An amide derivative and its preparation method and application

Abstract

The present invention discloses an amide derivative, its preparation method, and application, belonging to the field of pharmaceutical chemistry technology. Specifically, it relates to the application of an amide derivative represented by the following structural formula, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the compound, in the preparation of a TLR7 receptor agonist. TLR7 is a key target for immunotherapy and has important applications in the treatment of autoimmune diseases, such as allergic asthma and systemic lupus erythematosus. It also has potential application value in anti-chronic hepatitis B, anti-hepatitis B virus drugs, and various late-stage solid tumor drugs. The synthetic route adopted by the present invention uses inexpensive and readily available raw materials, commonly used reagents, mild reaction conditions, and simple post-processing, allowing for efficient and large-scale preparation of this type of compound. #imgabs0#

Description

Technical Field

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

[0002] TLR7 (toll like receptor 7) is a member of the Toll-like receptor (TLRs) family and is mainly responsible for recognizing single-stranded RNA of pathogens. TLR7 agonists activate immune cells such as plasmacytoid dendritic cells (pDC) and B cells by binding to TLR7, thereby triggering an immune response. TLR7 agonists are generally highly selective and can specifically activate TLR7 receptors, reduce nonspecific activation of other receptors, and reduce adverse reactions. At present, there are limited cases of TLR7 agonists being directly marketed as single drugs. However, TLR7 agonists may have some applications in combination therapy or as part of a drug combination, but these are usually not drugs with TLR7 agonists as the only active ingredient. In the Chinese market, the drugs currently on the market targeting TLR7 are mainly imiquimod, which is mainly used to treat genital warts.

[0003] In addition, many drugs and bioactive molecules contain amide structures, which play an important role in their pharmacological activities. The introduction of amide groups can change the solubility, stability, biodistribution and binding affinity of compounds to target molecules.

[0004] In general, amide derivatives, as important structural units, are not only widely used in the pharmaceutical field but also play a key role in other fields of chemical and biochemical research. Based on this, the present invention discloses an amide derivative that not only provides a new compound, but also enriches the structural types of TLR7 receptor agonists, providing potential possibilities for the treatment of diseases targeting TLR7. Summary of the Invention

[0005] The primary purpose of the present invention is to provide an amide derivative represented by the following structural formula or a pharmaceutically acceptable salt thereof.

[0006]

[0007] The present invention also provides a pharmaceutical composition comprising the amide derivative or a pharmaceutically acceptable salt thereof as an active ingredient, mixed with a pharmaceutically acceptable carrier or excipient, and prepared in a clinically acceptable dosage form. The pharmaceutically acceptable excipient refers to any diluent, adjuvant, and / or carrier used in the pharmaceutical field. The amide derivative or a pharmaceutically acceptable salt thereof of the present invention may be used in combination with other active ingredients, as long as they do not produce other toxic side effects.

[0008] The preparation method of the amide derivative comprises the following steps:

[0009]

[0010] Compound 1 was dissolved in THF and stirred, 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) and N,N-diisopropylethylamine (DIPEA) were added, and compound 2 was added to the solution. The mixture was stirred at room temperature for reaction. After the reaction, compound 3 was separated and purified to obtain compound 3.

[0011]

[0012] Compound 3 was dissolved in DCM, and HCl / dioxane was added under ice bath conditions (below 0°C). The mixture was heated to room temperature and stirred for reaction. After the reaction was completed, compound 4 was separated and purified to obtain compound 4.

[0013]

[0014] Compound 4 was dissolved in DCM, TEA was added, and then compound 5 was added under ice bath conditions (below 0°C). The reaction was stirred at room temperature. After the reaction was completed, compound 6 was separated and purified to obtain compound 6.

