WO2025206013A1 - High-purity 2-aminoethyl methacrylate hydrochloride, copolymer including said hydrochloride as structural unit, and method for producing 2-aminoethyl methacrylate hydrochloride - Google Patents

Abstract

This method for producing 2-aminoethyl methacrylate hydrochloride comprises a reaction step for reacting aminoethanol hydrochloride and methacrylic acid chloride and a drying step for drying 2-aminoethyl methacrylate hydrochloride obtained in the reaction step at not higher than 45°C.

Description

High-purity 2-aminoethyl methacrylate hydrochloride, copolymer containing said hydrochloride as a constituent unit, and method for producing 2-aminoethyl methacrylate hydrochloride

The present invention relates to high-purity 2-aminoethyl methacrylate hydrochloride, a copolymer containing this hydrochloride as a structural unit, and a method for producing 2-aminoethyl methacrylate hydrochloride.

The compound represented by the following formula (1) is useful as a raw material for polymers for contact lens surface treatment and polymers for contact lens treatment solutions, as described in Patent Documents 1 and 2, for example.

In formula (1), a:b=50:50 to 98:2, ^{R 1} is a hydrogen atom or a methyl group, and X ¹ is O or NH.

The compound represented by formula (1) above can be obtained, for example, by polymerizing a compound represented by formula (2) below with 2-aminoethyl methacrylate hydrochloride (AEMA) represented by formula (3) below using a radical polymerization method or the like.

In formula (2), ^R2 is a hydrogen atom or a methyl group, and ^X2 is O or NH.

AEMA can be produced, for example, by the method described in Non-Patent Document 1. Specifically, it can be obtained by reacting ethanolamine hydrochloride with methacrylic acid chloride, purifying the mixture with diethyl ether, filtering, and drying.

Journal of Polymer Science, Part A: Polymer Chemistry (2013), 51 (21), 4522-4529.

International Publication No. 2019/111838 International Publication No. 2021/070862

In the conventional manufacturing method (the manufacturing method described in Non-Patent Document 1), there was a step for which detailed conditions for the production of AEMA were not described. When AEMA was produced using this manufacturing method, a homopolymer of AEMA represented by the following formula (4) was produced as a by-product and was sometimes contained in the AEMA (AEMA composition). Removal of the homopolymer of AEMA from the AEMA was difficult, and there was a need to reduce the amount of by-product. In other words, the manufacturing method described in Non-Patent Document 1 made it difficult to consistently produce highly pure AEMA.

In formula (4), n is 20 to 30,000.

If AEMA homopolymer is contained in AEMA (in an AEMA composition), it is difficult to completely remove it by purification. When AEMA contaminated with AEMA homopolymer is used as a constituent unit of a compound (copolymer) represented by formula (1), the AEMA homopolymer is contaminated into the compound represented by formula (1). The contamination of the AEMA homopolymer into the compound represented by formula (1) may reduce the safety of the compound represented by formula (1). When the compound represented by formula (1) contaminated with AEMA homopolymer is used for contact lenses, the AEMA homopolymer may inhibit the reaction of the compound represented by formula (1) or the AEMA homopolymer may react, preventing the desired effect from being obtained.
In view of the above circumstances, an object of the present invention is to provide high-purity 2-aminoethyl methacrylate hydrochloride, a copolymer containing or essentially consisting of said hydrochloride as a constituent unit, and a method for producing 2-aminoethyl methacrylate hydrochloride.

The inventors conducted extensive research to achieve the above objective and discovered that high-purity AEMA can be obtained by drying AEMA under specific drying conditions during the AEMA production process.

That is, the present invention is as follows.
1. 2-aminoethyl methacrylate hydrochloride, having a content of homopolymer of 2-aminoethyl methacrylate hydrochloride of less than 2 mol %.
2. 2-aminoethyl methacrylate hydrochloride substantially free of homopolymers of 2-aminoethyl methacrylate hydrochloride.
3. The 2-aminoethyl methacrylate hydrochloride according to item 1 or 2 above, wherein when a solution prepared by dissolving the 2-aminoethyl methacrylate hydrochloride in ethanol at a concentration of 10 wt % is measured for transmittance at a wavelength of 555 nm using a spectrophotometer, the transmittance is 50% or more.
4. A method for producing 2-aminoethyl methacrylate hydrochloride according to item 1 above, comprising the following steps:
1) a reaction step of reacting 2-aminoethanol hydrochloride with methacrylic acid chloride; and
2) A drying step of drying the 2-aminoethyl methacrylate hydrochloride obtained in the reaction step 1) under reduced pressure at 45°C or less.
5. The manufacturing method according to item 4 above, wherein the reduced pressure is 100 hPa or less.
6. The production method according to item 4 or 5 above, further comprising a crystallization step between step 1) and step 2).
7. The method according to item 4 or 5 above, wherein the 2-aminoethyl methacrylate hydrochloride contains less than 2 mol % of a homopolymer of 2-aminoethyl methacrylate hydrochloride.
8. The production method according to item 4 or 5 above, wherein the 2-aminoethyl methacrylate hydrochloride does not substantially contain a homopolymer of 2-aminoethyl methacrylate hydrochloride.
9. A copolymer comprising 2-aminoethyl methacrylate hydrochloride as a structural unit according to item 1 or 2 above and a compound represented by formula (2) as a structural unit.

