KR102561912B1 - Cephalothin derivative or pharmaceutically acceptable salt thereof, process for the preparation thereof and composition for preventing hair loss or promoting hair growth comprising the same - Google Patents

Abstract

The present invention provides a cephalotene derivative or a pharmaceutically acceptable salt thereof, a method for preparing the same, and a composition for preventing hair loss or stimulating hair growth comprising the same.

Description

Cephalotin derivative or pharmaceutically acceptable salt thereof, method for preparing the same, and composition for preventing hair loss or promoting hair growth containing the same growth comprising the same}

The present invention relates to a cephalotene derivative or a pharmaceutically acceptable salt thereof, a method for preparing the same, and a composition for preventing hair loss or stimulating hair growth comprising the same.

The hair follicle, a physiological organ in the body that produces hair, is responsible for the generation of hair during postnatal development, the anagen phase in which the generated hair actively grows, the catagen phase in which hair degenerates, and when hair falls out. It repeats the hair cycle, which is divided into a telogen phase, which is maintained until, and an exogen phase, in which hair loss occurs, and is involved in the growth, maintenance, and loss of hair. The activation of hair follicles occurs by dermal papilla cells, and in particular, proliferation and differentiation of dermal papilla cells are primarily involved in the progress of the hair growth cycle and hair formation (Botchkarev VA, Kishimoto J, Molecular control of epithelial-mesenchymal interactions) during hair follicle cycling.J Iinvestig Dermatol Symp Proc 8, 46-55,2003). During the growth phase, when hair grows actively, dermal papilla cells actively proliferate and differentiate, and hair growth stops, and during the catagen, telogen, and hair loss phases where hair loss occurs, these cells die (Batchkarev V.A, Batchkareva N.V., Nakamura M, Noggin is required for induction of the hair follicle growth phase in postnatal skin. FASEB J 15, 2205-2214, 2001). In addition, cell division and migration near the dermal papilla are closely related to the growth of hair, and hair is newly generated from the dermal papilla in the growth phase. Cells are activated by various cytokines, hormones, etc. (Eva M.J. Peters, Desmond J Tobin, Migration of melanoblasts into developing murine hair follicle is accompanied by transient c-kit expression, J Histochem Cytochem, 50, 6751-766, 2002; Jahoda CA, Cell movement in the hair follicle dermis-more than a two-way street J Invest Dermatol, 1221, 1523-1747, 2003). Therefore, since hair growth and hair loss are closely related to the proliferation and death of dermal papilla cells, lengthening the growth period by inducing their proliferation or suppressing cell death and shortening the catagen, telogen, and hair loss periods will be a treatment plan to improve hair loss. .

The present inventors have completed a patent application by finding that cephalothin or a pharmaceutically acceptable salt thereof can be usefully used for preventing hair loss or promoting hair growth (Korean Patent Application No. 10-2019-0140991, 2019 Filed on November 6).

The inventors of the present invention conducted tests on various cephalotene derivatives in order to develop a compound capable of avoiding the side effects of antibiotic resistance induction due to lack of antibiotic activity while preventing hair loss or promoting hair growth. It was found that tin derivatives have excellent hair loss prevention or hair growth promoting activity while avoiding side effects due to antibiotic activity.

Accordingly, an object of the present invention is to provide the cephalotene derivative or a pharmaceutically acceptable salt thereof.

In addition, an object of the present invention is to provide a method for preparing the cephalotene derivative or a pharmaceutically acceptable salt thereof.

In addition, an object of the present invention is to provide a composition for preventing hair loss or stimulating hair growth comprising the cephalotene derivative or a pharmaceutically acceptable salt thereof as an active ingredient.

According to one aspect of the present invention, a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof is provided.

<Formula 1>

According to another aspect of the present invention, there is provided a method for preparing the compound of Formula 1 or a pharmaceutically acceptable salt thereof, comprising reacting 7-aminocephalosporanic acid and 2-thiophenecarbonyl chloride.

According to another aspect of the present invention, there is provided a composition for preventing hair loss or stimulating hair growth comprising the compound of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

It was found by the present invention that a specific cephalotene derivative (ie, the compound of Formula 1) or a pharmaceutically acceptable salt thereof has excellent anti-hair loss or hair growth promoting activity while avoiding side effects due to antibiotic activity. Therefore, the compounds and compositions including them according to the present invention can be usefully used for preventing hair loss or stimulating hair growth while fundamentally avoiding side effects such as induction of antibiotic resistance in the case of long-term administration or skin application.

