A cyclic pentapeptide compound, its preparation method and application

Abstract

This invention belongs to the field of cyclic peptide compound technology, and relates to a cyclic pentapeptide compound, its preparation method, and its applications. The chemical structural formula of the cyclic pentapeptide compound is: [Insert chemical formula here]. The cyclic pentapeptide compound provided by this invention not only possesses anti-inflammatory and antihistamine activities, but also exhibits no cellular toxicity, which is beneficial for its application in the cosmetics and pharmaceutical fields.

Description

Technical Field

[0001] This invention belongs to the field of cyclic peptide compound technology, and relates to a cyclic pentapeptide compound, its preparation method and application. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Histamine is a biogenic amine widely found in tissues such as skin, playing a key mediator role in allergic diseases and immune responses. Histamine activates its four G protein-coupled receptors (HIR-H4R), triggering multiple cascade pathways and ultimately amplifying the inflammatory response. Histamine HI receptors (H1R) have seven transmembrane structures (TMs) containing many conserved amino acid residues, some of which are important lipophilic sites. These lipophilic sites, along with other amino acid residues, form lipophilic cavities, which are the primary binding sites for H1 receptor antagonists. Activation of histamine H1 receptors (H1R) promotes vasodilation and increased capillary permeability, leading to plasma exudation, local tissue redness and swelling, and bronchial and gastrointestinal smooth muscle contraction, thus causing skin sensitivity and allergic reactions. Simultaneously, histamine can interact with TSLP (interleukin-7 (IL-7) cytokine), triggering an inflammatory response.

[0004] In recent years, factors such as increased air pollution, environmental changes, altered dietary structures, and improper use of cosmetics have led to a continuous rise in skin sensitivity, allergies, and inflammation. Therefore, developing ingredients with both anti-inflammatory and antihistamine properties has become an important research direction in the cosmetics and pharmaceutical industries. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a cyclic pentapeptide compound, its preparation method, and its application. The cyclic pentapeptide compound provided by the present invention not only possesses anti-inflammatory and antihistamine activities but also exhibits no cellular toxicity, which is beneficial for its application in the cosmetics and pharmaceutical fields.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows: Firstly, a cyclic pentapeptide compound with the following chemical structural formula: .

[0007] In a second aspect, a method for preparing the cyclic pentapeptide compound according to the first aspect of the present invention includes the step of preparing the cyclic pentapeptide compound according to the following reaction route; .

[0008] Thirdly, the use of the cyclic pentapeptide compound described in the first aspect of the present invention in the preparation of antihistamine products.

[0009] The beneficial effects of this invention are as follows: 1. The cyclic pentapeptide compound provided by this invention has the characteristic of high safety. Experiments show that the cyclic pentapeptide compound of this invention did not exhibit cytotoxicity to test cells at concentrations up to 50 ppm, indicating that the compound has good biosafety within its effective concentration range, providing a safety guarantee for its application in the fields of cosmetics and pharmaceuticals.

[0010] 2. The cyclic pentapeptide compound provided by this invention can significantly inhibit the expression of TSLP (thymic stromal lymphopoietin, interleukin-7 (IL-7) cytokine) gene, exerting anti-inflammatory and anti-allergic effects. Thus, it can intervene in the occurrence of allergic reactions by inhibiting the TSLP-mediated inflammatory pathway, thereby achieving anti-inflammatory and anti-allergic effects.

[0011] 3. The cyclic pentapeptide compounds of the present invention can significantly reduce the average fluorescence intensity of FITC-histamine at effective concentrations, indicating that the compounds can effectively competitively bind to the histamine H1 receptor, block the binding of histamine to the receptor, and thus achieve an antihistamine effect. Attached Figure Description

[0012] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0013] Figure 1 The cyclic pentapeptide prepared in Example 1 of this invention 1 H NMR spectrum. Figure 2 The cyclic pentapeptide prepared in Example 1 of this invention 13 C10 NMR spectrum. Figure 3 This is the mass spectrum of the cyclic pentapeptide prepared in Example 1 of the present invention. Figure 4 This is a bar chart of cell viability in Example 2 of the present invention.

