Use of N-methylatanine in the preparation of fungal inhibitors

Abstract

This invention provides the application of N-Methylatanine in the preparation of fungal inhibitors, belonging to the field of antifungal drug technology. The N-Methylatanine of this invention is of natural origin and has low toxicity, exhibiting IC50 activity against normal human lung epithelial cells. 50 The concentration is >40 μM; it exhibits significant antibacterial effects. At a concentration of 100 μM, its inhibition rates against *Epidermophyton floccosum*, *Trichophyton rubrum*, and *Microsporum gypseum* reached 99.385±0.8%, 102.17±1.956%, and 100±0.115%, respectively. This invention provides a new approach for the treatment of fungal infections of the skin in humans and animals.

Description

Technical Field

[0001] This invention belongs to the field of antifungal drug technology, and particularly relates to the application of N-Methylatanine in the preparation of fungal inhibitors. Background Technology

[0002] Skin and epidermal fungal infections are common infectious diseases in humans and animals, mainly caused by fungi such as Trichophyton, Epidermophyton, and Microsporum. Clinical manifestations include itching, scaling, inflammation, and even ulceration of the skin, severely impacting quality of life. Currently, most commonly used antifungal drugs are chemically synthesized preparations (such as terbinafine hydrochloride and ketoconazole). While they can inhibit fungal growth to some extent, long-term use can easily lead to increased fungal resistance. Furthermore, some drugs have side effects such as skin irritation and hepatotoxicity, limiting their applicability, especially to infants, pregnant women, and individuals with sensitive constitutions.

[0003] Naturally derived antifungal components have become a research hotspot due to their advantages such as low toxicity and good biocompatibility. However, there are few existing natural products that possess both high antifungal activity and low toxicity, and most natural components have narrow antifungal spectra or excessively high effective concentrations, making it difficult to meet clinical needs. Therefore, developing novel fungal inhibitors that are naturally derived, low in toxicity, have clear antifungal effects, and have a wide range of applications is of great significance for the treatment of fungal infections of the skin in humans and animals. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide the application of N-Methylatanine in the preparation of fungal inhibitors. N-Methylatanine is of natural origin and has low toxicity, with significant antibacterial effect, providing a new approach for the treatment of fungal infections of the skin in humans and animals.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] Application of N-Methylatanine in the preparation of fungal inhibitors.

[0007] Preferably, the concentration of N-Methylatanine used is 20 μM or higher.

[0008] Preferably, the fungi include human and animal epidermal infection fungi.

[0009] More preferably, the fungi include fungi of the genera Trichophyton, Epidermophyton, and Microsporum.

[0010] The present invention also provides a fungal inhibitor, the antifungal ingredient comprising N-Methylatanine and / or a pharmaceutically acceptable salt thereof.

[0011] Preferably, the concentration of N-Methylatanine and / or its pharmaceutically acceptable salt is 20 μM or higher.

[0012] Preferably, the dosage form includes solutions, suspensions, emulsions, sprays, ointments, creams, gels, powders, tablets, films, suppositories, or patches.

[0013] Preferably, it also includes pharmaceutically acceptable carriers or excipients.

[0014] More preferably, the carrier or excipient includes one or more of the following: additives, wetting agents, emulsifiers, suspending agents, preservatives, salts that affect osmotic pressure, buffers, or colorants.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The active ingredient of this invention, N-Methylatanine, can be extracted from plants of the Rutaceae family and is a natural product. Safety test results show that it has no adverse effects on the IC50 levels of normal human lung epithelial cells. 50 The concentration was >40 μM, which is much higher than that of the positive control drugs doxorubicin and paclitaxel, indicating that it has extremely low toxicity to normal cells, good biocompatibility, and can reduce the risk of side effects from long-term use.

[0017] N-Methylatanine exhibits a clear inhibitory effect on the main pathogenic fungi (Trichophyton, Epidermophyton, and Microsporum) causing epidermal infections in humans and animals. Experimental data show that at a concentration of 100 μM, its inhibition rates against Epidermophyton floccosum, Trichophyton rubrum, and Microsporum gypseum reached 99.385±0.8%, 102.17±1.956%, and 100±0.115%, respectively, approaching the antibacterial effect of the positive control drug terbinafine hydrochloride. Furthermore, it effectively inhibits some fungi at concentrations above 20 μM, meeting the concentration requirements for clinical application. Attached Figure Description

[0018] Figure 1 The NMR spectrum of N-Methylatanine is shown. Detailed Implementation

[0019] This invention provides the application of N-Methylatanine in the preparation of fungal inhibitors.

