Metronidazole derivative, preparation method, and use in preparation of drug

Abstract

A metronidazole derivative of formula I, a preparation method, and a use in preparation of a drug. The metronidazole derivative can effectively ameliorate the symptoms of rosacea. Moreover, compared with metronidazole, the metronidazole derivative has less medication dosage and low medication frequency, but still can achieve a better improvement effect. Therefore, the metronidazole derivative has the prospect of being developed into a drug for ameliorating the symptoms of rosacea. The preparation method for the metronidazole derivative involves simple steps and mild conditions, and is easy for industrial production.

Description

A metronidazole derivative, preparation method and application in drug preparation Technical Field

[0001] The present invention belongs to the field of pharmaceutical chemistry and relates to a metronidazole derivative, a preparation method and an application thereof in drug preparation. Background Art

[0002] Rosacea is a common chronic disease with various skin and ocular manifestations, including persistent central facial erythema, papules, pustules, and telangiectasia. Ocular manifestations include dryness, telangiectasia at the eyelid margin, and conjunctival congestion. Rosacea is most common in fair-skinned individuals, with a higher prevalence in women over 30 years old. The pathogenesis of rosacea remains under investigation, and treatment focuses on symptom control. The pathogenesis of rosacea remains unclear, but precipitating factors include abnormalities in innate immunity, inflammatory responses to skin microbes, ultraviolet damage, and vascular dysfunction.

[0003] Metronidazole is a broad-spectrum nitroimidazole antibiotic with anti-anaerobic properties and dual anti-demodex and anti-inflammatory properties. Topical metronidazole gel is effective for treating papulopustular lesions and rosacea erythema. Studies have shown that applying a cold compress of metronidazole sodium chloride injection to the affected area for 15 minutes can improve erythema, papules, and sensitivity. Because metronidazole is an antibiotic, its use alone can easily lead to skin resistance. Therefore, its use alone or for long periods of time is not recommended, and frequent application can also hinder patient compliance.

[0004] Summary of the Invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and to provide a metronidazole derivative, a preparation method and an application thereof in drug preparation.

[0006] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0007] A metronidazole derivative represented by formula I or a pharmaceutically acceptable salt thereof:

[0008] A method for preparing a metronidazole derivative represented by formula I, the synthetic route is as follows:

[0009] In the above route, J3 is the metronidazole derivative represented by Formula I; Z represents an amide condensation reaction of compound J1 and compound J2 to obtain the target compound J3.

[0010] Preferably, the molar ratio of compounds J1 and J2 is 1:2 to 1:2.2.

[0011] Preferably, the condensing agent for the amide condensation reaction is one or more of 1-hydroxy-7-azabenzotriazole, 1-hydroxybenzotriazole, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, O-benzotriazole-tetramethyluronium hexafluorophosphate and 1H-benzotriazole-1-yloxytripyrrolidino hexafluorophosphate.

[0012] Preferably, the base for the amide condensation reaction is one or more of N,N-diisopropylethylamine DIPEA and triethylamine TEA.

[0013] Preferably, the organic solvent for the amide condensation reaction is one or more of tetrahydrofuran, acetonitrile, N,N-dimethylformamide and dimethyl sulfoxide.

[0014] Use of the above-mentioned metronidazole derivative or a pharmaceutically acceptable salt thereof in the preparation of a drug for improving the symptoms of rosacea. Beneficial effects:

[0015] The present invention provides a metronidazole derivative that can effectively improve the symptoms of rosacea. Furthermore, compared with metronidazole, this metronidazole derivative requires a lower dosage and requires fewer applications, yet achieves unexpectedly superior improvement. Therefore, this metronidazole derivative has the potential to be developed into a drug for improving rosacea symptoms. The present invention also provides a method for preparing this metronidazole derivative, which features simple steps, mild conditions, and is amenable to industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is a time-release concentration curve of the J3 serum release experiment;

[0017] Figure 2 shows the intradermal metronidazole concentration curves corresponding to different time points in the skin pharmacokinetic experiment in J3 mice;

[0018] FIG3 shows the gross observation of LL-37-induced rosacea treated with low-dose J3, high-dose J3, and metronidazole;

[0019] Figure 4 shows HE staining images of skin sections from different groups in the pharmacodynamic experiment. DETAILED DESCRIPTION

[0020] The essential contents of the present invention are described in detail below with reference to the embodiments, but the protection scope of the present invention is not limited thereto.

