An acetyl hexapeptide-8 fatty acid derivative, and a preparation method and application thereof

Abstract

The present application relates to a kind of acetyl hexapeptide-8 fatty acid derivatives and its preparation method and application, belong to the field of cosmetics.The preparation method provided by the present application can obtain acetyl hexapeptide-8 fatty acid derivative with higher yield and purity, and the operation is simple, and the production cost is low.The long-chain fatty acid is introduced in the sequence structure of acetyl hexapeptide-8 in the present application, the hydrophobicity of structure is enhanced, and the transdermal ability is significantly enhanced.The propylene glycol is used as cosolvent in the present application, the transdermal ability of sample is further improved, and the bioavailability as cosmetic raw material is increased.

Description

Technical Field

[0001] This invention belongs to the field of cosmetic technology, specifically relating to an acetyl hexapeptide-8 fatty acid derivative, its preparation method, and its application. Background Technology

[0002] Acetyl hexapeptide-8 (Ac-EEMQRR-NH2), also known as Argireline, is a neurotransmitter peptide with the sequence Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2. It is a six-amino acid peptide chain derived from the N-terminus (positions 12-17) of the synaptosome-associated protein SNAP-25. It was first extracted from the single-celled Pseudomonas aeruginosa found in Antarctic glacial mud. Its structure is shown in the following formula:

[0003]

[0004] Acetyl hexapeptide-8, by mimicking the SNAP-25 protein fragment, competes with SNAP-25 for binding to vesicle-associated membrane protein (VAMP), affecting the formation and stability of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex. The SNARE complex is a vesicle fusion complex that releases neurotransmitters such as acetylcholine through exocytosis, inducing muscle contraction. Therefore, under the action of acetyl hexapeptide-8, vesicles cannot effectively bind to the membrane to release acetylcholine, blocking muscle electrical impulse signals, inhibiting neurotransmitter release, and relaxing facial muscles, thus smoothing dynamic wrinkles, static wrinkles, and fine lines.

[0005] Acetyl hexapeptide-8, a classic anti-aging cosmetic ingredient, has gained widespread recognition for its efficacy and safety. However, its transdermal delivery faces several challenges: firstly, its large molecular weight approaches the limit for passive diffusion through the skin; secondly, its strong hydrophilicity results in poor compatibility with the lipid environment of the stratum corneum; and finally, acetyl hexapeptide-8 readily exists in zwitterionic form at physiological pH, further weakening its transmembrane permeability. These factors collectively lead to poor transdermal absorption and low bioavailability.

[0006] Currently, methods to enhance the transdermal effect of peptides mainly include physical and chemical methods. Commonly used physical methods include iontophoresis and microneedling (CN114099383A), which have been proven to improve the transdermal effect of various drug molecules. However, physical methods have many drawbacks. They usually require specialized instruments, are costly, have poor dosage form flexibility, and can cause pain to varying degrees, requiring consideration of skin safety. Therefore, current reports mainly focus on chemical methods. Chemical methods include structural modification, the use of chemical penetrants, and coupling with transdermal peptides. Chinese patent publication CN115517999B reports an anti-aging composition, the main formulation of which is: 0.1-5 parts of antioxidant, 0.0001-0.05 parts of complex peptide, 1-10 parts of panthenol, and 0.3-2 parts of glycoside compound. Formulating a combination of multiple anti-aging peptides (acetyl octapeptide-3, acetyl hexapeptide-8, pentapeptide-3, tripeptide-1, and acetyl hexapeptide-1) with various chemical penetration enhancers can promote dermal collagen regeneration, fill and reduce fine lines, and achieve anti-aging and moisturizing effects in aesthetic medicine. However, the use of chemical penetration enhancers to attempt to open the skin barrier does not yield ideal transdermal results, and the use of multiple chemical transdermal enhancers can easily cause skin damage, inflammation, allergies, and systemic toxicity. Patent CN108714111B reports a method for preparing a membrane-penetrating peptide-acetyl hexapeptide nanoemulsion. The method involves first designing and artificially synthesizing the membrane-penetrating peptide-acetyl hexapeptide (Ac-EEMQRRYGRKKRRQRRR), and then preparing it into a nanoemulsion. This invention coordinates nanoliposome technology and membrane-penetrating peptide modification technology to improve the transdermal penetration and absorption of acetyl hexapeptide, while maintaining the stability of the peptide molecular structure and its biological activity. However, the coupling of transmembrane peptides significantly increases production costs and time, and the use of liposome nanotechnology has problems such as incomplete encapsulation, instability, and high production costs. Summary of the Invention

