A carbazole derivative and its application

Abstract

The present invention belongs to the field of biomedicine technology, and discloses a carbazole derivative and its application. The carbazole derivative is a structural formula shown in formula (1) or a salt thereof, and is applied in medicines or cosmetics for the treatment or prevention of freckles, pregnancy spots, chloasma, coffee spots, nevus, ultraviolet-induced pigmentation and hereditary dark skin, and has a significant effect. Formula (1).

Description

Technical Field

[0001] The invention relates to a carbazole derivative and application thereof, in particular to a carbazole derivative and application thereof in medicine or cosmetics for treating or preventing hyperpigmentation diseases, belonging to the technical field of biomedicine. Background Art

[0002] The color of human skin, eyes and hair all rely on the production of melanin, which is determined by its quantity, quality and distribution. Melanin plays a key role in protecting human skin from UV rays, as well as damage from various environmental pollutants and toxic chemicals. However, excessive melanin production can lead to some pigmentation diseases, such as freckles, melasma, post-inflammatory hyperpigmentation, and even cancer, so regulating melanin formation is very important for controlling pigmentation.

[0003] Globally, the existing technology for the treatment of diseases related to pigment metabolism disorders remains a very important but not fully met clinical need. Tyrosinase is the rate-limiting enzyme that regulates melanin synthesis. Inhibiting tyrosinase activity is the most commonly used method to inhibit melanin synthesis. For example, hydroquinone, arbutin and kojic acid have been widely used as cosmetic whitening agents. However, the above compounds all have certain toxic side effects, which limits their clinical application. Microphthalmia-related transcription factor (MITF) is another key regulatory factor in the melanin synthesis pathway. It can not only regulate the proliferation and survival of melanocytes, but also regulate the expression of tyrosinase as a transcription factor to promote melanin synthesis. For example, isoliquiritigenin and hesperidin can inhibit the synthesis of melanin by accelerating the degradation of MITF. However, due to the poor bioavailability of the above compounds, no in vivo studies have been conducted to verify their effects. Summary of the invention

[0004] The purpose of the present invention is to overcome the deficiencies in the prior art, provide a carbazole derivative and its application, and improve the application effect in medicines or cosmetics for treating or preventing hyperpigmentation diseases.

[0005] To achieve the above object, the present invention is implemented by adopting the following technical solutions:

[0006] In a first aspect, the present invention provides a carbazole derivative, characterized in that it is a carbazole derivative having a structural formula (1) or a salt thereof:

[0007]

[0008] Among them, R1 is H or F, R2 is H or F, and R3 is any one of N-furan-2-ylmethyl-N-(2-hydroxypropyl)-methanesulfonamide, N-(2-chloro-6-cyanophenyl)-acetamide, 1-(1,1-dioxo-isothiazolin-2-yl)-2-propanol, and 1-(2-hydroxypropyl)-imidazolin-2-one.

[0009] Furthermore, R1 is H, R2 is H, R3 is N-furan-2-ylmethyl-N-(2-hydroxypropyl)-methanesulfonamide, and formula (1) is N-(3-carbazole-9-yl-2-hydroxypropyl)-N-furan-2-ylmethylmethanesulfonamide (KL001).

[0010] Furthermore, R1 is H, R2 is H, R3 is N-(2-chloro-6-cyanophenyl)-acetamide, and formula (1) is 2-carbazole-9-yl-N-(2-chloro-6-cyanophenyl)-acetamide (KL044).

[0011] Furthermore, R1 is H, R2 is H, R3 is 1-(1,1-dioxo-isothiazoline-2-yl)-2-propanol, and formula (1) is 1-carbazole-9-yl-3-(1,1-dioxo-isothiazoline-2-yl)-2-propanol.

[0012] Furthermore, R1 is F, R2 is F, R3 is 1-(2-hydroxypropyl)-imidazoline-2-one, and formula (1) is 1-(3-(3,6-difluoro-carbazole-9-yl)-2-hydroxypropyl)-imidazoline-2-one.