(In formula (2), ^R2 is a hydrogen atom or a methyl group, and ^X2 is O or NH.)
10. A copolymer represented by formula (1):
The copolymer contains the structural units of 2-aminoethyl methacrylate hydrochloride described in the above item 1 or 2 and the structural unit of the compound represented by formula (2).

(In formula (1), a:b=50:50 to 98:2, R ¹ is a hydrogen atom or a methyl group, and X ¹ is O or NH.)

(In formula (2), ^R2 is a hydrogen atom or a methyl group, and ^X2 is O or NH.)

The present invention can provide high-purity 2-aminoethyl methacrylate hydrochloride and a method for producing said hydrochloride.

The method for producing 2-aminoethyl methacrylate hydrochloride of the present invention will be described in more detail below. High-purity 2-aminoethyl methacrylate hydrochloride obtained by the method for producing 2-aminoethyl methacrylate hydrochloride of the present invention is referred to as 2-aminoethyl methacrylate hydrochloride of the present invention. The method for producing 2-aminoethyl methacrylate hydrochloride of the present invention includes at least the following 1) reaction step and 2) drying step, but may also include other steps.
The purity of AEMA can be measured by ¹ H NMR and transmittance. ¹ H NMR can measure the AEMA homopolymer contained in the AEMA, while transmittance can evaluate even smaller amounts of impurities.
In this specification, when preferred numerical ranges (e.g., ranges of concentration or weight- or number-average molecular weight) are described in stages, the lower limit and upper limit of each numerical range can be freely combined. For example, in the description "preferably 10 or more, more preferably 20 or more, and preferably 100 or less, more preferably 90 or less," the "preferable lower limit: 10" and the "more preferable upper limit: 90" can be combined to obtain "10 or more and 90 or less." Furthermore, in the description "preferably 10 to 100, more preferably 20 to 90," the range can be similarly set to "10 to 90."

(Reaction step)
AEMA can be obtained by the reaction of 2-aminoethanol hydrochloride with methacrylic acid chloride.
In the reaction of 2-aminoethanol hydrochloride with methacrylic acid chloride, the amount of methacrylic acid chloride used is preferably 1.0 mol or more per mol of 2-aminoethanol hydrochloride, more preferably 1.1 to 5.0 mol, and even more preferably 1.2 to 2.0 mol. An amount of less than 1.0 mol is not preferred because aminoethanol hydrochloride remains, while an amount of more than 5.0 mol is not economical and may make it difficult to remove unreacted methacrylic acid chloride.
It is preferable to carry out the above reaction by melting 2-aminoethanol hydrochloride and not using a solvent. Therefore, the reaction temperature is preferably 80°C or higher, more preferably 85°C or higher, and even more preferably 90°C or higher. If the temperature is lower than 80°C, the reaction may not proceed sufficiently.
The reaction is preferably carried out without using a solvent, but a solvent can also be used. When a solvent is used, the solvent is preferably one that does not react with methacrylic acid chloride, such as dimethyl sulfoxide, N,N-dimethylformamide, or acetonitrile.
Since hydrogen chloride is generated as the reaction proceeds, it is preferable to carry out the reaction while removing the hydrogen chloride by blowing in an inert gas. Examples of the inert gas include nitrogen and argon, with nitrogen being preferred.
The reaction is preferably carried out by adding methacrylic acid chloride dropwise, which makes it easier to control the heat of reaction and the amount of hydrogen chloride generated by the reaction.
In the above reaction, a polymerization inhibitor may be added as needed. The polymerization inhibitor is not particularly limited as long as it is one that has been conventionally used as a polymerization inhibitor for radical polymerization. Examples include hydroquinone, hydroquinone monomethyl ether, 2-t-butylhydroquinone, 4-methoxyphenol, 2,6-di-t-butyl-4-methylphenol (BHT), phenothiazine, and the like. These polymerization inhibitors may be used alone or in combination of two or more. The amount of the polymerization inhibitor is not particularly limited, but is preferably 100 ppm to 50,000 ppm, more preferably 1,000 ppm to 40,000 ppm, and even more preferably 5,000 ppm to 30,000 ppm, relative to the mass of methacrylic acid chloride.
The reaction time, including the time for dropping methacrylic acid chloride, is preferably 12 hours or less, more preferably 8 hours or less, even more preferably 6 hours or less, particularly preferably 3 hours or less, and most preferably 2 hours or less. If the reaction time is too long, there is a risk of increasing the amount of by-products.
If the reaction mixture is cooled after completion of the reaction, it will solidify and become difficult to handle, so it is preferable to dilute it with a solvent. Preferred solvents include acetate esters such as methyl acetate, ethyl acetate, and butyl acetate, ketones such as acetone, methyl ethyl ketone, and methyl isobutyl ketone, and alcohols such as methanol, ethanol, 1-propanol, 2-propanol, and butanol, and two or more solvents may be used in combination.

