JP2018024818A - Random copolymer of n-vinyl imidazolidine-2-one compound and vinyl ester and cell culture material prepared therewith - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a random copolymer of an N-vinyl imidazolidine-2-one compound and a vinyl ester that is expected for application to a cell culture material and the like.SOLUTION: A random copolymer is composed of a repeating unit represented by formula (1), and a repeating unit of a vinyl ester having an acyl group (Ris H, a C1-12 alkyl group or a C1-12 acyl group).SELECTED DRAWING: None

Description

The present invention relates to a random copolymer of an N-vinylimidazolidin-2-one compound and vinyl esters which are expected to be applied as a cell culture material or a temperature-responsive polymer material.

In recent years, there has been a great deal of development related to temperature-responsive materials synthesized using stimuli-responsive materials, particularly N-isopropylacrylamide (NIPAAM). In the case of NIPAAM homopolymer, the lower critical solution temperature (LCST) is 32 ° C. near body temperature. At lower temperatures, NIPAAM homopolymer is soluble in water, but at higher temperatures it is insoluble in water. It has been known. Copolymers of NIPAAM and various comonomers have been synthesized, and their application to drug release control materials, chromatographic separation gels, cell culture beds and the like are being studied. In addition, there are reports that the LCST is adjusted by controlling the type and composition of the comonomer or the affinity for a specific protein is increased (Non-Patent Documents 1 to 4).

On the other hand, Patent Documents 1 and 2 describe N-imidazolidin-2-one derivatives and polymers thereof. Specifically, poly (1-vinylimidazolidin-2-one),
Poly (1-methyl-3-vinylimidazolidin-2-one), poly (1-ethyl-3-vinylimidazolidin-2-one) and poly (1-butyl-3-vinylimidazolidin-2-one)
2-one) or poly (1-methyl-3-vinylimidazolidin-2-one)
A bactericidal composition using N-vinylimidazolidine-2-in accordance with the present invention is shown.
There is no description of the synthesis of random copolymers of ON compounds and vinyl esters, and cell culture materials using the same.

JP 2001-172262 A.

JP 2001-172333 A.

Langmuir, vol. 14, pages 4657-4662 (1998).

Macromolecules, 33, 8312-8316 (2000).

Biomolecules, Vol. 5, 505-510 (2004).

Langmuir, 28, 16623-16737 (2012).

Provided is a random copolymer of an N-vinylimidazolidin-2-one compound and vinyl esters applicable as a cell culture material.

As a result of intensive studies to solve the above problems, the present inventors have used a random copolymer of an N-vinylimidazolidin-2-one compound having a specific structure and vinyl esters as a coating agent component. The present inventors have found that the carrier surface can be easily treated, and that the carrier treated with the coating agent can be used as a cell culture material, and the present invention has been completed.

  That is, the present invention provides the following general formula (1)

（式中、Ｒ^１は水素原子、炭素数１〜１２のアルキル基または炭素数１〜１２のアシル基
を表す。）で表される繰り返し単位と、下記一般式（２）

(Wherein R ¹ represents a hydrogen atom, an alkyl group having 1 to 12 carbon atoms or an acyl group having 1 to 12 carbon atoms), and the following general formula (2)

（式中、Ｒ^２は炭素数２〜１８のアシル基を表す。）で表される繰り返し単位からなるラ
ンダム共重合体（以下、本発明のランダム共重合体と称する。）に関するものであり、下
記一般式（３）

(Wherein R ² represents an acyl group having 2 to 18 carbon atoms), and is related to a random copolymer (hereinafter referred to as a random copolymer of the present invention). The following general formula (3)

（式中、Ｒ^１は水素原子、炭素数１〜１２のアルキル基または炭素数１〜１２のアシル基
を表す。）で表されるＮ−ビニルイミダゾリジン−２−オン化合物、および下記一般式（
４）

(Wherein R ¹ represents a hydrogen atom, an alkyl group having 1 to 12 carbon atoms, or an acyl group having 1 to 12 carbon atoms), and the following general formula (
4)

（式中、Ｒ^２は炭素数２〜１８のアシル基を表す。）で表されるビニルエステル類をラジ
カル重合させるＮ−ビニルイミダゾリジン−２−オン化合物とビニルエステル類とのラン
ダム共重合体の製造法に関するものである。さらに本発明のランダム共重合体を含むコー
ト剤、および該コート剤で担体表面を処理して得られる細胞培養用材料に関するものであ
る。

(Wherein R ² represents an acyl group having 2 to 18 carbon atoms) Random copolymer of an N-vinylimidazolidin-2-one compound and vinyl esters which undergo radical polymerization of vinyl esters represented by This relates to the manufacturing method. Furthermore, the present invention relates to a coating agent containing the random copolymer of the present invention, and a cell culture material obtained by treating a carrier surface with the coating agent.

In the repeating unit of the general formula (1) and the N-vinylimidazolidin-2-one compound of the general formula (3), the alkyl group having 1 to 12 carbon atoms represented by R ¹ has a branched and / or cyclic structure. Methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl, sec-butyl, tert-butyl, cyclobutyl, pentyl, neopentyl, isopentyl, tert-pentyl Group, 1-methylbutyl group, 1-ethylpropyl group, cyclobutylmethyl group, cyclopentyl group, hexyl group, 1-methylpentyl group, 4-methylpentyl group, 1-ethylbutyl group, 2-ethylbutyl group, cyclohexyl group, heptyl Group, 1-methylhexyl group, 1-ethylpentyl group,
Cyclohexylmethyl group, cycloheptyl group, octyl group, 1-methylheptyl group, 2-
Ethylhexyl group, nonyl group, decyl group, 3,7-dimethyloctyl group, undecyl group,
A dodecyl group etc. can be illustrated. The acyl group having 1 to 12 carbon atoms represented by R ¹ may have a branched and / or cyclic structure, and is a formyl group, acetyl group, propionyl group, butyryl group, isobutyryl group, pentanoyl group, 3-methylbutyryl group, 2 , 2-dimethylpropanoyl group, hexanoyl group, heptanoyl group, cyclohexanecarbonyl group, octanoyl group,
Nonanoyl group, decanoyl group, dodecanoyl group and the like can be exemplified. R ¹ is a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a tert-butyl group, an acetyl group, a propionyl group, or a butyryl group in terms of ease of synthesis and suitability for cell culture. Or an isobutyryl group, an ethyl group, a propyl group,
More preferably, they are an isopropyl group, an acetyl group, or a propionyl group.

