JP7660998B2 - Acyl neutral amino acid ester - Google Patents

Description

The present invention relates to acyl neutral amino acid esters, etc.

The stratum corneum has two functions: a moisturizing function that retains moisture inside, and a barrier function that prevents the intrusion of foreign substances (e.g., foreign bodies, viruses, and bacteria) from the outside world. Substances that affect the stratum corneum's functions include sebum, intercellular lipids, and natural moisturizing factors.

Profilaggrin stored in the granular layer of skin tissue undergoes dephosphorylation and is broken down into filaggrin during keratinization. Filaggrin migrates from the granular layer to the stratum corneum, where it binds to and aggregates with keratin fibers to form a keratin pattern. Filaggrin is then broken down into low molecular weight substances such as urocanic acid, which become natural moisturizing factors involved in water retention and UV absorption. Therefore, it is believed that the more the production of filaggrin is promoted, the greater the amount of the natural moisturizing factors that are its decomposition products, and the better the skin's moisturizing ability.

The keratinocytes that make up the stratum corneum and the intercellular lipids that exist between the keratinocytes prevent foreign substances from entering from the outside world. Therefore, if the number or size of the keratinocytes is insufficient, gaps will appear in the stratum corneum, reducing its barrier function.

Around keratinocytes, which contain structures formed by the aggregation of keratin such as keratin 10 and filaggrin, there exists the marginal zone, which is an extremely tough and huge insoluble structure that lines the cell membrane of keratinocytes. The main structural elements of the marginal zone are proteins such as involucrin produced in spinous cells and loricrin produced in granular cells, which are cross-linked by enzymes such as transglutaminase (TG) 1. Therefore, filaggrin, involucrin, transglutaminase 1, keratin 10, and loricrin are thought to strongly affect the formation of keratinocytes and are related to the barrier function of the skin.

It has been reported that the expression of filaggrin, involucrin, transglutaminase 1, keratin 10, and loricrin is promoted by certain peptides such as glycylproline (Patent Document 1).

International Publication No. 2014/175001

However, certain peptides such as glycylproline have a weak effect of promoting the expression of moisturizing-related proteins such as filaggrin, and are therefore thought to be insufficient in improving the moisturizing ability of the stratum corneum to retain moisture. Such certain peptides also have a weak effect of promoting the expression of proteins related to the skin's barrier function, and are thought to be insufficient in improving the barrier function that prevents the intrusion of foreign substances. In other words, such certain peptides are thought to be insufficient in improving keratinocyte function.

Furthermore, maintaining moisture in the stratum corneum is extremely important for skin health, and if moisture could be maintained not only by retaining moisture inside the stratum corneum but also by an action from outside the stratum corneum, it would be possible to more effectively maintain moisture in each layer. The function of improving the dryness of the stratum corneum and smoothing the stratum corneum, as well as the function of improving moisture retention (action outside the stratum corneum), are called emollient properties, but the specific peptides mentioned above are not known to have emollient properties.

The present invention aims to provide a compound that can improve the stratum corneum function and has excellent emollient properties.

As a result of extensive research, the inventors discovered that a specific acyl neutral amino acid ester is a promising ingredient that can improve stratum corneum function and has excellent emollient properties, and thus completed the present invention.

〔式中、
Ａは、炭素原子数２～１０の直鎖もしくは分岐鎖の炭化水素基であり、
Ｂは、炭素原子数２～１８の直鎖もしくは分岐鎖の炭化水素基であり、
Ｘは、炭素原子数１～５の直鎖もしくは分岐鎖の炭化水素基である。〕で表される化合物。
〔２〕前記式（１）で表される化合物が、下記式（２）：

〔式中、
ＡおよびＢはそれぞれ、前記式（１）のものと同じである。〕、
下記式（３）：

〔式中、
ＡおよびＢはそれぞれ、前記式（１）のものと同じである。〕、
下記式（４）：

〔式中、
ＡおよびＢはそれぞれ、前記式（１）のものと同じである。〕、あるいは
下記式（５）：

〔式中、
ＡおよびＢはそれぞれ、前記式（１）のものと同じである。〕のいずれか一つで表される化合物である、〔１〕の化合物。
〔３〕Ｂが、炭素原子数２～８の直鎖もしくは分岐鎖の炭化水素基である、〔１〕または〔２〕の化合物。
〔４〕Ｂが、炭素原子数２もしくは３の直鎖もしくは分岐鎖の炭化水素基である、〔３〕の化合物。
〔５〕Ａが、炭素原子数７～１０の直鎖もしくは分岐鎖の炭化水素基である、〔１〕～〔４〕のいずれかの化合物。
〔６〕Ａが、炭素原子数２～６の直鎖もしくは分岐鎖の炭化水素基である、〔１〕～〔４〕のいずれかの化合物。
〔７〕Ａ、ＢおよびＸにおける炭化水素基がアルキルである、〔１〕～〔６〕のいずれかの化合物。
〔８〕〔１〕～〔７〕のいずれかの化合物、および担体を含む、組成物。
〔９〕〔１〕～〔７〕のいずれかの化合物を含む、皮膚外用剤。
〔１０〕〔１〕～〔７〕のいずれかの化合物を含む、化粧料。 That is, the present invention is as follows.
[1] The following formula (1):

[Wherein,
A is a linear or branched hydrocarbon group having 2 to 10 carbon atoms;
B is a linear or branched hydrocarbon group having 2 to 18 carbon atoms;
and X is a straight or branched chain hydrocarbon group having 1 to 5 carbon atoms.
[2] The compound represented by the formula (1) is represented by the following formula (2):

[Wherein,
A and B are the same as those in the above formula (1).
The following formula (3):

[Wherein,
A and B are the same as those in the above formula (1).
The following formula (4):

[Wherein,
A and B are the same as those in the above formula (1), or the following formula (5):

[Wherein,
A and B are the same as those in the above formula (1), respectively.
[3] The compound according to [1] or [2], wherein B is a linear or branched hydrocarbon group having 2 to 8 carbon atoms.
[4] The compound of [3], wherein B is a straight-chain or branched-chain hydrocarbon group having 2 or 3 carbon atoms.
[5] The compound according to any one of [1] to [4], wherein A is a linear or branched hydrocarbon group having 7 to 10 carbon atoms.
[6] The compound according to any one of [1] to [4], wherein A is a linear or branched hydrocarbon group having 2 to 6 carbon atoms.
[7] The compound according to any one of [1] to [6], wherein the hydrocarbon groups in A, B and X are alkyl.
[8] A composition comprising a compound according to any one of [1] to [7] and a carrier.
[9] An external skin preparation comprising any one of the compounds according to [1] to [7].
[10] A cosmetic comprising any one of the compounds according to [1] to [7].