[0007] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0008] To address the shortcomings of existing technologies, the present invention aims to provide a method for preparing acetyl hexapeptide-8 fatty acid derivatives and their applications. By coupling the N-terminus of acetyl hexapeptide-8 with a long-chain fatty acid, its hydrophobic structure and transdermal absorption capacity are enhanced, enabling it to exert its efficacy safely and for a long time, thereby improving its bioavailability. The method provided by this invention is simple to operate, has low production costs, and achieves high yield and purity.

[0009] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0010] An acetyl hexapeptide-8 fatty acid derivative having the general structural formula shown in Formula I:

[0011] (Formula I);

[0012] Wherein, R is a straight-chain or branched C7~C19 alkyl group.

[0013] As a preferred embodiment of the acetyl hexapeptide-8 fatty acid derivative of the present invention, wherein: R in the general formula shown in Formula I is a straight-chain or branched C11~C15 alkyl group.

[0014] Another object of the present invention is to provide a method for preparing the acetyl hexapeptide-8 fatty acid derivative as described above, comprising the following steps:

[0015] A solid-phase synthetic peptide resin of the formula Z-Glu-Glu-Met-Gln-Arg-Arg-resin is provided, wherein resin is a resin and Z is an NH2-terminal protecting group;

[0016] A fatty acid and a condensing agent are added to the solid-phase synthetic peptide resin to react and obtain the R-Glu-Glu-Met-Gln-Arg-Arg-resin complex; wherein R is a C8~C20 fatty acid;

[0017] The complex was lysed in a lysis buffer, and the precipitated solid was centrifuged, purified, and lyophilized to obtain an acetyl hexapeptide-8 fatty acid derivative.

[0018] As a preferred embodiment of the preparation method of the acetyl hexapeptide-8 fatty acid derivative of the present invention, the solid-phase synthetic peptide resin of the formula Z-Glu-Glu-Met-Gln-Arg-Arg-resin is provided by: dissolving Z-Arg-OH and a condensing agent in a solvent, adding the pretreated resin, stirring, filtering under reduced pressure, and deprotecting to obtain a resin with the first amino acid attached; then coupling Z-Arg-OH, Z-Gln-OH, Z-Met-OH, Z-Glu-OH, and Z-Glu-OH sequentially using the same method to obtain the solid-phase synthetic peptide resin of Z-Glu-Glu-Met-Gln-Arg-Arg-resin.

[0019] In a preferred embodiment of the preparation method of the acetyl hexapeptide-8 fatty acid derivative of the present invention, the condensing agent is a mixed solution of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, diisopropylethylamine, and N,N dimethylformamide in a molar ratio of 1~3:2~4:5.

[0020] The molar ratio of the resin to Z-Arg-OH is 1:5~10; the molar ratio of the resin to the condensing agent is 1:5~10; and the condensation time is 30~60 minutes.

[0021] In a preferred embodiment of the preparation method of the acetyl hexapeptide-8 fatty acid derivative of the present invention, the molar ratio of the resin to Z-Arg-OH is 1:5; the molar ratio of the resin to the condensing agent is 1:5; and the condensation time is 60 minutes.

[0022] In a preferred embodiment of the preparation method of the acetyl hexapeptide-8 fatty acid derivative of the present invention, Z is an Fmoc group.

[0023] As a preferred embodiment of the preparation method of the acetyl hexapeptide-8 fatty acid derivative of the present invention, the method further includes the steps of swelling and deprotecting the resin. Specifically, the amino resin is added to a solid-phase synthesis syringe for swelling, washing, drying, and deprotection to obtain the pretreated resin.