[0013] In a second aspect, the present invention provides a compound comprising any one of the above-mentioned carbazole derivatives or its optical isomer or salt thereof, and a pharmaceutically acceptable carrier or excipient.

[0014] A use of any of the above-mentioned carbazole derivatives in medicines or cosmetics for treating or preventing hyperpigmentation diseases.

[0015] Furthermore, the pigmented diseases include freckles, pregnancy spots, chloasma, coffee spots, nevi, ultraviolet-induced pigmentation and hereditary dark skin.

[0016] Compared with the prior art, the beneficial effects achieved by the present invention are:

[0017] The carbazole derivatives provided by the present invention are used in medicines or cosmetics for treating or preventing hyperpigmentation diseases, and significantly inhibit melanin synthesis;

[0018] In vivo pharmacodynamic experiments further demonstrated that N-(3-carbazol-9-yl-2-hydroxypropyl)-N-furan-2-ylmethyl methanesulfonamide (KL001) can inhibit the pigmentation on the back of guinea pigs caused by UVB irradiation, and has the efficacy of treating hyperpigmentation diseases, especially melasma, post-inflammatory pigmentation and senile lentigo. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The effect of KL001 on the expression of Tyrosinase and MITF, key proteins in melanin synthesis, in human primary melanocytes, where A is a protein immunoblot image and B is the relative expression of the protein;

[0020] Figure 2 The effect of KL044 on the expression of Tyrosinase and MITF, key proteins in melanin synthesis, in human primary melanocytes, where A is a protein immunoblot image and B is the relative expression of the protein;

[0021] Figure 3 This is a comparison chart of the effects of KL001 on melanin synthesis in the back skin of guinea pigs;

[0022] Figure 4 This is a color difference value diagram of the effects of KL001 and the control group on the back skin of guinea pigs over time;

[0023] Figure 5 This is a comparison chart of the effects of KL001 on melanin synthesis in the back skin of guinea pigs;

[0024] Figure 6 This is a comparison chart of the effects of KL001 on the number of melanocytes in the back skin of guinea pigs. DETAILED DESCRIPTION

[0025] The present invention will be further described below in conjunction with the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and cannot be used to limit the protection scope of the present invention.

[0026] This example studies the effects of carbazole derivatives on melanin synthesis in human primary melanocytes, specifically involving the effects of four carbazole derivatives (purchased from MedChemExpress) on the proliferation of human primary melanocytes and the effects on melanin synthesis in human primary melanocytes.

[0027] Embodiment 1

[0028] 1. Effects of carbazole derivatives on proliferation of primary human melanocytes. The specific experimental process is as follows:

[0029] 100 μL / well (about 1×10 ) of human primary melanocytes were added to a 96-well plate. 4), and cultured in a 37°C 5% CO2 cell culture incubator for 24 hours;

[0030] The four carbazole derivatives (20 μmol / L) were added respectively, and cultured in a cell culture incubator at 37°C, 5% CO2 air and 100% humidity for 48 hours;

[0031] Add 50 μL of 1× MTT to each well and incubate at 37°C for 4 h to reduce MTT to formazan;

[0032] The supernatant was aspirated, and 150 μL DMSO was added to each well to dissolve the formazan, and the mixture was shaken on a plate shaker.

[0033] The optical density of each well was detected by an ELISA instrument at a wavelength of 490 nm, and the cell proliferation rate was calculated. The results are shown in Table 1.

[0034] Experimental results: Compared with the blank control group, the drug-treated group had no significant effect on the growth of normal human primary melanocytes (P>0.05).

[0035] From Table 1, it can be concluded that the four carbazole derivatives have no significant effect on the proliferation of human primary melanocytes and show low toxicity.