The compound of the present invention can enhance the production of various proteins related to the improvement of stratum corneum function (e.g., the moisturizing ability to retain moisture inside the stratum corneum, and the barrier ability of the stratum corneum to suppress the intrusion of foreign substances from the outside world).
The compounds of the present invention can also exhibit excellent emollient properties.
Therefore, the compound of the present invention can improve the water retention in the stratum corneum. For example, the improvement in the water retention in the stratum corneum can be achieved from the inside or outside of the stratum corneum, or both.
The compounds of the present invention can also be stably formulated with hydrophilic organic solvents and hydrophobic organic solvents (oils) within an effective concentration range.
The compound of the present invention can also exhibit transparent properties in a mixture of the compound of the present invention and a hydrophobic organic solvent (oil) and in a mixture of the compound of the present invention and a hydrophilic organic solvent, so that it can impart a sense of luxury to the appearance and can also avoid visual effects on the skin. Therefore, the compound of the present invention has an advantage that, for example, white residue caused by application can be reduced.

1 is a diagram showing the amplification amount (relative value to the amplification amount of the control) of each moisturizing-related protein gene of octanoylvaline (Example 3: C8Val), octanoylalanine (Example 4: C8Ala), glycylproline (Comparative Example 2: Gly-Pro), and valine (Comparative Example 3: Val). The moisturizing-related protein genes tested are, from the left, genes encoding filaggrin, involucrin, transglutaminase 1 (TG1), keratin 10, and loricrin (similar to FIG. 2). FIG. 2 is a diagram showing the amplification amount (relative value to the amplification amount of the control) of each moisturizing-related protein gene for octanoyl leucine (Example 5: C8Leu), octanoyl isoleucine (Example 6: C8Ile), octanoyl glycine (Example 7: C8Gly), glycylproline (Comparative Example 2: Gly-Pro), and valine (Comparative Example 3: Val). 3 is a diagram showing the amplification amount (relative value to the amplification amount of the control) of each moisturizing-related protein gene of butanoyl valine (Example 8: C4Val), hexanoyl valine (Example 9: C6Val), octanoyl valine (Example 10: C8Val), decanoyl valine (Example 11: C10Val), lauroyl (dodecanoyl) valine (Example 12: C12Val) and methanoyl valine (Example 13: C2Val). The moisturizing-related protein genes tested are, from the left, genes encoding filaggrin, involucrin, and transglutaminase 1 (TG1). FIG. 4 shows the amount of amplification of the filaggrin gene (relative to the amount of amplification of the control) for lauroyl valine (Example 14: C12Val), decanoyl valine (Example 15: C10Val), octanoyl valine (Example 16: C8Val), hexanoyl valine (Example 17: C6Val), butanoyl valine (Example 18: C4Val), and acetyl valine (Example 19: C2Val). FIG. 5 shows the amount of amplification of the filaggrin gene (relative to the amount of amplification of the control) for lauroylalanine (Example 20: C12Ala), decanoylalanine (Example 21: C10Ala), octanoylalanine (Example 22: C8Ala), hexanoylalanine (Example 23: C6Ala), and butanoylalanine (Example 24: C4Ala). FIG. 6 shows the absorbance of lauroyl valine (Comparative Example 4: C12Val), decanoyl valine (Example 25: C10Val), octanoyl valine (Example 26: C8Val), hexanoyl valine (Example 27: C6Val), butanoyl valine (Example 28: C4Val), and acetyl valine (Example 29: C2Val) in cells (percentage of the absorbance of the control). 7 shows HPLC (Shimadzu) chromatograms (measurement wavelength 210 nm) of N-octanoylalanine produced by hydrolysis of C8Ala-iPA in the presence or absence of esterase. In the chromatogram, a peak for N-octanoylalanine is observed at elution time 0.6 minutes, and a peak for C8Ala-iPA is observed at elution time 1.7 minutes. When C8Ala-iPA is reacted with esterase, the peak at 1.7 minutes decreases, and a new peak appears at 0.6 minutes. 8 shows HPLC (Shimadzu) chromatograms (measurement wavelength 210 nm) of N-octanoylalanine produced by hydrolysis of C8Ala-Et in the presence or absence of esterase. On the chromatogram, a peak for N-octanoylalanine is observed at elution time 0.6 minutes, and a peak for C8Ala-Et is observed at elution time 1.2 minutes. When C8Ala-Et is reacted with esterase, the peak at 1.2 minutes decreases, and a new peak appears at 0.6 minutes.

The present invention provides a compound represented by the above formula (1).

A is a straight or branched chain hydrocarbon group having 2 to 10 carbon atoms. The number of carbon atoms in the hydrocarbon group in A may be, for example, 5 to 10. The hydrocarbon group in A is saturated or unsaturated. Examples of such hydrocarbon groups include alkyl, alkenyl, and alkynyl.

When A is an alkyl, the alkyl is a straight-chain or branched-chain alkyl having 2 to 10 carbon atoms. Examples and preferred examples of the number of carbon atoms of the alkyl in A are the same as the number of carbon atoms of the hydrocarbon group in A described above. Examples of the alkyl in A include ethyl, propyl (e.g., n-propyl, isopropyl), butyl (e.g., n-butyl, iso-butyl, sec-butyl, tert-butyl), pentyl (e.g., n-pentyl, iso-pentyl, neo-pentyl, 1-ethylpropyl), hexyl (e.g., n-hexyl, iso-hexyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 2-ethylbutyl), heptyl (e.g., n-heptyl, 1-methylhexyl), , 2-methylhexyl, 3-methylhexyl, 1,1-dimethylpentyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl), octyl (e.g., n-octyl, 1-methylheptyl, 2-methylheptyl, 3-methylheptyl, 1,1-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 2-ethylhexyl), nonyl (e.g., n-nonyl, 3,5,5-trimethylhexyl), and decanyl (e.g., n-decanyl).

When A is alkenyl, the alkenyl is a straight-chain or branched-chain alkenyl having 2 to 10 carbon atoms. Examples and preferred examples of the number of carbon atoms of the alkenyl in A are the same as the number of carbon atoms of the hydrocarbon group in A described above. Examples of the alkenyl in A include ethenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, and decenyl.

When A is alkynyl, the alkynyl is a straight or branched alkynyl having 2 to 10 carbon atoms. Examples and preferred examples of the number of carbon atoms of the alkynyl in A are the same as the number of carbon atoms of the above-mentioned hydrocarbon group in A. Examples of the alkynyl in A include ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, and decynyl.

Preferably, A is a linear or branched alkyl having 2 to 10 carbon atoms. Examples and preferred examples of the number of carbon atoms of the alkyl in A are the same as the number of carbon atoms of the hydrocarbon group in A described above. Specific examples of such alkyl are as described above.