[0024] In a preferred embodiment of the preparation method of the acetyl hexapeptide-8 fatty acid derivative of the present invention, the amino resin is at least one of 2-CTC resin, Wang resin, and Rink-Amide-AM-resin resin, with a degree of substitution of 0.85~1.05 mmol / g, the solvent is dichloromethane, and the swelling time is 30~40 minutes.

[0025] In a preferred embodiment of the preparation method of the acetyl hexapeptide-8 fatty acid derivative of the present invention, the amino resin is Rink-Amide-AM-resin resin with a degree of substitution of 0.90 mmol / g, the solvent is dichloromethane, and the swelling time is 30 minutes.

[0026] In a preferred embodiment of the preparation method of the acetyl hexapeptide-8 fatty acid derivative of the present invention, the deprotecting agent is a piperidine N,N-dimethylformamide solution with a volume fraction of 5-20%, and the deprotection reaction time is 5-10 minutes.

[0027] In a preferred embodiment of the preparation method of the acetyl hexapeptide-8 fatty acid derivative of the present invention, the deprotecting agent is a 20% (v / v) piperidine N,N-dimethylformamide solution, and the deprotection reaction time is 5 minutes.

[0028] In a preferred embodiment of the preparation method of the acetyl hexapeptide-8 fatty acid derivative of the present invention, the fatty acid is one or a combination of several of octanoic acid, lauric acid, palmitic acid, and arachidic acid, and the molar ratio of resin to fatty acid is 1:2~10.

[0029] In a preferred embodiment of the preparation method of the acetyl hexapeptide-8 fatty acid derivative of the present invention, the reaction of adding fatty acid and condensing agent takes 30 to 60 minutes.

[0030] In a preferred embodiment of the preparation method of the acetyl hexapeptide-8 fatty acid derivative of the present invention, the lysis solution is trifluoroacetic acid, triisopropylsilane, 1,2-ethylenedithiol and water, in a volume ratio of 94:1:2~4:1~3.

[0031] In a preferred embodiment of the preparation method of the acetyl hexapeptide-8 fatty acid derivative of the present invention, the lysis solution is trifluoroacetic acid, triisopropylsilane, 1,2-ethylenedithiol and water, in a volume ratio of 94:1:2.5:2.5.

[0032] In a preferred embodiment of the preparation method of the acetyl hexapeptide-8 fatty acid derivative of the present invention, the cleavage is carried out at room temperature for 2 to 3 hours.

[0033] Another object of the present invention is to provide the use of the acetyl hexapeptide-8 fatty acid derivative as described above in the preparation of transdermal pharmaceuticals or cosmetics.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] (1) The synthesis method provided by the present invention can obtain acetyl hexapeptide-8 fatty acid derivatives with high yield and purity, and is simple to operate and has low production cost.

[0036] (2) Introducing long-chain fatty acids into the acetyl hexapeptide-8 sequence structure enhances the hydrophobicity of the structure, improves the transdermal absorption capacity of the sample, and improves its bioavailability. Attached Figure Description

[0037] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein:

[0038] Figure 1 The structural formulas of acetyl hexapeptide-8 and its fatty acid derivatives provided by the present invention are as follows: (a) is Ac-EEMQRR-NH2, (b) is C8-EEMQRR-NH2, (c) is C12-EEMQRR-NH2, (d) is C16-EEMQRR-NH2, and (e) is C20-EEMQRR-NH2.

[0039] Figure 2 This is a schematic diagram of the synthetic route for the acetyl hexapeptide-8 fatty acid derivative provided by the present invention.

[0040] Figure 3 The following are liquid chromatograms of acetyl hexapeptide-8 fatty acid derivatives in Examples 1-5, where (a) is the liquid chromatogram of Ac-EEMQRR-NH2, (b) is the liquid chromatogram of C8-EEMQRR-NH2, (c) is the liquid chromatogram of C12-EEMQRR-NH2, (d) is the liquid chromatogram of C16-EEMQRR-NH2, and (e) is the liquid chromatogram of C20-EEMQRR-NH2.

[0041] Figure 4 The cumulative transdermal curves of acetyl hexapeptide-8 fatty acid derivatives at different PG concentrations are shown, where (a) is 0% PG, (b) is 35% PG, (c) is 65% PG, and (d) is 100% PG. Detailed Implementation

[0042] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0043] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0044] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0045] Unless otherwise specified, all raw materials used in this invention are preferably commercially available products. Raw material source: Shanghai Aladdin Biochemical Technology Co., Ltd.

