6 and 13 was confirmed, and a saturated aqueous sodium hydrogencarbonate solution (452mL) was added. The mixed solution was washed once with hexane (565mL) to remove trityl methyl ether. The methanol solution was concentrated under reduced pressure to obtain a crude product containing compound 15. TLC analysis and¹H-NMR measurement confirmed that the obtained crude product contained the above-mentioned compounds 1, 9 and 16.

Next, methylene chloride (1.1L) was added to the crude product, and the mixture was washed three times with ion-exchanged water (1.1L) and 1 time with a saturated aqueous sodium chloride solution (1.1L) under 10 ℃. According to TLC analysis, disappearance of compounds 1, 9 and 13 was confirmed. Magnesium sulfate was added to the organic layer to dehydrate, followed by filtration. The filtrate was concentrated under reduced pressure to obtain a purified product containing compound 15 as a pale yellow transparent liquid.

Compound 15

¹H-NMR(CDCl₃TMS, internal standard); δ (ppm):

1.44(9H，s，-CH₂CH₂-COO-C(CH₃)₃)，

2.50(2H，t，-CH₂CH₂-COO-C(CH₃)₃)，

2.6(1H，t，H-(OCH₂CH₂)₁₂-OCH₂CH₂-)，

3.45-3.85(50H，m，H-(OCH₂CH₂)₁₂-OCH₂CH₂-)

yield: 118g (yield: 71%)

Examples 2 to 5, step (E)

In the formula (8), a is 12 and R²Synthesis of Compound 17 which is t-butyl

Compound 15(117g, 173mmol) and dichloromethane (469mL) were added to a reaction vessel equipped with a thermometer, nitrogen inlet tube and stirrer, and the compound was dissolved under a nitrogen atmosphere. Then, phthalimide (36g, 245mmol) and triphenylphosphine (64g, 244mmol) were added. After stirring at room temperature for 30 minutes, diisopropyl azodicarboxylate (42g, 208mmol) diluted with dichloromethane (118mL) was added dropwise, and the mixture was stirred for 1 hour. After 1 hour, TLC confirmed the disappearance of compound 15, the solvent was distilled off under reduced pressure, methanol (518mL) and ethylenediamine monohydrate (141mL, 1.73mol) were added, and the reaction was carried out at 40 ℃ for 3 hours. A6N aqueous hydrochloric acid solution was additionally added for neutralization, the solvent was distilled off under reduced pressure, and sodium chloride and chloroform (585mL) were added for extraction. Saturated aqueous sodium chloride (585mL) was added to the extract of the resultant, and two washes were performed. To the organic layer was added 1% aqueous sodium dihydrogen phosphate (585mL), and the mixture was extracted four times. Seven washes were performed while adding a mixed solvent of chloroform (293mL) and toluene (293mL) to the extracted aqueous solution. After an additional 5N aqueous sodium hydroxide solution was added for neutralization, sodium chloride was added and extraction was performed four times with dichloromethane (590 mL). Sodium sulfate was added to the organic layer for dehydration, followed by filtration. The filtrate was concentrated under reduced pressure to obtain compound 17 as a pale yellow transparent liquid.

Compound 17

¹H-NMR(CDCl₃TMS, internal standard); δ (ppm):

1.44(9H，s，-CH₂CH₂-COO-C(CH₃)₃)，

2.50(2H，t，-CH₂CH₂-COO-C(CH₃)₃)，

3.1(2H，t，H₂N-CH₂CH₂O-(CH₂CH₂O)₁₁-)，

3.45-3.85(48H，m，H₂N-CH₂CH₂O-(CH₂CH₂O)₁₁-CH₂CH₂-)

yield: 113g (yield:

97％)

examples 2 to 6, step (F)

Synthesis of Compound 18 in which a is 12 in formula (1)

Compound 17(50g, 74mmol) and 1M hydrochloric acid (250mL) were added to a reaction vessel equipped with a thermometer, a nitrogen inlet tube and a stirrer and the compound was dissolved, followed by stirring at 50 ℃ to 55 ℃ for 1 hour. After cooling to 15 ℃, the mixture was neutralized with 10M aqueous sodium hydroxide solution, and sodium chloride and dichloromethane (250mL) were added for washing. The aqueous layer was washed once more with dichloromethane (250mL), adjusted to pH 2 with 6M hydrochloric acid, and extracted four times with dichloromethane (250 mL). The recovered organic layers were mixed and dehydrated with sodium sulfate, and then filtered. The filtrate was concentrated under reduced pressure to obtain compound 18 as a pale yellow solid.

Compound 18

¹H-NMR(CD₃OD, internal TMS); δ (ppm):

2.56(2H，t，-CH₂CH₂-COOH)，

3.17(2H，t，H₂N-CH₂CH₂O-)，

3.6-3.9(48H，m，-CH₂CH₂O-(CH₂CH₂O)₁₁-CH₂CH₂-)

yield: 44g (yield: 91%)

Purity: 99.8% (HPLC-RI)

The HPLC measurement conditions for purity measurement are as follows.

The device comprises the following steps: alias Corporation

Column: inertsil ODS-3 (column size: 4.6 mm. times.25 cm, particle size 5 μm), manufactured by GL Science Inc

A detector: RI (Ri)

Developing solvent: methanol solution/5 mM ammonium acetate solution 27.5/72.5

Flow rate: 1.0 mL/min

Column temperature: 40 deg.C

Sample concentration: 2mg/mL

Injection amount: 50 μ L

(example 3)

Synthesis of Compound 40 in which a is 24 in formula (1)

(example 3-1)

In the formula (3), a is 12 and R¹Synthesis of Compound 19 which is trityl and L is methylsulfonyl

The reaction product containing compound 12 (compound 12: 90g, less than 0.11mol) and toluene (451mL) were charged into a reaction vessel equipped with a thermometer, a nitrogen inlet tube, and a stirrer and the product was dissolved under a nitrogen atmosphere, followed by addition of triethylamine (29mL, 0.21 mol). Methanesulfonyl chloride (14mL, 0.18mol) was added dropwise at 10 ℃, and the mixture was stirred at room temperature for 2 hours. After 2 hours, disappearance of compound 12 was confirmed by TLC analysis, 5% sodium dihydrogenphosphate aqueous solution (450mL) was added, and liquid phase separation was performed. The organic layer was washed once with 5% aqueous sodium dihydrogen phosphate (450mL), twice with saturated aqueous sodium bicarbonate (450mL), and with saturated aqueous sodium chloride (450mL)And washing once. Sodium sulfate was added to the organic layer for dehydration, followed by filtration. The filtrate was concentrated under reduced pressure to obtain a reaction product containing compound 19 as a pale yellow transparent liquid. Furthermore, by TLC analysis and¹the results of H-NMR measurement confirmed that the obtained reaction product contained the above-mentioned compounds 3, 6 and 13.

Compound 19

¹H-NMR(CDCl₃TMS, internal standard); δ (ppm):

3.07(3H，s，-OCH₂CH₂-O-SO₂CH₃)，

3.23(2H，t，(C₆H₅)₃C-OCH₂CH₂-, including those derived from compounds 3, 6 and 13)

3.45-3.85(44H，m，-OCH₂CH₂-(OCH₂CH₂)₁₀-OCH₂CH₂-, including those derived from compounds 3, 6 and 13),

4.37(2H，t，-OCH₂CH₂-O-SO₂CH₃)，

7.21-7.47(15H，m，(C₆H₅)₃C-OCH₂CH₂-, including those derived from compounds 3, 6 and 13)

Yield: 96g of

Preparation of Compound 19 according to example 3-1¹As a result of H-NMR measurement, it was confirmed that the contents of the compounds 3, 6 and 13 were about 15 mol% (compound 3: 6 mol%, compound 6: 5 mol%, compound 13: 4 mol%, rough estimate).

The calculation formula of the contents of compounds 3, 6 and 13 based on the δ 3.23 peak is represented by the following formula.

(((2-[δ4.32])/4H)/([δ4.32]/2H))×100(mol％)

As to the contents of the compounds 3 and 6, the values calculated in example 1-1 were applied.

Further, the reaction product 12 used in example 3-1 contained the compounds 3, 6 and 13 in an amount of about 15.9% by weight.

Example 3-2, step (A)

In the formula (4), a is 16 and R¹Synthesis of Compound 20 as a Tribenzyl group

Sodium hydride (6.3g) was placed in a reaction vessel equipped with a thermometer, a nitrogen inlet pipe, and an agitator. After nitrogen displacement, MeCN (192mL) was added and the mixture was cooled to 0 ℃. MeCN (96mL) was mixed with tetraethylene glycol 1(108g, 0.56mol) azeotropically dehydrated with toluene (53mL), and the mixed solution was added dropwise over 30 minutes. After completion of the dropwise addition, MeCN (96mL) was mixed with the reaction product containing compound 19 (compound 19: 96g, less than 0.11mol), and the mixed solution was added dropwise over 15 minutes. After completion of the dropwise addition, the reaction mixture was heated to 75 ℃ and stirred for 3 hours. After 3 hours, use¹H-NMR confirmed the disappearance of compound 19, and the mixture was cooled until the temperature became 40 ℃ or less. Saturated aqueous ammonium chloride (170mL) and hexane (146mL) were added to the reaction mixture solution and liquid phase separation was performed. The lower layer from which the hexane layer (upper layer) had been removed was concentrated under reduced pressure, and toluene (481mL) was added to the residue. The toluene solution was washed once with saturated aqueous ammonium chloride (260mL) and three times with saturated aqueous sodium chloride (480 mL). Sodium sulfate was added to the organic layer for dehydration, followed by filtration. The filtrate was concentrated under reduced pressure to obtain a reaction product containing compound 20 as a pale yellow transparent liquid. Furthermore, by TLC analysis and¹the results of H-NMR measurements confirmed that the obtained reaction products contained the above-mentioned compounds 3, 6, 13 and 21.