[0014] Figure 5 This is a bar graph of the average fluorescence intensity of FITC in Example 3 of the present invention; compared with the normal control group, ***p<0.001.

[0015] Figure 6 This is a typical graph of the average fluorescence intensity of FITC in Example 3 of the present invention.

[0016] Figure 7The graph shows the relative expression levels of the TSLP gene in Example 4 of this invention; compared with the model control group, *p<0.05, ***p<0.001. Detailed Implementation

[0017] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0018] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0019] A typical embodiment of the present invention provides a cyclic pentapeptide compound with the following chemical structural formula: .

[0020] Another embodiment of the present invention provides a method for preparing the above-mentioned cyclic pentapeptide compound, comprising the step of preparing the cyclic pentapeptide compound according to the following reaction route; .

[0021] In some embodiments, the cyclic pentapeptide compound is prepared using a liquid-phase synthesis method.

[0022] In some embodiments, the amino acid derivative H-Ser(tBu)-OH at position 1 undergoes dehydration condensation with the Fmoc-protected amino acid NHS ester at position 2, and the Fmoc protecting group is removed to obtain the dipeptide fragment H-Trp-Ser(tBu)-OH; then, the steps of dehydration condensation and removal of the Fmoc protecting group are repeated sequentially with the Fmoc-protected amino acid NHS ester at positions 3 to 5 to obtain the linear pentapeptide H-Arg(Pbf)-Met-Trp-Trp-Ser(tBu)-OH; the two ends of the linear pentapeptide are dehydrated and cyclized to obtain cyclic compound A; the Pbf and tBu protecting groups in cyclic compound A are removed to obtain the cyclic pentapeptide compound.

[0023] Specifically, HOSu / DIC is used as a condensing agent to activate the carboxyl groups of amino acids to obtain amino acid NHS esters. More specifically, HOSu and amino acids are dissolved, DIC is added dropwise at 15-25°C, and then the temperature is raised to 25-40°C for activation reaction. More specifically, the molar ratio of amino acids to HOSu is 1:1.1-1.3.

[0024] Specifically, the dehydration condensation temperature during the preparation of the linear pentapeptide is 25~35℃.

[0025] Specifically, during the preparation of the linear pentapeptide, the temperature for removing the Fmoc protecting group is 30~35℃.

[0026] Specifically, using the TOTU / Oxyma activation system, the carboxyl and amino terms of the linear pentapeptide H-Arg(Pbf)-Met-Trp-Trp-Ser(tBu)-OH are intramolecularly condensed. More specifically, this includes the following steps: Sodium hydride was reacted with Oxyma in DMF to obtain a DMF solution of sodium Oxyma salt; The linear pentapeptide H-Arg(Pbf)-Met-Trp-Trp-Ser(tBu)-OH was added to a portion of the Oxyma sodium salt DMF solution, and TEA and EA were added to dissolve and prepare a linear peptide solution. TOTU was added to another portion of the Oxyma sodium salt DMF solution, along with TEA and EA, and dissolved to prepare a TOTU solution. At a temperature of 25~35℃, a linear peptide solution is added dropwise to the TOTU solution to carry out the reaction, thus obtaining the product.

[0027] The linear peptide solution is divided into two parts. First, a portion of the linear peptide solution is added dropwise to the TOTU solution to allow the reaction to complete. Then, TOTU is added to the reacted material, and the other portion of the linear peptide solution is added dropwise until the reaction is complete. Preferably, the molar ratio of the linear pentapeptide H-Arg(Pbf)-Met-Trp-Trp-Ser(tBu)-OH in the linear peptide solution to TOTU in the TOTU solution is 1:2.7~3. Preferably, the molar ratio of the linear pentapeptide H-Arg(Pbf)-Met-Trp-Trp-Ser(tBu)-OH in the linear peptide solution to the added TOTU is 1:2.7~3.3.