In certain embodiments, the group in A may have 7 to 10 carbon atoms (preferably 8 to 10). When the group in A has 7 to 10 carbon atoms, the compound represented by the above formula (1) has the advantage of being able to significantly induce the expression of genes encoding various proteins related to the improvement of stratum corneum function. Examples of such proteins include proteins that can generate natural moisturizing factors in skin tissue (e.g., filaggrin, profilaggrin), and proteins that can be involved in skin barrier function (e.g., filaggrin, involucrin, transglutaminase 1, keratin 10, loricrin).

In another specific embodiment, the number of carbon atoms in the group in A may be 6 or less (i.e., 2 to 6). Even if the number of carbon atoms in the group in A is 6 or less, the compound represented by the above formula (1) has a partition coefficient (Log P) in the range of 1 to 4 and a molecular weight of 500 Da or less, and therefore has the advantages of excellent cost absorption and being able to be provided in the stratum corneum at a high concentration.

B is a straight or branched chain hydrocarbon group having 2 to 18 carbon atoms. The number of carbon atoms in the hydrocarbon group in B is preferably 2 to 15, more preferably 2 to 12, even more preferably 2 to 8, even more preferably 2 to 6, and particularly preferably 2 or 3. The hydrocarbon group in B is saturated or unsaturated. Examples of such hydrocarbon groups include alkyl, alkenyl, and alkynyl.

When B is an alkyl, the alkyl is a straight-chain or branched-chain alkyl having 2 to 18 carbon atoms. Examples and preferred examples of the number of carbon atoms of the alkyl in B are the same as the number of carbon atoms of the hydrocarbon group in B described above. Examples of the alkyl in B include ethyl, propyl (e.g., n-propyl, isopropyl), butyl (e.g., n-butyl, iso-butyl, sec-butyl, tert-butyl), pentyl (e.g., n-pentyl, iso-pentyl, neo-pentyl, 1-ethylpropyl), hexyl (e.g., n-hexyl, iso-hexyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 2-ethylbutyl), heptyl (e.g., n-heptyl, 1-methylhexyl, 2-methylhexyl, 3-methylhexyl, 1,1-dimethylpentyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl), octyl (e.g., n- octyl, 1-methylheptyl, 2-methylheptyl, 3-methylheptyl, 1,1-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 2-ethylhexyl), nonyl (e.g., n-nonyl, 3,5,5-trimethylhexyl), decanyl (e.g., n-decanyl), undecanyl (e.g., n-undecanyl), dodecanyl (e.g., n-dodecanyl), tridecanyl (e.g., n-tridecanyl), tetradecanyl (e.g., n-tetradecanyl), pentadecanyl (e.g., n-pentadecanyl), hexadecanyl (e.g., n-hexadecanyl), heptadecanyl (e.g., n-heptadecanyl), and octadecanyl (e.g., n-octadecanyl).

When B is alkenyl, the alkenyl is a straight or branched alkenyl having 2 to 18 carbon atoms. Examples and preferred examples of the number of carbon atoms of the alkenyl in B are the same as the number of carbon atoms of the hydrocarbon group in B described above. Examples of the alkenyl in B include ethenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, dodecenyl, tridecenyl, tetradecenyl, pentadecenyl, hexadecenyl, heptadecenyl, and octadecenyl.

When B is alkynyl, the alkynyl is a straight or branched alkynyl having 2 to 18 carbon atoms. Examples and preferred examples of the number of carbon atoms of the alkynyl in B are the same as the number of carbon atoms of the hydrocarbon group in B described above. Examples of the alkynyl in B include ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl, undecynyl, dodecynyl, tridecynyl, tetradecynyl, pentadecynyl, hexadecynyl, heptadecynyl, and octadecynyl.

Preferably, B is a linear or branched alkyl having 2 to 18 carbon atoms. Examples and preferred examples of the number of carbon atoms of the alkyl in B are the same as the number of carbon atoms of the hydrocarbon group in B described above. Specific examples of such alkyl are as described above.

X is a straight or branched chain hydrocarbon group having 1 to 5 carbon atoms. The number of carbon atoms in the hydrocarbon group in X may be, for example, 1 to 4. The hydrocarbon group in X is saturated or unsaturated. Examples of such hydrocarbon groups include alkyl, alkenyl, and alkynyl.

When X is an alkyl, the alkyl is a straight-chain or branched-chain alkyl having 1 to 5 carbon atoms. Examples and preferred examples of the number of carbon atoms of the alkyl in X are the same as the number of carbon atoms of the hydrocarbon group in X described above. Examples of the alkyl in X include methyl, ethyl, propyl (e.g., n-propyl, isopropyl), butyl (e.g., n-butyl, iso-butyl, sec-butyl, tert-butyl), and pentyl (e.g., n-pentyl, iso-pentyl, neo-pentyl, 1-ethylpropyl).

When X is alkenyl, the alkenyl is a straight-chain or branched-chain alkenyl having 2 to 5 carbon atoms. Examples and preferred examples of the number of carbon atoms of the alkenyl in X are the same as the number of carbon atoms of the above-mentioned hydrocarbon group in X. Examples of the alkenyl in X include ethenyl, propenyl, butenyl, and pentenyl.

When X is alkynyl, the alkynyl is a straight-chain or branched-chain alkynyl having 2 to 5 carbon atoms. Examples and preferred examples of the number of carbon atoms of the alkynyl in X are the same as the number of carbon atoms of the above-mentioned hydrocarbon group in X. Examples of the alkynyl in X include ethynyl, propynyl, butynyl, and pentynyl.

Preferably, X is a linear or branched alkyl having 1 to 5 carbon atoms. Examples and preferred examples of the number of carbon atoms of the alkyl in X are the same as the number of carbon atoms of the hydrocarbon group in X described above. Specific examples of such alkyl are as described above.

Preferably, the compound represented by the above formula (1) is a compound represented by any one of the above formulas (2) to (5). With respect to A and B in the compounds represented by the above formulas (2) to (5), the definitions, examples, preferred examples and specific examples of the hydrocarbon group (e.g., alkyl, alkenyl, alkynyl) and the number of carbon atoms are the same as those described above for formula (1).

In a specific embodiment, A and B in the compounds represented by the above formulas (2) to (5) may each independently have a different number of carbon atoms and a different hydrocarbon group. For example, A in the compounds represented by the above formulas (2) to (4) may be a linear or branched chain hydrocarbon group having 2 to 10 carbon atoms, and A in the compound represented by the above formula (5) may be a linear or branched chain hydrocarbon group having 6 or less carbon atoms, or 7 to 10 (preferably 8 to 10) carbon atoms. Also, A in the compounds represented by the above formulas (2) to (4) may be a linear or branched chain hydrocarbon group having 2 to 10 carbon atoms, and A in the compound represented by the above formula (5) may be a linear or branched chain hydrocarbon group having 7 to 10 (preferably 8 to 10) carbon atoms. Furthermore, A in the compounds represented by the above formulas (2) to (4) may be a linear or branched chain hydrocarbon group having 5 to 10 carbon atoms, and A in the compound represented by the above formula (5) may be a linear or branched chain hydrocarbon group having 7 to 10 (preferably 8 to 10) carbon atoms.