Compound 20

¹H-NMR(CDCl₃TMS, internal standard); δ (ppm):

2.78(1H，b，-C-(OCH₂CH₂)₁₆-OH)，

3.23(2H，t，(C₆H₅)₃C-OCH₂CH₂-, including those derived from compounds 3, 6, 13 and 21)

3.45-3.85(62H，m，-OCH₂CH₂-(OCH₂CH₂)₁₅-OH, including those derived from compounds 3, 6, 13 and 21),

7.21-7.47(15H，m，(C₆H₅)₃C-OCH₂CH₂-, including those derived from compounds 3, 6, 13 and 21)

Yield: 103g

(examples 3 to 3)

In the formula (3), a is 16 and R¹Synthesis of Compound 22 which is trityl and L is methylsulfonyl

The reaction product containing compound 20 (compound 20: 100g, less than 0.10mol) and toluene (500mL) were charged into a reaction vessel equipped with a thermometer, a nitrogen inlet tube, and a stirrer and the product was dissolved under a nitrogen atmosphere, followed by addition of triethylamine (20mL, 0.14 mol). Methanesulfonyl chloride (9.6mL, 0.12mol) was added dropwise at 10 ℃, and the mixture was stirred at room temperature for 2 hours. After 2 hours, disappearance of compound 14 was confirmed by TLC analysis, 5% sodium dihydrogenphosphate aqueous solution (500mL) was added, and liquid phase separation was performed. The organic layer was washed once with 5% aqueous sodium dihydrogen phosphate (500mL), twice with saturated aqueous sodium bicarbonate (500mL), and once with saturated aqueous sodium chloride (500 mL). Sodium sulfate was added to the organic layer for dehydration, followed by filtration. The filtrate was concentrated under reduced pressure to obtain a reaction product containing compound 22 as a pale yellow transparent liquid. Furthermore, by TLC analysis and¹the results of H-NMR measurements confirmed that the obtained reaction products contained the above-mentioned compounds 3, 6, 13 and 21.

Compound 22

¹H-NMR(CDCl₃TMS, internal standard); δ (ppm):

3.07(3H，s，-OCH₂CH₂-O-SO₂CH₃)，

3.23(2H，t，(C₆H₅)₃C-OCH₂CH₂-, including the compounds 3, 6, 13 and 21 derived from)

3.45-3.85(60H，m，-OCH₂CH₂-(OCH₂CH₂)₁₄-OCH₂CH₂-, including those derived from compounds 3, 6, 13 and 21),

4.37(2H，t，-OCH₂CH₂-O-SO₂CH₃)，

7.21-7.47(15H，m，(C₆H₅)₃C-OCH₂CH₂-, including those derived from compounds 3, 6, 13 and 21)

Yield: 106g of

Examples 3 to 4, step (A)

In the formula (4), a is 20 and R¹Synthesis of Compound 23 which is a Tribenzyl group

Sodium hydride (5.7g) was placed in a reaction vessel equipped with a thermometer, a nitrogen inlet tube, and an agitator, and after nitrogen substitution, MeCN (208mL) was added and the mixture was cooled to 0 ℃. MeCN (105mL) was mixed with tetraethylene glycol 1(97g, 0.50mol) azeotropically dehydrated with toluene (48mL), and the mixed solution was added dropwise over 30 minutes. After completion of the dropwise addition, MeCN (105mL) was mixed with the reaction product comprising compound 22 (compound 22: 109g, less than 0.10mol), and the mixed solution was added dropwise over 15 minutes. After completion of the dropwise addition, the reaction mixture was heated to 75 ℃ and stirred for 3 hours. After 3 hours, use¹H-NMR confirmed the disappearance of compound 22 and the mixture was cooled until the temperature became below 40 ℃. Saturated aqueous ammonium chloride (190mL) and hexane (159mL) were added to the reaction mixture solution and subjected to liquid phase separation. The lower layer from which the hexane layer (upper layer) had been removed was concentrated under reduced pressure, and toluene (524mL) was added to the residue. The toluene solution was washed once with saturated aqueous ammonium chloride solution (285mL) and three times with saturated aqueous sodium chloride solution (520 mL). Sodium sulfate was added to the organic layer for dehydration, followed by filtration. Concentrating the filtrate under reduced pressure to obtain a filtrate comprisingThe reaction product of compound 23 was a light yellow transparent liquid. Furthermore, by TLC analysis and¹the results of H-NMR measurements confirmed that the obtained reaction products contained the above-mentioned compounds 3, 6, 13, 21 and 24.

Compound 22

¹H-NMR(CDCl₃TMS, internal standard); δ (ppm):

2.64(1H，b，-C-(OCH₂CH₂)₂₀-OH)，

3.23(2H，t，(C-6H5)₃C-OCH₂CH₂-, including those derived from compounds 3, 6, 13, 21 and 24)

3.45-3.85(78H，m，-OCH₂CH₂-(OCH₂CH₂)₁₉-OH, including those derived from compounds 3, 6, 13, 21 and 24),

7.21-7.47(15H，m，(C₆H₅)₃C-OCH₂CH₂-, including those derived from compounds 3, 6, 13, 21 and 24)

Yield: 109g of

(examples 3 to 5)

In the formula (3), a is 20 and R¹Synthesis of Compound 25 which is trityl and L is methylsulfonyl

The reaction product containing compound 23 (compound 23: 107g, less than 0.094mol) and toluene (585mL) were charged into a reaction vessel equipped with a thermometer, a nitrogen inlet tube, and an agitator and the product was dissolved under a nitrogen atmosphere, followed by addition of triethylamine (18mL, 0.13 mol). Methanesulfonyl chloride (8.7mL, 0.11mol) was added dropwise at 10 ℃, and the mixture was stirred at room temperature for 2 hours. After 2 hours, disappearance of compound 14 was confirmed by TLC analysis, 5% sodium dihydrogenphosphate aqueous solution (535mL) was added, and liquid phase separation was performed. The organic layer was washed once with 5% aqueous sodium dihydrogen phosphate (535mL), twice with saturated aqueous sodium bicarbonate (535mL), and once with saturated aqueous sodium chloride (535 mL). To the direction ofSodium sulfate was added to the organic layer for dehydration, followed by filtration. The filtrate was concentrated under reduced pressure to obtain a reaction product containing compound 25 as a pale yellow transparent liquid. Furthermore, by TLC analysis and¹the results of H-NMR measurements confirmed that the obtained reaction products contained the above-mentioned compounds 3, 6, 13, 21 and 24.

Compound 25

¹H-NMR(CDCl₃TMS, internal standard); δ (ppm):

3.07(3H，s，-OCH₂CH₂-O-SO₂CH₃)，

3.23(2H，t，(C₆H₅)₃C-OCH₂CH₂-, including those derived from compounds 3, 6, 13, 21 and 24)

3.45-3.85(76H，m，-OCH₂CH₂-(OCH₂CH₂)₁₈-OCH₂CH₂-, including those derived from compounds 3, 6, 13, 21 and 24),

4.37(2H，t，-OCH₂CH₂-O-SO₂CH₃)，

7.21-7.47(15H，m，(C₆H₅)₃C-OCH₂CH₂-, including those derived from compounds 3, 6, 13, 21 and 24)

Yield: 113g

Examples 3 to 6, step (A)

In the formula (4), a is 24 and R¹Synthesis of Compound 26 as a Tribenzyl group

Sodium hydride (5.2g) was placed in a reaction vessel equipped with a thermometer, a nitrogen inlet tube, and an agitator, and after nitrogen substitution, MeCN (221mL) was added and the mixture was cooled to 0 ℃. MeCN (111mL) was mixed with tetraethylene glycol 1(88g, 0.46mol) azeotropically dehydrated with toluene (44mL), and the mixed solution was added dropwise over 30 minutes. After completion of the dropwise addition, MeCN (111mL) was mixed with a mixture comprising compound 25 (compound 2)5: 113g, less than 0.092mol) of the reaction products were mixed, and the mixed solution was added dropwise over 15 minutes. After completion of the dropwise addition, the reaction mixture was heated to 75 ℃ and stirred for 3 hours. After 3 hours, use¹H-NMR confirmed the disappearance of compound 25 and the mixture was cooled until the temperature became 40 ℃ or less. Saturated aqueous ammonium chloride (200mL) and hexane (168mL) were added to the reaction mixture solution and liquid phase separation was performed. The lower layer from which the hexane layer (upper layer) had been removed was concentrated under reduced pressure, and toluene (556mL) was added to the residue. The toluene solution was washed once with saturated aqueous ammonium chloride (300mL) and three times with saturated aqueous sodium chloride (555 mL). Sodium sulfate was added to the organic layer for dehydration, followed by filtration. The filtrate was concentrated under reduced pressure to obtain a reaction product containing compound 26 as a pale yellow transparent liquid. Furthermore, by TLC analysis and¹the results of H-NMR measurements confirmed that the obtained reaction products contained the above-mentioned compounds 3, 6, 13, 21, 24 and 27.