(Crystallization process)
The method for producing 2-aminoethyl methacrylate hydrochloride of the present invention preferably includes a crystallization step between the above-mentioned 1) reaction step and the below-described 2) drying step.
The crystallization step is not particularly limited and refers to a procedure for crystallizing, separating, and purifying 2-aminoethyl methacrylate hydrochloride containing impurities (such as AEMA homopolymers) and unreacted raw materials obtained in the above-mentioned 1) reaction step by cooling or heating. In addition, this step may include procedures such as extraction, purification, and filtration that are known per se.
Examples of good solvents used in crystallization include alcohols such as methanol, ethanol, 1-propanol, and 2-propanol, and two or more solvents may be used in combination. Examples of poor solvents include acetates such as methyl acetate, ethyl acetate, and butyl acetate, ketones such as acetone, methyl ethyl ketone, and methyl isobutyl ketone, hydrocarbons such as hexane and heptane, and aromatic compounds such as toluene, and two or more solvents may be used in combination.
To increase the degree of purification, the above crystallization may be carried out multiple times, or the crystals may be washed with the above solvent.
After crystallization, the precipitated crystals can be recovered by filtration, which may be performed by hot filtration, gravity filtration, vacuum (suction) filtration, pressure filtration, etc., with pressure filtration being preferred.
The crystallization step is not particularly limited, but examples thereof include the steps of crystallization → pressure filtration → obtaining first crude crystals → redissolution → hot filtration → crystallization → pressure filtration → obtaining second crude crystals → washing with a mixed solvent → obtaining third crude crystals.

(drying process)
The crystals recovered in the above 1) reaction step or the above crystallization step contain the organic solvent used in the crystallization, and are therefore ultimately dried at a temperature of 45° C. or less to obtain powdered AEMA. The drying temperature is preferably 20° C. or higher and 45° C. or lower, and more preferably 30° C. or higher and 40° C. or lower. If the temperature is lower than 20° C., the solvent may not be sufficiently removed, and if the temperature exceeds 45° C., a homopolymer of AEMA may be produced.
Drying is preferably carried out under reduced pressure. By reducing the pressure, the drying time can be shortened and the residual solvent can be reduced. There is no particular limitation on the pressure, but it is preferably 100 hPa or less, more preferably 50 hPa or less, even more preferably 20 hPa or less, and particularly preferably 10 hPa or less.
The drying time is not particularly limited, but is preferably 1 hour or more and 24 hours or less. If it is less than 1 hour, the solvent may not be sufficiently removed, and if it exceeds 24 hours, it is not efficient. For example, it is 1 hour to 10 hours, or 2 hours to 8 hours.
The loss on drying of AEMA is preferably 2.0 wt% or less, more preferably 1.0 wt%, even more preferably 0.5 wt%, particularly preferably 0.3 wt% or less, and most particularly preferably 0.2 wt% or less. If more than 2.0 wt% of volatile components (solvent and water) remain, the stability and handling properties may be deteriorated.

(2-aminoethyl methacrylate hydrochloride of the present invention)
The method for producing 2-aminoethyl methacrylate hydrochloride of the present invention can provide 2-aminoethyl methacrylate hydrochloride (composition) containing less than 2 mol%, 1.5 mol% or less, 1.0 mol% or less, 0.5 mol% or less, 0.3 mol% or less, 0.2 mol% or less, 0.1 mol% or less, 0.05 mol% or less, or 0.01 mol% of the AEMA homopolymer represented by formula (4).
In addition, the method for producing 2-aminoethyl methacrylate hydrochloride of the present invention can provide 2-aminoethyl methacrylate hydrochloride (composition) that is substantially free of the homopolymer of AEMA represented by formula (4). Note that "substantially free" means that the homopolymer is below the detection limit of known measuring devices.
The above mole % means the molar ratio of the AEMA homopolymer to the total number of moles of 2-aminoethyl methacrylate hydrochloride and impurities (particularly, the AEMA homopolymer).

Furthermore, the 2-aminoethyl methacrylate hydrochloride of the present invention exhibits high solubility in ethanol. When the transmittance of a 10 wt% ethanol solution at a wavelength of 555 nm is measured using a spectrophotometer, the transmittance is 50% or more, preferably 60% or more, more preferably 70% or more, even more preferably 80% or more, even more preferably 90% or more, most preferably 95% or more, and most preferably 97% or more.

(Compound represented by formula (2))
The monomer represented by formula (2) in the present invention can be produced by a known method, for example, by reacting a hydroxyl group-containing polymerizable monomer with 2-bromoethylphosphoryl dichloride in the presence of a tertiary base, and then reacting the compound obtained with a tertiary amine. Alternatively, a method may be used in which a cyclic compound is obtained by reacting a hydroxyl group-containing polymerizable monomer with a cyclic phosphorus compound, and then the cyclic compound is subjected to a ring-opening reaction with a tertiary amine.
When ^X2 is an oxygen atom, ^R2 is preferably a methyl group from the viewpoint of polymerizability and stability, and when ^X2 is NH, ^R2 is preferably a hydrogen atom from the viewpoint of polymerizability.
Suitable examples of the compound represented by formula (2) include 2-(meth)acryloyloxyethyl (2-(trimethylammonio)ethyl)phosphate in which ^X2 is an oxygen atom, and 2-(meth)acrylamidoethyl (2-(trimethylammonio)ethyl)phosphate in which ^X2 is NH, more preferably 2-methacryloyloxyethyl (2-(trimethylammonio)ethyl)phosphate (MPC) in which ^R2 is a methyl group and ^X2 is an oxygen atom, and 2-acrylamidoethyl (2-(trimethylammonio)ethyl)phosphate in which ^R2 is a hydrogen atom and ^X2 is NH, and from the viewpoint of availability, 2-methacryloyloxyethyl (2-(trimethylammonio)ethyl)phosphate is even more preferred.