The compounds represented by formulas (1) to (5) correspond to N-acyl neutral amino acid esters (neutral amino acid derivatives). The compounds represented by formulas (1) to (5) may be optical isomers (L-form or D-form) with respect to the nitrogen atom, the carbon atom of the carbonyl group, and the carbon atom to which X is bonded, or may be a mixture thereof. Preferably, the compounds represented by formulas (1) to (5) are L-forms.

The preparation of the compound of the present invention can be suitably carried out by reacting an N-acyl neutral amino acid and an alcohol. The reaction of an N-acyl neutral amino acid and an alcohol can be carried out by a general method for producing an ester from a carboxylic acid compound and an alcohol. Such reactions are well known in the art. For example, the reaction can be carried out by appropriately adding a catalyst. The reaction can also be carried out at an appropriate temperature (e.g., 15 to 200°C) for several hours (e.g., 0.5 to 8 hours). More specifically, the preparation of the compound of the present invention can be carried out by the method of Production Example A described below. The preparation of the compound of the present invention can also be carried out in the same manner as the method described in JP-A-11-240828, except that the types of N-acyl neutral amino acid and alcohol are changed in the method described in JP-A-11-240828. The compound of the present invention can be suitably purified by any purification method (e.g., extraction) used in the field of organic synthesis.

The compound of the present invention is useful as an agent for improving keratin function and a moisturizing agent, and for the development of products such as cosmetics, medicines, and quasi-drugs. The compound of the present invention may be provided in the form of a composition containing the compound of the present invention and a carrier (e.g., a carrier acceptable for cosmetics, medicines, or quasi-drugs). Examples of such carriers include thickeners, stabilizers, pH adjusters, preservatives, UV protection agents, fragrances, and dyes. The specific types and amounts of these components can be set appropriately. Such compositions can be prepared as compositions in any state. Examples of such compositions include liquid compositions, cream compositions, emulsion compositions, powder compositions, stick compositions, and sheet-impregnated compositions.

For example, the compounds of the present invention can be used by applying (e.g., spreading, administering) an effective amount (e.g., 0.001 mg to 10 g) to a desired site (e.g., skin, hair, body hair, scalp) of a subject (e.g., mammals such as humans, animals such as birds and reptiles). Preferably, the compounds of the present invention are applied to humans. The condition of the subject to which the compounds of the present invention are applied is a healthy condition or an abnormal condition (e.g., disease). Such abnormal conditions include, for example, rough skin, dry skin, scales, disturbances in turnover, and skin diseases (e.g., dermatitis such as atopic dermatitis).

The compound of the present invention may also be a cosmetic or topical agent. The cosmetic or topical agent of the present invention may be prepared in any form that can be applied to a desired site (e.g., skin, hair, scalp) according to a conventional method.

The compound of the present invention can be preferably used as a cosmetic or topical agent for the skin, hair, or scalp. Examples of the cosmetic or topical agent for the skin include milky lotion, lotion, cream, gel, beauty serum, and face mask. Examples of the cosmetic or topical agent for the hair include hair milky lotion, hair treatment, hair conditioner, shampoo, and hair lotion. Examples of the cosmetic or topical agent for the scalp include hair growth agents. Examples of preferred cosmetic agents include leave-on cosmetic agents, milky lotion, lotion, cream, gel, beauty serum, and face mask. Examples of preferred topical agents include ointments, creams, mousses, and gels.

The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples. All of the N-acyl neutral amino acids used as raw materials in the following production examples are L-form.

Preparation Example A: Synthesis of N-acyl neutral amino acid ester from alcohol and N-acyl neutral amino acid Ethanol (320 mL, 5.48 mol) or isopropyl alcohol (418 mL, 5.48 mol) was added with N-acyl neutral amino acid (0.14 mol) and mixed. p-Toluenesulfonic acid monohydrate (5.3 g, 0.03 mol) was added to the mixture, a reflux tube was attached to prevent the alcohol from evaporating, and the mixture was stirred at a bath temperature of 125°C for 3 hours. The product was concentrated under reduced pressure in vacuum, then diluted with ethyl acetate (600 mL) and washed four times with saturated sodium bicarbonate water (240 mL). The organic phase was further washed twice with ultrapure water (480 mL). Water was removed from the organic layer with sodium sulfate. Furthermore, sodium acid was removed by filtration, and the organic layer was concentrated under reduced pressure in vacuum to obtain the product (acyl neutral amino acid ester).

Production Example 1: Production of N-octanoylalanine isopropyl ester (C8Ala-iPA)

N-octanoylalanine (C8Ala, 30.00 g, 0.14 mol) was used as the N-acyl amino acid, and isopropyl alcohol (418 mL, 5.48 mol) was used as the alcohol. C8Val-iPA was obtained (yield 97%) by reacting according to the synthesis method of Example A.

¹ H-NMR (400 MHz, CDCl ₃ , r.t.): δ6.077-6.093 (1H, d, J=6.4Hz), 5.015-5.078 (1H, sep, 6.4Hz), 4.511-4.583 (1H, quint, J = 7.2Hz), 2.184-2.222 (2H, t, J =7.6Hz), 1.599-1.669 (2H, quint, J=7.2Hz), 1.372-1.390 (3H, d, J=7.2Hz) 1.244-1.298 (14H, m), 0.859-0.892 (3H, t, J=6.8Hz) ppm.

ESI-MS (positive) m/z 258.2[M+H] ⁺ ,280.1[M+Na] ⁺

Production Example 2: Production of N-octanoyl valine isopropyl ester (C8Val-iPA)

N-octanoyl valine (C8Val, 34.06 g, 0.14 mol) was used as the N-acyl amino acid, and isopropyl alcohol (418 mL, 5.48 mol) was used as the alcohol. The reaction was carried out for 8 hours according to the synthesis method of Example A to obtain C8Val-iPA (yield 93%).

¹ H-NMR (400 MHz, CDCl ₃ , r. 2Hz), 2.135-2.182 (1H, sex, J = 6.8Hz), 1.610-1.765 (2H, quint, J = 7.2Hz), 1.256-1.314 (14H, m), 0.860-0.949 (9H, m) ppm.

ESI-MS (positive) m/z 286.2[M+H] ⁺ ,308.1[M+Na] ⁺

Production Example 3: Production of N-octanoylglycine isopropyl ester (C8Gly-iPA)

N-octanoylglycine (C8Gly, 28.18 g, 0.14 mol) was used as the N-acyl amino acid, and isopropyl alcohol (418 mL, 5.48 mol) was used as the alcohol. C8Gly-iPA was obtained by reacting according to the synthesis method of Example A (yield 97%).

