TWI619710B - Synthesis and antiphotoaging activity of novel 3-ped4hpt derivatives - Google Patents

Abstract

The invention discloses a pharmaceutical composition for treating skin photoaging and comprising a triazole derivative of the formula I, and a method for preparing a triazole derivative of the formula I, which is obtained by reacting a starting compound of the formula II with zinc chloride. R ₁ , R ₂ and R _{3 of the} starting material of the formula II and the derivative of the formula I may each be a group such as hydrogen, halogen, methyl, ethyl, nitro, methoxy, amino or trihalomethyl, and n is 1 or 2. The derivative of formula I can inhibit the damage of skin fibroblasts caused by ultraviolet light A, reduce the expression of ROS, MMP-1 and/or SA-β-Gal of fibroblasts, and/or increase the potential of ATP and mitochondrial membrane , TIMP-1 and / or COL1α1 performance. Therefore, the derivative of the formula I has the potential to treat, treat and improve the photoaging of the skin, and is applied to industries such as medical treatment, medical beauty, beauty, skin care, skin conditioning, cosmetics, and makeup products.

Description

Synthesis and Anti-photoaging Activity Evaluation of 3-PED4HPT Derivatives

The present invention relates to a derivative, a preparation method thereof and use thereof, in particular to a triazole derivative, a preparation method thereof and use thereof for treating skin photoaging.

Photoaging of the skin is mainly caused by ultraviolet radiation in the sun, which causes dullness of the skin, sagging skin, wrinkles, decreased skin immunity, and is more likely to cause skin cancer. Even a small amount of ultraviolet light destroys collagen fibers (the main structural protein of the skin), causing some abnormal elastic hard proteins to accumulate, causing the skin to lose its elasticity.

The symptoms of skin photoaging are closely related to tissue changes and subtle structural changes in the connective tissue of the dermis. These changes include the breakdown of enzymes and the reduction of newly formed collagen, which causes wrinkles to prematurely on the skin. Changes in hyaluronic acid and proteoglycan matrices can also result in reduced cell water content and cell relaxation. Furthermore, UV-induced extracellular matrix recombination also strongly influences cell phenotypes, such as the ability of dermal fibroblasts to recombine (Rock and Fischer, Der Hautarzt, 2011, 62(8): 591-597.).

In addition, vitamins C and E have been used in the formulation of cosmeceuticals, but the concentration of vitamins C and E in cosmeceuticals is very low, and vitamins will be reduced when exposed to air and light. The stability of C and E, and the ester form or isomer form of vitamins C and E will not be absorbed or effectively metabolized by the skin, so vitamins C and E are resistant to erythema caused by ultraviolet rays, sunburn, sunburn There is no actual effect on chronic ultraviolet photoaging and skin cancer (Burke, Dermatologic Therapy. 2007, 20(5): 314-321.).

In addition, Pallela et al. (Pallela et al., Marine Drugs. 2010, 8(4): 1189-1202) disclose compounds for preventing light exposure and photoaging from marine organisms, such as Coralina pilulifera . The methanol extract can protect against oxidative stress caused by ultraviolet light A by protecting DNA and inhibiting matrix metalloproteinase (MMP) in human dermal fibroblasts. Pallela's paper also reveals anti-photo-aging substances obtained by marine organisms such as eckol, mycosporine-glycine, and palythene, but its disadvantage is that marine organisms such as algae need to be cultured, harvested, processed, extracted, etc., and the cost in industrial processes will be Quite high.

In view of the deficiencies in the prior art, the applicant of this case, after careful experimentation and research, and a perseverance spirit, finally conceived the case and can overcome the shortcomings of the prior art. The following is a brief description of the case.

In order to find novel anti-photoaging derivatives or compounds, the present invention also prepares a series of triazole derivatives capable of resisting photoaging in order to overcome the disadvantages of the prior art anti-photoaging composition instability or time-consuming and labor-intensive extraction. Things. Further, the series of triazole derivatives are 3-phenylethynyl-5,6-dihydroxy-4hydro-pyrrole[1,2-c][1,2,3]triazole derivatives ( 3-phenylethynyl-5,6-dihydro-4H-pyrrolo-[1,2-c][1,2,3]triazole, abbreviated as 3-PED4HPT). The synthesized triazole derivatives can effectively treat, treat and improve skin photoaging, and are applied to industries such as medical treatment, medical beauty, beauty, skin care, skin conditioning, cosmetics, and makeup products.