[0046] Example 1: Synthesis of Acetyl Hexapeptide-8

[0047] The synthesis of acetyl hexapeptide-8 includes the following steps:

[0048] (1) Resin swelling and deprotection: Take 300 mg of Rink-Amide-AM-resin resin into a solid-phase synthesis syringe, add DCM, stir well, let stand for 30 minutes, and then dry the DCM. Add DMF, stir well with a glass rod, let stand for 30 minutes, and then dry the DMF. Add a 20% (v / v) piperidine / DMF solution to the syringe, stir well with a glass rod, deprotect for 5 minutes, and then dry the solution. Wash three times each with DCM and DMF.

[0049] (2) Solid-phase peptide coupling reaction: Dissolve 5 equivalents of Fmoc-Arg(Pbf)-OH in 3 mL of HATU / DIPEA / DMF condensing agent solution (molar equivalent ratio HATU:DIPEA:DMF = 2:3:5), react for 30 minutes, filter to remove the reaction solution, and wash 3 times each with DCM and DMF. Repeat the above deprotection and condensation steps to couple Fmoc-Arg(Pbf)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Met-OH, Fmoc-Glu(OtBu)-OH, and Fmoc-Glu(OtBu)-OH in sequence.

[0050] (3) Acetylation reaction: Take 3 mL of acetylation reaction solution (acetic anhydride: pyridine: DMF = 5: 6: 89) into a syringe, react for 5 minutes, wash 3 times each with DCM and DMF, and then drain the liquid.

[0051] (4) Peptide cleavage: Transfer 4 mL of the prepared lysis buffer into the reactor and lyse for 2 hours. The lysis buffer consists of trifluoroacetic acid, triisopropylsilane, 1,2-ethylenedithiol, and water in a volume ratio of 94:1:4:1. Dry the lysis buffer, add about 3 mL of ice-cold ether, centrifuge for 2 minutes, and repeat the operation twice. Dry the remaining ether, purify, and freeze-dry to obtain acetyl hexapeptide-8 (Ac-EEMQRR-NH2).

[0052] The purification conditions were as follows: C18 column (4.6 × 150 mm); mobile phase A: ultrapure water (0.1% trifluoroacetic acid); mobile phase B: acetonitrile (0.1% trifluoroacetic acid); mobile phase separation ratio: 85:15; flow rate: 1 mL / min; detection wavelength: 220 nm; injection volume: 10 μL; run time: 20 min; column temperature: 30℃.

[0053] Example 2 Synthesis of Acetyl Hexapeptide-8 Fatty Acid (N-Octaic Acid) Derivatives

[0054] The synthesis method of the acetyl hexapeptide-8 fatty acid (octanoic acid) derivative is basically the same as that in Example 1, except that:

[0055] In step (3), 5 times the equivalent of the resin of octanoic acid is dissolved in 3 mL of HATU:DIPEA:DMF condensing agent solution (the molar equivalent ratio of HATU:DIPEA:DMF = 2:3:5), reacted for 60 minutes, filtered to remove the reaction solution, and washed 3 times each with DCM and DMF.

[0056] In step (4), 4 mL of the prepared lysis buffer was transferred to the reactor for lysis for 2 hours. The lysis buffer consisted of trifluoroacetic acid, triisopropylsilane, 1,2-ethylenedithiol, and water in a volume ratio of 94:1:4:1. The lysis buffer was dried, and about 3 mL of ice-cold diethyl ether was added. The mixture was centrifuged at 5000 r / min for 2 minutes, and the operation was repeated twice. The remaining diethyl ether was dried, purified, and lyophilized to obtain C8-EEMQRR-NH2.

[0057] The purification conditions described above were as follows: C18 column (4.6 × 150 mm); mobile phase A: ultrapure water (0.1% trifluoroacetic acid); mobile phase B: acetonitrile (0.1% trifluoroacetic acid); mobile phase separation ratio: 80:20; flow rate: 1 mL / min; detection wavelength: 220 nm; injection volume: 10 μL; run time: 20 min; column temperature: 30 ℃.