In the vinyl esters of the general formula (2) and the general formula (4), R ² has 2 to 18 carbon atoms.
An acetyl group, a propionyl group, a butyryl group, an isobutyryl group, a pentanoyl group, a 3-methylbutyryl group, a 2,2-dimethylpropanoyl group, a hexanoyl group,
Examples include heptanoyl group, cyclohexanecarbonyl group, octanoyl group, nonanoyl group, decanoyl group, dodecanoyl group, tetradecanoyl group, hexadecanoyl group, octadecanoyl group, benzoyl group and 4-tert-butylbenzoyl group. Propionyl group, butyryl group, isobutyryl group, pentanoyl group, 3-
A methylbutyryl group, 2,2-dimethylpropanoyl, hexanoyl group, cyclohexanecarbonyl group or benzoyl group is preferred.

When a random copolymer is produced from the N-vinylimidazolidin-2-one compound (3) and the vinyl ester (4), a polymer can be obtained simply and efficiently by using radical polymerization. More specific radical polymerization methods include known methods such as bulk polymerization using free radical polymerization, solution polymerization, and emulsion polymerization. Any amount of radical initiator can be added to initiate radical polymerization more efficiently. Examples of the radical initiator suitably used for the reaction include inorganic peroxides such as potassium persulfate and ammonium persulfate, and N, N, N ′, N′-tetramethylethylenediamine, which is called a polymerization accelerator, N, N— By using in combination with an amine compound such as dimethylparatoluidine, rapid polymerization is possible at a low temperature. Furthermore, organic peroxides such as dilauroyl peroxide, benzoyl peroxide, di-tert-butyl peroxide, tert-butyl hydroperoxide, cumene hydroperoxide as radical initiators, and α, α-azobisisobutyronitrile (AIBN) 2,2'-azobis (4-methoxy-2,4-
Examples thereof include azo compounds such as dimethylvaleronitrile) (V-70) and 1,1′-azobis (cyclohexane-1-carbonitrile) (V-40). Among these, an azo compound is preferably used from the viewpoint of suppressing side reactions.

The random copolymer of the present invention can be produced by mixing the N-vinylimidazolidin-2-one compound (3) and vinyl esters (4) in an arbitrary ratio and carrying out free radical polymerization, and is also reversible. It can also be produced by using an addition fragmentation chain transfer (RAFT) polymerization method.

Any proportion of N-vinylimidazolidin-2-one compound (3) and vinyl esters (
4) The mixture is subjected to RAFT polymerization in the presence of the radical polymerization initiator and the RAFT agent listed above.

The molar amount [M] of all monomers and the molar amount of radical species generated from the radical polymerization initiator [I
By controlling the ratio [M] / [I ·] and the monomer conversion, the molecular weight and molecular weight distribution of the random copolymer of the present invention can be controlled. Amount of RAFT agent used [R
[AFT] may be equal to or more than [I ·], and generally [RAFT] / [I ·].
] Is adjusted to 1 to 10, preferably 1.5 to 5. Monomer conversion is RAFT
Agent type, [RAFT] / [I ·] value, presence or absence of solvent, type, initial monomer concentration,
It can be arbitrarily controlled by the viscosity of the solution, the polymerization temperature, the polymerization time and the like. As a RAFT agent,
In general, RA of non-conjugated monomers such as dithiocarbamate compounds and dithiocarbonate compounds
A known material suitable for FT polymerization can be used, and more specifically, N-methyl-N-
Cyanomethyl phenyldithiocarbamate, cyanomethyl N, N-diphenyldithiocarbamate, cyanomethyl N-methyl-N-4-pyridyldithiocarbamate, ethoxycarbonylmethyl N, N-diphenyldithiocarbamate, ethoxydithiocarboxylic acid 1-
Cyanoethyl and the like can be exemplified, but depending on the type and combination of R ¹ and R ² R
The choice of AFT agent may vary. For selection of the RAFT agent, for example, Mac
ROMOLECULES, Vol. 45, 5321-5342 (2012), etc. may be referred to.

Usually, the random copolymer of the present invention can be produced with good reproducibility by performing polymerization in an inert gas atmosphere such as nitrogen gas or argon gas or under deoxygenation, and the polymerization reaction proceeds smoothly when a solvent is used. Examples of the solvent include water, methanol, ethanol, propanol, isopropyl alcohol, butanol, isobutanol, hexanol, pentane, cyclopentane, hexane, cyclohexane, heptane, octane, benzene, toluene, xylene, chlorobenzene, dichloromethane, chloroform, four Carbon chloride, 1,2-dichloroethane, acetone, methyl ethyl ketone, diethyl ether, cyclopentyl methyl ether, tetrahydrofuran, dioxane, ethyl acetate, acetonitrile, dimethyl sulfoxide, N, N-dimethylformamide (DMF), N, N-dimethylacetamide (DM
Ac), ammonia, trimethylamine, diethylamine, triethylamine, diisopropylamine, butylamine, dibutylamine, tributylamine, pyridine, picoline and the like can be used, but are not limited thereto. Also, two or more of these solvents can be mixed and used. The polymerization reaction normally proceeds smoothly in the range of 0 ° C to 100 ° C.