[0036] 2. Effects of carbazole derivatives on melanin synthesis in human primary melanocytes. The specific experimental process is as follows:

[0037] Human primary melanocytes (2 mL / well) (approximately 2 × 10 5 ), and cultured in a 37°C 5% CO2 cell culture incubator for 24 hours;

[0038] The four carbazole derivatives (20 μmol / L) were added respectively, and cultured in a cell culture incubator at 37°C, 5% CO2 air and 100% humidity for 48 hours;

[0039] 100 μL of non-denaturing lysis buffer (containing 1 nM PMSF) was added to each well and lysed on ice for 20 minutes. The cells treated with the above drugs were collected and centrifuged at 4°C, 12000 r / min for 20 minutes. The supernatant was taken and the protein was quantified by the BCA method to calculate the total protein content;

[0040] The lower melanin precipitate was added with 100 μL NaOH solution (1 mol / L, containing 10% DMSO), placed in a metal bath at 80°C for 2 hours for lysis, and the completely dissolved melanin was added to a 96-well plate at 100 μL / well.

[0041] The optical density of each well was detected by an ELISA instrument at a wavelength of 405 nm, and the cell proliferation rate was calculated. The results are shown in Table 1. In Table 1, compared with the blank control group, *P﹤0.05, **P﹤0.01, ***P﹤0.001.

[0042] Experimental results: Compared with the blank control group, the four carbazole derivatives can significantly inhibit the melanin synthesis of human primary melanocytes.

[0043] Experimental conclusion: The four carbazole derivatives can significantly inhibit the synthesis of melanin in human primary melanocytes, among which the compound N-(3-carbazole-9-yl-2-hydroxypropyl)-N-furan-2-ylmethyl methanesulfonamide (KL001) has the most obvious effect in inhibiting melanin synthesis.

[0044] Table 1: Effects of carbazole derivatives on proliferation rate and melanin synthesis of human primary melanocytes

[0045]

[0046] Embodiment 2

[0047] This example studies the effects of KL001 and KL044 on the expression of Tyrosinase and MITF, important regulatory proteins for melanin synthesis, in human primary melanocytes. The specific process is as follows:

[0048] Human primary melanocytes (2 mL / well) (approximately 2 × 10 5 ), and cultured in a 37°C 5% CO2 cell culture incubator for 24 hours;

[0049] Melanocytes were treated with α-MSH, and then different concentrations of KL001 or KL044 were added and cultured in a cell culture incubator at 37°C, 5% CO2 air and 100% humidity for 48 hours;

[0050] 100 μL of non-denaturing lysis buffer (containing 1 nM PMSF) was added to each well and lysed on ice for 20 minutes. The cells treated with the above drugs were collected and centrifuged at 4°C, 12000 r / min for 20 minutes. The supernatant was taken and the protein was quantified by the BCA method to calculate the total protein content;

[0051] Adjust the protein lysate to the same concentration, mix with an equal volume of 2x loading buffer, boil for 10 minutes, and perform SDS-polyacrylamide gel electrophoresis;

[0052] After electrophoresis, the proteins were transferred to PVDF membranes using a voltage of 100 V for 1 h and blocked with TBST (0.05% Tween-20) containing 5% skim milk for 1.5 h;

[0053] The membrane was incubated with MITF, Tyrosinase, and β-actin primary antibodies (1:1000) dissolved in TBST at 4°C overnight, washed 4 times with TBST for 7 minutes each time, and then HRP-labeled secondary antibodies were added to bind to the primary antibodies, and the membrane was incubated at room temperature for 1 hour; the membrane was removed and washed 4 times with TBST for 7 minutes each time.

[0054] For chemiluminescence detection, add the pre-mixed luminescent solution droplets onto the membrane and take photos using a gel imaging system. The results are analyzed using Bio-Rad's Quantity One software. Figure 1 The following table shows the effect of KL001 on the expression of Tyrosinase and MITF, key proteins in melanin synthesis in human primary melanocytes. Figure 2 The effect of KL044 on the expression of Tyrosinase and MITF, key proteins in melanin synthesis, in primary human melanocytes was investigated. Figure 1 , 2 It can be seen that compared with the α-MSH positive control group, KL001 and KL044 can significantly inhibit the protein expression of MITF and Tyrosinase in human primary melanocytes, and the difference is significant ( # P﹤0.05, ## P﹤0.01)

[0055] Experimental conclusion: KL001 and KL044 can inhibit melanin synthesis by inhibiting the expression of important proteins (MITF, Tyrosinase) in human primary melanocytes.