The present invention provides a triazole derivative of formula I, and a pharmaceutical composition for treating photoaging of the skin comprising a triazole derivative of formula I: Wherein R ₁ , R ₂ and R ₃ are each, but not limited to, hydrogen, halogen, methyl, ethyl, nitro, methoxy, amino or trihalomethyl. The halogen may be fluorine, chlorine, bromine or iodine, and the trihalomethyl group may be trifluoromethyl, trichloromethyl, tribromomethyl or triiodomethyl. The value of n is 1 or 2, and n = 1 indicates that the one carbon atom forms a pentacyclic structure with other carbon atoms and nitrogen atoms, and n = 2 indicates that the two carbon atoms form a six-ring structure with other carbon atoms and nitrogen atoms.

The invention also provides a method of treating photoaging comprising administering an effective amount of the pharmaceutical composition described above to an individual in need thereof. And will result in decreased expression of active oxides (ROS), matrix metalloproteinase-1 (MMP-1) and/or aging-related β-galactosidase (SA-β-gal) in individual cells, and/or The expression levels of adenosine triphosphate (ATP), mitochondrial membrane potential, tissue inhibitor of metalloproteinase-1 (TIMP-1), and/or type 1 collagen alpha 1 (COL1α1) increased. The purpose of the method of treating photoaging includes, but is not limited to, medical treatment, medical beauty, beauty, skin care, conditioning, and the like.

The present invention also provides a process for the preparation of a triazole derivative of the formula I, which comprises reacting a starting material of the formula II with zinc chloride to obtain the triazole derivative.

According to the above concept, the starting material was dissolved in dimethylformamide under a nitrogen atmosphere, and the reaction was carried out at 100 ° C for at least 12 hours. In a specific embodiment, It is carried out for 12 to 24 hours. After the reaction was completed, an aqueous solution was added to terminate the reaction.

The term "derivative" as used herein refers to a molecule in which a hydrogen atom or substituent is substituted with another atom or substituent to form another molecule. As used herein, the term "compound" refers to a molar ratio (or weight ratio) of two or more molecules at a fixed molar ratio and under appropriate reaction conditions. Another molecule.

The "3-PED4HPT derivative" or "triazole derivative" herein is completed by the production method disclosed in the embodiment, and the group of the specific compound may be further substituted by other groups, so that it can be prepared according to the spirit Other derivatives. Therefore, "3-PED4HPT derivative", "triazole derivative" and "derivative" herein can be used interchangeably.

The term "ultraviolet light" or "ultraviolet light" as used herein includes near ultraviolet (ultraviolet light A, wavelength 320-400 nm), medium ultraviolet light (ultraviolet light B, wavelength 290-320 nm), and far ultraviolet light (ultraviolet light C, wavelength 200- 280 nm or 200-290 nm), but specific embodiments of the invention may be applied, but are not limited to, ultraviolet light A, and ultraviolet light B and C may also be applied to the present invention.

The inventions set forth in the present disclosure will be fully understood from the following description of the embodiments of the present invention, which can be practiced by those skilled in the art. However, the implementation of the present invention is not limited by the following embodiments. Other embodiments may be devised by those skilled in the art in the light of the embodiments disclosed herein.

Experiment 1, Preparation of 3-PED4HPT Derivatives

In one embodiment, a starter (Formula III, 300 mg, 1.4 mmol) and 10 mL of dimethylformamide were added to a 100 mL round bottom vial and stirred for 5 minutes with nitrogen. Add zinc oxide (39 mg, 0.3 mmol) and heat to 100 ° C for 12 to 24 hours. At this time, the color of the round bottom bottle is changed from transparent white to opaque blue-black. After the reaction is completed, the reaction is completed. The reaction was terminated with a single aqueous solution (50 ml). After extracting with ethyl acetate, the residue was purified by column chromatography (yield: 0.040 to 0.633 mm, ethyl acetate / n-hexane: 1/100 to 1/3) to give 243 mg of the derivative 1 as a white solid. The rate is 81%, and the melting point is 67 ° C ~ 69 ° C. The reaction equation is as follows:

The nature of the derivative 1: ¹ H NMR (CDCl ₃ , 400 MHz): δ 7.56~7.51 (m, 2H), 7.54~7.32 (m, 3H), 4.35 (t, J = 7.6 Hz, 2H), 3.01 ( t, J = 7.4 Hz, 2H), 2.83 (quintet, J = 7.4 Hz, 2H). ¹³ C NMR (CDCl ₃ , 100 MHz). δ 144.12, 131.50 (2C), 128.52, 128.28 (2C), 123.98, 122.54, 92.67, 78.84, 46.85, 28.09, 20.79.