[0015] The present invention also provides the use of the amide derivative or its pharmaceutically acceptable salt, or a pharmaceutical composition comprising the derivative, in the preparation of a TLR7 receptor agonist. TLR7 is a key target for immunotherapy and has important applications in the treatment of autoimmune diseases, such as allergic asthma and systemic lupus erythematosus. At the same time, it has potential application value in anti-chronic hepatitis B, anti-hepatitis B virus drugs, and various advanced solid tumor drugs. Therefore, the amide derivative or its pharmaceutically acceptable salt, or a pharmaceutical composition comprising the derivative of the present invention can also be used as an immunostimulant in the preparation of antiviral, autoimmune disease, and anti-tumor drugs.

[0016] The present invention also provides the amide derivative or its pharmaceutically acceptable salt, or a pharmaceutical composition comprising the derivative, which has important application value in immunotherapy.

[0017] Beneficial effects of the present invention:

[0018] 1) The amide derivatives provided by this invention have potential applications as TLR7 receptor agonists, including the treatment of autoimmune diseases and advanced solid tumors. They also enrich the structural types of TLR7 receptor agonist drugs and can be used to treat allergic asthma, systemic lupus erythematosus, chronic hepatitis B, hepatitis B, and advanced solid tumors. They can also be combined with other drugs in combination therapies to achieve immunotherapeutic effects.

[0019] 2) The synthetic route adopted by the present invention uses cheap and readily available raw materials, the reagents used are commonly used reagents, the reaction conditions are mild, the post-processing is simple, and this type of compound can be prepared efficiently and on a large scale. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Compound 3 1 H NMR spectrum (400 MHz, CDCl3);

[0021] Figure 2 Compound 3 13 C NMR spectrum (101 MHz, CDCl3);

[0022] Figure 3 HRMS (ESI) spectrum of compound 3;

[0023] Figure 4 HRMS (ESI) spectrum of compound 4;

[0024] Figure 5 Compound 6 1 H NMR spectrum (400 MHz, CDCl3);

[0025] Figure 6 Compound 6 13 C NMR spectrum (101 MHz, CDCl3);

[0026] Figure 7 HRMS (ESI) spectrum of compound 6;

[0027] Figure 8 Toxicity test of compound 6 on normal human cell lines;

[0028] Figure 9 Immune activation assay of compound 6; (a) RAW264.7 cell line was stimulated with 10, 30, and 50 μM of compound 6, and its proliferation was detected by CCK8. DC2.4 cell line was stimulated with 10, 30, and 50 μM of compound 6, and the secretion levels of IFNα (b), IL-6 (c), and IFNβ (d) were detected by RT-qPCR;

[0029] Figure 10 Compound 6 activates the TLR-7 signaling pathway. DC2.4 cells were treated with compound 6 at a final concentration of 30 μM, and proteins were collected 15, 30, and 60 minutes after stimulation. Western blotting was used to analyze the expression levels of MYD88 and p-IRF7. DETAILED DESCRIPTION

[0030] Example 1

[0031] A method for preparing an amide derivative, comprising the following steps:

[0032]

[0033] Compound 1, 2-amino-5-phenylpyridine (300.00 mg, 1.76 mmol, 1.1 eq) was dissolved in THF (2.0 mL), HATU (731.07 mg, 1.92 mmol, 1.2 eq) and DIPEA (414.17 mg, 3.2 mmol, 2.0 eq) were added, and compound 2, 1-tert-butoxycarbonyl-4-piperidinylacetic acid (389.83 mg, 1.60 mmol, 1.0 eq) was added to the solution, and the mixture was stirred at room temperature overnight. TLC monitoring showed that the raw material spots disappeared and new spots were generated. THF was dried and water was added. The aqueous phase (40 mL) was extracted with EtOAc (20 mL × 3 times). The organic phases were combined, dried over Na2SO4, concentrated under reduced pressure, and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 2:1, v / v). f =0.5, to obtain a purple solid (250.00 mg, 39.45%). Figure 1-3 shown.

