Metronidazole derivative, preparation method and application of metronidazole derivative in medicine preparation

Abstract

The invention discloses a metronidazole derivative, a preparation method and application in medicine preparation. The invention provides a metronidazole derivative. The metronidazole derivative can effectively improve the symptoms of rosacea. Moreover, compared with metronidazole, the metronidazole derivative has the advantages that the dosage is reduced, the medication frequency is reduced, but a better improvement effect can be obtained, and the effect is unexpected. Therefore, the metronidazole derivative has the prospect of being developed into the medicine for improving the acne rosacea symptom. The invention also provides a preparation method of the metronidazole derivative, which has the advantages of simple steps, mild conditions and easiness in industrial production.

Description

Technical Field

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

[0002] Rosacea is a common chronic disease with various skin or eye manifestations, such as persistent erythema, papules, pustules, telangiectasia in the central part of the face, and dryness, eyelid margin telangiectasia, conjunctival congestion, etc. in the eyes. Rosacea is most common in people with light skin color, and the prevalence is higher in women over 30 years old. At present, the pathogenesis remains to be further studied, and the treatment mainly focuses on controlling symptoms. The pathogenesis of rosacea is not clear, and the triggering factors include innate immune abnormalities, inflammatory reactions of skin microorganisms, ultraviolet damage, and vascular dysfunction, etc.

[0003] Metronidazole is a nitroimidazole antibiotic with a broad-spectrum anti-anaerobic effect and has dual effects of anti-demodex and anti-inflammatory. Topical metronidazole gel is effective in treating papulopustular skin lesions and rosacea erythema. Studies have shown that cold compress with metronidazole sodium chloride injection on the affected area for 15 minutes can improve erythema, papules, and sensitive symptoms. Since metronidazole is an antibiotic, when using metronidazole gel alone, it is easy to cause drug resistance in the skin. Therefore, it is not recommended to use it alone or for a long time, and frequent application is also not conducive to the compliance of patients. Summary of the Invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a metronidazole derivative, a preparation method thereof, and an application thereof in the preparation of drugs.

[0005] The above purpose of the present invention is achieved by the following technical solutions:

[0006] A metronidazole derivative of formula I or a pharmaceutically acceptable salt thereof:

[0007]

[0008] A preparation method of a metronidazole derivative of formula I, and the synthetic route is as follows:

[0009]

[0010] In the above route, J3 is the metronidazole derivative of formula I; Z represents carrying out an amide condensation reaction on compound J1 and compound J2 to obtain the target compound J3.

[0011] Preferably, the molar ratio of compound J1 to compound J2 is 1:2 to 1:2.2.

[0012] Preferably, 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, and 1H-benzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate.

[0013] Preferably, the base for the amide condensation reaction is one or more of N,N-diisopropylethylamine (DIPEA) and triethylamine (TEA).

[0014] Preferably, the organic solvent for the amide condensation reaction is one or more of tetrahydrofuran, acetonitrile, N,N-dimethylformamide, and dimethyl sulfoxide.

[0015] 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.

[0016] Beneficial effects:

[0017] The present invention provides a metronidazole derivative, which can effectively improve the symptoms of rosacea. Moreover, compared with metronidazole, the dosage and frequency of administration of this metronidazole derivative are reduced, but better improvement effects can be obtained, and this effect is unexpected. Therefore, this metronidazole derivative has the prospect of being developed into a drug for improving the symptoms of rosacea. The present invention also provides a preparation method of this metronidazole derivative, which has simple steps, mild conditions, and is easy for industrial production. Description of the drawings

[0018] Figure 1 It is the time-release concentration curve of the J3 serum release experiment;

[0019] Figure 2 It is the intradermal metronidazole concentration curve corresponding to different time points in the J3 mouse skin pharmacokinetics experiment;

[0020] Figure 3 It is the gross observation diagram of J3 low dose, J3 high dose and metronidazole in the treatment of LL-37-induced rosacea;

[0021] Figure 4 It is the HE staining diagram of skin sections of different groups in the pharmacodynamic experiment. Detailed implementation manners

[0022] The following specifically introduces the substantive content of the present invention in combination with examples, but does not limit the protection scope of the present invention thereto.