Other 3-PED4HPT derivatives having the structural formula of Formula I can be prepared according to the spirit of the foregoing experimental methods, as shown in Table 1 below.

In addition, the present invention also synthesizes other 3-PED4HPT derivatives according to the above reaction equation, including the derivative 9 (C ₁₃ H ₁₂ N ₄ ), the derivative 10 (C ₁₁ H ₁₁ N ₃ , R ₁ =R ₂ =R ₃ =H, n=2) and derivative 11 (C ₁₁ H ₁₁ N ₃ ), the structural formulas are as follows:

In addition to the use of zinc chloride, a Lewis acid such as ferric chloride, aluminum chloride or boron trifluoride may be used in the reaction.

Experiment 2, cell survival rate

In order to determine the degree of photoaging resistance of cells, fibroblasts (1×10 ⁶ cells/ml) were irradiated with different doses of ultraviolet light A, and the radiation doses were 0, 4, 8, 12 and 16 joules/cm 2 , respectively. The irradiation time of one unit of ultraviolet light A (joules per square centimeter) is 3.5 minutes, and the irradiation time of ultraviolet light A of, for example, 4 joules/cm 2 is 14 minutes. Please refer to Fig. 1, which is a graph showing the relationship between the radiation dose of ultraviolet light A irradiated to the fibroblasts and the cell survival rate. In Fig. 1, the dose of ultraviolet A radiation of 8 to 16 J/cm ² will significantly inhibit cell viability (compared with the control group of 0 J/cm ² ), and the lowest UV which has an inhibitory effect on cell survival rate. The dose of light A was 8 J/cm ² . The second lowest ultraviolet A radiation dose was 4 J/cm ² , and the intermediate value of 4 J/cm ² and 8 J/cm ² was 6 J/cm ² . Further, when the dose of the ultraviolet light A was equal to or lower than 6 J/cm ² , the type change of the fibroblasts was not observed under the microscope (results not shown). Therefore, ultraviolet light A having an intensity of 6 J/cm ^{2 was} used as the maximum radiation dose in the subsequent experiments.

Experiment 3, the amount of intracellular adenosine triphosphate (ATP)

This experiment is to determine the intracellular ATP expression related to cellular energy, metabolic regulation, cell signaling and other physiological mechanisms to determine the physiological mechanism of cells. The generally cultured fibroblasts (1 x 10 ⁶ cells/ml) were treated with different 5 μM 3-PED4HPT derivatives for 4 hours, and then subjected to 6 J/cm ² intensity, 21 minutes of ultraviolet light A. The intracellular relative ATP content of the experimental group was calculated from the intracellular ATP content of the group not treated with ultraviolet light A (0 J/cm ² , control group), and is shown as a percentage. Please refer to Fig. 2, which is the ATP performance of the fibroblasts treated with the 3-PED4HPT derivative of the present invention and ultraviolet light A, wherein each experimental group was subjected to three independent experiments, ^* p <0.05 and ^** p <0.01 indicates statistically significant significance compared with the control group. In Fig. 2, almost all of the groups in the fibroblasts treated with the 3-PED4HPT derivative and then treated with ultraviolet light A showed a significant increase in ATP expression (with 6 J/cm ² UV A). The group processed is compared). Therefore, the 3-PED4HPT derivative of the present invention is indeed resistant to photoaging problems caused by ultraviolet A irradiation. Subsequent experiments were carried out with derivatives 7 and 11 with the highest ATP performance.

Experiment 4: Determination of active oxides (ROS) and mitochondrial membrane potential (△Ψmt)

The active oxide and mitochondrial membrane potentials are determined using flow cytometry techniques well known in the art. In the determination of active oxides, fibroblasts (1 × 10 ⁶ cells/ml) were treated with different 5 μM 3-PED4HPT derivatives for 4 hours, followed by 6 J/cm ² intensity, 21 minutes of UV A, after The activity was also measured by flow cytometry by staining with 2',7'-dichlorfluorescein-diacetate (DCFH-DA). In the determination of mitochondrial membrane potential, fibroblasts received the same 3-PED4HPT derivative and UV-A treatment, followed by iodo-3,3'-dihexyloxycarbocyanine (3,3'- Dihexyloxacarbocyanine iodide, DiOC ₆ ) staining, the mitochondrial membrane potential was measured immediately by flow cytometry.