(Method for producing a compound represented by formula (1))
The compound represented by formula (1) can be obtained by copolymerizing the structural unit 2-aminoethyl methacrylate hydrochloride of the present invention with the structural unit compound represented by formula (2). For example, the compound represented by formula (1) is a polymerization reaction product (copolymer) of the polymerizable unsaturated groups of the respective structural units.
The polymerization reaction can be carried out by radical polymerization (for example, known methods such as bulk polymerization, suspension polymerization, emulsion polymerization, and solution polymerization) in the presence of a radical polymerization initiator in an atmosphere substituted with an inert gas such as nitrogen, carbon dioxide, argon, or helium, or in an inert gas atmosphere. From the viewpoint of purification, solution polymerization is preferred. The copolymer can be purified by a common purification method such as reprecipitation, dialysis, or ultrafiltration.
Examples of the radical polymerization initiator include an azo-based radical polymerization initiator, an organic peroxide, and a persulfate.
Examples of azo radical polymerization initiators include 2,2'-azobis(2-methylpropionamidine) dihydrochloride (V-50), 2,2-azobis(2-diaminopropyl) dihydrochloride, 2,2-azobis(2-(5-methyl-2-imidazolin-2-yl)propane) dihydrochloride, 4,4'-azobis(4-cyanovaleric acid), 2,2-azobisisobutylamide dihydrate, 2,2-azobis(2,4-dimethylvaleronitrile), 2,2-azobisisobutyronitrile (AIBN), etc. It is more preferable to use 2,2'-azobis(2-methylpropionamidine) dihydrochloride (V-50).
Examples of organic peroxides include t-butyl peroxyneodecanoate (Perbutyl (registered trademark) ND), benzoyl peroxide, diisopropyl peroxydicarbonate, t-butylperoxy-2-ethylhexanoate, t-butyl peroxypivalate, t-butyl peroxydiisobutyrate, lauroyl peroxide, and succinic acid peroxide (succinyl peroxide).
Examples of persulfate include ammonium persulfate, potassium persulfate, and sodium persulfate.
These radical polymerization initiators can be used alone or in combination of two or more. The amount of the polymerization initiator used is usually 0.001 to 10 parts by mass, preferably 0.01 to 5.0 parts by mass, per 100 parts by mass of the monomer composition of the copolymer (compound represented by formula (1)).

The polymerization reaction can be carried out in the presence of a solvent. The solvent can be one that dissolves the monomer composition, which is each structural unit, but does not react with it. Examples of the solvent include water, alcoholic solvents such as methanol, ethanol, n-propanol, and isopropanol, ketone solvents such as acetone, methyl ethyl ketone, and diethyl ketone, ester solvents such as ethyl acetate, linear or cyclic ether solvents such as ethyl cellosolve, tetrahydrofuran, and N-methylpyrrolidone, and nitrogen-containing solvents such as acetonitrile and nitromethane. Water, alcohol, or a mixture thereof is preferred, and water is more preferred.
The weight average molecular weight of the compound represented by formula (1) obtained above is 10,000 to 2,000,000, preferably 100,000 to 1,000,000, more preferably 150,000 to 600,000, and even more preferably 200,000 to 450,000.
The molar ratio of a to b in formula (1) can be set arbitrarily, but is preferably a:b=50:50 to 98:2, more preferably a:b=80:20 to 95:5, and even more preferably a:b=88:12 to 94:6.

The present invention will be explained in detail below using examples, but the present invention is not limited to the following examples.

<Measurement of AEMA homopolymer content>
The measurements were carried out according to the following procedure.
^1H NMR measurement of AEMA was carried out. The measurement conditions are described below. The area value of the peak derived from AEMA observed near 6.2 ppm in the obtained ^1H NMR spectrum was designated as A _m , and the area value of the broad peak derived from the AEMA homopolymer observed near 0.4 to 1.0 ppm was designated as A _p . The content of the compound represented by formula (4) was calculated according to the following calculation formula (1):
((A _p /3) / (A _m + (A _p /3))) × 100 ... Formula (1)
3: Number of protons in peak area A _p ¹ H NMR measurement conditions Apparatus: ECS400 (manufactured by JEOL Ltd.)
Solvent: deuterated dimethyl sulfoxide Sample concentration: 3%
Number of times accumulated: 32

<Loss on drying measurement method (method for measuring residual solvent amount)>
The measurements were carried out according to the following procedure.
The tare of the screw cap tube was weighed, and this weight was designated as ^W1 . 1 g of AEMA was weighed into this screw cap tube, and the sum of the weights of the screw cap tube and AEMA was designated as ^W2 . This was dried in an oven at 125°C for 4 hours. After drying, the screw cap tube was capped and cooled to room temperature in a desiccator. After cooling, it was removed from the desiccator, the cap was removed, and the tube was weighed, and this weight was designated as ^W3 . The loss on drying (%) was calculated using the following formula (2):
(W ² −W ³ )/(W ² −W ¹ )×100 ... Calculation formula (2)