[0028] Specifically, the lysis buffer for removing the Pbf and tBu protecting groups from the ring A compound consists of H2O, anisole, Tis, ethylene dithiol, and TFA. The molar ratio of H2O, anisole, Tis, and ethylene dithiol is 18–22:9–11:2.7–3.3:2.7–3.3. The ratio of H2O to TFA is 18–22:22–26 mol / L.

[0029] A third embodiment of the present invention provides the application of the above-mentioned cyclic pentapeptide compound in the preparation of antihistamine products.

[0030] In some embodiments, the product is a pharmaceutical or cosmetic product.

[0031] In some embodiments, the product is a product for treating or relieving histamine-induced skin sensitivity, skin allergies, and / or skin inflammation.

[0032] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments and comparative examples.

[0033] Example 1 Synthesis of the cyclic pentapeptide compound H1RH-114 1. Synthesis of Fmoc-Trp-Osu Add 10.5 mol of Fmoc-Trp-OH and 12 mol of HoSu to the reactor, then add 14 L of DCM and stir to dissolve. Slowly add 12.5 mol of DIC dropwise in a 25°C water bath. After the addition is complete, raise the water bath temperature to 35°C and stir for 40 minutes. The reactor temperature is maintained at 35°C. Take a sample every 20 minutes to check if the remaining raw material is below 0.2%. If not, continue stirring until the requirement is met, then add 4 L of heptane and mix well.

[0034] 40 L of n-heptane was added to a precipitation tank, and the above-mentioned active ester solution was added dropwise while stirring at 25 °C, resulting in the precipitation of a large amount of white solid. After the addition was complete, the mixture was stirred for 60 min, filtered, and the filter cake was washed twice and dried under vacuum at 40 °C to constant weight.

[0035] 2. Synthesis of Fmoc-Met-Osu Add 5 mol of Fmoc-Met-OH and 5.75 mol of HoSu to the reactor, then add 6-8 L of DCM and stir to dissolve. Slowly add 6 mol of DIC dropwise in a 25°C water bath. After the addition is complete, raise the water bath temperature to 35°C and stir for 60 min. The reactor temperature is maintained at 35°C. Take a sample every 20 min to check if the remaining raw material is below 0.5%. If not, continue stirring until the requirement is met, then add 2 L of n-heptane and mix well.

[0036] 20 L of n-heptane was added to a precipitation tank, and the above-mentioned active ester solution was added dropwise while stirring at 25 °C, resulting in the precipitation of a large amount of white solid. After the addition was complete, the mixture was stirred for 60 min, filtered, and the filter cake was washed twice and dried under vacuum at 40 °C to constant weight.

[0037] 3. Synthesis of Fmoc-Arg(Pbf)-Osu Fmoc-Arg(Pbf)-OH (6 mol) and HoSu (7.5 mol) were added to the reactor, followed by 15 L of DCM and stirred until dissolved. DIC (7.8 mol) was then rapidly added dropwise in a 20°C water bath. After the addition was complete, the water bath temperature was raised to 25°C and the mixture was stirred for 20 min, with the reactor temperature maintained below 30°C. Immediately after the reaction time was complete, 6 L of MTBE was added and mixed thoroughly.

[0038] Heptane (30 L) and MTBE (12 L) were added to a precipitation tank, and the above-mentioned active ester solution was added dropwise while stirring at 25 °C, resulting in the precipitation of a large amount of white solid. After the addition was complete, the mixture was stirred for 20 min, filtered, and the filter cake was washed twice and dried under vacuum at 40 °C to constant weight.

[0039] 4. Synthesis of H-Trp-Ser(tBu)-OH Add isopropanol (10 L), H-Ser(tBu)-OH (5.5 mol, 1.1 eq), and TEA (5.5 mol, 1.1 eq) to the reaction vessel and stir until homogeneous.