[0058] Example 3 Synthesis of Acetyl Hexapeptide-8 Fatty Acid (Lauryl Acid) Derivatives

[0059] The synthesis method of the acetyl hexapeptide-8 fatty acid (lauric acid) derivative is basically the same as that in Example 1, except that:

[0060] In step (3), 5 times the amount of lauric acid equivalent to the resin is dissolved in 3 mL of HATU / DIPEA / DMF condensing agent solution (the molar equivalent ratio of HATU:DIPEA:DMF = 2:3:5), reacted for 60 minutes, filtered to remove the reaction solution, and washed 3 times each with DCM and DMF.

[0061] In step (4), 4 mL of the prepared lysis buffer was transferred to the reactor for lysis for 2 hours. The lysis buffer consisted of trifluoroacetic acid, triisopropylsilane, 1,2-ethylenedithiol, and water in a volume ratio of 94:1:4:1. The lysis buffer was dried, and about 3 mL of ice-cold ether was added. The mixture was centrifuged at 5000 r / min for 2 minutes, and the operation was repeated twice. The remaining ether was dried, purified, and lyophilized to obtain C12-EEMQRR-NH2.

[0062] The purification conditions described above were as follows: C18 column (4.6 × 150 mm); mobile phase A: ultrapure water (0.1% trifluoroacetic acid); mobile phase B: acetonitrile (0.1% trifluoroacetic acid); mobile phase separation ratio: 60:40; flow rate: 1 mL / min; detection wavelength: 220 nm; injection volume: 10 μL; run time: 20 min; column temperature: 30 ℃.

[0063] Example 4 Synthesis of Acetyl Hexapeptide-8 Fatty Acid (Palmic Acid) Derivatives

[0064] The synthesis method of the acetyl hexapeptide-8 fatty acid (palmitic acid) derivative is basically the same as that in Example 1, except that:

[0065] In step (3), palmitic acid equivalent to 5 times that of the resin is dissolved in 3 mL of HATU / DIPEA / DMF condensing agent solution (the molar equivalent ratio of HATU:DIPEA:DMF = 2:3:5), reacted for 60 minutes, filtered to remove the reaction solution, and washed 3 times each with DCM and DMF.

[0066] In step (4), 4 mL of the prepared lysis buffer was transferred to the reactor for lysis for 2 hours. The lysis buffer consisted of trifluoroacetic acid, triisopropylsilane, 1,2-ethylenedithiol, and water in a volume ratio of 94:1:4:1. The lysis buffer was dried, and about 3 mL of ice-cold ether was added. The mixture was centrifuged at 5000 r / min for 2 minutes, and the operation was repeated twice. The remaining ether was dried, purified, and lyophilized to obtain C16-EEMQRR-NH2.

[0067] The purification conditions described above were as follows: C18 column (4.6 × 150 mm); mobile phase A: ultrapure water (0.1% trifluoroacetic acid); mobile phase B: acetonitrile (0.1% trifluoroacetic acid); mobile phase separation ratio: 40:60; flow rate: 1 mL / min; detection wavelength: 220 nm; injection volume: 10 μL; run time: 20 min; column temperature: 30 ℃.

[0068] Example 5 Synthesis of Acetyl Hexapeptide-8 Fatty Acid (Arachidic Acid) Derivatives

[0069] The synthesis method of the acetyl hexapeptide-8 fatty acid (arachidic acid) derivative is basically the same as that in Example 1, except that:

[0070] In step (3), 5 times the amount of arachidic acid equivalent to the resin is dissolved in 3 mL of HATU / DIPEA / DMF condensing agent solution (the molar equivalent ratio of HATU:DIPEA:DMF = 2:3:5), reacted for 60 minutes, filtered to remove the reaction solution, and washed 3 times each with DCM and DMF.

[0071] In step (4), 4 mL of the prepared lysis buffer was transferred to the reactor for lysis for 2 hours. The lysis buffer consisted of trifluoroacetic acid, triisopropylsilane, 1,2-ethylenedithiol, and water in a volume ratio of 94:1:4:1. The lysis buffer was dried, and about 3 mL of ice-cold ether was added. The mixture was centrifuged at 5000 r / min for 2 minutes, and the operation was repeated twice. The remaining ether was dried, purified, and lyophilized to obtain C20-EEMQRR-NH2.