<Purity measurement by transmittance>
The measurements were carried out according to the following procedure.
Preparation of measurement solution 0.3 g of AEMA and 2.7 g of ethanol (ultra-dehydrated) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) were weighed into a screw tube and stirred for 10 minutes to prepare a measurement solution.
Transmittance Measurement: Ethanol (ultra-dehydrated) was placed in a cell with an optical path length of 1 cm, and background measurement was performed in the wavelength range of 400 to 750 nm. The measurement solution was then transferred to a cell with an optical path length of 1 cm, and spectrum measurement was performed in the wavelength range of 400 to 750 nm to evaluate the transmittance at 555 nm. Other conditions were as follows:
Transmittance measurement conditions Equipment: UV-visible spectrophotometer V-650 (manufactured by JASCO Corporation)
Response: Medium
Band width: 2.0 nm
Scanning speed: 200 nm/min
The purity based on transmittance was evaluated as follows: the smaller the score, the higher the purity.
Transparency 75 or more: Score "1"
Transmittance 50 or more but less than 75: Score "2"
Transmittance 0 or more and less than 50: Score "3"

<Measurement of weight average molecular weight>
The obtained copolymer was dissolved in ion-exchanged water to a concentration of 0.1 wt %, and the weight average molecular weight was measured by gel permeation chromatography (GPC).
Column: Shodex (GSM-700)
Mobile phase: 0.1 mol/L aqueous sodium sulfate solution Standard substance: Pullulan Measuring equipment: Differential refractive index RI-8020 (manufactured by Tosoh Corporation)
Calculation method for weight average molecular weight: Molecular weight calculation program (GPC program for SC-8020)
Flow rate: 1.0 mL per minute
Injection volume: 100μL
Column oven temperature: 40°C
Measurement time: 30 minutes

<Cytotoxicity test using rabbit corneal epithelial cells (SIRC cells)>
Cytotoxicity tests using SIRC cells were performed with reference to ISO 10993-5:2009 and N. Tani et al. Toxicology in vitro 13 (1999) 175-187, and cell viability was measured and evaluated.

Preparation of SIRC cell culture medium: 5 mL of antibiotic/antimycotic solution (100x) was added to a 500 mL bottle of Dulbecco's Modified Eagle's (DMEM) medium. 50 mL of sterilized fetal bovine serum (FBS, heat-inactivated) was then added after thawing at 4°C to prepare a cell culture medium.
Dulbecco's modified Eagle's medium (DMEM) was manufactured by Sigma-Aldrich Japan, antibiotic/antimycotic (100x) was penicillin/streptomycin (100x) manufactured by Sigma-Aldrich Japan, and sterilized fetal bovine serum (FBS) was manufactured by Japan Biotest Laboratory.

・Culturing of SIRC cells 9 mL of cell culture medium and 1 mL of cell suspension were added to a sterile dish, and the cells were cultured in a _CO2 incubator for 48 hours or more to proliferate. The cells in the dish were observed under a microscope to confirm the increase in cell number and their state (whether they had died or were floating without adhering).

The cell suspension was adjusted to a concentration of 1 x ¹⁰ cells/mL using cell culture medium. 100 µL of the adjusted cell suspension was dispensed into a 96-well plate using a micropipette, and the cells were cultured in a _CO2 incubator for 24 hours.

Preparation of Test Substances Test substances were prepared by dissolving a 10 w/v % solution of each copolymer and biochemical grade sodium lauryl sulfate (SDS) as a positive control in a cell culture medium.
The prepared test substance was serially diluted to 100 w/v%, 75 w/v%, 50 w/v%, 25 w/v%, 12.5 w/v%, and 0 w/v% on a 96-well plate and used for evaluation.
Biochemical grade sodium lauryl sulfate was manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., and Dulbecco's phosphate buffered saline was manufactured by Sigma-Aldrich Japan.

Exposure to test substance The SIRC cell culture medium was removed from the 96-well plate, and the test substance prepared above was added to the 96-well plate that had been cultured for 24 hours at 200 μL/well, and the plates were exposed to CO 2 for 24 hours in a CO ₂ incubator.

Cytotoxicity evaluation using the Neutral Red (NR) method: NR was dissolved in ion-exchanged water to a concentration of 5 mg/mL to prepare an NR stock solution. The NR stock solution was diluted 100-fold with cell culture medium to prepare an NR medium. The 96-well plate exposed to the test substance was removed, and the medium was discarded. Next, 100 μL of NR medium was added per well, and the cells were cultured in a _CO2 incubator for 3 hours to allow the NR to be incorporated into the cells.
The 96-well plate was removed from the _CO2 incubator and the NR medium was removed. 100 μL/well of PBS was added to the 96-well plate. After the PBS was removed, 100 μL/well of NR extract solution (50% ethanol, 49% ion-exchanged water, and 1% acetic acid) was added using a micropipette. The plate was stirred for 5 minutes on a shaker to extract NR from the cells, and the absorbance at 540 nm was measured using a plate reader.
It was confirmed that the contact concentration of SDS resulting in a cell viability of 50% was approximately 0.01% by mass, and the cell viability and IC ₅₀ value after treatment with the test substance were calculated using the following formula.
Neutral red manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. was used.
Formula for calculating cell viability: Cell viability (%) = (absorbance of test substance treatment - absorbance of blank) / (absorbance of medium treatment - absorbance of blank) x 100
Calculation formula for _IC50 value: _IC50 value = 10 ^{(log10(A/B) x (50-C)/(D-C) + log10(B))}
A: High test substance concentrations (w/v%) that border on a cell viability of 50%
B: Low test substance concentrations (w/v%) that border the cell viability of 50%
C: Cell viability in B. D: Cell viability in A. The absorbance of the medium treatment is the absorbance when only medium is added to the wells seeded with cells.