Compound 26

¹H-NMR(CDCl₃TMS, internal standard); δ (ppm):

2.66(1H，b，-C-(OCH₂CH₂)₂₄-OH)，

3.23(2H，t，(C₆H₅)₃C-OCH₂CH₂-, including those derived from compounds 3, 6, 13, 21, 24 and 27)

3.45-3.85(94H，m，-OCH₂CH₂-(OCH₂CH₂)₂₃-OH, including those derived from compounds 3, 6, 13, 21, 24 and 27),

7.21-7.47(15H，m，(C₆H₅)₃C-OCH₂CH₂-, including those derived from compounds 3, 6, 13, 21, 24 and 27)

Yield: 115g

Examples 3 to 7, step (B)

In the formula (6), a is 24, R¹Is trityl, and R²Synthesis of Compound 28 which is t-butyl

The reaction product containing compound 28 (compound 28: 5.02g, less than 3.79mmol) and dichloromethane (25mL) were charged to a reaction vessel equipped with a thermometer, nitrogen inlet tube and stirrer, and the preparation was dissolved under nitrogen atmosphere, followed by addition of powdered potassium hydroxide (128mg, 2.28 mmol). After cooling to 5 ℃ tert-butyl acrylate (R in formula (5)) was added dropwise²Compound which is tert-butyl, 1.1mL, 7.58mmol), and the mixture was reacted at 5 ℃ for 4 hours. After the reaction, a saturated aqueous ammonium chloride solution (10mL) was added and liquid phase separation was performed. The organic layer was washed once with saturated aqueous sodium chloride (15 mL). The organic layer was concentrated under reduced pressure to obtain a reaction product containing compound 28 as a pale yellow transparent liquid. Furthermore, by TLC analysis and¹the results of H-NMR measurements confirmed that the obtained reaction products contained the above-mentioned compounds 3, 6, 13, 21, 24 and 27.

Compound 28

¹H-NMR(CDCl₃TMS, internal standard); δ (ppm):

1.44(9H，s，-CH₂CH₂-COO-C(CH₃)₃)，

2.49(2H，t，-CH₂CH₂-COO-C(CH₃)₃)，

3.23(2H，t，(C₆H₅)₃C-OCH₂CH₂-, including those derived from compounds 3, 6, 13, 21, 24 and 27),

3.45-3.85(96H，m，-OCH₂CH₂-(OCH₂CH₂)₂₃-OCH₂CH₂-, including those derived from compounds 3, 6, 13, 21, 24 and 27),

7.21-7.47(15H，m，(C₆H₅)₃C-OCH₂CH₂-, including those derived from compounds 3, 6, 13, 21, 24 and 27)

Yield: 5.31g

Examples 3 to 8, step (C)

In the formula (7), a is 24 and R²Synthesis of Compound 29 which is t-butyl

The reaction product containing compound 28 (compound 28: 5.01g, less than 3.46mmol) and methanol (25mL) were charged into a reaction vessel equipped with a thermometer, nitrogen inlet tube and stirrer, and the preparation was dissolved under a nitrogen atmosphere. Thereafter, p-toluenesulfonic acid monohydrate (329mg, 1.72mmol) and hexane (20mL) were added. After stirring at room temperature for 30 minutes, the hexane layer was removed, hexane (13mL) was added again, and the mixture was stirred for 30 minutes. After four times of the same operation, by¹As a result of H-NMR measurement, it was confirmed that compounds 28, 3, 6, 13, 21, 24 and 27 disappeared, and a saturated aqueous solution of sodium hydrogencarbonate (11mL) was added. The mixed solution was washed once with hexane (13mL) to remove trityl methyl ether. The methanol solution was concentrated under reduced pressure and 20% aqueous sodium chloride solution (10mL) and methylene chloride (10mL) were added, followed by liquid phase separation. Magnesium sulfate was added to the organic layer to dehydrate, followed by filtration. The filtrate was concentrated under reduced pressure to obtain a reaction product containing compound 29 as a pale yellow transparent liquid. Furthermore, by TLC analysis and¹the results of H-NMR measurements confirmed that the obtained reaction products contained the above-mentioned compounds 1, 9, 16, 30, 31 and 32.

Compound 29

¹H-NMR(CDCl₃TMS, internal standard); δ (ppm):

1.44(9H，s，-CH₂CH₂-COO-C(CH₃)₃)，

2.50(2H，t，-CH₂CH₂-COO-C(CH₃)₃)，

2.7(1H，b，H-(OCH₂CH₂)₂₄-OCH₂CH₂-)，

3.45-3.85(98H，m，H-(OCH₂CH₂)₂₄-OCH₂CH₂-, including those derived from compounds 1, 9, 16, 30, 31 and 32)

Yield: 3.13g

Examples 3 to 9, step (E)

In the formula (8), a is 24 and R²Synthesis of Compound 33 which is t-butyl

The reaction product containing compound 29 (compound 29: 3.00g, less than 2.49mmol) and dichloromethane (12mL) were charged to a reaction vessel equipped with a thermometer, nitrogen inlet tube and stirrer and the preparation dissolved under a nitrogen atmosphere. Then, phthalimide (641mg, 4.36mmol) and triphenylphosphine (1.14g, 4.35mmol) were added. After stirring at room temperature for 30 minutes, diisopropyl azodicarboxylate (756mg, 3.74mmol) diluted with dichloromethane (3mL) was added dropwise, and the mixture was stirred for 3 hours. After 3 hours, disappearance of compound 29 was confirmed by TLC analysis, the solvent was distilled off under reduced pressure, methanol (13mL) and ethylenediamine monohydrate (2mL, 24.8mol) were added, and the reaction was carried out at 40 ℃ for 4 hours. A 6N aqueous hydrochloric acid solution was additionally added for neutralization, the solvent was distilled off under reduced pressure, sodium chloride and chloroform (15mL) were added for extraction, and washing was performed twice while adding a saturated aqueous sodium chloride solution (15mL) to the extract. To the organic layer was added 1% aqueous sodium dihydrogen phosphate solution (15mL), and the mixture was extracted four times. Seven washes were performed while adding a mixed solvent of chloroform (8mL) and toluene (8mL) to the extracted aqueous solution. After adding a further 5N aqueous sodium hydroxide solution for neutralization, sodium chloride was added and extraction was carried out four times with dichloromethane (15 mL). Sodium sulfate was added to the organic layer for dehydration, followed by filtration. The filtrate was concentrated under reduced pressure to obtain a reaction product containing compound 33 as a pale yellow transparent liquid. Furthermore, by TLC analysis and¹the results of H-NMR measurements confirmed that the obtained reaction products contained the above-mentioned compounds 34, 35, 36, 37, 38 and 39.

Compound 33

¹H-NMR(CDCl₃Internal standard of

TMS)；δ(ppm)：

1.44(9H，s，-CH₂CH₂-COO-C(CH₃)₃)，

2.50(2H，t，-CH₂CH₂-COO-C(CH₃)₃)，

3.1(2H，t，H₂N-CH₂CH₂O-(CH₂CH₂O)₂₃-)，

3.45-3.85(96H，m，H₂N-CH₂CH₂O-(CH₂CH₂O)₂₃-CH₂CH₂-, including those derived from compounds 34, 35, 36, 37, 38 and 39)

Yield: 2.45g

Examples 3 to 10, Steps (F) and (G)

Synthesis of Compound 40 in which a is 24 in formula (1)

The reaction product containing compound 33 (compound 33: 1.22g, less than 1.00mmol) and 1M hydrochloric acid (6mL) were added to a reaction vessel equipped with a thermometer, nitrogen inlet tube and stirrer and the preparation dissolved, followed by stirring at 50 ℃ to 55 ℃ for 2 hours. After 2 hours, disappearance of compound 33 was confirmed by TLC. The mixture was cooled to room temperature.

Then, a 10M aqueous sodium hydroxide solution was added to the reaction solution to adjust the pH to 10, and washing was performed twice while adding dichloromethane (6 mL). The solution was adjusted to pH 2 with 6M hydrochloric acid and, after addition of sodium chloride, extracted three times with dichloromethane (6 mL). The recovered organic layers were mixed and dehydrated with sodium sulfate, and then filtered. The filtrate was concentrated under reduced pressure to obtain compound 40(1.16g) as a pale yellow solid. After tetrahydrofuran (5mL) was added to the resulting compound 40 for dissolution, the solution was cooled to 10 ℃ and hexane (24mL) cooled to 10 ℃ was added to perform crystallization. The crystals were collected by filtration and washed with hexane (10mL) cooled to 10 ℃. The crystals were collected by filtration and dried in vacuo to give compound 40 as white powder crystals.

Compound 40

¹H-NMR(CD₃OD, internal TMS); δ (ppm):

2.56(2H，t，-CH₂CH₂-COOH)，

3.17(2H，t，H₂N-CH₂CH₂O-)，

3.6-3.9(96H，m，-CH₂CH₂O-(CH₂CH₂O)₂₃-CH₂CH₂-)

yield: 893mg

(example 4)

For calculation of the content of R in formula (4) when removing a compound having trityl groups at both terminals from a reaction product comprising Compound 5 obtained in example 1-2¹The derivatization of compound 42 was carried out with the content of compound 5 in which a is 8 in the trityl group-containing compound.

In the formula (3), a is 8 and R¹Synthesis of Compound 41 which is trityl and L is tosyl

The reaction product containing compound 5 (compound 5: 5g, less than 8.2mmol) and dichloromethane (25mL) were added to a reaction vessel equipped with a thermometer, a nitrogen inlet tube and a stirrer, and the product was dissolved under a nitrogen atmosphere, triethylamine (1.2mL, 8.6mmol), 4-dimethylaminopyridine (100mg, 0.82mmol) and TsCl (1.4g, 7.3mmol) were added, and the mixture was stirred at room temperature for 4 hours. After 4 hours, by¹H-NMR analysis confirmed the disappearance of TsCl, and 1M aqueous hydrochloric acid (25mL) was added and liquid phase separation was performed. The organic layer was washed once with 1M aqueous hydrochloric acid (25mL), twice with saturated aqueous sodium bicarbonate (25mL), and once with saturated aqueous sodium chloride (25 mL). Sodium sulfate was added to the organic layer for dehydration, followed by filtration. The filtrate was concentrated under reduced pressure to obtain a reaction product containing compound 41 as a pale yellow transparent liquid.Furthermore, by TLC analysis and¹H-NMR measurement confirmed that the obtained reaction product contained the above-mentioned compounds 3 and 6.