[0034] 1 H NMR (400MHz, CDCl3): δ / ppm=8.49(d,J=2.0Hz,1H),8.28(d,J=4.0Hz,1H),7.93-7.91(m,1H),7.57-7.54(m,2H),7.46(t,J=8.0Hz,2H),7.40-7.3 6(m,1H),4.11(s,2H),2.74(t,J=12.0Hz,2H),2.34(d,J=8.0Hz,2H),2. 12-2.04(m,1H),1.77(d,J=12.0Hz,2H),1.46(s,9H),1.26-1.20(m,2H).

[0035] 13 C NMR (101MHz, CDCl3): δ / ppm=154.82,146.01,137.39,136.88,133.10,129.09,127.84,126.79,113.72,79.41,44.69,33.50,31.95,28.47.

[0036] HRMS(ESI)calculated for C 23 H 29 N3O3[M+H +]:396.2282,found 396.2274.

[0037]

[0038] Compound 3,4-(2-oxo-2-((5-phenylpyridin-2-yl)amino)ethyl)piperidine-1-carboxylic acid tert-butyl ester (200.00 mg, 505.69 μmol, 1.0 eq) was dissolved in DCM (1.5 mL), 4M HCl / Dioxane (1.5 mL) was added under ice-cooling, and the mixture was warmed to room temperature and stirred for 2 h. TLC monitoring showed that the starting material spot disappeared. The solvent and HCl gas were evaporated under reduced pressure to obtain a white solid (120.00 mg, 80.34%). Mass spectrum is shown as follows Figure 4 shown.

[0039] HRMS(ESI)calculated for C 18 H 21 N3O[M+H + ]:296.1757,found 296.1754.

[0040]

[0041] Compound 4, N-(5-phenylpyridin-2-yl)-2-(piperidin-4-yl)acetamide (100.0 mg, 338.54 μmol, 1.0 eq) was dissolved in DCM (1.0 mL), and TEA (102.77 mg, 1.02 mmol, 3.0 eq) was added. Compound 5, benzenesulfonyl chloride (65.77 mg, 372.79 μmol, 1.1 eq) was then added under ice-bath conditions. The mixture was stirred at room temperature for 12 h. TLC monitoring showed that the starting material spot disappeared and new spots were generated. The aqueous phase (40 mL) was extracted with EtOAc (20 mL × 3 times). The organic phases were combined and dried over Na2SO4. The organic phases were concentrated under reduced pressure and purified by silica gel column chromatography (dichloromethane:methanol=100:1, v / v). f =0.4, and a white solid (110.00 mg, 74.60%) was obtained. Figure 5-7 shown.

[0042] 1H NMR (400MHz, CDCl3): δ / ppm=8.48(d,J=2.0Hz,1H),8.22(d,J=8.0Hz,2H),7.91-7.88(m,1H),7.77-7.75(m,2H),7.60-7.58(m,1H),7.54d,J=8.0Hz ,4H),7.46(t,J=8.0Hz,2H),7.40-7.38(m,1H),3.84-3.79(m,2H),2.34-2 .27(m,4H),1.84(d,J=12.0Hz,2H),1.43-1.37(m,2H),1.16-1.08(m,1H).

[0043] 13 C NMR (101MHz, CDCl3): δ / ppm=169.85,150.23,145.97,137.32,136.87,136.17,133.13, 132.74,129.11,129.01,127.89,127.65,126.77,113.77,46.25,43.90,32.50,31.27.

[0044] HRMS(ESI)calculated for C 21 H 16 ClN3O3S[M+H + ]:436.1689,found 436.1682.

[0045] Example 2

[0046] 1. Toxicity test of compound 6

[0047] To clarify the safety of compound 6, we used human normal skin keratinocyte cell line Hacat and human healthy skin fibroblast cell line HSF to evaluate its toxicity. The corresponding cells were seeded in 96-well cell culture plates and cultured at 37°C and 5% CO2 for 24 hours. Compound 6 was added to different culture wells to a final concentration of 10, 30, and 50 μM, and a negative control was set up. The mixture was thoroughly mixed and placed in an incubator for further 6 hours. 10 μL of CCK8 solution was added to each well, and the cells were incubated in the incubator for 1 hour. The absorbance value at 450 nm was read using a microplate reader. The experimental results showed that within the concentration range of 10 to 50 μM, compound 6 did not produce toxic effects on normal cells ( Figure 8 ).