[0021] Where specific techniques or conditions are not specified in the examples, the experiments were carried out according to those described in the literature in the field or according to the product instructions. Reagents or instruments used without manufacturer identification are all conventional products available through regular channels. The experimental methods in the following examples are all conventional methods unless otherwise specified. The experimental materials used in the following examples are all commercially available products unless otherwise specified.

[0022] The synthetic route of the target compound of the present invention is as follows:

[0023] Example 1: Bis(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)azelaate (Preparation of J3)

[0024] J1 (100 mg, 0.52 mmol) was dissolved in DMF, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (218 mg, 1.144 mmol), 1-hydroxybenzotriazole (154 mg, 1.144 mmol), J2 (195 mg, 1.144 mmol), and 4-dimethylaminopyridine (63.44 mg, 0.52 mmol) were added. The reaction mixture was stirred at room temperature overnight. After completion of the reaction, the mixture was extracted three times with equal volumes of ethyl acetate and water. The organic layers were combined and concentrated under low pressure to remove the solvent. The residue was purified by silica gel column chromatography (elution system: dichloromethane:methanol = 100:1) to obtain J3 as a white solid (223 mg, yield: 80.4%, purity: 97.77%). 1 H NMR(300MHz,DMSO-d6)δ8.02(s,2H), 4.61 (t, J = 5.1Hz, 4H), 4.38 (t, J = 5.0Hz, 4H), 2.48 (s, 6H), 2.23 (t, J = 7.4Hz, 4H), 1.43 (p, J = 7.3Hz, 4H), 1.18 (d, J = 4.0Hz, 6H).

[0025] Example 2: Compound J3 serum release experiment

[0026] The release experiment used rat serum as the incubation fluid at a compound concentration of 1 μM / ml (using polyethylene glycol 400 for solubilization). The mixture was then incubated at 37°C in a metal thermostat. Twenty μl of the incubation fluid at different time points was added to a methanol solution containing an internal standard and shaken evenly until a white precipitate formed. The supernatant was centrifuged at low temperature (12,000 rpm) for 5 minutes, and the J3 and metronidazole concentrations at different time points were determined by HPLC. After 2 hours, the residual amount of compound J3 was 0.046 μM / ml, and the metronidazole concentration was 1.81 μM / ml, indicating that J3 contributed to 95.4% of the release. The concentration curve is shown in Figure 1.

[0027] Example 3: Mouse skin pharmacokinetic experiment

[0028] Compound J3 and metronidazole are prepared into gel preparations. Metronidazole is prepared according to the concentration of metronidazole gel already on the market (0.044mmol / ml). J3 is prepared as a transparent gel preparation with a concentration of 0.022mmol / ml. The two gels differ only in compound. Before the experiment, Balb / c mice were shaved on the back and randomly divided into metronidazole experimental group and J3 experimental group. Each experimental group carried out 3 parallel experiments. The prepared gel preparation was evenly applied to the back of the mice in equal amounts and bandaged with gauze to prevent scratches. The back skin of the mice (1cm*1cm) was taken at 2h, 4h, 6h, 8h, 10h, and 24h respectively, rinsed three times with normal saline and removed most of the moisture with a paper towel. The skin weight was weighed and added to the chromatographic methanol solvent at 50mg / ml. The supernatant was extracted and stored at low temperature for standby use after cryogenic grinding.

[0029] The supernatant extracted above was diluted and prepared as an analytical sample for LCMS-MS analysis to obtain the time-concentration curve shown in Figure 2, and the area under the curve is shown in Table 1.

[0030] Table 1 Area under the curve

[0031] Assuming J3 fully releases metronidazole, equal masses of 0.022 mmol / ml J3 gel and 0.044 mmol / ml metronidazole gel should deliver the same amount of metronidazole. However, as shown in "Example 2: Compound J3 Serum Release Experiment," J3 gel does not fully release metronidazole. Even so, in this example, J3 gel released 1.348 times more metronidazole into the skin than metronidazole gel. This remarkable and unexpected effect of J3 is truly remarkable.

[0032] Example 4: Anti-rosacea pharmacodynamics experiment

[0033] Balb / c female mice (5-6 weeks old) were purchased from the Xipulbikai Biotechnology Center in Xuanwu District, Nanjing. All animal experiments were approved by the Animal Ethics Committee of China Pharmaceutical University. The mice were shaved on their backs and injected with LL-37 at a concentration of 640 μM every 12 hours. After five injections, significant rosacea erythema developed.