The ratio of the repeating unit (1) to the repeating unit (2) in the random copolymer of the present invention is not particularly limited as long as the carrier surface coating or cell culture is possible, but generally the repeating unit (1): Repeating unit (2) = 1: 9 to 9: 1 (weight ratio), preferably 2: 8 to
8: 2. This ratio can be obtained by measuring a nuclear magnetic resonance spectrum such as ¹ H-NMR and calculating the number average value from the integral value of the spectrum derived from each repeating unit, and converting it to a weight ratio.

The molecular weight of the random copolymer of the present invention can be used according to the measuring method such as weight average molecular weight, number average molecular weight, viscosity average molecular weight. 1 for weight average molecular weight (Mw)
It is preferably from 000 to 1,000,000, and more preferably from 5,000 to 500,000 from the viewpoint of control of the properties of the polymer and processability. Although there is no restriction | limiting in particular in molecular weight distribution (PD), it is preferable that it is the range of about 1-20 in general, and it is more preferable that it is the range of 1-5 from a viewpoint of the uniformity of a polymer. As a method for calculating the molecular weight, gel filtration chromatography (G) converted based on standard samples such as polystyrene and polyethylene glycol
PC) method, viscosity method, light scattering method and the like can be mentioned.

A coating agent can be prepared by dissolving the random copolymer of the present invention in a solvent. The solvent used in the coating agent is not particularly limited as long as the solvent can be removed after the random copolymer of the present invention is dissolved and applied to the surface of the carrier, but water, methanol, ethanol, propanol, isopropyl alcohol, butanol, Hexanol, pentane, cyclopentane, hexane,
Cyclohexane, heptane, octane, benzene, toluene, xylene, chlorobenzene, dichloromethane, chloroform, carbon tetrachloride, 1,2-dichloroethane, acetone, methyl ethyl ketone, diethyl ether, cyclopentyl methyl ether, tetrahydrofuran, dioxane, acetonitrile, dimethyl sulfoxide, DMF, N, N-dimethylacetamide, N-methylpyrrolidin-2-one, ethylene glycol, 2-methoxyethanol, 2-butoxyethanol and the like can be used, but are not limited thereto. Also, two or more of these solvents can be mixed and used.

The concentration of the coating agent is not particularly limited as long as it does not affect the coating, but in general, the concentration of the random copolymer of the present invention is preferably adjusted to 0.01 to 20% by weight.

The surfaces of various shapes of carriers such as petri dishes, bags, sponge-like porous substrates, granular porous substrates, nonwoven fabrics, woven fabrics, and other fiber substrates made of various materials such as glass and resin are used in the present invention. A cell culture material can be prepared by treating with a coating agent containing a random copolymer. There is no restriction | limiting in particular in the processing method of a coating agent, A well-known method can be used, For example, a dip method, a spray method, a casting method, a spin cast method, an inkjet printing method etc. can be used.

By treating the carrier surface of various materials and shapes with the coating agent containing the random copolymer of the present invention, the influence of these carrier surfaces on the cells is reduced, cell culture, culture cell storage,
Since convenience of handling such as collection and transfer of cultured cells can be improved, application as biocompatible materials and cell culture materials is expected.

Hereinafter, although an example is given and the present invention is explained still in detail, the present invention is not limited to these. The molecular weight of the obtained polymer was determined from the GPC results. GL-7400 (detector: GL-7456, column (4)): TSK
gel Super H5000, H4000 × 2, H2000, column temperature: 40 ° C., developing solvent: 0.01 M LiCl in DMF solution, standard polystyrene conversion) was used. The structure of the random copolymer and the copolymerization ratio are AVANCEI manufactured by Bruker-Biospin.
It was determined from the result of ¹ H-NMR measurement using II-400.

  Reference example 1

２００ｍＬナスフラスコにイミダゾリジン−２−オン１０．３ｇ（１２０ｍｍｏｌ）お
よびプロピオン酸無水物１００ｍＬを加えて、１５０℃で１時間撹拌した。室温まで放冷
して析出した固体を回収し、少量の冷エタノールで洗浄することにより、無色固体の１−
プロピオニルイミダゾリジン−２−オン１３．０ｇ（収率：７６．２％）を得た。^１Ｈ−
ＮＭＲ（４００ＭＨｚ，ＣＤＣｌ_３，ｐｐｍ），δ：１．６６（３Ｈ，ｔ，Ｊ＝７．４Ｈ
ｚ），２．９３（２Ｈ，ｑ，Ｊ＝７．４Ｈｚ），３．５０（２Ｈ，ｔ，Ｊ＝８．１Ｈｚ）
，３．９６（２Ｈ，ｔ，Ｊ＝８．１Ｈｚ），５．６８（１Ｈ，ｂｓ）．ＥＩ−ＭＳ，ｍ／
ｚ：１４２（Ｍ）^＋，１２７（Ｍ−ＣＨ_３）^＋，１１３（Ｍ−Ｃ_２Ｈ_５）^＋，１００．Ｉ
Ｒ（ｎｅａｔ，ｃｍ^−１），ν：３２３０（ｗ），３１２９（ｗ），２９８３（ｗ），２
８９８（ｗ），１７３４（ｍ），１６７０（ｍ），１３７７（ｍ），１２６５（ｍ），１
１５９（ｍ），１０６２（ｍ），１０２６（ｍ），９４１（ｍ）。