[0023] For those without specific techniques or conditions noted in the examples, they shall be in accordance with the techniques or conditions described in the literature in this field or in accordance with the product specifications. For reagents or instruments without the manufacturer noted, they are all conventional products that can be obtained through regular channels. The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all commercially available products unless otherwise specified.

[0024] The synthetic route of the target compound of the present invention is as follows:

[0025]

[0026] Example 1: Preparation of bis(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl) azelate (J3)

[0027] Dissolve J1 (100 mg, 0.52 mmol) in DMF solution, and add 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). Stir the reaction mixture at room temperature overnight. After monitoring the reaction by TLC until completion, add an equal volume of ethyl acetate and water for liquid-liquid extraction 3 times. Combine the organic layers and concentrate under reduced pressure to remove the solvent. The residue is purified by silica gel column chromatography (elution system: dichloromethane:methanol = 100:1) to obtain the final product J3 as a white solid (223 mg, yield: 80.4%, purity 97.77%). 1 1H NMR (300 MHz, DMSO-d6) δ 8.02 (s, 2H), 4.61 (t, J = 5.1 Hz, 4H), 4.38 (t, J = 5.0 Hz, 4H), 2.48 (s, 6H), 2.23 (t, J = 7.4 Hz, 4H), 1.43 (p, J = 7.3 Hz, 4H), 1.18 (d, J = 4.0 Hz, 6H).

[0028] Example 2: Serum release experiment of compound J3

[0029] The release experiment used rat serum as the incubation solution. The incubation compound concentration was 1 μM / ml (using polyethylene glycol 400 to assist dissolution). Subsequently, it was incubated at a constant temperature in a 37°C metal constant temperature incubator. 20 μl of the incubation solution at different time points was added to the methanol solution containing the internal standard and shaken evenly. A white precipitate appeared, and after low-temperature centrifugation (12,000 / min) for 5 min, the supernatant was taken, and the concentrations of J3 and metronidazole at different time points were detected by HPLC; after 2 h, the remaining amount of compound J3 was 0.046 μM / ml, and the metronidazole concentration was 1.81 μM / ml, that is, the percentage of J3 participating in the release was 95.4%. The concentration curve is as Figure 1 shown.

[0030] Example 3: Pharmacokinetics experiment of drugs on mouse skin

[0031] Compound J3 and metronidazole were prepared into gel preparations. Metronidazole was prepared according to the concentration of commercially available metronidazole gel (0.044 mmol / ml), and J3 was prepared into a transparent gel preparation with a concentration of 0.022 mmol / ml. The two gels differed only in the compound. Before the experiment, the hair on the back of Balb / c mice was shaved and randomly divided into groups, namely the metronidazole experimental group and the J3 experimental group. Each experimental group had 3 parallel experiments. The prepared gel preparations were evenly applied to the back of the mice in equal amounts and bandaged with gauze at the same time to prevent rubbing. Mouse back skin (1 cm * 1 cm) was taken at 2 h, 4 h, 6 h, 8 h, 10 h, and 24 h respectively, rinsed three times with normal saline and most of the moisture was removed with a tissue. The skin weight was weighed and added to the chromatographic methanol solvent at 50 mg / ml, and the supernatant was extracted after low-temperature grinding and stored at low temperature for later use.

[0032] The above-extracted supernatant was diluted and prepared into an analytical sample for LCMS-MS analysis to obtain a time-concentration curve as shown in Figure 2 shown, and the area under the curve is shown in Table 1.