[0034] (1) Mice with obvious rosacea erythema were randomly divided into four groups: a model group (no drug treatment), a low-dose group (0.044 mmol / g J3 gel applied once daily), a high-dose group (0.088 mmol / g J3 gel applied once daily), and a metronidazole group (0.044 mmol / g metronidazole gel applied twice daily). The changes in the erythema on the back of the mice after drug administration were observed and recorded. J3 gel and metronidazole gel differed only in their compounds.

[0035] (2) After three days of continuous administration, dorsal tissue samples (0.5 cm x 0.5 cm) were collected from the mice and divided into two sections. One section was fixed with formalin and paraffin wax to examine the entire skin of the rosacea-like lesions. The sections were then sliced ​​into 4 μm thick slices. The sections were stained with hematoxylin and eosin (H&E) and the tissue morphology was observed under a Leica upright fluorescence microscope (DM2500). The other section was stored at -80°C.

[0036] (3) The cryopreserved tissue was obtained and ground evenly with PBS solution using a homogenizer. ELISA experiments were performed and the levels of IL-1β and TNF-α in the skin tissue were detected using a full-wavelength microplate reader (Thermo scientific varioskan flash).

[0037] ELISA analysis compared the effects of the model group, metronidazole group, J3 low-dose group, J3 high-dose group, and blank group on IL-1β and TNF-α levels. Experimental results are presented as mean ± SD (three independent tests). Statistical analysis of IL-1β inflammatory factor levels: P < 0.0001 (metronidazole group vs. model group), P < 0.0001 (J3 low-dose group vs. model group), P < 0.0001 (J3 high-dose group vs. model group), P < 0.0001 (blank group vs. model group); P < 0.05 (J3 high-dose group vs. metronidazole group), P < 0.005 (J3 low-dose group vs. metronidazole group). Statistical analysis of TNF-α inflammatory factor levels: P<0.0001 (metronidazole group vs. model group), P<0.0001 (J3 low-dose group vs. model group), P<0.0001 (J3 high-dose group vs. model group), P<0.0001 (blank group vs. model group); P<0.005 (J3 high-dose group vs. metronidazole group), P<0.05 (J3 low-dose group vs. metronidazole group).

[0038] The results are shown in Figures 3 and 4 and Table 2. Compared with the model group, J3 gel effectively improved rosacea symptoms in a dose-dependent manner. Compared with the metronidazole group, which applied 0.044 mmol / g of J3 gel once daily, the J3 low-dose group, which applied 0.044 mmol / g of J3 gel twice daily, achieved better improvement despite a lower dosage and fewer applications (results showed that levels of IL-1β and TNF-α inflammatory factors in the J3 low-dose group were significantly lower than those in the model group and also lower than those in the metronidazole group). This unexpected effect was achieved.

[0039] Table 2 IL-1β and TNF-α contents in the skin of different experimental groups

[0040] The purpose of the above embodiments is to specifically introduce the essential content of the present invention, but those skilled in the art should know that the protection scope of the present invention should not be limited to this specific embodiment.

Claims

1. A metronidazole derivative of formula I or a pharmaceutically acceptable salt thereof:

2. A preparation method of a metronidazole derivative represented by Formula I, characterized in that, The synthetic route is as follows: In the above route, J3 is the metronidazole derivative shown in Formula I; Z represents the amide condensation reaction of compound J1 and compound J2 to obtain the target compound J3.

3. The preparation method according to claim 2, characterized in that: The molar ratio of compound J1 to compound J2 is 1:2 to 1:2.

2.

4. The preparation method according to claim 2, wherein: The condensing agent for the amide condensation reaction is one or more of 1-hydroxy-7-azabenzotriazole, 1-hydroxybenzotriazole, 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 1H-benzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate.

5. The preparation method according to claim 2, characterized in that: The base for the amide condensation reaction is one or more of N,N-diisopropylethylamine (DIPEA), 4-dimethylaminopyridine (DMAP), and triethylamine (TEA).

6. The preparation method according to claim 2, wherein: The organic solvent for the amide condensation reaction is one or more of tetrahydrofuran, acetonitrile, N,N-dimethylformamide, and dimethyl sulfoxide.

7. Use of the metronidazole derivative according to claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a drug for improving the symptoms of rosacea.

Images