[0040] Weigh out 5 mol of Fmoc-Trp-Osu and 3.5 mol of TEA (0.7 eq), add 4 L of EA, prepare a solution at 35 °C, and add it dropwise to the above reaction vessel while stirring. Control the dropping time to 10-15 min. After the dropping is completed, react for 120 min. Take a sample every 20 min to check if the raw material is less than 0.5% remaining. Otherwise, continue stirring until the requirements are met.

[0041] After the reaction was complete, water (11 kg) and saturated sodium chloride solution (12 kg) were added, followed by EA (15 L). Hydrochloric acid was added to adjust the pH to approximately 4, and the mixture was stirred for 2 minutes, then separated. For the organic phase, water (10 kg) and saturated sodium chloride solution (8 kg) were added, and hydrochloric acid was added to adjust the pH to approximately 4. The mixture was stirred for 2 minutes, then separated. For the organic phase, saturated sodium chloride solution (5 kg) was added, followed by a small amount of solid sodium chloride (30 g). The mixture was stirred and washed for 5 minutes, then separated. The temperature inside the reactor was controlled at 30℃ throughout the extraction process. A Fmoc-Trp-Ser (tBu)-OH solution was obtained.

[0042] Add DBU (5.5 eq) to the reactor. If solids precipitate, add isopropanol (1-3 L). If it still does not dissolve, concentrate and distill off part of the EA until dissolved. Stir the reaction at 35°C for 60 min. Take a sample to check if the remaining raw material is less than 0.1%. Otherwise, continue stirring the reaction. After the reaction is complete, add acetic acid (5.8 eq) to neutralize the DBU and adjust the pH to approximately 7.

[0043] 5. Synthesis of H-Trp-Trp-Ser(tBu)-OH TEA (2.0 eq, 10 mol), Fmoc-Trp-Osu (5.4 mol), NMM (2.5 mol, 0.5 eq), and EA (1 L) were added to the reactor to prepare a solution. This solution was then added dropwise to the reactor and stirred until the reaction was complete within 10 minutes. After the addition was complete, the reaction was allowed to proceed for 120 minutes. Sampling was started after 10 minutes, and samples were taken every 20 minutes until the remaining raw material was below 0.5%. Otherwise, the reaction was continued until the requirements were met. After the reaction was completed, 50 mL of ammonia water and 2 L of water were mixed and added to the reactor and stirred for 2 minutes to quench the reaction. Water (13 kg) and saturated sodium chloride solution (8 kg) were added, and hydrochloric acid was added to adjust the pH to approximately 4.5. EA (4 L) was added to make up the difference, and the mixture was stirred and washed for 2 minutes before separating the liquid and retaining the organic phase.

[0044] Add DBU (6 eq) to the reactor. If solid precipitates, add water until dissolved. Stir at 30°C for 60 min. Take a sample to check if the remaining raw material is less than 0.1%; otherwise, continue stirring. Add water (7 kg) and saturated sodium chloride solution (7 kg). Add hydrochloric acid while stirring to adjust the pH to approximately 3.5. Add sodium chloride solid (2 kg) and heat to 40°C while stirring to dissolve until a small amount of sodium chloride remains insoluble (the aqueous phase is saturated). Separate the liquid and retain the organic phase. Add water (11 kg) and saturated sodium chloride solution (5 kg), stir, wash, and separate the liquid, retaining the organic phase. Add saturated sodium chloride solution (5 kg) again, stir, wash, and separate the liquid, discarding the aqueous phase to obtain H-Trp-Trp-Ser (tBu)-OH·HCl solution. Concentrate under reduced pressure until the remaining solution volume is 11 L.

[0045] Add three times the volume of n-heptane to the precipitation tank, then add the above reaction solution dropwise. Stir to allow crystals to precipitate for 60 minutes, filter, wash the filter cake twice, and dry under vacuum. Dry at 50°C to constant weight.

[0046] 6. Synthesis of H-Met-Trp-Trp-Ser(tBu)-OH Add H-Trp-Trp-Ser(tBu)-OH·HCl (5mol) and isopropanol (8L) to the reactor and stir to dissolve. Add TEA (2.0q), heat at 30℃ and add Fmoc-Met-Osu (1.07eq), and add 4L EA to aid dissolution.