Figure 1 shows the results of evaluating the proliferation efficiency of dermal papilla cells according to the treatment of each concentration of the compound of Formula 1.
Figure 2 shows the expression of representative factors (Wnt5α, GSK3β, β-catenin, etc.) that mediate Wnt/β-catenin signaling known to function in hair follicle regeneration and growth promotion after treatment with the compound of Formula 1 in dermal papilla cells at different concentrations. It shows the result of measuring the amount and the expression level of VEGF, a cell growth factor, by immunoblot analysis.

The present invention provides a compound represented by Formula 1 below or a pharmaceutically acceptable salt thereof.

<Formula 1>

The compound of Formula 1 has a structure in which a thienylacetamido group of cephalothin (a compound of Formula 2 below) is modified into a thiophenecarbonylamido group. According to the present invention, the compound of Formula 1 or a pharmaceutically acceptable salt thereof (eg, the sodium salt of the compound of Formula 1) has excellent hair loss prevention or hair growth promoting activity while avoiding side effects due to antibiotic activity it turned out Therefore, the compound of Formula 1 or a pharmaceutically acceptable salt thereof can be usefully used for preventing hair loss or promoting hair growth.

<Formula 2>

The present invention also provides a method for preparing the compound of Formula 1 or a pharmaceutically acceptable salt thereof. The production method of the present invention includes the step of reacting 7-aminocephalosporanic acid and 2-thiophenecarbonyl chloride.

The reaction between 7-aminocephalosporanic acid and 2-thiophenecarbonyl chloride may be carried out in a polar solvent (eg, a mixed solvent of water and acetone) in the presence of a base such as triethylamine. The reaction can be carried out at low temperature, eg on an ice-bath. The obtained product, that is, the compound of Formula 1, can be isolated by conventional methods such as extraction using an organic solvent (eg, ethyl acetate), purification using column chromatography, and the like. If necessary, the compound of Formula 1 may be converted into a metal salt of the compound of Formula 1 by reacting with a metal such as sodium.

The present invention also provides a composition for preventing hair loss or promoting hair growth comprising the compound of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

The composition for preventing hair loss or stimulating hair growth of the present invention may be prepared in the form of a pharmaceutical composition or cosmetic composition containing the compound of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient. For example, the composition of the present invention may be in the form of a pharmaceutical composition for preventing hair loss or promoting hair growth comprising the compound of Formula 1 or a pharmaceutically acceptable salt thereof (eg, the sodium salt of the compound of Formula 1) as an active ingredient. can In addition, the composition of the present invention may be in the form of a cosmetic composition for preventing hair loss or promoting hair growth comprising the compound of Formula 1 or a pharmaceutically acceptable salt thereof (eg, sodium salt of the compound of Formula 1).

The pharmaceutical composition may include a pharmaceutically acceptable carrier, and may be formulated into parenteral formulations such as solutions, suspensions, emulsions, lotions, ointments, and lyophilized formulations according to conventional methods. Preferably, it may be formulated into a formulation for transdermal administration such as an external solution, emulsion, ointment, and the like. The pharmaceutically acceptable carrier includes an aqueous diluent or solvent such as phosphate buffered saline, purified water, and sterile water, and includes a non-aqueous diluent or solvent such as propylene glycol, polyethylene glycol, and olive oil. In addition, wetting agents, fragrances, preservatives, etc. may be included as needed. The dose of the compound of Formula 1 or a pharmaceutically acceptable salt thereof contained in the pharmaceutical composition may be appropriately selected by a person skilled in the art according to the condition of the patient, the severity of the disease, the route and duration of administration. The compound of Formula 1 or a pharmaceutically acceptable salt thereof may be administered (eg, applied) to the scalp in an amount of 0.01 to 5 mg, preferably 0.1 to 1 mg per day, for example, the administration may be administered once a day or divided into several times.

The cosmetic composition may be prepared in various forms according to conventional cosmetic preparation methods. For example, the cosmetic composition of the present invention may be prepared in the form of a cosmetic product containing the compound of Formula 1 or a salt thereof, shampoo, treatment, hair lotion, hair cream, hair gel, scalp essence, tonic, etc. It can be used by diluting with a normal cleansing liquid, astringent liquid and moisturizing liquid. In addition, the cosmetic composition may include conventional adjuvants such as stabilizers, solubilizers, vitamins, pigments, and fragrances commonly used in the field of cosmetic compositions. In the cosmetic composition of the present invention, the content of the compound of Formula 1 or a pharmaceutically acceptable salt thereof is an amount effective to achieve the effect of preventing hair loss or promoting hair growth, for example, 0.0005 to 0.5% by weight based on the total weight of the composition. content, preferably about 0.005 to 0.1% by weight.