The following scores were set as the criteria for assessing safety.
_IC50 value of 50 or more: score "1" (indicating extremely excellent safety)
_IC50 value: 25 or more and less than 50: score "2" (indicating excellent safety)
_IC50 value 12.5 or more and less than 25: score "3" (no safety issues)
_IC50 value 0 or more and less than 12.5: score "4" (more detailed safety testing required)

<Example 1-1>
Synthesis of AEMA by the Production Method of the Present Invention A four-neck flask equipped with a thermometer, stirring blade, dropping funnel, and reflux condenser was charged with 50.0 g (0.51 mol) of 2-aminoethanol hydrochloride and 1.02 g of phenothiazine (approximately 12,800 ppm by mass relative to methacrylic acid chloride), and stirring was initiated. The temperature was raised to 90°C to melt the 2-aminoethanol hydrochloride. 79.8 g (0.76 mol) of methacrylic acid chloride was added dropwise over 3 hours to carry out the reaction. After completion of the reaction, the mixture was cooled to approximately 60°C, and 530 g of ethyl acetate was added. After the addition of ethyl acetate, the mixture was further cooled to room temperature to carry out crystallization, and crude crystals (A) were obtained by pressure filtration using No. 5A filter paper (manufactured by Toyo Roshi Kaisha, Ltd.) at a pressure of 0.02 MPa with nitrogen. To the obtained crude crystals (A), 1.02 g of phenothiazine, 140 g of 2-propanol, and 370 g of ethyl acetate were added, and the mixture was heated to 80 ° C. and dissolved. After confirming dissolution, insoluble matter was removed by hot pressure filtration using No. 5A filter paper at a pressure of 0.02 MPa with nitrogen, and the filtrate was recovered. The filtrate was cooled to room temperature and then further cooled to -5 ° C. to perform crystallization, and No. 5A filter paper was used to obtain crude crystals (B) by pressure filtration using nitrogen at a pressure of 0.02 MPa. The obtained crude crystals (B) were washed with a mixed solvent of 70 g of 2-propanol and 185 g of ethyl acetate, and filtered to obtain crude crystals (C).
The obtained crude crystals (C) were dried at 35° C. under reduced pressure (20 hPa) for 5 hours to obtain the target AEMA. The loss on drying was 0.3 wt %.
Analysis by ¹ H NMR confirmed that the compound represented by formula (4) was AEMA, with no detectable compound.
Furthermore, transmittance measurement revealed that the transmittance at 555 nm was 81%.

<Example 1-2>
A four-neck flask equipped with a thermometer, stirring blade, dropping funnel, and reflux condenser was charged with 100.0 g (1.02 mol) of 2-aminoethanol hydrochloride and 2.04 g of phenothiazine (approximately 12,800 ppm by mass relative to methacrylic acid chloride), and stirring was started. The mixture was heated to 92 ° C. to melt the 2-aminoethanol hydrochloride. 159.6 g (1.52 mol) of methacrylic acid chloride was added dropwise over 5 hours, and the reaction was carried out. After the reaction was completed, the mixture was cooled to approximately 60 ° C., and 530 g of ethyl acetate was added. After the addition of ethyl acetate, crystallization was carried out by further cooling to room temperature, and crude crystals (A) were obtained by pressure filtration using No. 5A filter paper at a pressure of 0.02 MPa with nitrogen. 2.04 g of phenothiazine, 280 g of 2-propanol, and 740 g of ethyl acetate were added to the obtained crude crystals (A), and the mixture was heated to 80 ° C. and dissolved. After confirming dissolution, No. 5A filter paper was used to remove insoluble matter by hot pressure filtration at a pressure of 0.02 MPa with nitrogen, and the filtrate was recovered. The filtrate was cooled to room temperature and then further cooled to -5 ° C. to perform crystallization, and No. 5A filter paper was used to obtain crude crystals (B) by pressure filtration at a pressure of 0.02 MPa with nitrogen. The obtained crude crystals (B) were washed with a mixed solvent of 140 g of 2-propanol and 370 g of ethyl acetate and filtered to obtain crude crystals (C).
The obtained crude crystals (C) were dried at 40°C for 8 hours under reduced pressure (80 hPa) to obtain the target AEMA. The loss on drying was 0.6 wt%. Analysis by ^1H NMR confirmed that the AEMA was the compound represented by formula (4), and no detectable compound was found.
Furthermore, transmittance measurement revealed that the transmittance at 555 nm was 70%.