Compound 41

¹H-NMR(CDCl₃TMS, internal standard); δ (ppm):

2.45(3H，s，-OSO₂-phenyl-CH₃)，

3.23(2H，t，(C₆H₅)₃C-OCH₂CH₂-, including those derived from compounds 3 and 6),

3.45-3.85(28H，m，-OCH₂CH₂-(OCH₂CH₂)₆-OCH₂CH₂-, including those derived from compounds 3 and 6),

4.16(2H，t，-OCH₂CH₂-OSO₂-phenyl-CH₃)，

7.21-7.47(15H，m，(C₆H₅)₃C-OCH₂CH₂-, including those derived from compounds 3 and 6),

7.35(2H，d，-OSO₂-phenyl-CH₃)，

7.80(2H，d，-OSO₂-phenyl-CH₃)

Yield: 6.1g

Synthesis of Compound 42 having an 8-Unit glycol chain Length having a hydroxyl group at one end and a tosyl group at one end

Next, the reaction product containing compound 42 (compound 42: 6.1g, less than 8.0mmol) and methanol (25mL) were charged to a reaction vessel equipped with a thermometer, nitrogen inlet pipe, and stirrer. After dissolving the compound under a nitrogen atmosphere, p-toluenesulfonic acid monohydrate (0.78g, 4.1mmol) and hexane (20mL) were added. After stirring at room temperature for 30 minutes, the hexane layer was removed, hexane (13mL) was added again, and the mixture was stirred for 30 minutes. After six identical operations have been carried out, by¹As a result of H-NMR measurement, disappearance of compounds 42, 3 and 6 was confirmed, and a saturated aqueous sodium hydrogencarbonate solution (10mL) was added. The mixed solution was washed twice with hexane (13mL) to remove trityl methyl ether. The methanol solution was concentrated under reduced pressure, and dichloromethane (25mL) was added for extraction. Magnesium sulfate was added to the organic layer to dehydrate, followed by filtration. The filtrate was concentrated under reduced pressure to obtain compound 42 as a pale yellow transparent liquid.

Compound 42

¹H-NMR(CDCl₃TMS, internal standard); δ (ppm):

2.45(3H，s，-OSO₂-phenyl-CH₃)，

2.73(1H，t，H-(OCH₂CH₂)₈-)，

3.45-3.85(30H，m，-(OCH₂CH₂)₇-OCH₂CH₂-)，

4.16(2H，t，-(OCH₂CH₂)₇-OCH₂CH₂-OSO₂-phenyl-CH₃)，

7.35(2H，d，-OSO₂-phenyl-CH₃)，

7.80(2H，d，-OSO₂-phenyl-CH₃)

Yield: 3.0g

Purity: 99.7% (HPLC-RI)

The HPLC measurement conditions for purity measurement are as follows.

The device comprises the following steps: alias Corporation

Column: inertsil ODS-3 (column size: 4.6 mm. times.25 cm, particle size 5 μm), manufactured by GL Science Inc

A detector: RI (Ri)

Developing solvent: methanol solution/5 mM ammonium acetate 50/50

Flow rate: 0.6 mL/min

Column temperature: 40 deg.C

Sample concentration: 0.2mg/mL

Injection amount: 40 μ L

The purity value is the ratio of the peak area of compound 43 to the total peak area detected over a retention time of 10 to 40 minutes.

(example 5)

For calculation of the content of R in formula (4) at the time of removing the compound having trityl groups at both terminals from the reaction product containing the compound 12 obtained in example 2-2¹Derivatization of compound 44 was performed with the content of compound 12 in which a is 12 in the trityl-based compound.

In the formula (3), a is 12 and R¹Synthesis of Compound 43 which is trityl and L is tosyl

The reaction product containing compound 12 (compound 12: 5g, less than 6.3mmol) and dichloromethane (25mL) were added to a reaction vessel equipped with a thermometer, a nitrogen inlet tube, and a stirrer, and the product was dissolved under a nitrogen atmosphere, triethylamine (0.93mL, 6.6mmol), 4-dimethylaminopyridine (77mg, 0.63mmol), and TsCl (0.97g, 5.4mmol) were added, and the mixture was stirred at room temperature for 4 hours. After 4 hours, by¹H-NMR analysis confirmed the disappearance of TsCl, and 1M aqueous hydrochloric acid (25mL) was added and liquid phase separation was performed. The organic layer was washed once with 1M aqueous hydrochloric acid (25mL), twice with saturated aqueous sodium bicarbonate (25mL), and once with saturated aqueous sodium chloride (25 mL). Sodium sulfate was added to the organic layer for dehydration, followed by filtration. The filtrate was concentrated under reduced pressure to obtain a reaction product containing compound 43 as a pale yellow transparent liquid. Furthermore, by TLC analysis and¹the results of H-NMR measurement confirmed that the obtained reaction product contained the above-mentioned compounds 3, 6 and 13.

Compound 43

¹H-NMR(CDCl₃TMS, internal standard); δ (ppm):

2.45(3H，s，-OSO₂-phenyl-CH₃)，

3.23(2H，t，(C₆H₅)₃C-OCH₂CH₂-, including the compounds 3, 6 and13 derived from those of the group consisting of),

3.45-3.85(44H，m，-OCH₂CH₂-(OCH₂CH₂)₁₀-OCH₂CH₂-, including those derived from compounds 3, 6 and 13),

4.16(2H，t，-OCH₂CH₂-OSO₂-phenyl-CH₃)，

7.21-7.47(15H，m，(C₆H₅)₃C-OCH₂CH₂-, including those derived from compounds 3, 6 and 13),

7.35(2H，d，-OSO₂-phenyl-CH₃)，

7.80(2H，d，-OSO₂-phenyl-CH₃)

Yield: 6.1g

Next, the reaction product containing compound 43 (compound 43: 6.1g, less than 6.5mmol) and methanol (25mL) were charged into a reaction vessel equipped with a thermometer, nitrogen inlet pipe and stirrer. After dissolving the compound under a nitrogen atmosphere, p-toluenesulfonic acid monohydrate (0.60g, 3.2mmol) and hexane (20mL) were added. After stirring at room temperature for 30 minutes, the hexane layer was removed, hexane (13mL) was added again, and the mixture was stirred for 30 minutes. After six identical operations have been carried out, by¹As a result of H-NMR measurement, disappearance of compounds 43, 3, 6 and 13 was confirmed, and a saturated aqueous sodium hydrogencarbonate solution (10mL) was added. The mixed solution was washed twice with hexane (13mL) to remove trityl methyl ether. The methanol solution was concentrated under reduced pressure and dichloromethane (25mL) was added, followed by extraction. Magnesium sulfate was added to the organic layer to dehydrate, followed by filtration. The filtrate was concentrated under reduced pressure to obtain compound 44 as a pale yellow transparent liquid.

Compound 44

¹H-NMR(CDCl₃TMS, internal standard); δ (ppm):

2.45(3H，s，-OSO₂-phenyl-CH₃)，

2.73(1H，t，H-(OCH₂CH₂)₈-)，

3.45-3.85(46H，m，-(OCH₂CH₂)₁₁-OCH₂CH₂-)，

4.16(2H，t，-(OCH₂CH₂)₇-OCH₂CH₂-OSO₂-phenyl-CH₃)，

7.35(2H，d，-OSO₂-phenyl-CH₃)，

7.80(2H，d，-OSO₂-phenyl-CH₃)

Yield: 3.3g

Purity: 99.7% (HPLC-RI)

The HPLC measurement conditions for purity measurement are as follows.

The device comprises the following steps: alias Corporation

Column: inertsil ODS-3 (column size: 4.6 mm. times.25 cm, particle size 5 μm), manufactured by GL Science Inc

A detector: RI (Ri)

Developing solvent: methanol solution/5 mM ammonium acetate 55/45

Flow rate: 0.6 mL/min

Column temperature: 40 deg.C

Sample concentration: 0.2mg/mL

Injection amount: 40 μ L

The purity value is the ratio of the peak area of compound 44 to the total peak area detected over a retention time of 11 to 40 minutes.

(comparative example 1, production method for obtaining compound 10 via tosylation step) compound 10 in which a is 8 in formula (1) was synthesized by the production method described in patent document 4.

Comparative example 1-1

In the formula (3), a is 4 and R¹Synthesis of Compound 41 which is trityl and L is tosyl

Tetraethyleneglycol 1(200mL, 1.15mol) and toluene (50mL) were added to a reaction vessel equipped with a thermometer, a nitrogen inlet tube, and a stirrer, and the compounds were dissolved under a nitrogen atmosphere and then azeotropically dehydrated at 110 ℃ to 120 ℃. After azeotropic dehydration, the mixture was cooled, pyridine (18ml, 0.22mol) and trityl chloride (TrtCl, 40g, 0.14mol) were added, and the mixture was stirred at room temperature for 3 hours. After 3 hours, disappearance of TrtCl was confirmed by TLC, and ion-exchanged water (200mL) was added. Toluene (100mL) was added to the resulting mixed solution to conduct liquid phase separation, and the organic layer was washed once with a mixed solution of ion-exchanged water (80mL) and a saturated aqueous sodium chloride solution (20mL), once with a 1M aqueous hydrochloric acid solution (50mL), and four times with a saturated aqueous sodium chloride solution (50 mL). Sodium sulfate was added to the obtained organic layer, dried and filtered. Toluene (50mL) was added to the filtrate, and azeotropically dehydrated three times to obtain a reaction product containing compound 2 as a pale yellow transparent liquid. Further, it was confirmed by ESI-MS measurement that the obtained reaction product contained the above-mentioned compound 3.