Hereinafter, the present invention will be described in detail through examples and test examples. However, these examples and test examples are for exemplifying the present invention, and the scope of the present invention is not limited to these examples and test examples.

Example 1: Preparation of Cephalotene Derivatives (Compound of Formula 1)

<Formula 1>

After dissolving 7-aminocephalosporanic acid (0.50 g, 1.84 mmol) in a mixed solvent of water (5 mL) and acetone (5 mL), the pH was adjusted to 8 with triethylamine, and the ice-bath was placed. 2-thiophenecarbonyl chloride (0.22 mL, 2.20 mmol) was added dropwise while stirring on a -bath). After stirring the reaction mixture for 1.5 hours, the solvent was removed by distillation under reduced pressure. Water and ethyl acetate were added to the obtained residue and extracted twice. The combined organic layers were washed with saturated brine, dried over anhydrous MgSO ₄ , and the solids were removed by filtration and the filtrate was distilled under reduced pressure. The resulting residue was purified by silica gel column chromatography (methanol : dichloromethane = 1:20, (v/v)) to obtain the compound (0.17 g, 23.5%) as a light brown solid.

R _f 0.50 (methanol:dichloromethane = 1:2);

^1H -NMR (400 MHz, DMSO- d6 ) δ 2.01 (s, 3H) _, 3.24 (d, J = 17.2 Hz, 1H), 3.51 (d, J = 17.2 Hz, 1H), 4.82 (d, J = 12.4 Hz, 1H), 4.99 (d, J = 12.4 Hz, 1H), 5.06 (d, J = 4.4 Hz, 1H), 5.68 (dd, J = 8.0, 4.8 Hz, 1H), 6.16 (dd, J = 5.2, 3.6 Hz, 1H), 7.82 (dd, J = 5.2, 1.2 Hz, 1H), 8.01 (dd, J = 4.4, 1.2 Hz, 1H), 9.37 (d, J = 8.0 Hz, 1H);

¹³ C-NMR (100 MHz, DMSO- d6 ) 20.7, 25.1, 57.5 _, 59.1, 64.2, 128.1, 131.9, 138.3, 161.6, 162.8, 164.9, 170.5 ppm.

Test Example 1: Culture of human dermal papilla cells

Human-derived dermal papilla cells are Cell Applications, Inc. (USA) was purchased and used. The purchased cells were morphologically confirmed by microscopic observation, and DMEM medium (Dulbecco's Modified Eagle's medium) containing 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin/streptomycin, amphotericin) was used. (Hyclone, USA) was used to culture at 37°C, 5% CO ₂ incubator. After 24 hours of cell culture, the old medium was removed, washed with phosphate-buffered saline (PBS), and then the same new medium was added and further cultured.

Test Example 2: Effect of cephalotene derivatives on dermal papilla cell proliferation

The dermal papilla cells cultured in Test Example 1 were seeded at 5 X 10 ³ cells/well in each well of a 96-well plate coated with collagen for cell culture only, and cultured for 24 hours by adding the same medium. Cells were observed under a microscope, and after removing the existing medium, they were washed with PBS. Thereafter, for cell starvation, a new DMEM medium containing 1% FBS and antibiotics was added, followed by additional culture for 24 hours, and then the medium was removed and washed with PBS. When a new culture medium was added, the compound of Formula 1 prepared in Example 1 was treated at different concentrations (10, 50, 100, 250, 500, 750, 1000, 1500 μM), and then 37°C, 5% CO ₂ Incubated for 24 hours in an incubator. As a control, the same volume of PBS was treated. Each experimental group was simultaneously performed in 3 wells under the same conditions. Cell proliferation efficacy was evaluated by measuring absorbance at a wavelength of 450 nm using a WST-1 assay (Ez-cytox Cell Viability Assay Kit, Daeilbio, Korea) 24 hours after each sample treatment. The results are shown in FIG. 1 . As can be seen from the results of Figure 1, it was observed that cell proliferation was significantly increased in the experimental group to which the compound of Formula 1 was added, and at a concentration between 100 and 750 μM, the cell proliferation effect was more than 50% compared to the control group. was

By treating dermal papilla cells with beta-lactam compounds such as penicillin, ampicillin, cefazolin, cefadroxil, cephalexin, and cephradine at the same concentration, As a result of observing cell proliferation by the same method, these compounds had no significant effect on dermal papilla cell proliferation (data not shown).