To a 200 mL eggplant flask, 10.3 g (120 mmol) of imidazolidin-2-one and 100 mL of propionic anhydride were added and stirred at 150 ° C. for 1 hour. After cooling to room temperature, the precipitated solid is collected and washed with a small amount of cold ethanol to give a colorless solid 1-
Propionylimidazolidin-2-one 13.0 g (yield: 76.2%) was obtained. ¹ H-
NMR (400 MHz, CDCl ₃ , ppm), δ: 1.66 (3H, t, J = 7.4H
z), 2.93 (2H, q, J = 7.4 Hz), 3.50 (2H, t, J = 8.1 Hz)
, 3.96 (2H, t, J = 8.1 Hz), 5.68 (1H, bs). EI-MS, m /
z: 142 (M) ⁺ , 127 (M-CH ₃ ) ⁺ , 113 (M-C ₂ H ₅ ) ⁺ , 100. I
R (neat, cm ⁻¹ ), ν: 3230 (w), 3129 (w), 2983 (w), 2
898 (w), 1734 (m), 1670 (m), 1377 (m), 1265 (m), 1
159 (m), 1062 (m), 1026 (m), 941 (m).

アルゴン雰囲気下、１００ｍＬのシュレンク管に１−プロピオニルイミダゾリジン−２
−オン２．８６ｇ（２０．１ｍｍｏｌ）、ヨウ化銅（Ｉ）０．１９ｇ（１．０ｍｍｏｌ）
、Ｎ，Ｎ’−ジメチルエチレンジアミン０．２１ｍＬ（２．０ｍｍｏｌ），炭酸カリウム
５．５３ｇ（４０．０ｍｍｏｌ）および１Ｍの臭化ビニルのＴＨＦ溶液４０．０ｍＬ（４
０．０ｍｍｏｌ）を加えて、７０℃で７時間撹拌した。反応液をセライトで濾過して濾液
を濃縮した。アルミナカラムクロマトグラフィー（展開液：クロロホルム）で精製するこ
とにより、無色固体の１−プロピオニル−３−ビニルイミダゾリジン−２−オン（ＶＩｍ
）２．８３ｇ（収率：８３．６％）を得た。^１Ｈ−ＮＭＲ（４００ＭＨｚ，ＣＤＣｌ_３，
ｐｐｍ），δ：１．１７（３Ｈ，ｔ，Ｊ＝７．４Ｈｚ），２．９６（２Ｈ，ｑ，Ｊ＝７．
４Ｈｚ），３．５７（２Ｈ，ｔ，Ｊ＝８．２Ｈｚ），３．９３（２Ｈ，ｔ，Ｊ＝８．２Ｈ
ｚ），４．３３（１Ｈ，ｄｄ，Ｊ＝１．１，１５．９Ｈｚ），４．４６（１Ｈ，ｄｄ，Ｊ
＝１．１，９．０Ｈｚ），６．９９（１Ｈ，ｄｄ，Ｊ＝９．０，１５．９Ｈｚ）．ＥＩ−
ＭＳ，ｍ／ｚ：１６８（Ｍ）^＋，１３９（Ｍ−Ｃ_２Ｈ_５）^＋，１１１（Ｍ−ＣＯＣ_２Ｈ_５
）^＋．ＩＲ（ｎｅａｔ，ｃｍ^−１），ν：３１１１（ｗ），２９７９（ｗ），１７１６（
ｍ），１６９１（ｍ），１６３３（ｍ），１４０２（ｍ），１３６０（ｍ），１３６０（
ｍ），１２３０（ｍ），１０４９（ｍ）。

1-propionylimidazolidine-2 in a 100 mL Schlenk tube under argon atmosphere
-2.86 g (20.1 mmol) of ON, 0.19 g (1.0 mmol) of copper (I) iodide
N, N′-dimethylethylenediamine 0.21 mL (2.0 mmol), potassium carbonate 5.53 g (40.0 mmol) and 1M vinyl bromide in THF 40.0 mL (4
0.0 mmol) was added and stirred at 70 ° C. for 7 hours. The reaction solution was filtered through celite, and the filtrate was concentrated. By purifying with alumina column chromatography (developing solution: chloroform), colorless solid 1-propionyl-3-vinylimidazolidin-2-one (VIm
) 2.83 g (yield: 83.6%) was obtained. ¹ H-NMR (400 MHz, CDCl ₃ ,
ppm), δ: 1.17 (3H, t, J = 7.4 Hz), 2.96 (2H, q, J = 7.
4 Hz), 3.57 (2H, t, J = 8.2 Hz), 3.93 (2H, t, J = 8.2H)
z), 4.33 (1H, dd, J = 1.1, 15.9 Hz), 4.46 (1H, dd, J
= 1.1, 9.0 Hz), 6.99 (1H, dd, J = 9.0, 15.9 Hz). EI-
MS, m / z: 168 (M) ⁺ , 139 (M-C ₂ H ₅ ) ⁺ , 111 (M-COC ₂ H ₅
) ⁺ . IR (neat, cm ⁻¹ ), ν: 3111 (w), 2979 (w), 1716 (
m), 1691 (m), 1633 (m), 1402 (m), 1360 (m), 1360 (
m), 1230 (m), 1049 (m).