Compound 2

¹H-NMR(CDCl₃TMS, internal standard); δ (ppm):

2.4(1H，t，-C-(OCH₂CH₂)₄-OH)，

3.23(2H，t，(C₆H₅)₃C-OCH₂CH₂-, including one derived from Compound 3)

3.45-3.85(14H，m，-OCH₂CH₂-(OCH₂CH₂)₃-OH, including the one derived from Compound 3),

7.21-7.47(15H，m，(C₆H₅)₃C-OCH₂CH₂-, including one derived from Compound 3)

MS (ESI +): compound 2454.5 [ M + NH4] +, compound 3696.9 [ M + NH4] +

Yield: 63.8g

Next, the reaction product containing Compound 2 (Compound 2: 62.8g, less than 0.14mol) and toluene (314mL) were charged to a reaction vessel equipped with a thermometer, nitrogen inlet tube, and stirrer. After dissolution under a nitrogen atmosphere, triethylamine (24mL, 0.17mol) was added. Methanesulfonyl chloride (12.2mL, 0.16mol) was added dropwise at 10 ℃, and the mixture was stirred at room temperature for 2 hours. After 2 hours, disappearance of compound 2 was confirmed by TLC analysis, and 1M aqueous hydrochloric acid (314mL) was added followed by liquid phase separation. The organic layer was washed once with 1M aqueous hydrochloric acid (314mL), twice with saturated aqueous sodium bicarbonate (314mL), and once with saturated aqueous sodium chloride (314 mL). Sodium sulfate was added to the organic layer for dehydration, followed by filtration. The filtrate was concentrated under reduced pressure to obtain a reaction product containing compound 4 as a pale yellow transparent liquid. Further, it was confirmed by ESI-MS measurement that the obtained reaction product contained the above-mentioned compound 3.

Compound 4

¹H-NMR(CDCl₃TMS, internal standard); δ (ppm):

2.98(3H，s，-O-CH₂CH₂-O-SO₂CH₃)，

3.23(2H，t，(C₆H₅)₃C-OCH₂CH₂-, including one derived from Compound 3)

3.45-3.85(12H，m，-OCH₂CH₂-(OCH₂CH₂)₂-OCH₂CH₂-, including one derived from Compound 3)

4.33(2H，t，-OCH₂CH₂-O-SO₂CH₃)，

7.21-7.47(15H，m，(C₆H₅)₃C-OCH₂CH₂-, including one derived from Compound 3)

MS (ESI +): compound 4532.4 [ M + NH4] +, compound 3696.8 [ M + NH4] +

Yield: 70.4g

Preparation of Compound 4 according to comparative example 1-1¹As a result of H-NMR measurement, it was confirmed that Compound 3 was contained in an amount of about 4.8 mol%.

The calculation formula of the content of compound 3 based on the δ 3.23 peak is represented by the following formula.

(((2-[δ4.32])/4H)/([δ4.32]/2H))×100(mol％)

Further, the reaction product 2 obtained in comparative example 1-1 contained compound 3 in an amount of about 6.9 wt%.

Comparative examples 1 and 2

In the formula (4), a is 8 and R¹Synthesis of Compound 5 which is a Tribenzyl group

Sodium hydride (7.8g) was placed in a reaction vessel equipped with a thermometer, a nitrogen inlet tube, and an agitator, and after nitrogen substitution, MeCN (141mL) was added and the mixture was cooled to 0 ℃. MeCN (70mL) was mixed with tetraethylene glycol 1(213g, 1.10mol) azeotropically dehydrated with toluene (107mL), and the mixed solution was added dropwise over 30 minutes. After the dropwise addition was completed, MeCN (70mL) was mixed with the reaction product containing compound 4 (compound 4: 70.4g, less than 0.14mol), and the mixed solution was added dropwise over 15 minutes. After completion of the dropwise addition, the reaction mixture was heated to 80 ℃ and stirred for 3 hours. After 3 hours, use¹H-NMR confirmed the disappearance of Compound 4, and the mixture was cooled until the temperature became 40 ℃ or less. The reaction mixture solution was concentrated under reduced pressure, and toluene (352mL) was added to the residue. The toluene solution was washed twice with saturated aqueous ammonium chloride (352mL) and three times with saturated aqueous sodium chloride (352 mL). Sodium sulfate was added to the organic layer for dehydration, followed by filtration. The filtrate was concentrated under reduced pressure to obtain a reaction product containing compound 5 as a pale yellow transparent liquid. Further, it was confirmed by ESI-MS measurement that the obtained reaction product contained the above-mentioned compounds 3 and 6.

Compound 5

¹H-NMR(CDCl₃TMS, internal standard); δ (ppm):

2.52(1H，t，-C-(OCH₂CH₂)₈-OH)，

3.23(2H，t，(C₆H₅)₃C-OCH₂CH₂-, including those derived from compounds 3 and 6)

3.45-3.85(30H，m，-OCH₂CH₂-(OCH₂CH₂)₇-OH, including those derived from compounds 3 and 6),

7.21-7.47(15H，m，(C₆H₅)₃C-OCH₂CH₂-, including those derived from compounds 3 and 6)

MS (ESI +): compound 5630.8 [ M + NH4] +, compound 3696.8 [ M + NH4] +, compound 61048.4 [ M + NH4] +

Yield: 82.4g

Comparative examples 1 to 3

In the formula (3), a is 8 and R¹Synthesis of Compound 41 which is trityl and L is tosyl

Tetrahydrofuran (200mL) was added to the reaction product containing Compound 5 (Compound 5: 77.8g, less than 0.14mol) in a reactor equipped with a thermometer, nitrogen inlet and stirrer, and the product was dissolved under a nitrogen atmosphere. The solution was then cooled to 0 ℃. Aqueous sodium hydroxide (20g, 0.5mol/60mL) was added and the mixture was stirred at 0 ℃ for 20 minutes. To the reaction mixture was added dropwise a tosyl chloride/tetrahydrofuran solution (30g, 0.16 mmol/60 mL) for 30 minutes, and the mixture was stirred at 0 ℃ for 1.5 hours. After 1.5 hours, after confirming the disappearance of compound 5 by TLC, the mixture was stirred at room temperature for 12.5 hours to disappear the excess tosyl chloride. After 12.5 hours, the disappearance of tosyl chloride was confirmed by TLC and ion-exchanged water (30mL) and ether (50mL) were added. The mixture was washed once with saturated aqueous sodium bicarbonate (50mL) and three times with saturated aqueous sodium chloride (50 mL). Sodium sulfate was added to the organic layer for dehydration, followed by dehydration. The filtrate was concentrated under reduced pressure to obtain a reaction product containing compound 41 as a pale yellow transparent liquid. Further, it was confirmed by ESI-MS measurement that the obtained reaction product contained the above-mentioned compounds 3 and 6.

Compound 41

MS (ESI +): compound 41785.2 [ M + NH4] +, compound 3697.0 [ M + NH4] +, compound 61048.7 [ M + NH4] +

Yield: 86.1g (yield: 88%)

Comparative examples 1 to 4

Synthesis of Compound 42 having a hydroxyl group at one end and a tosyl group at one end and having an ethylene glycol chain Length of 8 units

A reaction product containing Compound 41 (Compound 41: 85.6g, less than 0.144mol), methanol (20mL), palladium on carbon (Pd/C, 2g) was charged into a reaction vessel equipped with a thermometer, a nitrogen inlet, and a stirrer, hydrogen substitution was performed, and the mixture was stirred at room temperature for 18 hours. After 18 hours, disappearance of compound 42 was confirmed by TLC and Pd/C was removed by filtration through celite. Ion-exchanged water (130mL) was added to the filtrate, and the triphenylmethane formed was filtered. Since triphenylmethane remained in the filtrate, it was washed five times with hexane (100mL) to remove triphenylmethane. The methanol/ion-exchanged aqueous layer was concentrated under reduced pressure to give a crude product containing compound 41. Then, methylene chloride (120mL) was added to the crude product and the mixture was washed three times with ion-exchanged water (100mL) and twice with a saturated aqueous sodium chloride solution (100mL) under 20 ℃. Sodium sulfate was added to the organic layer, dried and filtered. The filtrate was concentrated under reduced pressure to obtain a purified product of compound 42 as a pale yellow transparent liquid.

Compound 42

Purification of the product

¹H-NMR(CDCl₃TMS, internal standard); δ (ppm):

2.45(3H，s，-OSO₂-phenyl-CH₃)，

2.73(1H，t，H-(OCH₂CH₂)₈-)，

3.45-3.85(30H，m，-(OCH₂CH₂)₇-OCH₂CH₂-)，

4.16(2H，t，-(OCH₂CH₂)₇-OCH₂CH₂-OSO₂-phenyl-CH₃)，

7.35(2H，d，-OSO₂-phenyl-CH₃)，

7.80(2H，d，-OSO₂-phenyl-CH₃)

MS (ESI +): compound 42542.4 [ M + NH4] +

Crude product

MS (ESI +): compound 42542.4 [ M + NH4] +, compound 1212.7 [ M + NH4] +, compound 9564.5 [ M + NH4] +

Yield: 52.0g (yield: 89%)

Purity: 96.7% (HPLC-RI)

The HPLC measurement conditions for purity measurement are as follows.

The device comprises the following steps: alias Corporation

Column: inertsil ODS-3 (column size: 4.6 mm. times.25 cm, particle size 5 μm), manufactured by GL Science Inc

A detector: RI (Ri)

Developing solvent: methanol solution/5 mM ammonium acetate 50/50, flow rate: 0.6 mL/min

Column temperature: 40 deg.C

Sample concentration: 0.2mg/mL

Injection amount: 40 μ L

The purity value is the ratio of the peak area of compound 42 to the total peak area detected over a retention time of 10 to 40 minutes.