ESI-MS (positive) m/z 244.1[M+H] ⁺ ,266.1[M+Na] ⁺

Production Example 4: Production of N-octanoyl isoleucine isopropyl ester (C8Ile-iPA)

N-octanoylisoleucine (C8Ile, 36.03 g, 0.14 mol) was used as the N-acyl amino acid, and isopropyl alcohol (418 mL, 5.48 mol) was used as the alcohol. C8Ile-iPA was obtained by reacting according to the synthesis method of Production Example A (yield 71%).

ESI-MS (positive) m/z 300.2[M+H] ⁺ ,322.2[M+Na] ⁺

Production Example 5: Production of N-octanoyl leucine isopropyl ester (C8Leu-iPA)

N-octanoylleucine (C8Leu, 36.03 g, 0.14 mol) was used as the N-acyl amino acid, and isopropyl alcohol (418 mL, 5.48 mol) was used as the alcohol. The reaction was carried out according to the synthesis method of Example A to obtain C8Leu-iPA (yield 97%).

ESI-MS (positive) m/z 300.2[M+H] ⁺ ,322.2[M+Na] ⁺

Production Example 6: Production of N-lauroyl isoleucine isopropyl ester (C12Ile-iPA)

N-lauroylisoleucine (C12Ile, 43.89 g, 0.14 mol) was used as the N-acyl amino acid, and isopropyl alcohol (418 mL, 5.48 mol) was used as the alcohol. C12Ile-iPA was obtained (yield 85%) by reacting according to the synthesis method of Production Example A.

ESI-MS (positive) m/z 356.3 [M+H] ⁺ ,378.2 [M+Na] ⁺

Production Example 7: Production of N-octanoylalanine ethyl ester (C8Ala-Et)

N-octanoylalanine (C8Ala, 30.00 g, 0.14 mol) was used as the N-acyl amino acid, and ethanol (320 mL, 5.48 mol) was used as the alcohol. The reaction was carried out according to the synthesis method of Example A to obtain C8Val-Et (yield 90%).

^1H -NMR (400MHz, CDCl ₃ , rt.): δ6.063-6.078 (1H, d, J = 6.0Hz), 4.552-4.624 (1H, quint, J = 7.2Hz), 4.176-4.230 (2H, q, J=7.2Hz), 2.186-2.224 (2H, t, J=7.6Hz), 1.599-1.670 (2H, quint, J=7.2Hz), 1.389-1.407 (3H, d, J=7.2Hz) 1.267-1.302 (11H, m), 0.860-0.892 (3H, m) ppm.

ESI-MS (positive) m/z 244.2[M+H] ⁺ ,266.1[M+Na] ⁺

Production Example 8: Production of N-octanoyl valine ethyl ester (C8Val-Et)

N-octanoylvaline (C8Val, 34.06 g, 0.14 mol) was used as the N-acyl amino acid, and ethanol (320 mL, 5.48 mol) was used as the alcohol. The reaction was carried out for 8 hours according to the synthesis method of Example A to obtain C8Val-Et (yield 93%).

¹ H-NMR (400 MHz, CDCl ₃ , r. 2.121-2.202 (1H, sex, J = 6.8Hz), 1.610-1.681 (2H, quint, J = 7.2Hz), 1.268-1.303 (11H, m), 0.860-0.952 (9H, m) ppm.

ESI-MS (positive) m/z 272.2[M+H] ⁺ ,294.1[M+Na] ⁺

Production Example 9: Production of N-octanoylglycine ethyl ester (C8Gly-Et)

N-octanoylglycine (C8Gly, 28.18 g, 0.14 mol) was used as the N-acyl amino acid, and ethanol (320 mL, 5.48 mol) was used as the alcohol. The reaction was carried out according to the synthesis method of Example A to obtain C8Gly-Et (yield 96%).

ESI-MS (positive) m/z 230.2[M+H] ⁺ ,252.1[M+Na] ⁺

Production Example 10: Production of N-octanoyl isoleucine ethyl ester (C8Ile-Et)

N-octanoylisoleucine (C8Ile, 36.03 g, 0.14 mol) was used as the N-acyl amino acid, and ethanol (320 mL, 5.48 mol) was used as the alcohol. The reaction was carried out according to the synthesis method of Example A to obtain C8Ile-Et (yield 97%).

ESI-MS (positive) m/z 286.2[M+H] ⁺ ,308.2[M+Na] ⁺

Production Example 11: Production of N-octanoyl leucine ethyl ester (C8Leu-Et)

N-octanoylleucine (C8Leu, 36.03 g, 0.14 mol) was used as the N-acyl amino acid, and ethanol (320 mL, 5.48 mol) was used as the alcohol. The reaction was carried out according to the synthesis method of Example A to obtain C8Gly-Et (yield 98%).

ESI-MS (positive) m/z 286.2[M+H] ⁺ ,308.2[M+Na] ⁺

Production Example 12: Production of N-lauroyl isoleucine ethyl ester (C12Ile-Et)

N-lauroylisoleucine (C12Ile, 43.89 g, 0.14 mol) was used as the N-acyl amino acid, and ethanol (320 mL, 5.48 mol) was used as the alcohol. The reaction was carried out according to the synthesis method of Example A to obtain C12Ile-Et (yield 91%).

ESI-MS (positive) m/z 342.3[M+H] ⁺ ,364.3[M+Na] ⁺

Production Example 13: Production of N-lauroyl leucine ethyl ester (C12Leu-Et)

N-lauroylleucine (C12Leu, 43.89 g, 0.14 mol) was used as the N-acyl amino acid, and ethanol (320 mL, 5.48 mol) was used as the alcohol. The reaction was carried out according to the synthesis method of Example A to obtain C12Leu-Et (yield 88%).

ESI-MS (positive) m/z 342.3[M+H] ⁺ ,364.3[M+Na] ⁺

Production Example 14: Production of N-lauroyl valine ethyl ester (C12Val-Et)

N-lauroylvaline (C12Val, 41.92 g, 0.14 mol) was used as the N-acyl amino acid, and ethanol (320 mL, 5.48 mol) was used as the alcohol. The reaction was carried out for 8 hours according to the synthesis method of Example A to obtain C12Val-Et (yield 87%).

ESI-MS (positive) m/z 328.3[M+H] ⁺ ,350.2[M+Na] ⁺

Production Example 15: Production of N-decanoyl valine isopropyl ester (C10Val-iPA)

N-decanoyl valine (C10Val, 38 g, 0.14 mol) was used as the N-acyl amino acid, and isopropyl alcohol (418 mL, 5.48 mol) was used as the alcohol. C10Val-iPA was obtained (yield 98%) by reacting according to the synthesis method of Example A.

ESI-MS (positive) m/z 314.5[M+H] ⁺ ,336.4[M+Na] ⁺

Production Example 16: Production of N-decanoylalanine isopropyl ester (C10Ala-iPA)

N-decanoylalanine (C10Ala, 34.6 g, 0.14 mol) was used as the N-acyl amino acid, and isopropyl alcohol (418 mL, 5.48 mol) was used as the alcohol. The reaction was carried out according to the synthesis method of Example A to obtain C10Ala-iPA (yield 97%).