[0047] Measure 0.8 eq of NMM, add 1 L of EA to prepare a solution, and add it dropwise to the reactor while stirring. The dropwise addition time is controlled at 10 min. After the addition is complete, react for 120 min. Sampling begins 10 min later, and samples are taken every 20 min until the remaining raw material is below 0.5%. Otherwise, continue stirring until the requirement is met. After the reaction is complete, mix 50 mL of ammonia and 2 L of water, add the mixture to the reactor, and stir for 5 min to quench the reaction.

[0048] DBU (5.5 eq) was added to the reactor, and the mixture was stirred at 30°C for 60 min. Samples were taken for testing until deprotection was complete. After the reaction was completed, EA (13 L), water (8 kg), and saturated sodium chloride solution (8 kg) were added. Hydrochloric acid was added under stirring to adjust the pH to approximately 3.5. Sodium chloride solid (2 kg) was added, and the mixture was heated to 35°C and stirred until a small amount of sodium chloride remained insoluble (the aqueous phase was saturated). The mixture was separated, and the organic phase was retained. THF (5 L), water (8 kg), and saturated sodium chloride solution (8 kg) were added under heating at 45°C. The mixture was stirred, washed, and separated, and the organic phase was retained. Saturated sodium chloride solution (5 kg) was added again, and the mixture was stirred, washed, and separated. The aqueous phase was discarded to obtain H-Met-Trp-Trp-Ser(tBu)-OH.HCl solution.

[0049] 7. Synthesis of H-Arg(Pbf)-Met-Trp-Trp-Ser(tBu)-OH TEA (2.0 eq) was added to the above reaction solution, and Fmoc-Arg(Pbf)-Osu (1.07 eq) was added while heating at 30°C.

[0050] Measure 0.8 eq of NMM, add 1 L of EA to prepare a solution, and add it dropwise to the reactor while stirring. The dropwise addition time is controlled at 10 min. After the addition is complete, react for 120 min. Start sampling after 10 min, and take samples every 20 min to check if the raw material remains below 0.5%. Otherwise, continue stirring until the requirement is met. After the reaction is complete, mix 50 ml of ammonia water and 2 L of water and add it to the reactor while stirring and quenching for 5 min.

[0051] Add DBU (6 eq) to the reactor. If solid precipitates, add water until dissolved. Stir at 35°C for 60 min. Take a sample to check if the remaining raw material is less than 0.1%; otherwise, continue stirring. Add water (7 kg) and saturated sodium chloride solution (7 kg). Add hydrochloric acid while stirring to adjust the pH to approximately 3.5. Add sodium chloride solid (2 kg) and heat at 35°C while stirring to dissolve until a small amount of sodium chloride remains insoluble (the aqueous phase is saturated). Separate the liquid and retain the organic phase. Add water (11 kg) and saturated sodium chloride solution (5 kg), stir, wash, and separate the liquid, retaining the organic phase. Add saturated sodium chloride solution (5 kg) again, stir, wash, and separate the liquid, discarding the aqueous phase to obtain H-Arg(Pbf)-Met-Trp-Trp-Ser (tBu)-OH·HCl solution. Concentrate under reduced pressure until the remaining solution volume is 10-12 L.

[0052] Add 3 times the volume of n-heptane to a precipitation tank, then add the above reaction solution dropwise. Stir to crystallize for 60 minutes, filter, wash the filter cake twice, and dry under vacuum. Dry at 50°C to constant weight.

[0053] 8. Cycloning (3 mol) Weigh 3.6 mol of sodium hydride and mix it with 36 L of DMF. Slowly add 4.2 mol of Oxyma to dissolve and react, obtaining a sodium Oxyma salt DMF solution. Note that a large amount of hydrogen gas will be released. Leave 10% in the reaction flask, add 21 mol of TEA, and add 24 L of DMF to the reaction flask. Heat the mixture to 30°C and set aside for later use.