[0072] The purification conditions described above were as follows: C18 column (4.6 × 150 mm); mobile phase A: ultrapure water (0.1% trifluoroacetic acid); mobile phase B: acetonitrile (0.1% trifluoroacetic acid); mobile phase separation ratio: 60:40; flow rate: 1 mL / min; detection wavelength: 220 nm; injection volume: 10 μL; run time: 20 min; column temperature: 30 ℃.

[0073] Example 6: Yield and Purity Test of Acetyl Hexapeptide-8 Fatty Acid Derivatives (C8-C20)

[0074] Following the methods described in Examples 1-5, acetyl hexapeptide-8 fatty acid derivatives (C8-C20) with different carbon chain lengths were synthesized, and the yield and purity test results are shown in Table 1.

[0075] Table 1

[0076]

[0077] As shown in Table 1, under the same synthesis and separation methods, different acetyl hexapeptide-8 fatty acid derivatives can achieve high yields and purities, with yields exceeding 60% and purities maintaining above 95%, which is considered high within the field. Table 1 also shows that as the carbon chain of the coupled fatty acid lengthens, steric hindrance increases, leading to a decrease in synthesis yield.

[0078] Example 7

[0079] Based on Example 3, the degree of resin substitution was adjusted, and the yield and purity test results obtained under different degrees of resin substitution are shown in Table 2.

[0080] Table 2

[0081]

[0082] As shown in Table 2, yield, purity, and resin substitution degree exhibit a significant positive correlation. As the resin substitution degree increases from 0.80 mmol / g to 1.00 mmol / g, the yield rises sharply from 35.6% to a peak of 79.2%, an increase of over 120%. For every 0.10 mmol / g increase in substitution degree, purity increases by approximately 1.2 percentage points, from 96.3% to 98.3%. Optimizing the resin substitution degree not only improves yield but also simultaneously improves product purity.

[0083] Example 8

[0084] Based on Example 3, the volume fraction of piperidine was adjusted, and the yield and purity test results are shown in Table 3.

[0085] Table 3

[0086]

[0087] As shown in Table 3, the yield is positively correlated with the volume fraction of piperidine. As the volume fraction of piperidine increases from 5% to 20%, the yield jumps dramatically from 22.7% to 75.6%, an increase of over 230%. The purity exhibits a pattern of initial slight fluctuations followed by a significant increase with the change in the volume fraction of piperidine. When the volume fraction increases from 5% to 10%, the purity decreases slightly; however, when the volume fraction further increases to 20%, the purity jumps dramatically.

[0088] The yield reached 98.3%, the peak value for this group of experiments. A piperidine volume fraction of 20% was the optimal parameter, under which both yield and purity reached their highest values.

[0089] Example 9

[0090] Based on Example 3, the amino acid condensation reaction time was adjusted, and the yield and purity test results are shown in Table 4.

[0091] Table 4

[0092]

[0093] As shown in Table 4, the yield exhibited a fluctuating trend of first decreasing and then increasing with the extension of reaction time. When the reaction time was extended from 30 minutes to 60 minutes, the yield decreased slightly from 77.6% to 73.1%; when extended further to 90 minutes, the yield rebounded and reached a peak of 78.6%, slightly higher than the yield at 30 minutes. The trend of purity change was highly consistent with that of yield, also showing a characteristic of first decreasing and then increasing. At 60 minutes, the purity dropped to its lowest value of 95.2%; at 90 minutes, the purity rose to a peak of 97.7%.

[0094] In summary, the optimal reaction time for amino acid condensation is 30 minutes.

[0095] Example 10

[0096] Based on Example 3, the fatty acid coupling reaction time was adjusted, and the yield and purity test results are shown in Table 5.

[0097] Table 5

[0098]

[0099] As shown in Table 5, as the reaction time increased from 30 minutes to 90 minutes, the yield increased significantly from 37.8% to a peak of 85.9%, an increase of over 127%. The purity showed a steady upward trend with the extension of the reaction time, indicating that extending the reaction time not only improves the yield but also reduces unreacted raw materials or by-product residues, thereby simultaneously optimizing the purity of the product.