  Reference example 2

５０ｍＬナスフラスコにＮ−イソプロピルエチレンジアミン１０ｍＬ（８１ｍｍｏｌ）
、尿素４．９ｇ（８１ｍｍｏｌ）およびエチレングリコール５．４ｍＬを加えて、１３０
℃で１時間、さらに１８０℃で４時間撹拌した。減圧下でエチレングリコールを留去し、
残渣をシリカゲルカラムクロマトグラフィー（展開液：クロロホルム／メタノール＝１０
／１）で精製することにより、無色固体の１−イソプロピルイミダゾリジン−２−オン７
．７ｇ（収率：７３％）を得た。^１Ｈ−ＮＭＲ（４００ＭＨｚ，ＣＤＣｌ_３，ｐｐｍ），
δ：１．１３（６Ｈ，ｄ，Ｊ＝６．８Ｈｚ），３．３９（４Ｈ，ｍ），４．１４（１Ｈ，
ｓｅｐ，Ｊ＝６．８Ｈｚ），５．２７（１Ｈ，ｂｒｓ）．ＥＩ−ＭＳ，ｍ／ｚ：１２８（
Ｍ）^＋，１１３（Ｍ−ＣＨ_３）^＋．ＩＲ（ｎｅａｔ，ｃｍ^−１），ν：３１９７（ｗ），
３０８９（ｗ），２９７６（ｍ），２９２９（ｍ），１６８５（ｍ），１４８５（ｍ），
１４２７（ｍ），１２６５（ｍ），１２２７（ｍ），１１２６（ｗ），１１０１（ｗ），
１０７６（ｗ）。

N-isopropylethylenediamine 10 mL (81 mmol) in a 50 mL eggplant flask
, Urea 4.9 g (81 mmol) and ethylene glycol 5.4 mL were added
The mixture was stirred at 1 ° C. for 1 hour and further at 180 ° C. for 4 hours. Ethylene glycol is distilled off under reduced pressure,
The residue was subjected to silica gel column chromatography (developing solution: chloroform / methanol = 10
/ 1) to give colorless solid 1-isopropylimidazolidin-2-one 7
. 7 g (yield: 73%) was obtained. ¹ H-NMR (400 MHz, CDCl ₃ , ppm),
δ: 1.13 (6H, d, J = 6.8 Hz), 3.39 (4H, m), 4.14 (1H,
sep, J = 6.8 Hz), 5.27 (1H, brs). EI-MS, m / z: 128 (
_{^{M) +, 113 (M-}} CH 3) +. IR (neat, cm ⁻¹ ), ν: 3197 (w),
3089 (w), 2976 (m), 2929 (m), 1685 (m), 1485 (m),
1427 (m), 1265 (m), 1227 (m), 1126 (w), 1101 (w),
1076 (w).

アルゴン雰囲気下、１００ｍＬのシュレンク管に１−イソプロピルイミダゾリジン−２
−オン５．１２７ｇ（４０．００ｍｍｏｌ）、ヨウ化銅（Ｉ）３７９ｍｇ（１．９９ｍｍ
ｏｌ）、炭酸カリウム１１．０７ｇ（８０．０６ｍｍｏｌ）、Ｎ，Ｎ’−ジメチルエチレ
ンジアミン０．４３ｍＬ（４．０ｍｍｏｌ）、臭化ビニル５．６ｍＬ（８０ｍｍｏｌ）お
よびトルエン４０ｍＬを加えて、９０℃で１７時間撹拌した。反応液をセライトで濾過し
て濾液を濃縮し、残渣を減圧蒸留（８０℃／１０Ｐａ）することにより、無色液体の１−
イソプロピル−３−ビニルイミダゾリジン−２−オン（ＶＩｍ−ｉＰ）４．３３９ｇ（収
率：７０．３４％）を得た。^１Ｈ−ＮＭＲ（４００ＭＨｚ，ＣＤＣｌ_３，ｐｐｍ），δ：
１．１５（６Ｈ，ｄ，Ｊ＝６．８Ｈｚ），３．３８〜３．５１（４Ｈ，ｍ），４．０４（
１Ｈ，ｄ，Ｊ＝１５．９Ｈｚ），４．１５（１Ｈ，ｄ，Ｊ＝９．０Ｈｚ），４．２１（１
Ｈ，ｓｅｐ，Ｊ＝６．８Ｈｚ），７．０１（１Ｈ，ｄｄ，Ｊ＝９．０，１５．９Ｈｚ）．
ＥＩ−ＭＳ，ｍ／ｚ：１５４（Ｍ）^＋，１２５（Ｍ−ＣＨ_３）^＋，１１１（Ｍ−Ｃ_２Ｈ_５
）^＋．ＩＲ（ｎｅａｔ，ｃｍ^−１），ν： ２９７２（ｗ），２９３７（ｗ），２８７５
（ｗ），１６９７（ｍ），１６２４（ｍ），１４８５（ｍ），１４１９（ｍ），１３３５
（ｗ），１２６７（ｍ），１２３２（ｍ），１１２６（ｗ），１０６８（ｗ），１０３４
（ｗ），９８０（ｗ）。

1-Isopropylimidazolidine-2 in a 100 mL Schlenk tube under argon atmosphere
-ON 5.127 g (40.00 mmol), copper (I) iodide 379 mg (1.99 mm)
ol), 11.07 g (80.06 mmol) of potassium carbonate, 0.43 mL (4.0 mmol) of N, N′-dimethylethylenediamine, 5.6 mL (80 mmol) of vinyl bromide, and 40 mL of toluene, and the mixture was added at 90 ° C. Stir for hours. The reaction mixture was filtered through celite, the filtrate was concentrated, and the residue was distilled under reduced pressure (80 ° C./10 Pa) to give a colorless liquid 1-
4.339 g (yield: 70.34%) of isopropyl-3-vinylimidazolidin-2-one (VIm-iP) was obtained. ¹ H-NMR (400 MHz, CDCl ₃ , ppm), δ:
1.15 (6H, d, J = 6.8 Hz), 3.38 to 3.51 (4H, m), 4.04 (
1H, d, J = 15.9 Hz), 4.15 (1H, d, J = 9.0 Hz), 4.21 (1
H, sep, J = 6.8 Hz), 7.01 (1H, dd, J = 9.0, 15.9 Hz).
EI-MS, m / z: 154 (M) ⁺ , 125 (M-CH ₃ ) ⁺ , 111 (M-C ₂ H ₅
) ⁺ . IR (neat, cm ⁻¹ ), ν: 2972 (w), 2937 (w), 2875
(W), 1697 (m), 1624 (m), 1485 (m), 1419 (m), 1335
(W), 1267 (m), 1232 (m), 1126 (w), 1068 (w), 1034
(W), 980 (w).