[0048] 2. Immune activation function detection of compound 6

[0049] To clarify the immune activation function of compound 6, we first tested its ability to promote the proliferation of mouse macrophages RAW264.7. The results showed that 30μM and 50μM of compound 6 could effectively promote the proliferation of RAW264.7 ( Figure 9 a). Cytokines are important indicators of immune activation. TLR-7 is a pattern recognition receptor widely expressed in natural immune cells. Its activation can promote the secretion of interferon in the early stage of infection and achieve an antiviral effect. To clarify the effect of compound 6 on cytokine secretion, we selected the DC2.4 dendritic cell line derived from mouse bone marrow for cytokine secretion detection. As the most powerful professional antigen-presenting cell in the body, DC plays an indispensable bridge role in immune activation. RT-qPCR results showed that compound 6 can significantly promote the secretion of type I interferon by DC ( Figure 9 b, 9c). In addition, 10μM and 50μM of compound 6 could significantly inhibit the secretion of IL-6 ( Figure 9 d), indicating that it can activate antiviral immune responses without triggering the secretion of inflammatory factors, and has good application potential.

[0050] 3. Compound 6 can activate TLR-7 downstream signaling pathway

[0051] To clarify that the immune activation function of compound 6 is achieved through the TLR-7 signaling pathway, we performed WB detection on the TLR-7 downstream signaling. The results showed that 15 minutes after compound 6 treatment, the TLR-7 downstream adaptor protein MYD88 began to be recruited and reached a peak at 30 minutes. At the same time, the phosphorylation level of IRF7, which regulates interferon gene transcription in the downstream of the TLR-7 signaling pathway, was significantly increased within 30 minutes after compound 6 treatment ( Figure 10 ), consistent with previous experimental results showing that compound 6 promotes interferon secretion. These results suggest that compound 6's immune activation function is achieved through activation of TLR-7, and it is expected to be used as a new immunostimulant in antiviral, autoimmune disease, and anti-tumor research.

Claims

1. An amide derivative or a pharmaceutically acceptable salt thereof, characterized in that: The structural formula of the amide derivative is:

2. A pharmaceutical composition, characterized in that The amide derivative or a pharmaceutically acceptable salt thereof according to claim 1 is used as an active ingredient and mixed with a pharmaceutically acceptable carrier or excipient to prepare a clinically acceptable dosage form.

3. The method for preparing the amide derivative or the pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The following steps are involved: Compound 1 was dissolved in THF and stirred, HATU and DIPEA were added, and compound 2 was added to the solution. The mixture was stirred at room temperature for reaction. After the reaction, compound 3 was obtained by separation and purification. Compound 3 was dissolved in DCM, and HCl / dioxane was added under ice bath conditions. The mixture was heated to room temperature and stirred for reaction. After the reaction was completed, compound 4 was obtained by separation and purification. Compound 4 was dissolved in DCM, TEA was added, and then compound 5 was added under ice bath conditions. The reaction was stirred at room temperature. After the reaction was completed, compound 6 was obtained by separation and purification.

4. Use of the amide derivative according to claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a TLR7 receptor agonist.

5. Use of the amide derivative according to claim 1 or a pharmaceutically acceptable salt thereof as an immunostimulant in the preparation of antiviral, autoimmune disease and antitumor drugs.

6. Use of the pharmaceutical composition according to claim 2 in the preparation of a TLR7 receptor agonist.

7. Use of the pharmaceutical composition according to claim 2 as an immunostimulant in the preparation of antiviral, autoimmune disease and antitumor drugs.

Images