[0054] Weigh 3 mol of linear pentapeptide, add the remaining 90% Oxyma sodium salt DMF solution, add 4.5 mol of TEA to dissolve until clear, add 60 L of EA and mix well, divide into two equal portions, and heat to 30°C and keep warm.

[0055] The reactor temperature was controlled at 30℃. 9 mol TOTU was added with stirring, followed by 60 L EA and mixed thoroughly. Half of the linear peptide solution was immediately added dropwise over 15 minutes at a uniform rate. After stopping the addition and stirring for 10 minutes, another 9 mol TOTU was added to the reactor and stirred until dissolved. The remaining linear peptide solution was then added dropwise over 15 minutes at a uniform rate. Stirring continued for 10 minutes, and samples were taken every 10 minutes until the remaining raw material was below 0.2%. If the reaction was still incomplete after 30 minutes, TOTU was added again until the reaction was complete.

[0056] Add 60 L of EA and 180 L of 10% sodium chloride solution, adjust the pH to approximately 3 with hydrochloric acid, wash and separate the layers, retaining the organic phase. Add 70 L of THF and 180 L of 5% (mass fraction) sodium chloride solution while stirring at 45°C, adjust the pH to approximately 8 with sodium carbonate solid, wash and separate the layers, retaining the organic phase. If solid precipitates, add THF as needed and increase the temperature, but the reactor temperature should not exceed 50°C. Add another 180 L of 5% (mass fraction) sodium chloride solution to the organic phase, adjust the pH to approximately 8 with sodium carbonate solid, wash and separate the layers again, retaining the organic phase. Finally, add 120 L of saturated sodium chloride solution, wash and separate the layers, retaining the organic phase.

[0057] Concentrate the organic phase to 10 L while maintaining the temperature at 45 °C. If a significant amount of solid precipitates and cannot be completely dissolved by heating, evaporate the solvent to dryness. Dissolve the solid in 5 L of THF by heating to 50 °C until clear, then add 5 L of EA and mix.

[0058] Add 5 times the volume of MTBE to a precipitation tank, then add the above solution dropwise. After the addition is complete, add 2 times the volume of n-heptane to accelerate precipitation. Stir to allow crystals to precipitate for 30 minutes, then filter. Wash the filter cake twice and dry it under vacuum. Dry it at 45-55℃ under vacuum to constant weight to obtain a yellow solid.

[0059] 9. Pyrolysis (3 mol) Preparation of pyrolysis solution: H2O / benzyl sulfide / Tis / ethylenedithiol = 20eq / 10eq / 3eq / 3eq = 60mol / 30mol / 9mol / 9mol. Measure the above solution and add it to the reactor. Add 24L of TFA and mix well for later use. Control the temperature inside the reactor at 15℃.

[0060] Weigh 3 mol of cyclic peptide and slowly add it to the reaction vessel until completely dissolved. Stir the reaction vessel for 35 minutes.

[0061] Add 5 times the volume of MTBE to a precipitation tank, add the above reaction solution dropwise, stir to precipitate crystals for 30 minutes, filter, wash the filter cake twice, and dry it under vacuum. Dry it at 50℃ under vacuum to constant weight to obtain crude H1RH-114.

[0062] 10. Purification and freeze-drying Dissolution: Weigh 50g of H1RH-114 crude peptide, add 750mL of methanol and 1.75L of water, stir and sonicate until completely dissolved. If the solution is turbid, add acetonitrile dropwise until the solution is clear, then filter and set aside.

[0063] purification: Column: DAC200, with Daiso C18 SP-120-10-ODS-P packing material and a packing weight of 5 kg; Phase A: 0.03% acetic acid / H2O; Phase B: ACN.

[0064] Elution gradient:

[0065] The retention time of the main product is approximately 55–70 min, depending on the actual chromatogram. The target component is collected for intermediate-scale analysis, retaining the main component with a purity ≥95%. Its structural characterization is as follows: Figure 1 , Figure 2 and Figure 3 As shown. The sequence is shown in SEQ ID NO:1.