  Example 1

参考例１で得られた１−プロピオニル−３−ビニルイミダゾリジン−２−オン（ＶＩｍ
）（０．５０５ｇ，３．００ｍｍｏｌ）と安息香酸ビニル（ＶＢ）（０．４４４ｇ，３．
００ｍｍｏｌ）をナスフラスコに秤量し、ここにＡＩＢＮ（９．９ｍｇ，６０μｍｏｌ）
を加えて、ＤＭＡｃ２．０ｍＬに溶解した。凍結脱気を行い、封管した後、６０℃で８７
時間撹拌した。２００ｍＬのジエチルエーテルに投入して無色粉末を０．７７８ｇ回収し
た。Ｍｗ：１０２２００、ＰＤ：４．９９、収率：８２．０％。^１Ｈ−ＮＭＲスペクトル
を図１に示した。図１のＶＩｍ由来の重合体に帰属される０．９４ｐｐｍ付近のピークの
積分値とＶＢ由来の重合体に帰属される７．８２ｐｐｍ付近のピークの積分値の比から組
成比を求めたところｘ／ｙ＝４７／５３であった。

1-propionyl-3-vinylimidazolidin-2-one obtained in Reference Example 1 (VIm
) (0.505 g, 3.00 mmol) and vinyl benzoate (VB) (0.444 g, 3.
00 mmol) was weighed into an eggplant flask, where AIBN (9.9 mg, 60 μmol)
Was dissolved in 2.0 mL of DMAc. After freezing and degassing and sealing, 87 ° C. at 87 ° C.
Stir for hours. Into 200 mL of diethyl ether, 0.778 g of colorless powder was recovered. Mw: 102200, PD: 4.99, yield: 82.0%. ^{The 1} H-NMR spectrum is shown in FIG. The composition ratio was determined from the ratio of the integral value of the peak near 0.94 ppm attributed to the polymer derived from VIm in FIG. 1 and the integral value of the peak near 7.82 ppm attributed to the polymer derived from VB. / Y = 47/53.

  Example 2

参考例１で得られた１−プロピオニル−３−ビニルイミダゾリジン−２−オン（ＶＩｍ
）（０．５０５ｇ，３．００ｍｍｏｌ）とヘキサン酸ビニル（ＶＨ）（０．４２７ｇ，３
．００ｍｍｏｌ）をナスフラスコに秤量し、ここにＶ−４０（１４．７ｍｇ，６０μｍｏ
ｌ）を加えて、ＤＭＡｃ２．０ｍＬに溶解した。凍結脱気を行い、封管した後、８０℃で
４２時間撹拌した。２００ｍＬのヘキサンに投入して無色粉末を０．６１０ｇ回収した。
Ｍｗ：３６６００、ＰＤ：２．８６、収率：６５．５％。^１Ｈ−ＮＭＲスペクトルを図２
に示した。図２のＶＩｍ由来の重合体に帰属される１．１０ｐｐｍ付近のピークの積分値
とＶＨ由来の重合体に帰属される０．８９ｐｐｍ付近のピークの積分値の比から組成比を
求めたところｘ／ｙ＝５３／４７であった。

1-propionyl-3-vinylimidazolidin-2-one obtained in Reference Example 1 (VIm
) (0.505 g, 3.00 mmol) and vinyl hexanoate (VH) (0.427 g, 3
. 00 mmol) was weighed into an eggplant flask, where V-40 (14.7 mg, 60 μmo)
l) was added and dissolved in 2.0 mL of DMAc. After freeze degassing and sealing, the mixture was stirred at 80 ° C. for 42 hours. Into 200 mL of hexane, 0.610 g of colorless powder was recovered.
Mw: 36600, PD: 2.86, yield: 65.5%. ^{The 1} H-NMR spectrum is shown in FIG.
It was shown to. The composition ratio was determined from the ratio of the integrated value of the peak near 1.10 ppm attributed to the polymer derived from VIm in FIG. 2 to the integrated value of the peak near 0.89 ppm attributed to the polymer derived from VH. / Y = 53/47.

  Example 3

参考例２で得られた１−イソプロピル−３−ビニルイミダゾリジン−２−オン（ＶＩｍ
−ｉＰ）（０．４６３ｇ，３．００ｍｍｏｌ）と安息香酸ビニル（ＶＢ）（０．４４４ｇ
，３．００ｍｍｏｌ）をナスフラスコに秤量し、ここにＡＩＢＮ（１８．５ｍｇ，６０μ
ｍｏｌ）を加えて、ＤＭＡｃ２．０ｍＬに溶解した。凍結脱気を行い、封管した後、６０
℃で８６時間撹拌した。２００ｍＬのジエチルエーテルに投入して淡黄色粉末を０．６３
０ｇ回収した。Ｍｗ：３４７００、ＰＤ：２．０５、収率：６９．５％。^１Ｈ−ＮＭＲス
ペクトルを図３に示した。図３のＶＩｍ−ｉＰ由来の重合体に帰属される０．９３ｐｐｍ
付近のピークの積分値とＶＢ由来の重合体に帰属される７．８５ｐｐｍ付近のピークの積
分値の比から組成比を求めたところｘ／ｙ＝４５／５５であった。

1-Isopropyl-3-vinylimidazolidin-2-one (VIm) obtained in Reference Example 2
-IP) (0.463 g, 3.00 mmol) and vinyl benzoate (VB) (0.444 g
, 3.00 mmol) was weighed into an eggplant flask where AIBN (18.5 mg, 60 μm) was weighed.
mol) was added and dissolved in 2.0 mL of DMAc. After freeze degassing and sealing, 60
Stir at 86 ° C. for 86 hours. Put in 200 mL of diethyl ether and add 0.63 of pale yellow powder.
0 g was recovered. Mw: 34700, PD: 2.05, yield: 69.5%. ^{The 1} H-NMR spectrum is shown in FIG. 0.93 ppm attributed to the polymer derived from VIm-iP in FIG.
When the composition ratio was determined from the ratio of the integral value of the nearby peak and the integral value of the peak near 7.85 ppm attributed to the polymer derived from VB, it was x / y = 45/55.