ESI-MS (positive) m/z 285.4 [M+H] ⁺ , 307.4 [M+Na] ⁺

Production Example 17: Production of N-decanoyl leucine isopropyl ester (C10Leu-iPA)

N-decanoyl leucine (C10Leu, 39.9 g, 0.14 mol) was used as the N-acyl amino acid, and isopropyl alcohol (418 mL, 5.48 mol) was used as the alcohol. The reaction was carried out according to the synthesis method of Example A to obtain C10Leu-iPA (yield 97%).

ESI-MS (positive) m/z 328.5 [M+H] ⁺ , 350.3 [M+Na] ⁺

Production Example 18: Production of N-hexanoyl valine ethyl ester (C6Val-Et)

N-hexanoyl valine (C6Val, 30.14 g, 0.14 mol) was used as the N-acyl amino acid, and ethanol (320 mL, 5.48 mol) was used as the alcohol. The reaction was carried out for 8 hours according to the synthesis method of Example A to obtain C6Val-Et (yield 82%).

ESI-MS (positive) m/z 244.3[M+H] ⁺ ,266.2[M+Na] ⁺

Reference Production Examples 1 to 3: Production of N-lauryl valine isopropyl ester (C12 Val-iPA), N-lauryl alanine isopropyl ester (C12 Ala-iPA), and N-lauryl alanine ethyl ester (C12 Ala-Et) N-lauryl valine isopropyl ester (C12 Val-iPA), N-lauryl alanine isopropyl ester (C12 Ala-iPA), and N-lauryl alanine ethyl ester (C12 Ala-Et) were produced according to the description in JP-A-11-240828.

Example 1: Evaluation of acyl neutral amino acid esters (1) Evaluation of compatibility with hydrophobic organic solvents (oil) 1 g of each sample was weighed out, added to 5 g of liquid paraffin, and mixed by stirring for 10 minutes at 25° C. After stirring was stopped, the mixture was left to stand for 10 minutes at 25° C., and the state of separation of each sample and liquid paraffin was visually confirmed, and evaluation was performed according to the following criteria.
Very favorable (A): No separation was observed, and the solution was clear. Preferred (B): Almost no separation was observed, and the solution was clear. Not very favorable (C): Localized separation occurred, and the solution became cloudy. Not favorable (D): Clear separation occurred.

(2) Evaluation of compatibility with hydrophilic organic solvent 0.5 g of each sample was weighed out, added to 10 g of 1,3-butylene glycol, and mixed by stirring for 10 minutes at 25° C. After stirring was stopped, the mixture was allowed to stand for 10 minutes at 25° C., and the state of separation of each sample and 1,3-butylene glycol was visually confirmed, and evaluation was performed according to the following criteria.
Very favorable (A): No separation was observed, and the solution was clear. Preferred (B): Almost no separation was observed, and the solution was clear. Not very favorable (C): Localized separation occurred, and the solution became cloudy. Not favorable (D): Clear separation occurred.

(3) Property Evaluation Each compound was cooled to 4° C. for one day, and then allowed to stand at room temperature (25° C.) for three days. After three days, the properties of each compound were confirmed at room temperature (25° C.).
Preferred (A): The property is liquid at room temperature (25° C.). Therefore, it is easy to prepare a formulation by simply mixing with other liquid ingredients at room temperature without heating.
Unfavorable (B): The substance is in a solid or crystalline form at room temperature (25° C.), and it is difficult to prepare a formulation by simply mixing it with other liquid ingredients at room temperature without heating.

(4) Evaluation of Emollient Properties Next, emollient properties were evaluated. The backs of the hands of five expert panelists were washed with soap and then dried, and 200 mg of each evaluation sample was added thereto and spread evenly on the skin. After drying for 15 minutes at 25°C, the addition of each sample was evaluated according to the following criteria as to whether the dryness of the skin was improved and the skin was smoothed, and whether the moisturizing property was improved. Based on the average evaluation score of the panelists, the emollient properties during use were evaluated as follows.

<Evaluation of emollient properties during use>
3 points: Skin was very well conditioned, and moisturizing properties were improved very significantly. 2 points: Skin was well conditioned, and moisturizing properties were improved significantly. 1 point: Skin was not well conditioned, and moisturizing properties were not improved much. 0 points: Skin was not well conditioned at all, and moisturizing properties were not improved at all.

An average score of 2.4 or more by the expert panelists is an A.
1.5 or more but less than 2.4 is B,
0.5 or more but less than 1.5 is C,
Less than 0.5 is D
It was decided.

As a result, the acyl neutral amino acid ester could be stably blended with hydrophobic organic solvents (oils) and hydrophilic organic solvents (Table 1). Therefore, it was demonstrated that the acyl neutral amino acid ester is an excellent oil agent.
The acyl neutral amino acid ester also facilitated the preparation of the formulation (Table 1). Thus, the acyl neutral amino acid ester was shown to have excellent handling properties.
The acyl neutral amino acid ester also had excellent emollient properties (Table 1). Therefore, it was demonstrated that the acyl neutral amino acid ester can improve the retention of moisture in the stratum corneum from outside the stratum corneum through its emollient action (Table 1).

Example 2: Evaluation of hydrolysis of acyl neutral amino acid ester by esterase 5 mg of acyl neutral amino acid ester was added to 1x PBS buffer, and 2.15 μl (0.2 wt%) of Triton-X100 (manufactured by Aldrich) was added, followed by uniform mixing by ultrasonic irradiation. A buffer solution of esterase (Esterase from porcine liver, manufactured by Sigma-Aldrich) was added to the mixture so that esterase was 5 Units per molecule of acyl amino acid, and the mixture was mixed by stirring. This mixture was shaken and stirred for 24 hours in a shaker heated to 30°C to react. The reaction product was centrifuged with a centrifuge at 12000xg, and the supernatant was analyzed by high performance liquid chromatography (manufactured by Shimadzu). As a control, only buffer was added instead of the esterase solution. The esterase used (Esterase from porcine liver) was used as a model of the esterase enzyme present in human skin [Bonina, F. P. et al., Eur. J. Pharm. Sci. 14, 123-134 (2001); Wong, O. et al., Int. J. Pharm., 52, 191-202 (1989); Vavrova K. et al., J. Control Release. 104, 41-49 (2005)].

When the ester bond of the acyl neutral amino acid ester is hydrolyzed by esterase, the peak area of the acyl neutral amino acid ester decreases on the HPLC chromatogram, and a new peak is observed at the same elution time as the acyl neutral amino acid. The appearance of this peak allows us to conclude that the acyl neutral amino acid ester has been hydrolyzed by esterase to produce the acyl neutral amino acid.

The test substances used were the compounds produced in Production Examples 1 to 18 and Reference Production Examples 1 to 3.