[0020] The chemical formula of N-Methylatanine of this invention is 4-methoxy-1-methyl-3-(3-methyl-2-butenyl)-2(1H)-quinolinone, which has a quinolone structural core, and its structural formula is shown in formula (I):

[0021]

[0022] The NMR spectrum of N-Methylatanine is as follows: Figure 1 As shown, in 1 H-NMR (CDCl3) spectrum δ H 1.68 (3H, d, J =2.1 Hz) and 1.80 (3H, d, J = 1.8 Hz) represents the two methyl signals at the isopentenyl terminus, 3.40 (2H, d, J = 7 Hz) represents the methylene signal on the isopentenyl segment, 3.73 (3H, s, N-Me), 3.91 (3H, s, O-Me), and 5.24 (1H, m) represent the hydrogen signals of the double bond on the isopentenyl group, among which 7.25, 7.37, 7.53, and 7.83 are typical hydrogen signals on the benzene ring of a 2-quinolone; 13 C-NMR (CDCl3) spectrum δ C 164.37 is the carbonyl carbon signal. δ C 160.76 is the methoxy carbon signal. δ C 30.15 is the N-methyl carbon signal. δ C 62.08 is the oxymethyl carbon signal.

[0023] This invention does not have a specific limitation on the source of N-Methylatanine. It can be obtained through market channels or extracted and prepared from Rutaceae plants (such as the dried, nearly mature fruit of Evodia rutaecarpa or Lepidium apetalum).

[0024] In this invention, the concentration of N-Methylatanine used is preferably 20 μM or more, more preferably 20 to 200 μM, and even more preferably 100 μM.

[0025] In this invention, the preferred fungi include fungi that cause skin infections in humans and animals, more preferably including fungi of the genus Trichophyton, genus Epidermophyton, and genus Microsporum, and more preferably Epidermophyton floccosum (…). 絮状表皮癣菌 ), Trichophyton rubrum ( 红色毛癣菌 ) and Microsporum gypseum ( 石膏样小孢子菌 ).

[0026] The present invention also provides a fungal inhibitor whose antifungal components include N-Methylatanine and / or its pharmaceutically acceptable salts.

[0027] In this invention, the concentration of N-Methylatanine and / or its pharmaceutically acceptable salt is preferably 20 μM or more, more preferably 20 to 200 μM, and even more preferably 100 μM.

[0028] In this invention, the preferred dosage forms of fungal inhibitors include solutions, suspensions, emulsions, sprays, ointments, creams, gels, powders, tablets, films, suppositories, or patches. More preferably, the fungal inhibitors also include pharmaceutically acceptable carriers or excipients; more preferably, they include one or more of the following: adjuvants, wetting agents, emulsifiers, suspending agents, preservatives, salts affecting osmotic pressure, buffers, or colorants.

[0029] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0030] Example 1

[0031] An N-Methylatanine external solution: using 30 mL of anhydrous ethanol and 65 mL of purified water as solvents, 0.05 g of benzalkonium chloride (preservative) and 5 mL of glycerin (wetting agent) as excipients, N-Methylatanine is added to a final concentration of 100 μM to prepare an N-Methylatanine solution.

[0032] Example 2

[0033] An N-Methylatanine topical ointment: using 80g of petrolatum and 10g of lanolin as a base, 5g of glyceryl stearate (emulsifier) ​​and 0.1g of ethylparaben (preservative) as excipients, and adding N-Methylatanine to a concentration of 20μM, an N-Methylatanine ointment is prepared.

[0034] Example 3

[0035] An N-Methylatanine topical gel: using 2g of carbomer 940 as the base, 90g of purified water as the solvent, and 1g of triethanolamine (neutralizing agent), 5g of glycerin (humectant), and 0.3g of phenoxyethanol (preservative) as excipients, N-Methylatanine hydrochloride is added to a concentration of 200μM to prepare an N-Methylatanine gel.

[0036] Experimental Example 1

[0037] Safety tests of N-Methylatanine

[0038] 1. Cell seeding: Prepare a single-cell suspension (human normal lung epithelial cells BEAS-2B, purchased from Meisen CTCC) using culture medium (DMEM) containing 10% fetal bovine serum. Seed 5000 cells per well into a 96-well plate, with a volume of 100 μL per well. The cells should be seeded and cultured 12-24 hours in advance.

[0039] 2. Add the solution of the compound to be tested: N-Methylatanine was dissolved in DMSO and sieved at 40, 8, 1.6, 0.32 and 0.064 μM, with a final volume of 200 μL per well. Each treatment had 3 replicates.