[0100] Example 11

[0101] Based on Example 3, the fatty acid coupling reaction temperature was adjusted, and the yield and purity test results are shown in Table 6.

[0102] Table 6

[0103]

[0104] As shown in Table 6, within the reaction temperature range of 25–40℃, the yield initially decreased slightly, then increased significantly, reaching a peak of 80.2% at 40℃. When the temperature exceeded 40℃, the yield showed a continuous decreasing trend with increasing temperature, reaching a minimum of 29.6% at 75℃. The purity fluctuated relatively little overall, remaining stable within the range of 94.4–98.9%; the peak purity occurred at 30℃ (98.9%).

[0105] Example 12

[0106] Based on Example 3, the condensing agent formulation was adjusted, and the yield and purity test results are shown in Table 7.

[0107] Table 7

[0108]

[0109] As shown in Table 7, the yield is significantly correlated with the proportion of each component in the feed ratio, exhibiting an overall trend of first increasing and then decreasing. Specifically, when the proportion of HATU remains constant, the yield gradually increases from 33.6% to 63.8% as the proportion of DMF decreases and the proportion of DIPEA increases. When the proportion of DMF is fixed at 1, increasing the proportion of HATU raises the yield from 63.8% to a peak of 75.3%. Continuing to increase the proportion of DIPEA, however, causes the yield to decrease to 59.6%. Therefore, the ratio of HATU:DIPEA:DMF = 2:2:1 yields the highest yield in this experiment.

[0110] Except for the HATU:DIPEA:DMF ratio of 1:1:8, which yielded only 93.2% purity, the purity of other formulations could reach over 95%. When the proportion of DIPEA was too high, the purity decreased slightly. The peak purity occurred in the ratio of 2:2:1, which had the highest yield.

[0111] Example 13

[0112] Based on Example 3, the volume ratio of the lysis solution and the lysis time were adjusted, and the yield and purity test results are shown in Table 8.

[0113] Table 8

[0114]

[0115] Table 8 shows that the yield is affected by both the pyrolysis liquid volume ratio and the pyrolysis time. When the pyrolysis time is fixed at 3 hours, the yield continuously increased from 26.2% to 62.9% when the pyrolysis liquid volume ratio was adjusted from 90:2:4:4 to 94:1:4:1; subsequently, when adjusted to 94:1:2.5:2.5, the yield slightly decreased to 61.6%. With a fixed pyrolysis liquid volume ratio, shortening the pyrolysis time significantly improves the yield. The purity fluctuated very little, remaining stable between 96.2% and 98.3%, indicating that adjustments to the pyrolysis liquid volume ratio and pyrolysis time have a negligible impact on product purity.

[0116] The optimal experimental conditions were a pyrolysis liquid volume ratio of 94:1:2.5:2.5 plus a pyrolysis time of 2 hours. Under these conditions, the yield reached a peak of 83.2%, while the purity remained at a high level of 98.2%, achieving the dual optimization of yield and purity.

[0117] Example 14 Transdermal transdermal assay of acetyl hexapeptide-8 and related fatty acid derivatives

[0118] To test the transdermal permeability of acetyl hexapeptide-8 fatty acid derivatives, the following experiments were conducted:

[0119] Experimental samples: Acetyl hexapeptide-8 and its fatty acid derivatives as described in Examples 7-11.

[0120] Experimental Methods: A Franz vertical diffusion cell was used. The abdominal skin of SD rats was fixed between the receiving and diffusion cells. PBS buffer was used as the receiving solution. The dermal layer of the skin was placed face down on the diffusion cell. The peptide sample to be tested was dissolved in pure water. Approximately 2 mL of the solution was injected into the drug delivery cell. During the experiment, the water bath temperature was maintained at 37 ± 0.5 ℃, and the stirring speed was 300 rpm. 1 mL samples were taken from the receiving cell at 1, 3, 6, 9, 12, and 24 h, and an equal volume of blank receiving solution was immediately added. The sample content was analyzed using high-performance liquid chromatography (HPLC). The results of the 24-hour transdermal transdermal assay of acetyl hexapeptide-8 and its fatty acid derivatives under pure water conditions are shown in Table 9.