[0033] Table 1 Area under the curve

[0034] Metronidazole gel J3 gel Area under the curve (AUC) 2.254 3.039

[0035] Assuming that J3 completely releases metronidazole, the metronidazole provided by 0.022 mmol / ml J3 gel and 0.044 mmol / ml metronidazole gel of the same mass should be the same. However, according to "Example 2: Serum release experiment of compound J3", J3 gel cannot completely release metronidazole. Even so, in this example, the metronidazole released by J3 gel into the skin is as high as 1.348 times that of metronidazole gel released into the skin. This effect of J3 is very amazing and unexpected.

[0036] Example 4: Pharmacodynamics experiment against rosacea

[0037] Balb / c female mice (5 - 6 weeks old) for experiments were purchased from Xipu Bikai Biotechnology Center, Xuanwu District, Nanjing. All animal experiments were approved by the Animal Ethics Committee of China Pharmaceutical University. Before the experiment, the hair on the backs of the mice was shaved, and LL-37 with a concentration of 640 μM was injected once every 12 h. After 5 injections, obvious rosacea erythema appeared.

[0038] (1) The mice with obvious rosacea erythema induced were randomly divided into 4 groups, namely: model group (without drug treatment), low-dose group (applying 0.044 mmol / g of J3 gel once a day), high-dose group (applying 0.088 mmol / g of J3 gel once a day), and metronidazole group (applying 0.044 mmol / g of metronidazole gel twice a day); the changes in the erythema on the backs of the mice after drug administration were observed and recorded. Among them, J3 gel and metronidazole gel only differed in the compound.

[0039] (2) After continuous drug administration for three days, the back tissues (0.5 cm * 0.5 cm) of the mice were taken and divided into two parts. One part was fixed with formalin, and all the skin with rosacea-like lesions was fixed with paraffin and cut into 4-μm thick slices. The slices were stained with hematoxylin and eosin (H&E) and the tissue morphology was observed under a Leica upright fluorescence microscope (DM2500); the other part was stored at -80 °C.

[0040] (3) The tissues stored at low temperature were taken, ground evenly with a homogenizer using PBS solution, and ELISA experiments were carried out. The contents of IL-1β and TNF-α in the skin tissues were detected using a full-wavelength microplate reader (Thermo scientific varioskan flash).

[0041] The ELISA analysis was used to compare the effects of the model group, metronidazole group, low-dose J3 group, high-dose J3 group and blank group on the levels of IL-1β and TNF-α. The experimental results are shown as the mean ± SD (three independent tests). Statistical analysis of the IL-1β inflammatory factor level: P<0.0001 (metronidazole group vs model group), P<0.0001 (low-dose J3 group vs model group), P<0.0001 (high-dose J3 group vs model group), P<0.0001 (blank group vs model group); P<0.05 (high-dose J3 group vs metronidazole group), P<0.005 (low-dose J3 group vs metronidazole group). Statistical analysis of the TNF-α inflammatory factor level: P<0.0001 (metronidazole group vs model group), P<0.0001 (low-dose J3 group vs model group), P<0.0001 (high-dose J3 group vs model group), P<0.0001 (blank group vs model group); P<0.005 (high-dose J3 group vs metronidazole group), P<0.05 (low-dose J3 group vs metronidazole group).

[0042] The results are as Figure 3 , 4 and Table 2 show. Compared with the model group, J3 gel can effectively improve the symptoms of rosacea, and shows a dose-dependent relationship; compared with the metronidazole group applying 0.044 mmol / g metronidazole gel twice a day, the low-dose J3 group applying 0.044 mmol / g J3 gel once a day has a reduced dosage and frequency of administration, but can obtain better improvement effects (the results show that the levels of IL-1β and TNF-α inflammatory factors in the low-dose J3 group are much lower than those in the model group and are also lower than those in the metronidazole group at the same time), and this effect is unexpected.

[0043] Table 2 Content table of IL-1β and TNF-α in the skin of different experimental groups

[0044]

[0045] The role of the above embodiments is to specifically introduce the substantial 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 represented by 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 represented by 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, characterized in that: 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, characterized in that: 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 or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a drug for improving the symptoms of rosacea.

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