Test Example 3: Evaluation of Wnt/β-catenin signaling activation

Immunoblot analysis to determine the efficacy of the compound of Formula 1 on Wnt / β-catenin signaling known to affect the regeneration and growth promotion of dermal papilla cells and the expression of VEGF, a representative growth factor in cells Immunoblot analysis was performed. Dermal papilla cells were seeded at 3 X 10 ⁵ cells/well in each well of a 6-well plate containing DMEM medium and cultured in a 37°C, 5% CO ₂ incubator. After 24 hours, the compound of Formula 1 was treated at different concentrations (0.1, 1, 10, 100, 500 μM), cultured for 24 hours, and then the medium was removed and washed with PBS. The cells were lysed with RIPA buffer (Sigma-Aldrich, USA) containing a protease inhibitor cocktail (Sigma-Aldrich, USA) and a phosphatase inhibitor cocktail, and centrifuged. . SDS-PAGE was performed on the same amount of proteins obtained from the supernatant, and anti-Wnt5α antibody (Abcam, USA), anti-GSK3β antibody (Cell Signaling Technology, USA), anti-phospho-GSK3β antibody (Cell Signaling Technology , USA), anti-β-catenin antibody (Cell Signaling Technology, USA), anti-active-β-catenin antibody (Cell Signaling Technology, USA), anti-VEGF antibody (Abcam, USA), anti-β-actin antibody (Santa Cruz Biotechnology, USA) was used for immunoblot analysis. As can be seen from the results of FIG. 2, the protein amounts of Wnt5α, GSK3β, and β-catenin, which are representative factors mediating Wnt/β-catenin signaling, were increased in the group treated with the compound of Formula 1 compared to the control group. As a result, It was confirmed that the expression level of VEGF, a cell growth factor, also increased significantly. On the other hand, there was no significant difference in β-actin protein between the control group and the compound treated group of Formula 1.

Test Example 4: Evaluation of antimicrobial activity of the compound of Formula 1 against microorganisms

In order to evaluate the change in the antibiotic activity of the compound of Formula 1 against microorganisms, a disc diffusion assay was performed for Escherichia coli. In order to suppress the contamination of other microorganisms in the air during the experiment, a plasmid vector pET28a (Novagen, Germany) carrying a kanamycin resistance gene was introduced into the E. coli strain DH5α (Novagen, Germany) and used to evaluate antibiotic activity. E. coli strain DH5α into which pET28a was introduced was inoculated into LB broth (Luria-Bertani broth, BD Life Sciences, USA) and cultured in a shaking incubator at 37° C. and 180 rpm for 4 hours. After E. coli was smeared on an agar plate made of LB Agar (Luria-Bertani Agar, BD Life Sciences, USA), a disk containing the compound of Formula 1 was placed on the plate and cultured in a 37° C. incubator for 24 hours. Disks to which kanamycin, ampicillin, and cephalothin were respectively added were used as positive controls, and disks to which equal volumes of PBS and DMSO (dimethyl sulfoxide) were added instead of antibiotics were used as negative controls. The treatment concentration of the antibiotic was set according to the WHO disc potency standard, and the basic concentration of the compound of Formula 1 was set equal to the concentration having the antibiotic activity of the conventional cephalothin. Treatment concentrations of each antibiotic and compound of Formula 1 are shown in Table 1 below. Changes in antibiotic activity were evaluated at the basic concentration and at 2-, 5-, and 10-fold concentrations of the basic concentration.

Antibiotic Basic concentration (1X) 2X 5X 10X kanamycin 30ug/10ul - - - ampicillin 10ug/10ul - - - cephalotene 30ug/10ul 60ug/10ul 150ug/10ul 300ug/10ul Compound of Formula 1 30ug/10ul 60ug/10ul 150ug/10ul 300ug/10ul

The antibiotic ability of each material was compared by measuring the clear zone diameter shown on the agar plate, and the results are shown in Table 2 below.

matter Clear zone diameter (mm) 1X processing 2X processing 5X processing 10X processing PBS 0 - - - DMSO 0 - - - kanamycin 0 - - - ampicillin 8 - - - cephalotene 13 16 22 25 Compound of Formula 1 0 0 0 0

As can be seen from the results of Table 2, there was no antibiotic activity in the PBS and DMSO-treated groups used as negative controls, and no clear zone was formed in the kanamycin-treated group in the E. coli strain into which the plasmid carrying the kanamycin resistance gene was introduced. In the cephalotene-treated group, the clear zone diameter increased according to the treatment concentration. In contrast, in the group treated with the compound of Formula 1, antibiotic activity was not shown even at the basic concentration, as well as at 2, 5, and 10 times the basic concentration.

Claims (5)

delete delete delete A composition for preventing hair loss or stimulating hair growth, comprising a compound of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
<Formula 1>
The composition for preventing hair loss or stimulating hair growth according to claim 4, wherein the pharmaceutically acceptable salt of the compound of Formula 1 is the sodium salt of the compound of Formula 1.