Please refer to Fig. 3(A) and Fig. 3(B), which are the percentage of (A) ROS production rate and (B) mitochondria after treatment of fibroblasts with 3-PED4HPT derivative and/or ultraviolet light A. Percentage of membrane potential. Compared with the group treated only with ultraviolet light A, the active oxide of fibroblasts treated with 5 μM of the derivative 7 or 11 and treated with ultraviolet light A decreased, but the mitochondrial membrane potential increased. Since the exposure of cells or organisms to external environmental pressures such as ultraviolet rays or heat sources will result in an increase in the production of active oxides, resulting in decreased cellular antioxidant capacity, damage to cellular structures, and aging (ie, in an oxidative stress state), the present invention is derived. The substance 7 or 11 can protect the fibroblasts by reducing the damage caused by the ultraviolet A irradiation. In addition, when the cells are subjected to external pressure and enter the apoptosis program, the proteins that promote apoptosis are transferred to the mitochondria, and the permeability and integrity of the mitochondrial membrane are destroyed. The mitochondrial membrane is permeable. The mitochondrial permeability transition pore (MPTP) is opened to lower the membrane potential of the mitochondria. As can be seen from Fig. 3(B), the mitochondrial membrane potential of the derivative 7 or 11 of the present invention is higher than that of the ultraviolet light A group, indicating that the fibroblast is protected from external pressure, and the ultraviolet light A is effectively lowered. Cell damage caused by irradiation.

Experiment 5: Determination of MMP-1, TIMP-1 and COL1α1 gene expression

In the mechanism of cell aging, the excessive expression of MMP-1 will cause the extracellular network structure (composed of collagen, elastin, etc.) to disintegrate, causing skin cells to age, so MMP-1 is collagen breakdown and aging. symbols of. TIMP-1 binds to MMP-1 to form a complex to regulate and inhibit the expression of MMP-1. COL1α1 is a composition constituting type I collagen (also known as pro-α1 chain), which together with another pro-α1 chain and a pro-α2 chain translated by COL1α2 gene constitutes type I collagen, Type I collagen is the largest amount of collagen in an organism (such as human), so cells or organisms can be known by measuring MMP-1, TIMP-1 and COL1α1. The body is affected by external pressure or drugs.

In Experiment 5, fibroblasts (1 x 10 ⁶ cells/ml) were treated with different 5 μM 3-PED4HPT derivatives for 4 hours, followed by 6 J/cm ² intensity, 21 minutes of UV A. The fibroblast RNA was then extracted and matrix metalloproteinase-1 (MMP-1), tissue inhibitor of metalloproteinase-1 (TIMP-1) and type 1 collagen were determined by quantitative real-time polymerase chain reaction (qRT-PCR). The gene expression of protein α1 (COL1α1) was used to analyze the effects of 3-PED4HPT derivatives and UV-A on the photoaging mechanism of skin.

Please refer to Fig. 4(A) to Fig. 4(C), which are (A) MMP-1, (B) TIMP-1 and (C) after treatment of fibroblasts with 3-PED4HPT derivative or ultraviolet light A. The mRNA expression of COL1α1 indicates that the derivative 7 or 11 can cause a decrease in the expression of MMP-1 mRNA, an increase in the expression of TIMP-1 mRNA, and an increase in the expression of COL1α1 mRNA. In general, the derivative 7 or 11 protects the fibroblast from ultraviolet radiation, thereby preventing or reducing the occurrence of photoaging of the skin.

Experiment 6. Age-related β-galactosidase (SA-β-gal) activity

Senescence-associated β-galactosidase (SA-β-gal) decomposes galactoside in aging cells into monosaccharides at pH 6.0, so SA-β-gal can be used as aging and aging. The sign of the cell. In the cytochemistry test in vitro, the activity of SA-β-gal in cells or organisms is determined by measuring whether X-gal (5-bromo-4-chloro-3-indolyl β-D-galactoside) is The SA-β-gal cleavage results in a blue precipitate, such as the method disclosed by Lee et al. (Lee et al., Aging Cell, 2006.5(2): 187-195) or the modified method.

Fibroblasts (1 x 10 ⁶ cells/ml) were treated with different 5 μM 3-PED4HPT derivatives for 4 hours, followed by 6 J/cm ² intensity, 21 minutes of UV A. Next, the cell culture medium was removed, the fibroblasts were washed with PBS, and 3.7% of the formalin-fixed cells were added. Freshly prepared dye solution (1 mg/mL X-gal, 5 mM K ₃ Fe[CN] ₆ , 5 mM K ₄ Fe[CN] in PBS (pH 6.0) or citrate buffer (pH 4.5) ₆ , 2 mM MgCl ₂ ) and cells were cultured overnight at 37 ° C, after which the cells were washed with water, and the amount of blue precipitate was observed under a microscope (Lee et al., Aging Cell, 2006.5 (2): 187-195).