In the presence of esterase, C6Val-Et, C8Ala-iPA, C8Val-iPA, C8Gly-iPA, C8Ile-iPA, C8Leu-iPA, C8Ala-Et, C8Val-Et, C8Gly-Et, C8Ile-Et, C8Leu-Et, C12Ile-Et, C10Ala-iPA, C10Val-iPA, C10Leu-iPA, C1 2Ala-Et, C12Ala-iPA, C12Val-Et, C12Val-iPA, C12Ile-iPA, and C12Leu-Et were all hydrolyzed to produce C6Val, C8Ala, C8Val, C8Gly, C8Ile, C8Leu, C10Ala, C10Val, C10Leu, C12Ala, C12Val, C12Ile, and C12Leu.

Examples 3 to 7 and Comparative Examples 2 to 3: Comparison of amplification amount of genes of moisturizing-related proteins, etc. by octanoyl neutral amino acids Human normal epidermal keratinocytes NHEK (NB) (Kurabo Industries, Ltd.) were pre-cultured in HuMedia-KG2 (Kurabo Industries, Ltd.) and adjusted to a concentration of ³⁰ x 104 cells/mL, and 2 mL of the adjusted culture solution (i.e., 60 x ¹⁰⁴ cells/well) was added to a 6-well plate and cultured at 37°C for 1 day. Samples (octanoyl valine (Example 3: C8Val), octanoyl alanine (Example 4: C8Ala), octanoyl leucine (Example 5: C8Leu), octanoyl isoleucine (Example 6: C8Ile), octanoyl glycine (Example 7: C8Gly), glycylproline (Comparative Example 2: Gly-Pro), and valine (Comparative Example 3: Val)) were added to the concentrations shown in each figure and reacted for 48 hours. After the reaction, total RNA was extracted from the cells, reverse transcribed, and the amount of amplification of target genes (filaggrin, involucrin, transglutaminase (TG) 1, keratin 10, and loricrin) was measured by real-time PCR (n=2), and compared with the amount of amplification of the control (no addition). GAPDH was used as a correction gene. The calculation was performed using the comparative CT method, the primers and probes used were TaqMan® Gene Expression, and the fluorescent dye used was FAM. The results are shown in Figures 1 and 2.

As is clear from Figures 1 and 2, the amount of amplification of each target gene in Examples 3 to 7 was higher than in Comparative Examples 2 and 3. These results indicate that acyl neutral amino acids can promote the production of moisturizing-related proteins.

Examples 8 to 13: Comparison of amplification amount of genes such as moisturizing-related proteins by acyl valine The same procedure as in Example 3 was carried out except that butanoyl valine (Example 8: C4Val), hexanoyl valine (Example 9: C6Val), octanoyl valine (Example 10: C8Val), decanoyl valine (Example 11: C10Val), lauroyl valine (Example 12: C12Val) and acetyl valine (Example 13: C2Val) were used as samples and that filaggrin, involucrin, and transglutaminase (TG) 1 were used as target genes. The results are shown in FIG. 3.

As is clear from Figure 3, the amount of amplification of each target gene was higher in all of Examples 8 to 13 compared to the control. These results indicate that acyl neutral amino acids or salts thereof having an acyl group with 2 to 12 carbon atoms can promote the production of moisturizing-related proteins regardless of the type of acyl group.

Examples 14 to 24: Comparison of the amount of filaggrin gene amplification by acyl valine or acylalanine Human normal epidermal keratinocytes NHEK (NB) (Kurabo Industries, Ltd.) were pre-cultured in HuMedia-KG2 (Kurabo Industries, Ltd.) and adjusted to a concentration of ¹⁵ x 104 cells/mL, and 2 mL of the adjusted culture solution (i.e., 30 x ¹⁰⁴ cells/well) was added to a 6-well plate and cultured at 37°C for 1 day. Samples (lauroyl valine (Example 14: C12Val), decanoyl valine (Example 15: C10Val), octanoyl valine (Example 16: C8Val), hexanoyl valine (Example 17: C6Val), butanoyl valine (Example 18: C4Val), acetyl valine (Example 19: C2Val), lauroyl alanine (Example 20: C12Ala), decanoyl alanine (Example 21: C10Ala), octanoyl alanine (Example 22: C8Ala), hexanoyl alanine (Example 23: C6Ala), butanoyl alanine (Example 24: C4Ala)) were added to the concentrations shown in each figure and reacted for 24 hours. After the reaction, total RNA was extracted from the cells, reverse transcription was performed, and the amount of amplification of the target gene (filaggrin) was measured by real-time PCR (n=2), and the amount of amplification of the control (no addition) was set to 100% and compared with this value. GAPDH was used as a correction gene. Calculation was performed using the comparative CT method, the primers and probes were TaqMan (registered trademark) Gene Expression, and the fluorescent dye was FAM.

The following evaluations were performed according to the amount of amplification. The results are shown in Tables 2 and 3 and Figures 4 and 5.
Very favorable (A): 200% or more. Preferred (B): 100% or more but less than 200%. Not very favorable (C): 50% or more but less than 100%. Not favorable at all (D): Less than 50%.

Considering the results of Examples 2 to 24 above (Tables 2, 3, Figures 1 to 5, and 7 and 8), it is believed that the acyl neutral amino acid ester can improve the expression-promoting effect of moisturizing-related proteins such as filaggrin, and therefore can improve the moisturizing ability of retaining moisture inside the stratum corneum. In addition, it is believed that the acyl neutral amino acid ester can improve the expression-promoting effect of proteins related to the barrier function of the skin, and therefore can improve the barrier function that inhibits the intrusion of foreign substances. Furthermore, considering these results as well as the result of Example 1 (Table 1), it is believed that the acyl neutral amino acid ester has excellent handleability and can fully demonstrate the ability to improve stratum corneum function both by its action inside the stratum corneum and its emollient action from outside the stratum corneum.

Examples 25 to 29: Evaluation of cytotoxicity Human normal epidermal keratinocytes NHEK (NB) (Kurabo Industries, Ltd.) were pre-cultured in HuMedia-KG2 (Kurabo Industries, Ltd.) and adjusted to a concentration of ¹⁵ x 104 cells/mL, and 2 mL of the adjusted culture solution (i.e., 30 x ¹⁰⁴ cells/well) was added to a 6-well plate and cultured at 37°C for 1 day. Samples (lauroyl valine (Comparative Example 4: C12Val), decanoyl valine (Example 25: C10Val), octanoyl valine (Example 26: C8Val), hexanoyl valine (Example 27: C6Val), butanoyl valine (Example 28: C4Val), acetyl valine (Example 29: C2Val) were added to the concentrations shown in each figure and reacted for 24 hours. Thereafter, the medium was discarded and replaced with 2 mL/well of Neutral Red solution, and the cells were further cultured for 2 hours. The Neutral Red solution used was prepared by diluting Neutral Red (Kurabo) 1/100 with a mixture of HuMedia-KG2 medium/physiological saline (2:1). The cells stained with the Neutral Red solution were washed once with PBS (-), and then incubated in 1% CaCl ₂ The cells were fixed with 1.5 mL/well of 1% formalin solution for 1 minute, and then 1.5 mL/well of 1% acetic acid/50% ethanol solution was added and extracted at room temperature for 20 minutes, and the absorbance at 540/630 nm was measured using a plate reader.