[0056] Embodiment 3

[0057] This example conducts an experiment on the effect of KL001 on ultraviolet-induced pigmentation on the back of guinea pigs. The specific operation process is as follows:

[0058] After the guinea pigs were adapted to feeding (10, half male and half female), the hair on the back of the guinea pigs was shaved with an electric shaver, and the skin on the back of the guinea pigs was irradiated with an ultraviolet light therapy device (Sigma, SH4B) with a total area of ​​6*6cm 2 .

[0059] The lamp is 15cm away from the guinea pig's back, and the one-time irradiation dose is 500mJ / cm 2 , processed for one week.

[0060] One week after irradiation of the guinea pig's back skin, stable pigmentation was formed locally. The irradiated area was divided into two medication areas, one was smeared with KL001 (1.5%), and the other was smeared with blank matrix. Each medication area was a circle with a diameter of 1 cm. The two medication areas were separated by 0.5 cm. The medication was applied twice a day.

[0061] The treatment lasted for three weeks, and the short hair was shaved with an electric shaver before medication. The color changes of various parts of the guinea pig back skin were measured with a colorimeter (Tri-En, CR-400) before ultraviolet irradiation, 1 week after irradiation, 1 week, 2 weeks, and 3 weeks after medication. The color difference value = each measurement value - the measurement value before ultraviolet irradiation, and photos were taken for objective comparison.

[0062] Four weeks later, skin tissues were obtained, fixed with 4% paraformaldehyde solution, and routinely sectioned in paraffin.

[0063] The Masson-Fontana amine silver staining method was used to determine the changes in melanin in the back skin of guinea pigs, and the immunohistochemical S-100 method was used to detect the changes in the number of melanocytes in the back skin. Figure 3 , Figure 4 , Figure 5 , Figure 6 As shown, Figure 4 Compared with the control group, * P<0.05. Figure 3-Figure 6 It was found that compared with the blank control group, KL001 inhibited the synthesis of melanin in the back skin of guinea pigs induced by ultraviolet rays, and had no significant effect on the number of melanocytes in the skin.

[0064] The experimental conclusion of this example is that KL001 reduces ultraviolet-induced melanin deposition on the back skin of guinea pigs.

[0065] The present invention provides a use of a carbazole organism or a pharmaceutically acceptable salt thereof in preparing a medicine / cosmetic for treating and / or preventing hyperpigmentation diseases, in particular, a use of a carbazole organism or a pharmaceutically acceptable salt thereof in preparing a medicine / cosmetic for treating and / or preventing freckles, pregnancy spots, chloasma, coffee spots, nevus, ultraviolet-induced pigmentation, and hereditary dark skin.

[0066] In vitro experiments have shown that the above four carbazole derivatives can significantly inhibit melanin synthesis; in vivo pharmacodynamic experiments have further proved that N-(3-carbazole-9-yl-2-hydroxypropyl)-N-furan-2-ylmethyl methanesulfonamide (KL001) can inhibit the pigmentation of guinea pig backs caused by UVB irradiation, and has the efficacy of treating hyperpigmentation diseases, especially chloasma, post-inflammatory pigmentation and senile freckles.

[0067] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An application of a carbazole derivative, characterized in that: Application in the preparation of drugs or cosmetics for treating or preventing hyperpigmentation diseases; the carbazole derivative is of the structural formula (1) or its salt: Wherein, R1 is H or F, R2 is H or F, and R3 is The dashed line is the point of connection to the carbazole N atom.

2. The use according to claim 1, characterized in that: The formula (1) is 3. The use according to claim 1, characterized in that: The formula (1) is 4. The use according to claim 1, characterized in that: The formula (1) is 5. The use according to claim 1, characterized in that: The formula (1) is 6. The use according to claim 1, characterized in that: The hyperpigmented disease is selected from freckles, pregnancy spots, chloasma, coffee spots, nevus, ultraviolet-induced pigmentation or hereditary dark skin.

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