[0066] Example 2 Cytotoxicity Detection The cytotoxicity of H1RH-114 prepared in Example 1 against human immortalized keratinocytes was detected using the MTT assay. The experimental steps were as follows: 1. Seed cells in 96-well plates and incubate at 37 °C with 5% CO2 for 24 h.

[0067] The experimental groups are as follows:

[0068] 2. Sample feeding: Fresh culture medium containing the corresponding concentration of sample (H1RH-114 prepared in Example 1) was added to the sample group. The normal control group and the blank control group were replaced with fresh culture medium and incubated at 37 °C and 5% CO2 for 24 h.

[0069] 3. After incubation, add MTT solution to each well and continue incubation for 4 hours.

[0070] 4. Remove the culture medium, add DMSO solution, shake to mix, and measure the absorbance at 490 nm.

[0071]

[0072] OD 490 : Absorbance value at 490 nm.

[0073] Test results as follows Figure 4 As shown, compared with the normal control group (100%), the H1RH-114 prepared in Example 1 in the sample group had a cell viability value greater than 90% in the concentration range of 50 ppm, indicating that the H1RH-114 prepared in Example 1 has no cytotoxicity in the concentration range of 50 ppm.

[0074] Example 3 Antihistamine efficacy Using the stable cell line HEK293-HRH1-OE, we observed whether H1RH-114 prepared in Example 1 could physically bind to histamine receptors and exert a competitive inhibition of histamine-receptor binding, thus evaluating its antihistamine efficacy. The experimental groups are as follows:

[0075] Construction of stable HRH1 cell line: HEK293 cells in the logarithmic growth phase were transfected with HRH1 overexpression plasmid for about 48 h, followed by puromycin selection to obtain the stable HRH1-expressing cell line HEK293-HRH1-OE.

[0076] Preparation of fluorescent probes: FITC solution and histamine solution were mixed at a molar ratio of 1:2 and incubated with stirring for 4 h at room temperature in the dark. The pH of the system was then neutralized to terminate the reaction. The reaction solution was placed in a dialysis bag, and PBS was used as the dialysate. Dialysis was performed at 4°C in the dark for 24–48 h, with the dialysate being replaced during this period, until no obvious fluorescence was observed under 488 nm excitation. The reaction solution was collected, and the concentration was adjusted by fluorescence detection. The solution was then stored at 4°C in the dark for later use.

[0077] HEK293-HRH1-OE cell line was seeded into 6-well plates and incubated at 37°C with 5% CO2 for 24 h.

[0078] H1 receptor binding efficiency determination: HEK293-HRH1-OE cells in the logarithmic growth phase were pretreated for 30 min according to the above experimental groups, followed by the addition of FITC-histamine and incubation in the dark for 30 min. After the reaction, the liquid was discarded, and the cells were washed 3 times (to remove unbound fluorescent histamine). The cells were fixed and stained with DAPI. After the treatment, the fluorescence intensity of each group of cells was observed under a fluorescence microscope. The H1 receptor binding efficiency of the samples was evaluated by quantifying the average fluorescence intensity using ImageJ.

[0079]

[0080] In the formula, B represents the average fluorescence intensity of FITC.

[0081] The results are as follows Figures 5-6 As shown, compared with the normal control group, the average fluorescence intensity of FITC in the positive control group was significantly reduced, indicating that the experimental system is effective and reliable. Compared with the normal control group, the average fluorescence intensity of FITC in the sample group was significantly reduced at concentrations of 5 ppm, 20 ppm, and 50 ppm, with improvement rates of 24%, 33%, and 22%, respectively, indicating that H1RH-114 prepared in Example 1 has antihistamine properties.