Comparative examples 1 to 5

Synthesis of ethylene glycol chain Length Compound 45 having tosyl group at one end and t-butyl ester group at one end and having 8 units

Compound 45(1.0g, 1.91mmol), tert-butyl acrylate (1.8mL,19mmol) and toluene (25mL) were added to a reaction vessel equipped with a thermometer, nitrogen inlet tube and stirrer, and dissolved under a nitrogen atmosphere. After this time, the mixture was cooled to 0 ℃, powdered potassium hydroxide (53mg, 0.9mmol) was added, and the mixture was allowed to react at 0 ℃ for 1 hour. After the reaction, ion-exchanged water (20mL) was added and liquid phase separation was performed. The organic layer was washed once with saturated aqueous sodium chloride (20 mL). The organic layer was concentrated under reduced pressure to obtain compound 45 as a pale yellow transparent liquid.

Compound 45

¹H-NMR(CDCl₃TMS, internal standard); δ (ppm):

1.45(9H，s，(CH₃)₃C-O-CO-CH₂CH₂-)，

2.45(3H，s，-OSO₂-phenyl-CH₃)，

2.50(2H，t，(CH₃)₃C-O-CO-CH₂CH₂-)，

3.45-3.85(32H，m，-CH₂CH₂-(OCH₂CH₂)₇-OCH₂CH₂-)，

4.16(2H，t，-OCH₂CH₂-OSO₂-phenyl-CH₃)，

7.34(2H，d，-OSO₂-phenyl-CH₃)，

7.80(2H，d，-OSO₂-phenyl-CH₃)

MS (ESI +): compound 45670.6 [ M + NH4] +

Yield: 1.06g (yield: 85%)

Comparative examples 1 to 6

Synthesis of Compound 46 having a phthalimido group at one end and a t-butyl ester group at one end and having an ethylene glycol chain Length of 8 units

After compound 45(1.06g,1.60mmol) and acetonitrile (25mL) were added to a reaction vessel equipped with a thermometer, a nitrogen inlet tube, and a stirrer and the compounds were dissolved under a nitrogen atmosphere, phthalimide potassium salt (520mg, 2.80mmol) was added, and the mixture was stirred at 80 ℃ for 8 hours. Disappearance of compound 45 was confirmed by NMR, and the reaction solution was concentrated. After dichloromethane (7mL) was added and the solids were filtered, the filtrate was washed once with 0.1M aqueous sodium hydroxide (7mL) and once with saturated aqueous sodium chloride (10 mL). Sodium sulfate was added to the organic layer for dehydration, followed by filtration. The filtrate was concentrated under reduced pressure to obtain compound 46 as a pale yellow transparent liquid.

Compound 46

¹H-NMR(CDCl₃TMS, internal standard); δ (ppm):

1.45(9H，s，(CH₃)₃C-O-CO-CH₂CH₂-)，

2.50(2H，t，(CH₃)₃C-O-CO-CH₂CH₂-)，

3.45-3.85(32H，m，-CH₂CH₂-(OCH₂CH₂)₇-OCH₂CH₂-)，

3.90(2H，t，-OCH₂CH₂-a phthalimide compound),

7.71(2H, dd, -phthalimide),

7.80(2H, dd, phthalimide)

MS (ESI +): compound 46945.7 [ M + NH4] +

Yield: 965mg (yield: 95%)

Comparative examples 1 to 7

In the formula (8), a is 8 and R²Synthesis of Compound 9 which is t-butyl

Compound 46(510mg,0.80mmol), ethanol (10mL) and hydrazine monohydrate (334mg,6.70mmol) were added to a reaction vessel equipped with a thermometer, a nitrogen inlet tube and a stirrer, and reacted at 85 ℃ for 45 minutes. After cooling to room temperature, to dissolve the precipitated white solid, 12% potassium carbonate aqueous solution (5mL) was added and then the mixed solution was concentrated under reduced pressure. Subsequently, ion-exchanged water (3mL) was added, concentrated hydrochloric acid (0.6mL) was added to adjust the pH to 3, and the solid was filtered. The filtrate was washed three times with dichloromethane and the aqueous layer was saturated by addition of sodium chloride. The aqueous solution was extracted five times with dichloromethane, and the organic layer was dehydrated with sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to obtain compound 9 as a pale yellow transparent liquid.

Compound 9

¹H-NMR(CDCl₃TMS, internal standard); δ (ppm):

1.44(9H，s，-CH₂CH₂-COO-C(CH₃)₃)，

2.50(2H，t，-CH₂CH₂-COO-C(CH₃)₃)，

3.1(2H，t，H₂N-CH₂CH₂O-(CH₂CH₂O)₇-)，

3.45-3.85(32H，m，H₂N-CH₂CH₂O-(CH₂CH₂O)₇-CH₂CH₂-)

yield: 400mg (yield: 99%)

Comparative examples 1 to 8

Synthesis of Compound 10 in which a is 8 in formula (1)

After compound 9(400mg, 0.80mmol) and 1M hydrochloric acid (0.5mL) were added to a reaction vessel equipped with a thermometer, a nitrogen inlet tube and a stirrer, the compound was dissolved and then the mixture was stirred at 55 ℃ for 5 hours. After cooling to 15 ℃, 10M aqueous sodium hydroxide solution was added to adjust the pH to 5. To the solid obtained by twice azeotropic dehydration of water with toluene (5mL) was added dichloromethane (10mL), and the mixture was filtered. The filtrate was concentrated under reduced pressure to obtain compound 10 as a pale yellow transparent liquid.

Compound 10

¹H-NMR(CD₃OD, internal TMS); δ (ppm):

2.56(2H，t，-CH₂CH₂-COOH)，

3.17(2H，t，H₂N-CH₂CH₂O-)，

3.6-3.9(32H，m，-CH₂CH₂O-(CH₂CH₂O)₇-CH₂CH₂-)

yield: 320mg (yield: 83%)

Purity: 95.6% (HPLC-RI)

The HPLC measurement conditions for purity measurement are as follows.

The device comprises the following steps: alias Corporation

Column: inertsil ODS-3 (column size: 4.6 mm. times.25 cm, particle size 5 μm), manufactured by GL Science Inc

A detector: RI (Ri)

Developing solvent: methanol solution/5 mM ammonium acetate 15/85,

flow rate: 1.0 mL/min

Column temperature: 40 deg.C

Sample concentration: 1mg/mL

Injection amount: 50 μ L

(comparative example 2, production method for obtaining compound 18 via tosylation step) compound 18 in formula (1) in which a is 12 was synthesized by the production method described in patent document 4.

Comparative example 2-1

In the formula (3), a is 8 and R¹Synthesis of Compound 11 which is trityl and L is methylsulfonyl

The reaction product containing compound 5 (compound 5: 72.7g, less than 0.12mol) and toluene (350mL) were charged into a reaction vessel equipped with a thermometer, a nitrogen inlet tube, and a stirrer and the product was dissolved under a nitrogen atmosphere, followed by addition of triethylamine (20mL, 0.14 mol). Methanesulfonyl chloride (10mL, 0.13mol) was added dropwise at 10 ℃, and the mixture was stirred at room temperature for 2 hours. After 2 hours, disappearance of compound 5 was confirmed by TLC analysis, 1M aqueous hydrochloric acid solution (100mL) was added, and liquid phase separation was performed. The organic layer was washed once with 1M aqueous hydrochloric acid (100mL), twice with saturated aqueous sodium bicarbonate (100mL), and once with saturated aqueous sodium chloride (100 mL). Sodium sulfate was added to the organic layer for dehydration, followed by filtration. The filtrate was concentrated under reduced pressure to obtain a reaction product containing compound 11 as a pale yellow transparent liquid. Further, it was confirmed by ESI-MS measurement that the obtained reaction product contained the above-mentioned compounds 3 and 6.

Compound 11

¹H-NMR(CDCl₃TMS, internal standard); δ (ppm):

3.07(3H，s，-OCH₂CH₂-O-SO₂CH₃)，

3.23(2H，t，(C₆H₅)₃C-OCH₂CH₂-, including those derived from compounds 3 and 6)

3.45-3.85(28H，m，-OCH₂CH₂-(OCH₂CH₂)₆-OCH₂CH₂-, including those derived from compounds 3 and 6),

4.37(2H，t，-OCH₂CH₂-O-SO₂CH₃)，

7.21-7.47(15H，m，(C₆H₅)₃C-OCH₂CH₂-, including those derived from compounds 3 and 6)

MS (ESI +): compound 11708.3 [ M + NH4] +, compound 3696.4 [ M + NH4] +, compound 61048.5 [ M + NH4] +

Yield: 80.1g

Preparation of Compound 11 according to comparative example 2-1¹As a result of H-NMR measurement, it was confirmed that the contents of the compounds 3 and 6 were about 9.5 mol% (chemical formula)Compound 3: 4.8 mol%, compound 6: 4.7 mol%, rough estimate).

The calculated formula for the content of compounds 3 and 6 based on the delta 3.23 peak is represented by the following formula.

(((2-[δ4.32])/4H)/([δ4.32]/2H))×100(mol％)

As for the content of compound 3, the value calculated in comparative example 1-1 was applied.

Further, the reaction product 5 used in comparative example 2-1 contained the compounds 3 and 6 in an amount of about 11.7 wt%.