[0040] 3. Color development: After culturing at 37℃ for 48h, discard the culture medium in the wells and add 20μL of MTS solution and 100μL of culture medium to each well; set up 3 blank replicates (a mixture of 20μL of MTS solution and 100μL of culture medium), and continue incubation for 2~4h to allow the reaction to proceed fully before measuring the light absorbance.

[0041] 4. Colorimetric analysis: Select a wavelength of 492nm, use a multi-functional microplate reader (MULTISKAN FC) to read the absorbance values ​​of each well, record the results, and after data processing, plot the cell inhibition rate with the compound number as the x-axis and the cell inhibition rate as the y-axis.

[0042] 5. Positive control compounds: Two positive control compounds, doxorubicin (Dox) and paclitaxel (Taxol), were included in each experiment. Cell growth curves were plotted with concentration on the x-axis and cell viability on the y-axis. The IC50 of the compounds was calculated using the Reed-Muench method. 50 value.

[0043] Experimental results:

[0044] As shown in Table 1, the half-maximal inhibitory concentration (IC50) of N-Methylatanine against normal human lung epithelial cells (BEAS-2B) is... 50 The IC50 value was >40 μM, while the positive control drugs doxorubicin and paclitaxel had an IC50 value >40 μM. 50 The concentrations were 0.615±0.009 μM and 5.990±0.625 μM, respectively. This indicates that N-Methylatanine has significantly lower toxicity to normal cells than traditional chemotherapy drugs and exhibits good biocompatibility within its effective antibacterial concentration range, providing important safety evidence for its clinical application as a topical or local antifungal agent.

[0045] Table 1. IC50 of different compounds on normal human lung epithelial cells 50 value

[0046]

[0047] Experimental Example 2

[0048] Antibacterial activity test of N-Methylatanine

[0049] 1. Test strains: *Epidermophyton floccosum*, *Trichophyton rubrum*, and *Microsporum gypseum* were purchased from the Medical Fungi Preservation Center of the Chinese Academy of Medical Sciences; *Epidermophyton floccosum* ( 絮状表皮癣菌 CBS 566.94, Trichophyton rubrum ( 红色毛癣菌 ATCC 4438, Microsporum gypseum ( 石膏样小孢子菌 CBS 118893.

[0050] 2. Experimental materials: Terbinafine hydrochloride and DMSO were purchased from Sigma-Aldrich; agar powder was purchased from Scientific Research Special.

[0051] 3. Experimental Methods and Procedures: Take a 96-well culture plate, dilute the sample to be tested with a high-concentration stock solution, and the final concentration is 5 × 10⁻⁶. 5 Fungal culture was added to each well containing CFU / mL dilutions of 3, 15, and 100 μM, with a final concentration of 5 × 10⁻⁶. 5 The culture was incubated at CFU / mL at 25℃ for 5 days, and the absorbance was measured at 625 nm using a microplate reader. A blank control of the culture medium, a fungal control, and a terbinafine hydrochloride positive control were also included in the experiment.

[0052] Inhibition rate = (1 - absorbance of drug group / absorbance of control group) × 100%.

[0053] Experimental results:

[0054] As shown in Table 2, the positive control drug terbinafine hydrochloride showed near 100% inhibition against *Epidermophyton floccosum*, *Trichophyton rubrum*, and *Microsporum gypseum* at a low concentration (0.03 μM), with the inhibition rate gradually decreasing with decreasing concentration. N-Methylatanine exhibited extremely strong inhibitory effects against all three fungi at a concentration of 100 μM, with inhibition rates of 99.385 ± 0.8% (*Epidermophyton floccosum*), 102.17 ± 1.956% (*Trichophyton rubrum*), and 100 ± 0.115% (*Microsporum gypseum*), comparable to the antibacterial effect of high-concentration terbinafine hydrochloride. At a concentration of 20 μM, the inhibition rate against *Microsporum gypseum* still reached 77.339 ± 0.697%, demonstrating targeted inhibitory activity against this type of fungus; the inhibitory effect was weaker at low concentrations (≤4 μM). The above results confirm that N-Methylatanine has a significant inhibitory effect on epidermal infection-related fungi at concentrations above 20 μM, and can achieve highly efficient antibacterial activity at a concentration of 100 μM, verifying its practical application value as a fungal inhibitor.

[0055] Table 2. Inhibitory effects of different concentrations of compounds on fungal infections.

[0056]

[0057] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of N-Methylatanine in the preparation of fungal inhibitors, characterized in that, The concentration of N-Methylatanine used is 20-200 μM; the fungi are Epidermophyton floccosum, Trichophyton rubrum, and Microsporum gypseum.

2. The application according to claim 1, characterized in that, The concentration of N-Methylatanine used is 100 μM.

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