[0121] Table 9

[0122]

[0123] As can be seen from Table 9, under the same test conditions, C12-EEMQRR-NH2 exhibited the highest performance.

[0124] The cumulative transdermal absorption rate over 24 hours was approximately 2.73 times that of the parent peptide Ac-EEMQRR-NH2; C16-EEMQRR-NH2 was the second highest, approximately 2.53 times that of the parent peptide Ac-EEMQRR-NH2. This demonstrates that the introduction of fatty acids significantly improved the transdermal absorption capacity of the samples.

[0125] Example 15: Exploration of Transdermal Transdermal Optimization of Acetyl Hexapeptide-8 and Related Fatty Acid Derivatives

[0126] To further enhance the transdermal permeability of acetyl hexapeptide-8 fatty acid derivatives, different concentrations of propylene glycol (PG) were added as a co-solvent to the drug delivery tank. The experimental method was similar to that in Example 9, except that:

[0127] The test peptide samples were dissolved at 35%, 65%, and 100% concentrations, and approximately 2 mL of solution was injected into the drug delivery pool. The experimental results are shown in Table 10, and the 24-hour transdermal curves are as follows: Figure 3 As shown.

[0128] Table 10

[0129]

[0130] As shown in Table 10, with the addition of PG, C16-EEMQRR-NH2 gradually exhibited excellent transdermal effects, reaching a maximum transdermal absorption rate of 853.25±25 μg·cm⁻¹ under 100% PG conditions. -2 It is approximately 5.8 times that of Ac-EEMQRR-NH2 under the same conditions, significantly improving the transdermal effect.

[0131] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing an acetyl hexapeptide-8 fatty acid derivative, characterized in that: include, Z-Arg-OH and a condensing agent were dissolved in a solvent, and the pretreated resin was added. The mixture was stirred, filtered under reduced pressure, and deprotected to obtain a resin with the first amino acid attached. Then, Z-Arg-OH, Z-Gln-OH, Z-Met-OH, Z-Glu-OH, and Z-Glu-OH were sequentially coupled using the same method to obtain a solid-phase synthetic peptide resin of Z-Glu-Glu-Met-Gln-Arg-Arg-resin, where resin is the resin, Z is the NH2-terminal protecting group, and Z is the Fmoc group. Fatty acids and a condensing agent are added to the solid-phase synthetic peptide resin and reacted at a reaction temperature of 25-40°C to obtain an R-Glu-Glu-Met-Gln-Arg-Arg-resin complex; the condensing agent is a mixed solution of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, diisopropylethylamine, and N,N-dimethylformamide in a molar ratio of 2:2:1 or 2:3:

5. The complex was lysed in a lysis buffer to precipitate a solid, which was then centrifuged, purified, and lyophilized to obtain an acetyl hexapeptide-8 fatty acid derivative. The lysis buffer consisted of trifluoroacetic acid, triisopropylsilane, 1,2-ethylenedithiol, and water in a volume ratio of 94:1:2~4:1~3. The general structural formula of the acetyl hexapeptide-8 fatty acid derivative is shown in Formula I: (Formula I); Wherein, R is a straight-chain or branched C7~C19 alkyl group; The resin is Rink-Amide-AM-resin resin with a degree of substitution of 1~1.05 mmol / g.

2. The method for preparing the acetyl hexapeptide-8 fatty acid derivative according to claim 1, characterized in that: The molar ratio of the resin to Z-Arg-OH is 1:5~10; the molar ratio of the resin to the condensing agent is 1:5~10; and the condensation time is 30~60 min.

3. The method for preparing the acetyl hexapeptide-8 fatty acid derivative according to claim 1, characterized in that: The fatty acid is one or a combination of several of the following: octanoic acid, lauric acid, palmitic acid, and arachidic acid. The molar ratio of resin to fatty acid is 1:2 to 10.

4. The method for preparing the acetyl hexapeptide-8 fatty acid derivative according to claim 1, characterized in that: The pyrolysis was carried out at room temperature for 2-3 hours.

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