Please refer to Fig. 5, which is a graph comparing the number of aged cells after treatment of fibroblasts with 3-PED4HPT derivative or ultraviolet light A. Fibroblasts irradiated by ultraviolet light A showed strong SA-β-gal activity, while fibroblasts treated with derivative 7 or derivative 11 and irradiated with ultraviolet light A effectively inhibited SA-β. -gal activity, indicating that the 3-PED4HPT derivative of the present invention protects fibroblasts from photoaging problems caused by ultraviolet light A.

In summary, the novel triazole derivative (3-PED4HPT) synthesized in the present invention can reduce the expression of active oxide, matrix metalloproteinase-1 and/or aging-related β-galactosidase in skin fibroblasts. And/or increase the amount of adenosine triphosphate, mitochondrial membrane potential, tissue inhibitor of metalloproteinase-1, and/or type 1 collagen alpha1. The series of triazole derivatives synthesized by the above synthetic method have the potential to treat, treat and improve skin photoaging, and are applied to industries such as medical treatment, medical beauty, beauty, skin care, skin conditioning, cosmetics, and makeup products.

The invention is a difficult and innovative invention, and has profound industrial value, and is submitted in accordance with the law. In addition, the present invention may be modified by those skilled in the art without departing from the scope of the appended claims.

Figure 1 is a graph showing the relationship between the radiation dose of ultraviolet light A irradiated to fibroblasts and cell viability.

Figure 2 is a treatment of the 3-PED4HPT derivative and ultraviolet light A of the present invention. ATP expression after maternal cells.

Figure 3 (A) is the percentage of ROS production rate after treatment of fibroblasts with 3-PED4HPT derivative or UV light A.

Figure 3 (B) is the percentage of mitochondrial membrane potential after treatment of fibroblasts with 3-PED4HPT derivative or UV A.

Fig. 4(A) shows the mRNA expression level of MMP-1 after treatment of fibroblasts with 3-PED4HPT derivative or ultraviolet light A.

Figure 4 (B) shows the mRNA expression level of TIMP-1 after fibroblasts were treated with 3-PED4HPT derivative or ultraviolet light A.

Figure 4 (C) shows the mRNA expression of COL1α1 after treatment of fibroblasts with 3-PED4HPT derivative or ultraviolet light A.

Figure 5 is a graph comparing the number of aged cells after treatment of fibroblasts with 3-PED4HPT derivative or ultraviolet light A.

Claims (9)

A pharmaceutical composition for treating photoaging of the skin comprising a triazole derivative of the formula I: Wherein R ₁ , R ₂ and R ₃ are each selected from the group consisting of hydrogen, halogen, methyl, ethyl, nitro, methoxy, amino and trihalomethyl, and n is 1 or 2 . a triazole derivative of the formula I, Wherein R ₁ , R ₂ and R ₃ are each selected from the group consisting of hydrogen, halogen, methyl, ethyl, nitro, methoxy, amino and trihalomethyl, and n is 1 or 2 . The derivative according to claim 2, wherein the halogen is one selected from the group consisting of fluorine, chlorine, bromine and iodine. The derivative according to claim 2, wherein the trihalomethyl group is one selected from the group consisting of trifluoromethyl, trichloromethyl, tribromomethyl and triiodomethyl. A process for the preparation of a derivative according to claim 2, comprising: reacting a starting material of formula II with zinc chloride at 100 ° C for at least 12 hours to obtain the triazole derivative, The preparation method of claim 5, wherein the starting material is dissolved in dimethylformamide under a nitrogen atmosphere. The preparation method according to claim 5, wherein the preparation method further comprises: adding an aqueous solution to terminate the reaction. A use of a triazole derivative of the formula I obtained according to the preparation method described in claim 5 of the patent application for the preparation of a medicament for treating skin photoaging. The use according to claim 8, wherein the drug causes intracellular active oxide (ROS), matrix metalloproteinase-1 (MMP-1) and aging-related β-galactosidase (SA-) in the skin. The expression level of at least one of β-gal) is decreased, or the adenosine triphosphate (ATP), the mitochondrial membrane potential, the tissue inhibitor of metalloproteinase-1 (TIMP-1), and the first type collagen α1 of the cells are decreased. The amount of expression of at least one of (COL1α1) increases.