<Example 1-3>
A four-neck flask equipped with a thermometer, stirring blade, dropping funnel, and reflux condenser was charged with 50.0 g (0.51 mol) of 2-aminoethanol hydrochloride and 1.02 g of phenothiazine (approximately 12,800 ppm by mass relative to methacrylic acid chloride), and stirring was started. The mixture was heated to 90 ° C. to melt the 2-aminoethanol hydrochloride. 79.8 g (0.76 mol) of methacrylic acid chloride was added dropwise over 2 hours, and the reaction was carried out. After the reaction was completed, the mixture was cooled to approximately 60 ° C., and 530 g of ethyl acetate was added. After the addition of ethyl acetate, crystallization was carried out by further cooling to room temperature, and crude crystals (A) were obtained by pressure filtration using No. 5A filter paper at a pressure of 0.02 MPa with nitrogen. 1.02 g of phenothiazine, 140 g of 2-propanol, and 370 g of ethyl acetate were added to the obtained crude crystals (A), and the mixture was heated to 80 ° C. and dissolved. After confirming dissolution, No. 5A filter paper was used to remove insoluble matter by hot pressure filtration at a pressure of 0.02 MPa with nitrogen, and the filtrate was recovered. The filtrate was cooled to room temperature and then further cooled to -5 ° C. to perform crystallization, and No. 5A filter paper was used to obtain crude crystals (B) by pressure filtration at a pressure of 0.02 MPa with nitrogen. The obtained crude crystals (B) were washed with a mixed solvent of 70 g of 2-propanol and 185 g of ethyl acetate and filtered to obtain crude crystals (C).
The obtained crude crystals (C) were dried at 40°C for 2 hours under reduced pressure (10 hPa) to obtain the target AEMA. The loss on drying was 0.2 wt%. Analysis by ^1H NMR confirmed that the AEMA was the compound represented by formula (4), and no detectable compound was found.
Furthermore, transmittance measurement revealed that the transmittance at 555 nm was 90%.

<Example 1-4>
A four-neck flask equipped with a thermometer, stirring blade, dropping funnel, and reflux condenser was charged with 50.0 g (0.51 mol) of 2-aminoethanol hydrochloride and 1.02 g of phenothiazine (approximately 12,800 ppm by mass relative to methacrylic acid chloride), and stirring was started. The mixture was heated to 90 ° C. to melt the 2-aminoethanol hydrochloride. 79.8 g (0.76 mol) of methacrylic acid chloride was added dropwise over 2 hours, and the reaction was carried out. After the reaction was completed, the mixture was cooled to approximately 60 ° C., and 530 g of ethyl acetate was added. After the addition of ethyl acetate, crystallization was carried out by further cooling to room temperature, and crude crystals (A) were obtained by pressure filtration using No. 5A filter paper at a pressure of 0.02 MPa with nitrogen. 1.02 g of phenothiazine, 140 g of 2-propanol, and 370 g of ethyl acetate were added to the obtained crude crystals (A), and the mixture was heated to 80 ° C. and dissolved. After confirming dissolution, No. 5A filter paper was used to remove insoluble matter by hot pressure filtration at a pressure of 0.02 MPa with nitrogen, and the filtrate was recovered. The filtrate was cooled to room temperature and then further cooled to -5 ° C. to perform crystallization, and No. 5A filter paper was used to obtain crude crystals (B) by pressure filtration at a pressure of 0.02 MPa with nitrogen. The obtained crude crystals (B) were washed with a mixed solvent of 70 g of 2-propanol and 185 g of ethyl acetate and filtered to obtain crude crystals (C).
The obtained crude crystals (C) were dried at 25°C for 5 hours under reduced pressure (40 hPa) to obtain the target AEMA. The loss on drying was 1.0 wt%. Analysis by ^1H NMR confirmed that the AEMA was AEMA, with no detectable compound represented by formula (4).
Furthermore, transmittance measurement revealed that the transmittance at 555 nm was 97%.

<Comparative Example 1-1>
Crude crystals of AEMA (C) were obtained in the same manner as in Example 1-2.
The obtained crude crystals of AEMA (C) were dried under reduced pressure (30 hPa) for 4 hours at 50° C. Analysis by ¹ H NMR confirmed that the crude crystals contained 3.2 mol % of the AEMA homopolymer represented by formula (4).
Furthermore, when the sample was dissolved in ethanol in order to measure the transmittance, a precipitate was observed, and therefore the transmittance measurement was not carried out.

<Comparative Example 1-2>
Crude crystals of AEMA (C) were obtained in the same manner as in Example 1-2.
The obtained crude crystals of AEMA (C) were dried under reduced pressure (40 hPa) for 4 hours at 60° C. Analysis by ¹ H NMR confirmed that the crude crystals contained 5.5 mol % of the AEMA homopolymer represented by formula (4).
Furthermore, when the sample was dissolved in ethanol in order to measure the transmittance, a precipitate was observed, and therefore the transmittance measurement was not carried out.

<Example 2-1>
Synthesis of a compound represented by formula (1): In the compound represented by formula (2), ^R2 is a hydrogen atom and ^X2 is an oxygen atom. 50.0 g (0.17 mol) of 2-methacryloyloxyethyl (2-(trimethylammonio)ethyl)phosphate (MPC) (manufactured by NOF Corporation) and 3.1 g (0.02 mol) of 2-aminoethyl methacrylate hydrochloride (AEMA) synthesized in Example 1-1 were dissolved in 212.0 g of distilled water. This solution was placed in a four-neck flask equipped with a reflux condenser, a nitrogen inlet tube, a thermometer, and a stirring blade. The solution was heated to 65°C, and 0.36 g of 2,2'-azobis(2-methylpropionamidine) dihydrochloride (V-50) (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.) was added, and nitrogen bubbling was initiated at 0.3 L/min. The temperature of this solution was raised to 70°C and maintained for 120 minutes. After the reaction was completed, the mixture was cooled to room temperature, purified by dialysis using a dialysis membrane, and freeze-dried to obtain a white powder. The concentration of the compound represented by formula (1) obtained was adjusted to 10 w/v % for ease of handling.
The molecular weight was confirmed by GPC and was found to be 330,000.
The molar ratio of a to b in the compound represented by formula (1) was 90:10.