Comparative examples 2 and 2

In the formula (4), a is 12 and R¹Synthesis of Compound 12 as a Tribenzyl group

Sodium hydride (6.6g) was placed in a reaction vessel equipped with a thermometer, a nitrogen inlet tube, and an agitator, and after nitrogen substitution, MeCN (200mL) was added and the mixture was cooled to 0 ℃. MeCN (50mL) was mixed with tetraethylene glycol 1(180g, 0.93mol) azeotropically dehydrated with toluene (50mL), and the mixed solution was added dropwise over 30 minutes. After completion of the dropwise addition, MeCN (50mL) was mixed with the reaction product containing compound 11 (compound 11: 80.1g, less than 0.12mol), and the mixed solution was added dropwise over 15 minutes. After completion of the dropwise addition, the reaction mixture was heated to 80 ℃ and stirred for 3 hours. After 3 hours, use¹H-NMR confirmed the disappearance of compound 11, and the mixture was cooled until the temperature became 40 ℃ or less. The reaction mixture solution was concentrated under reduced pressure, and toluene (200mL) was added to the residue. The toluene solution was washed twice with saturated aqueous ammonium chloride solution (100mL) and three times with saturated aqueous sodium chloride solution (100 mL). Sodium sulfate was added to the organic layer for dehydration, followed by filtration. The filtrate was concentrated under reduced pressure to obtain a reaction product containing compound 12 as a pale yellow transparent liquid. In addition, by ESI-MS and¹H-NMR measurement confirmed that the obtained reaction product contained the above-mentioned compounds 3, 6 and 13.

Compound 12

¹H-NMR(CDCl₃TMS, internal standard); δ (ppm):

2.56(1H，t，-C-(OCH₂CH₂)₁₂-OH)，

3.23(2H，t，(C₆H₅)₃C-OCH₂CH₂-, including those derived from compounds 3, 6 and 13)

3.45-3.85(46H，m，-OCH₂CH₂-(OCH₂CH₂)₁₁-OH, including those derived from compounds 3, 6 and 13),

7.21-7.47(15H，m，(C₆H₅)₃C-OCH₂CH₂-, including those derived from compounds 3, 6 and 13)

MS (ESI +): compound 12806.4 [ M + NH4] +, compound 3696.8 [ M + NH4] +, compound 61048.1 [ M + NH4] +, compound 131400.9 [ M + NH4] +

Yield: 85.4g

Comparative examples 2 to 3

In the formula (3), a is 12 and R¹Synthesis of Compound 43 which is trityl and L is tosyl

The reaction product containing compound 12 (compound 12: 57.3g, less than 72.7mmol) and dichloromethane (280mL) were charged to a reaction vessel equipped with a thermometer, nitrogen inlet tube and stirrer, the product was dissolved under a nitrogen atmosphere, triethylamine (10mL, 73mol), 4-dimethylaminopyridine (888mg, 7.27mmol) and TsCl (12.5g, 65.5mmol) were added, and the mixture was stirred at room temperature for 4.5 hours. After 4.5 hours, by¹H-NMR analysis confirmed the disappearance of TsCl, and 1M aqueous hydrochloric acid (150mL) was added and liquid phase separation was performed. The organic layer was washed once with 1M aqueous hydrochloric acid (150mL), twice with saturated aqueous sodium bicarbonate (150mL), and once with saturated aqueous sodium chloride (150 mL). Sodium sulfate was added to the organic layer for dehydration, followed by filtration. Concentrating the filtrate under reduced pressure to obtain a concentrate comprising the compoundThe reaction product of substance 43 is a pale yellow transparent liquid. Further, it was confirmed by ESI-MS measurement that the obtained reaction products contained the above-mentioned compounds 3, 6 and 13.

Compound 43

MS (ESI +): compound 43960.3 [ M + NH4] +, compound 3696.3 [ M + NH4] +, compound 61048.2 [ M + NH4] +, compound 131400.8 [ M + NH4] +

Yield: 69.1g (yield: 101%)

Comparative examples 2 to 4

Synthesis of Compound 44 having a hydroxyl group at one end and a tosyl group at one end and having a glycol chain length of 12 units

The reaction product containing compound 43 (compound 43: 69.1g, less than 73.3mmol) and methanol (550L) were charged to a reaction vessel equipped with a thermometer, nitrogen inlet pipe and stirrer. After dissolving the compound under a nitrogen atmosphere, p-toluenesulfonic acid monohydrate (6.97g, 36.7mmol) and hexane (200mL) were added. After stirring at room temperature for 30 minutes, the hexane layer was removed, hexane (200mL) was added again, and the mixture was stirred for 30 minutes. After six identical operations have been carried out, by¹As a result of H-NMR measurement, disappearance of compounds 43, 3, 6 and 13 was confirmed, and a saturated aqueous sodium hydrogencarbonate solution (200mL) was added. The mixed solution was washed twice with hexane (200mL) to remove trityl methyl ether. The methanol solution was concentrated under reduced pressure, and methylene chloride (200mL) was added and the mixture was washed three times with ion-exchanged water (200mL) and once with a saturated aqueous sodium chloride solution (200mL) under conditions of 20 ℃ or lower. Magnesium sulfate was added to the organic layer to dehydrate, followed by filtration. The filtrate was concentrated under reduced pressure to obtain compound 44 as a pale yellow transparent liquid.

Compound 44

Purification of the product

¹H-NMR(CDCl₃TMS, internal standard); δ (ppm):

2.45(3H，s，-OSO₂-phenyl-CH₃)，

2.73(1H，t，H-(OCH₂CH₂)₈-)，

3.45-3.85(46H，m，-(OCH₂CH₂)₁₁-OCH₂CH₂-)，

4.16(2H，t，-(OCH₂CH₂)₇-OCH₂CH₂-OSO₂-phenyl-CH₃)，

7.35(2H，d，-OSO₂-phenyl-CH₃)，

7.80(2H，d，-OSO₂-phenyl-CH₃)

MS (ESI +): compound 44718.3 [ M + NH4] +

Crude product

MS (ESI +): compound 44718.3 [ M + NH4] +, compound 1212.3 [ M + NH4] +, compound 9564.5 [ M + NH4] +, compound 16916.4 [ M + NH4] +

Yield: 30.3g (yield: 59%)

Purity: 94.9% (HPLC-RI)

The HPLC measurement conditions for purity measurement are as follows.

The device comprises the following steps: alias Corporation

Column: inertsil ODS-3 (column size: 4.6 mm. times.25 cm, particle size 5 μm), manufactured by GL Science Inc

A detector: RI (Ri)

Developing solvent: methanol solution/5 mM ammonium acetate 55/45

Flow rate: 0.6 mL/min

Column temperature: 40 deg.C

Sample concentration: 0.2mg/mL

Injection amount: 40 μ L

The purity value is the ratio of the peak area of compound 44 to the total peak area detected over a retention time of 11 to 40 minutes.

Comparative examples 2 to 5

Synthesis of ethylene glycol chain Length Compound 47 having tosyl group at one end and t-butyl ester group at one end and having 12 units

Compound 44(4.96g, 7.08mmol), tert-butyl acrylate (3.09mL,21.2mmol) and toluene (100mL) were added to a reaction vessel equipped with a thermometer, a nitrogen inlet tube and a stirrer, and dissolved under a nitrogen atmosphere. Thereafter, the mixture was cooled to 0 ℃, followed by addition of powdered potassium hydroxide (199mg, 3.54mmol), and the mixture was allowed to react at 0 ℃ for 1 hour. After the reaction, a saturated aqueous ammonium chloride solution (50mL) was added and liquid phase separation was performed. The organic layer was washed once with saturated aqueous sodium chloride (50 mL). Sodium sulfate was added to the organic layer for dehydration, followed by filtration. The filtrate was concentrated under reduced pressure to obtain compound 47 as a pale yellow transparent liquid.

Compound 47

¹H-NMR(CDCl₃TMS, internal standard); δ (ppm):

1.45(9H，s，(CH₃)₃C-O-CO-CH₂CH₂-)，

2.45(3H，s，-OSO₂-phenyl-CH₃)，

2.50(2H，t，(CH₃)₃C-O-CO-CH₂CH₂-)，

3.45-3.85(48H，m，-CH₂CH₂-(OCH₂CH₂)₁₁-OCH₂CH₂-)，

4.16(2H，t，-OCH₂CH₂-OSO₂-phenyl-CH₃)，

7.34(2H，d，-OSO₂-phenyl-CH₃)，

7.80(2H，d，-OSO₂-phenyl-CH₃)

MS (ESI +): compound 47847.0 [ M + NH4] +

Yield: 5.43g (yield: 93%)

Comparative examples 2 to 6

Synthesis of ethylene glycol chain Length Compound 48 having a phthalimido group at one end and a t-butyl ester group at one end and having 12 units

After compound 47(5.43g,6.55mmol) and acetonitrile (45mL) were added to a reaction vessel equipped with a thermometer, a nitrogen inlet tube, and a stirrer and the compounds were dissolved under a nitrogen atmosphere, phthalimide potassium salt (1.58g, 8.52mmol) was added, and the mixture was stirred at 80 ℃ for 18 hours. Disappearance of compound 48 was confirmed by NMR, and the reaction solution was concentrated. After dichloromethane (50mL) was added to the residue and the solid was filtered, the filtrate was washed once with 0.1M aqueous sodium hydroxide solution (50mL) and once with saturated aqueous sodium chloride solution (50 mL). Sodium sulfate was added to the organic layer for dehydration, followed by filtration. The filtrate was concentrated under reduced pressure to obtain compound 48 as a pale yellow transparent liquid.