  Example 4

参考例１で得られた１−プロピオニル−３−ビニルイミダゾリジン−２−オン（ＶＩｍ）（０．６０５ｇ，３．６０ｍｍｏｌ）と安息香酸ビニル（ＶＢ）（０．５３３ｇ，３．６０ｍｍｏｌ）をナスフラスコに秤量し、ここにＡＩＢＮ（１０．７ｍｇ，７２．０μｍｏｌ）を加えて、ＤＭＡｃ２．４ｍＬに溶解した。凍結脱気を行い、封管した後、６０℃で２４時間撹拌した。２４０ｍＬのジエチルエーテルに投入して無色粉末を０．９３８ｇ回収した。Ｍｗ：４５５００、ＰＤ：２．６６、収率：８２．４％。^１Ｈ−ＮＭＲスペクトルを図４に示した。図４のＶＩｍ由来の重合体に帰属される０．９４ｐｐｍ付近のピークの積分値とＶＢ由来の重合体に帰属される７．８２ｐｐｍ付近のピークの積分値の比から組成比を求めたところｘ／ｙ＝４９／５１であった。

1-propionyl-3-vinylimidazolidin-2-one (VIm) (0.605 g, 3.60 mmol) and vinyl benzoate (VB) (0.533 g, 3.60 mmol) obtained in Reference Example 1 were mixed with eggplant. The flask was weighed, and AIBN (10.7 mg, 72.0 μmol) was added thereto and dissolved in 2.4 mL of DMAc. After freeze degassing and sealing, the mixture was stirred at 60 ° C. for 24 hours. Into 240 mL of diethyl ether, 0.938 g of colorless powder was recovered. Mw: 45500, PD: 2.66, yield: 82.4%. ^{The 1} H-NMR spectrum is shown in FIG. The composition ratio was determined from the ratio of the integral value of the peak near 0.94 ppm attributed to the polymer derived from VIm in FIG. 4 to the integral value of the peak near 7.82 ppm attributed to the polymer derived from VB. / Y = 49/51.

  Example 5

参考例１で得られた１−プロピオニル−３−ビニルイミダゾリジン−２−オン（ＶＩｍ）（０．７０６ｇ，４．２０ｍｍｏｌ）と安息香酸ビニル（ＶＢ）（０．２６７ｇ，１．８０ｍｍｏｌ）をナスフラスコに秤量し、ここにＡＩＢＮ（９．９ｍｇ，６０μｍｏｌ）を加えて、ＤＭＡｃ３．０ｍＬに溶解した。凍結脱気を行い、封管した後、６０℃で２４時間撹拌した。３００ｍＬのジエチルエーテルに投入して無色粉末を０．８６０ｇ回収した。Ｍｗ：３４０００、ＰＤ：２．５６、収率：８８．４％。^１Ｈ−ＮＭＲスペクトルを図１に示した。図１のＶＩｍ由来の重合体に帰属される０．９４ｐｐｍ付近のピークの積分値とＶＢ由来の重合体に帰属される７．８２ｐｐｍ付近のピークの積分値の比から組成比を求めたところｘ／ｙ＝６７／３３であった。

1-propionyl-3-vinylimidazolidin-2-one (VIm) (0.706 g, 4.20 mmol) obtained in Reference Example 1 and vinyl benzoate (VB) (0.267 g, 1.80 mmol) were mixed with eggplant. The flask was weighed and AIBN (9.9 mg, 60 μmol) was added thereto and dissolved in 3.0 mL of DMAc. After freeze degassing and sealing, the mixture was stirred at 60 ° C. for 24 hours. Into 300 mL of diethyl ether, 0.860 g of colorless powder was recovered. Mw: 34000, PD: 2.56, yield: 88.4%. ^{The 1} H-NMR spectrum is shown in FIG. The composition ratio was determined from the ratio of the integral value of the peak near 0.94 ppm attributed to the polymer derived from VIm in FIG. 1 and the integral value of the peak near 7.82 ppm attributed to the polymer derived from VB. / Y = 67/33.

  Example 6

参考例１で得られた１−プロピオニル−３−ビニルイミダゾリジン−２−オン（ＶＩｍ）（０．３０３ｇ，１．８０ｍｍｏｌ）と安息香酸ビニル（ＶＢ）（０．６２２ｇ，４．２０ｍｍｏｌ）をナスフラスコに秤量し、ここにＡＩＢＮ（９．９ｍｇ，６０μｍｏｌ）を加えて、ＤＭＡｃ２．０ｍＬに溶解した。凍結脱気を行い、封管した後、６０℃で２４時間撹拌した。２００ｍＬのジエチルエーテルに投入して無色粉末を０．７０５ｇ回収した。Ｍｗ：３８５００、ＰＤ：４．９９、収率：７６．２％。^１Ｈ−ＮＭＲスペクトルを図１に示した。図１のＶＩｍ由来の重合体に帰属される０．９４ｐｐｍ付近のピークの積分値とＶＢ由来の重合体に帰属される７．８２ｐｐｍ付近のピークの積分値の比から組成比を求めたところｘ／ｙ＝３３／６７であった。