[0082] Example 4 Anti-inflammatory effects TSLP interacts with histamine. TSLP is an interleukin-7 (IL-7)-like cytokine, mainly produced in epithelial cells, smooth muscle cells, keratinocytes, stromal cells, fibroblasts, mast cells, monocytes / macrophages, granulocytes, and dendritic cells, triggering inflammatory responses. Using human immortalized keratinocytes (HaCaT), the relative expression level of the TSLP gene was measured after treating the cells with H1RH-114 prepared in Example 1 to evaluate whether H1RH-114 has anti-inflammatory efficacy. The experimental steps are as follows: 1. Seed cells in 6-well plates and incubate at 37 °C with 5% CO2 for 24 h.

[0083] The experimental groups are as follows:

[0084] 2. After incubation, fresh culture medium was added to the normal control group, fresh culture medium containing histamine was added to the model control group, and fresh culture medium containing histamine and sample (H1RH-114 prepared in Example 1) was added to the sample group. The incubation was continued at 37 °C and 5% CO2 for 24 h.

[0085] 3. Total RNA was extracted from each experimental group, cDNA was synthesized, and the gene expression of β-actin and the target gene was detected by q-PCR.

[0086] 4. Using β-actin as an internal reference for gene expression, calculate the relative RNA expression level of the target gene.

[0087]

[0088]

[0089]

[0090]

[0091] In the formula, B represents the relative expression level of the TSLP gene.

[0092] The results are as follows Figure 7 As shown, compared with the normal control group, the relative expression level of the TSLP gene in the model control group was significantly upregulated, indicating that the test stimulation conditions of this embodiment are effective. Compared with the model control group, the relative expression level of the TSLP gene in the sample group was significantly downregulated at concentrations of 5 ppm and 20 ppm, with improvement rates of 40% and 19%, respectively, indicating that H1RH-114 prepared in Example 1 has anti-inflammatory effects at concentrations of 5 ppm and 20 ppm.

[0093] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A cyclic pentapeptide compound, characterized in that, Its chemical structural formula is: 。 2. A method for preparing the cyclic pentapeptide compound according to claim 1, characterized in that, This includes the steps of preparing the cyclic pentapeptide compound according to the following reaction route; 。 3. The preparation method according to claim 2, characterized in that, The cyclic pentapeptide compound was prepared by liquid-phase synthesis.

4. The preparation method according to claim 2, characterized in that, The amino acid derivative H-Ser(tBu)-OH at position 1 undergoes dehydration condensation with the Fmoc-protected amino acid NHS ester at position 2, and the Fmoc protecting group is removed to obtain the dipeptide fragment H-Trp-Ser(tBu)-OH; then, the dehydration condensation and Fmoc protecting group removal steps are repeated sequentially with the Fmoc-protected amino acid NHS esters at positions 3 to 5 to obtain the linear pentapeptide H-Arg(Pbf)-Met-Trp-Trp-Ser(tBu)-OH; the two ends of the linear pentapeptide are dehydrated and cyclized to obtain cyclic compound A; the Pbf and tBu protecting groups in cyclic compound A are removed to obtain the cyclic pentapeptide compound.

5. The preparation method according to claim 4, characterized in that, The carboxyl groups of amino acids were activated by using HOSu / DIC as a condensing agent to obtain amino acid NHS esters.

6. The preparation method according to claim 4, characterized in that, Dissolve HOSu and amino acids, add DIC dropwise at 15~25℃, and then heat to 25~40℃ for activation reaction; Alternatively, the molar ratio of amino acids to HOSu is 1:1.1~1.

3.

7. The preparation method according to claim 4, characterized in that, Using the TOTU / Oxyma activation system, the carboxyl terminus and amino terminus of the linear pentapeptide H-Arg(Pbf)-Met-Trp-Trp-Ser(tBu)-OH were intramolecularly condensed. Alternatively, the lysis solution for removing the Pbf and tBu protecting groups from the ring A compound consists of H2O, anisole, Tis, ethylene dithiol, and TFA.

8. The use of the cyclic pentapeptide compound of claim 1 in the preparation of antihistamine products.

9. The application as described in claim 8, characterized in that, The product in question is a drug or cosmetic.

10. The application as described in claim 8, characterized in that, The product is for treating or relieving skin sensitivity, skin allergies, and / or skin inflammation caused by histamine.

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