Compound 48

¹H-NMR(CDCl₃TMS, internal standard); δ (ppm):

1.45(9H，s，(CH₃)₃C-O-CO-CH₂CH₂-)，

2.50(2H，t，(CH₃)₃C-O-CO-CH₂CH₂-)，

3.45-3.85(48H，m，-CH₂CH₂-(OCH₂CH₂)₁₁-OCH₂CH₂-)，

3.90(2H，t，-OCH₂CH₂-a phthalimide compound),

7.71(2H, dd, -phthalimide),

7.80(2H, dd, phthalimide)

MS (ESI +): compound 48828.1 [ M + NH4] +

Yield: 4.26g (yield: 81%)

Comparative examples 2 to 7

In the formula (8), a is 12 and R²Synthesis of Compound 17 which is t-butyl

Compound 48(4.26g,5.30mmol), ethanol (60mL), and hydrazine monohydrate (3.86mL,79.5mmol) were added to a reaction vessel equipped with a thermometer, a nitrogen inlet tube, and a stirrer, and the reaction was carried out at 85 ℃ for 1 hour. After cooling to room temperature, to dissolve the precipitated white solid, 12% potassium carbonate aqueous solution (5mL) was added and then the mixed solution was concentrated under reduced pressure. Ion-exchanged water (20mL) was added to the residue, pH was adjusted to 3 by adding concentrated hydrochloric acid, and the solid was filtered. Sodium chloride was added to the filtrate and extracted twice with dichloromethane (20 mL). The organic layer was dehydrated with sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to obtain compound 17 as a pale yellow transparent liquid.

Compound 17

¹H-NMR(CDCl₃TMS, internal standard); δ (ppm):

1.44(9H，s，-CH₂CH₂-COO-C(CH₃)₃)，

2.50(2H，t，-CH₂CH₂-COO-C(CH₃)₃)，

3.1(2H，t，H₂N-CH₂CH₂O-(CH₂CH₂O)₁₁-)，

3.45-3.85(48H，m，H₂N-CH₂CH₂O-(CH₂CH₂O)₁₁-CH₂CH₂-)

yield: 3.13g (yield: 88%)

Comparative examples 2 to 8

Synthesis of Compound 18 in which a is 12 in formula (1)

After compound 17(3.13g, 4.65mmol) and 1M hydrochloric acid (3mL) were added to a reaction vessel equipped with a thermometer, a nitrogen inlet tube and a stirrer, the compound was dissolved and then the mixture was stirred at 55 ℃ for 2 hours. After cooling to 15 ℃, the mixture was diluted with ion-exchanged water (5mL) and washed three times with dichloromethane (10 mL). To the aqueous layer was added 2M aqueous sodium hydroxide solution to adjust the pH to 9. The aqueous solution was washed three times with dichloromethane (10mL) and the aqueous layer was saturated by the addition of sodium chloride. The aqueous solution was extracted three times with chloroform (10mL), and the organic layer was dehydrated with sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to obtain compound 18 as a pale yellow transparent liquid.

Compound 18

¹H-NMR(CD₃OD, internal TMS); δ (ppm):

2.56(2H，t，-CH₂CH₂-COOH)，

3.17(2H，t，H₂N-CH₂CH₂O-)，

3.6-3.9(48H，m，-CH₂CH₂O-(CH₂CH₂O)₁₁-CH₂CH₂-)

yield: 2.32g (yield: 76%)

Purity: 98.0% (HPLC-RI)

The HPLC measurement conditions for purity measurement are as follows.

The device comprises the following steps: allence manufactured by Waters Corporation,

column: inertsil ODS-3 (column size: 4.6 mm. times.25 cm, particle size 5 μm), manufactured by GL Science Inc,

a detector: the amount of the RI,

developing solvent: methanol solution/5 mM ammonium acetate 27.5/72.5,

flow rate: 1.0 mL/min of the reaction solution,

column temperature: 40 deg.C

Sample concentration: 2mg/mL of the solution is added,

injection amount: 50 μ L

Comparative example 3

Calculation formula of content of Compounds 3, 6 and 13 having trityl groups at both ends in reaction product containing Compound 12 obtained in comparative example 2-2

In the formula (3), a is 12 and R¹Is trityl and L isSynthesis of methanesulfonyl Compound 19

The reaction product containing compound 12 (compound 12: 25g, less than 317mmol) and toluene (125mL) were charged to a reaction vessel equipped with a thermometer, nitrogen inlet tube and stirrer and the product dissolved under nitrogen atmosphere, followed by addition of triethylamine (5.3mL, 38 mmol). Methanesulfonyl chloride (2.7mL, 35mmol) was added dropwise at 10 ℃, and the mixture was stirred at room temperature for 2 hours. After 2 hours, disappearance of compound 12 was confirmed by TLC analysis, 1M aqueous hydrochloric acid solution (50mL) was added, and liquid phase separation was performed. The organic layer was washed once with 1M aqueous hydrochloric acid (50mL), twice with saturated aqueous sodium bicarbonate (50mL), and once with saturated aqueous sodium chloride (50 mL). Sodium sulfate was added to the organic layer for dehydration, followed by filtration. The filtrate was concentrated under reduced pressure to obtain a reaction product containing compound 19 as a pale yellow transparent liquid. Further, it was confirmed by ESI-MS measurement that the obtained reaction products contained the above-mentioned compounds 3, 6 and 13.

Compound 19

¹H-NMR(CDCl₃TMS, internal standard); δ (ppm):

3.07(3H，s，-OCH₂CH₂-O-SO₂CH₃)，

3.23(2H，t，(C₆H₅)₃C-OCH₂CH₂-, including those derived from compounds 3, 6 and 13)

3.45-3.85(44H，m，-OCH₂CH₂-(OCH₂CH₂)₁₀-OCH₂CH₂-, including those derived from compounds 3, 6 and 13),

4.37(2H，t，-OCH₂CH₂-O-SO₂CH₃)，

7.21-7.47(15H，m，(C₆H₅)₃C-OCH₂CH₂-, including those derived from compounds 3, 6 and 13)

MS (ESI +): compound 19884.9 [ M + NH4] +, compound 3696.8 [ M + NH4] +, compound 61049.4 [ M + NH4] +, compound 131401.3 [ M + NH4] +

Yield: 26g

According to the 1HNMR measurement of compound 19 of comparative example 3, the contents of compounds 3, 6 and 13 were confirmed to be about 9.8 mol% (compound 3: 4.8 mol%, compound 6: 4.7 mol%, compound 13: 0.3 mol%, rough estimate).

The calculation formula of the contents of compounds 3, 6 and 13 based on the δ 3.23 peak is represented by the following formula.

(((2-[δ4.32])/4H)/([δ4.32]/2H))×100(mol％)

As to the contents of the compounds 3 and 6, the values calculated in comparative examples 1-1 and 2-1 were applied.

Further, the reaction product 12 used in comparative example 3 contained compounds 3, 6 and 13 in an amount of about 9.7 wt%.

Comparison of the total yield of Compound 10 Using Compound 5 of heterotypic monodisperse polyethylene glycol having hydroxyl group and trityl group at both terminals, respectively, as the starting Material until heterotypic monodisperse polyethylene glycol having amino group and carboxyl group at both terminals, respectively, is obtained

Table 1 shows the total yield when formula (1) is produced from formula (4) in the case where a is 8 or 12.

[ Table 1]

Value of a	Examples	Comparative example
			8	64％	58％
12	72％	34％

The total yield was calculated according to the following formula.

Case a is 8:

(value obtained by multiplying the yields in the respective steps from Compound 5 to Compound 10 obtained) × (pure content of Compound 10 with a being 8)/(pure content of Compound 5 with a being 8)

Case a is 12:

(value obtained by multiplying the yields in the respective steps from Compound 12 to Compound 18 obtained) × (pure content of Compound 18 wherein a is 12)/(pure content of Compound 12 wherein a is 12)

In the formula for determining the total yield, the pure content of the compound 5 in which a is 8 is a value obtained by multiplying a value obtained by subtracting the contents of the compounds 3 and 6 having trityl groups at both terminals from the amount of the reaction product containing the compound 5 obtained in example 1-2 by the purity of the compound 42 obtained in example 4. Further, it is a value obtained by multiplying a value obtained by subtracting the contents of the compounds 3 and 6 having trityl groups at both terminals from the amount of the reaction product containing the compound 5 obtained in comparative example 1-2 by the purity of the compound 42 obtained in comparative example 1-4.

In the formula for determining the total yield, the pure content of the compound 12 in which a is 12 is a value obtained by multiplying a value obtained by subtracting the content of the compounds 3, 6 and 13 having trityl groups at both terminals from the amount of the reaction product containing the compound 12 obtained in example 2-2 by the purity of the compound 44 obtained in example 5. Further, it is a value obtained by multiplying a value obtained by subtracting the contents of the compounds 3, 6 and 13 having trityl groups at both terminals from the amount of the reaction product containing the compound 5 obtained in comparative example 2-2 by the purity of the compound 44 obtained in comparative example 2-4.

In the formula for determining the total yield, the pure contents of compound 10 with a being 8 and compound 18 with a being 12 correspond to the purity determined by HPLC, respectively.

In the conventional preparation method, a hydroxyl group is temporarily converted into a tosyl group to purify an impurity containing trityl groups at both terminals, which are different in chain length and have a specific molecular weight, formed as a byproduct in the chain length extension step. On the other hand, by removing impurities without this step, the number of steps is smaller than before, and the presence of residual unreacted raw materials and the formation of reaction byproducts, which may cause a reduction in yield, can be suppressed, so that the yield can be improved.

Industrial applicability

According to the present invention, a novel process for producing a highly pure hetero-monodisperse polyethylene glycol having an amino group and a carboxyl group at each of both terminals thereof can be provided.

While the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes or modifications can be made therein without departing from the spirit and scope thereof.

The present application is based on japanese patent application (japanese patent application No. 2019-065528) filed on 29/3/2019, which is incorporated herein by reference in its entirety.