1-propionyl-3-vinylimidazolidin-2-one (VIm) (0.303 g, 1.80 mmol) obtained in Reference Example 1 and vinyl benzoate (VB) (0.622 g, 4.20 mmol) were mixed with eggplant. The flask was weighed, and AIBN (9.9 mg, 60 μmol) was added thereto and dissolved in 2.0 mL of DMAc. After freeze degassing and sealing, the mixture was stirred at 60 ° C. for 24 hours. Into 200 mL of diethyl ether, 0.705 g of colorless powder was recovered. Mw: 38500, PD: 4.99, yield: 76.2%. ^{The 1} H-NMR spectrum is shown in FIG. The composition ratio was determined from the ratio of the integral value of the peak near 0.94 ppm attributed to the polymer derived from VIm in FIG. 1 and the integral value of the peak near 7.82 ppm attributed to the polymer derived from VB. / Y = 33/67.

  Example 7

0.02 g of the random copolymer obtained in Example 1 was dissolved in 2 mL of DMF to obtain 1 wt% D.
An MF solution was prepared and used as a coating agent. A coating agent was applied to a round cover glass (manufactured by Matsunami Glass Kogyo Co., Ltd.) having a diameter of 15 mm by a spin coating method and then dried.

Subsequently, the cover glass was fitted into a Corning 24-well plate and used as a substrate for cell culture evaluation. Mouse connective tissue-derived cells (L929 cells) were seeded at a density of 1.9 × 10 ⁴ cells / well. As the medium, Dulbecco-Fork modified Eagle minimum essential medium (DMEM medium) containing 10 vol% fetal bovine serum was used, and 1.0 mL was added per well. The cells were cultured at 37 ° C. and a CO ₂ concentration of 5%, and observed with a 10 × 10-fold inverted phase contrast microscope (Olympus IX73) every predetermined time. The initial cell density of 100 / mm ^2, cell adhesion density 3 days reached 460 pieces / mm ^2, a cover glass treated with coating agent acted effectively as a material for cell culture.

Example 8
Evaluation was performed in the same manner as in Example 7 except that the random copolymer obtained in Example 2 was used. The initial cell density of 100 / mm ^2, cell adhesion density 3 days reached 475 / mm ^2, a cover glass treated with coating agent acted effectively as a material for cell culture.

Example 9
Evaluation was performed in the same manner as in Example 7 except that the random copolymer obtained in Example 3 was used. The initial cell density of 100 / mm ^2, cell adhesion density 3 days reached 460 pieces / mm ^2, a cover glass treated with coating agent acted effectively as a material for cell culture.

Example 10
Evaluation was performed in the same manner as in Example 7 except that the random copolymer obtained in Example 4 was used. The initial cell density of 100 / mm ^2, cell adhesion density 3 days reached 470 pieces / mm ^2, a cover glass treated with coating agent acted effectively as a material for cell culture.

Example 11
Evaluation was performed in the same manner as in Example 7 except that the random copolymer obtained in Example 5 was used. The initial cell density of 100 / mm ^2, cell adhesion density 3 days reached 480 / mm ^2, a cover glass treated with coating agent acted effectively as a material for cell culture.

Example 12
Evaluation was performed in the same manner as in Example 7 except that the random copolymer obtained in Example 6 was used. The initial cell density of 100 / mm ^2, cell adhesion density 3 days reached 450 / mm ^2, a cover glass treated with coating agent acted effectively as a material for cell culture.

By coating the surface of the carrier of various materials and shapes with the random copolymer of the present invention, the influence on the cell of the surface of the carrier is reduced, and the culture of the cell, storage of the cultured cell, collection of the cultured cell are reduced. And the convenience of handling such as transfer can be improved, and therefore, application as biocompatible materials and cell culture materials is expected.

¹ H-NMR spectrum chart (CDCl ₃ solution) of the random copolymer obtained in Example 1. ¹ H-NMR spectrum chart (CDCl ₃ solution) of the random copolymer obtained in Example 2. ¹ H-NMR spectrum chart (CDCl ₃ solution) of the random copolymer obtained in Example 3. ¹ H-NMR spectrum chart (CDCl ₃ solution) of the random copolymer obtained in Example 4. ¹ H-NMR spectrum chart (CDCl ₃ solution) of the random copolymer obtained in Example 5. ¹ H-NMR spectrum chart (CDCl ₃ solution) of the random copolymer obtained in Example 6.

Claims (6)

The following general formula (1)

(Wherein R ¹ represents a hydrogen atom, an alkyl group having 1 to 12 carbon atoms or an acyl group having 1 to 12 carbon atoms), and the following general formula (2)

(In the formula, R ² represents an acyl group having 2 to 18 carbon atoms). The random copolymer according to claim 1, wherein R ¹ is an alkyl group having 2 to 3 carbon atoms or an acyl group having 2 to 3 carbon atoms, and R ² is an acyl group having 3 to 7 carbon atoms. Polymer. The following general formula (3)

(Wherein R ¹ represents a hydrogen atom, an alkyl group having 1 to 12 carbon atoms, or an acyl group having 1 to 12 carbon atoms), and the following general formula (
4)

(Wherein R ² represents an acyl group having 2 to 18 carbon atoms) Random copolymer of an N-vinylimidazolidin-2-one compound and vinyl esters which undergo radical polymerization of vinyl esters represented by Manufacturing method. The random copolymer according to claim 3, wherein R ¹ is an alkyl group having 2 to 3 carbon atoms or an acyl group having 2 to 3 carbon atoms, and R ² is an acyl group having 3 to 7 carbon atoms. Production method of polymer.   The coating agent containing the random copolymer of Claim 1 or 2.   A cell culture material obtained by treatment with the coating agent according to claim 5.

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