<Examples 2-2 to 2-4>
A compound represented by formula (1) was synthesized in the same manner as in Example 2-1, except that the conditions were changed as shown in Table 2.
The molar ratio of a to b in the compound represented by formula (1) synthesized in Example 2-2 was 95:5.
The molar ratio of a to b in the compound represented by formula (1) synthesized in Example 2-3 was 92:8.
The molar ratio of a to b in the compound represented by formula (1) synthesized in Example 2-4 was 85:15.

<Comparative Examples 2-1 and 2-2>
A compound represented by formula (1) was synthesized in the same manner as in Example 2-1, except that the conditions were changed as shown in Table 2.
The molar ratio of a to b in the compound represented by formula (1) synthesized in Comparative Example 2-1 was 90:10.
The molar ratio of a to b in the compound represented by formula (1) synthesized in Comparative Example 2-2 was 90:10.

Example 3 and Comparative Example 3
In order to evaluate the safety of the compound represented by formula (1), cytotoxicity tests using SIRC cells were carried out (Examples 3-1 to 3-4, Comparative Examples 3-1 and 3-2). The results are shown in Table 3.

<Evaluation Results of Examples>
In Examples 1-1 to 1-4, as shown in the results in Table 1, AEMA with an AEMA homopolymer content of 2% or less (particularly, below the detection limit) could be obtained by drying at 45°C or less in the drying step. In addition, by lowering the pressure in the drying step, the loss on drying could be reduced by drying for a short time.
Using the AEMA obtained in Examples 1-1 to 1-4 as a raw material, compounds represented by formula (1) were synthesized in Examples 2-1 to 2-4. When these were evaluated in Examples 3-1 to 3-4, it was confirmed that the compounds represented by formula (1) had high _IC50 values and were highly safe.

<Evaluation Results of Comparative Examples>
As shown in the results in Table 1, in Comparative Examples 1-1 and 1-2, the drying temperature in the drying step was set to 50° C. or higher, resulting in an AEMA homopolymer content of 3.2% or higher.
Using the AEMA obtained in Comparative Examples 1-1 and 1-2 as a raw material, compounds represented by formula (1) were synthesized in Comparative Examples 2-1 and 2-2. When these compounds were evaluated in Comparative Examples 3-1 and 3-2, it was confirmed that the _IC50 values were low and that more detailed verification of safety was necessary.

The manufacturing method of the present invention can produce highly pure 2-aminoethyl methacrylate hydrochloride.

Although the present invention has been described in detail and with reference to specific embodiments, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the invention.
This application is based on a Japanese patent application (Patent Application No. 2024-53758) filed on March 28, 2024, the contents of which are incorporated herein by reference.

Claims (10)

2-aminoethyl methacrylate hydrochloride, containing less than 2 mol% of 2-aminoethyl methacrylate hydrochloride homopolymer. 2-aminoethyl methacrylate hydrochloride, substantially free of homopolymers of 2-aminoethyl methacrylate hydrochloride. The 2-aminoethyl methacrylate hydrochloride according to claim 1 or 2, wherein when a solution of the 2-aminoethyl methacrylate hydrochloride dissolved in ethanol at a concentration of 10 wt % is measured for transmittance at a wavelength of 555 nm using a spectrophotometer, the transmittance is 50% or more. A method for producing 2-aminoethyl methacrylate hydrochloride according to claim 1, comprising the following steps:
1) a reaction step of reacting 2-aminoethanol hydrochloride with methacrylic acid chloride; and
2) A drying step of drying the 2-aminoethyl methacrylate hydrochloride obtained in the reaction step 1) under reduced pressure at 45°C or less. The manufacturing method described in claim 4, wherein the reduced pressure is 100 hPa or less. The manufacturing method described in claim 4 or 5, which includes a crystallization step between step 1) and step 2). The manufacturing method described in claim 4 or 5, wherein the 2-aminoethyl methacrylate hydrochloride contains less than 2 mol% of a homopolymer of 2-aminoethyl methacrylate hydrochloride. The manufacturing method described in claim 4 or 5, wherein the 2-aminoethyl methacrylate hydrochloride is substantially free of homopolymers of 2-aminoethyl methacrylate hydrochloride. A copolymer comprising the 2-aminoethyl methacrylate hydrochloride of claim 1 or 2 as a structural unit and a compound represented by formula (2) as a structural unit.
(In formula (2), ^R2 is a hydrogen atom or a methyl group, and ^X2 is O or NH.) A copolymer represented by formula (1),
The copolymer contains the structural unit of 2-aminoethyl methacrylate hydrochloride according to claim 1 or 2 and the structural unit of the compound represented by formula (2).
(In formula (1), a:b=50:50 to 98:2, R ¹ is a hydrogen atom or a methyl group, and X ¹ is O or NH.)
(In formula (2), ^R2 is a hydrogen atom or a methyl group, and ^X2 is O or NH.)