The following evaluations were performed according to the cell viability, and the results are shown in Table 4 and FIG.
Very favorable (A): 95% or more. Preferred (B): 75% or more but less than 95%. Not very favorable (C): 50% or more but less than 75%. Not favorable at all (D): Less than 50%.

The results of Examples 14 to 24 show that each acyl neutral amino acid can amplify the filaggrin gene. Furthermore, the results of Examples 25 to 29 and Comparative Example 4 show that the acyl neutral amino acids and acyl neutral amino acid esters of Examples 25 to 29 have low cytotoxicity, whereas the acyl neutral amino acid of Comparative Example 4 has high cytotoxicity.

Example 30: Improvement of profilaggrin production rate by octanoyl valine (C8Val) First, human normal epidermal keratinocytes NHEK (NB) (Kurabo Industries, Ltd.) were cultured in HuMedia-KG2 (Kurabo Industries, Ltd.) at 37°C, 5% CO2, and saturated water vapor. The confluent cells were seeded in a 6-well plate at 15 x ¹⁰⁴ (cells/well). After culturing at 37°C for 1 day, the medium was replaced with HuMedia-KG2 medium supplemented with 100 μM octanoyl valine (C8Val) and 1.3 mM _CaCl2 , and the medium was replaced in the same manner every 1 to 2 days, and the culture was continued for 6 days. After culturing, the medium was removed, the wells were washed twice with cold PBS (-), and 200 μL/well of extract (M-PER (Thermo Fisher Scientific)) containing protease inhibitor and phosphatase inhibitor (EzRIPA Lysis (ATTO)) was added to each well, and the wells were shaken at 200 rpm for 5 minutes in a shaker (TAITEC). The cells were scraped with a scraper, and 200 μL of a denaturant (EzApply (ATTO)) was added to the total amount of liquid including the precipitate, and the wells were heated and shaken at 1100 rpm and 100 ° C. for 5 minutes in a shaker (Bioer Technology). The resulting solution was centrifuged at 10,000 G in a centrifuge (manufactured by Tommy Seiko Co., Ltd.) at 4° C. for 10 minutes, and the supernatant was used as a sample for Western blotting.

Next, 20 μL of each Western blot sample (profilaggrin) or 5 μL of a 50-fold diluted solution of the Western blot sample prepared in (1) (GAPDH) was applied to an e-PAGEL 7.5% (ATTO) per gel lane, and electrophoresis was performed for 1 hour at a constant current of 21 mA in an electrophoresis device (ATTO). After electrophoresis, the gel was transferred to a PVDF membrane, washed with TBS-T (ATTO), and blocked with EzBlock BSA (ATTO). Next, an antigen-antibody reaction was carried out using a primary antibody solution (Anti Filaggrin (AKH1) antibody (manufactured by Santa Cruz Biotechnology) (profilaggrin), Anti GAPDH (6C5) antibody (manufactured by Santa Cruz Biotechnology) (GAPDH)). Labeling was performed with a secondary antibody (Anti-IgG (H+L), Mouse, Goat-poly, HRP (manufactured by KPL)), and the band intensity (250-400 kDa (profilaggrin), 37 kDa (GAPDH)) was quantified using an image analyzer (manufactured by Amersham) by the chemiluminescence method (n=2). The expression level of profilaggrin was normalized by the expression level of GAPDH, and the production level of the control (no addition of octaenoylvaline (C8Val)) was set as 100% for comparison.

The results obtained were evaluated as follows and are shown in Table 5.
Very favorable (A): 250% or more. Preferred (B): 150% or more but less than 250%. Not very favorable (C): 100% or more but less than 150%. Not favorable at all (D): Less than 100%.

From the above, it was revealed that octanoylvaline (C8Val) improves the production of profilaggrin protein.

Example 31: Evaluation of partition coefficient of acyl neutral amino acid ester The partition coefficient (LogP) of acyl neutral amino acid ester was calculated using chemical structural formula calculation software called ChemSketch (manufactured by Advanced Chemistry Development, Inc. (ACD/Labs)).

From Table 6, it can be seen that the LogP values of acyl neutral amino acid esters such as C6Ala-iPA, C6Val-Et, and C6Ile-Et are in the range of 1 to 4, and have superior properties for transdermal absorption (Marc Schneider, M. et al., Dermatoendocrinol., 1, 197-206 (2009)).

The compounds of the present invention are useful as keratinocyte function improving agents and moisturizing agents, as well as for the development of cosmetics, pharmaceuticals, quasi-drugs, and other products.

Claims (10)

A skin topical preparation comprising a compound selected from the following (I) to (IV) and an organic solvent :
(I) N-octanoylisoleucine isopropyl ester, and N-octanoylisoleucine ethyl ester;
(II) N-octanoyl leucine ethyl ester;
(III) N-octanoylalanine isopropyl ester (excluding those contained in N-cocoylalanine isopropyl ester), and N-decanoylalanine isopropyl ester (excluding those contained in N-cocoylalanine isopropyl ester) ; and (IV) N-hexanoylvaline ethyl ester, N-octanoylvaline ethyl ester, and N-decanoylvaline isopropyl ester. The topical skin preparation according to claim 1, wherein the compound is a compound selected from (I). The topical skin preparation according to claim 1, wherein the compound is the compound (II). The topical skin preparation according to claim 1, wherein the compound is a compound selected from (III). The topical skin preparation according to claim 1, wherein the compound is a compound selected from (IV). A cosmetic composition comprising a compound selected from the following (I) to (IV) and an organic solvent :
(I) N-octanoylisoleucine isopropyl ester, and N-octanoylisoleucine ethyl ester;
(II) N-octanoyl leucine ethyl ester;
(III) N-octanoylalanine isopropyl ester (excluding those contained in N-cocoylalanine isopropyl ester), and N-decanoylalanine isopropyl ester (excluding those contained in N-cocoylalanine isopropyl ester) ; and (IV) N-hexanoylvaline ethyl ester, N-octanoylvaline ethyl ester, and N-decanoylvaline isopropyl ester. The cosmetic preparation according to claim 6 , wherein the compound is a compound selected from (I). The cosmetic preparation according to claim 6 , wherein the compound is the compound (II). The cosmetic preparation according to claim 6 , wherein the compound is a compound selected from the group (III). The cosmetic preparation according to claim 6 , wherein the compound is a compound selected from the group (IV).

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