Chlorogenic acid amide derivatives, and preparation method and application thereof

Abstract

The application provides a chlorogenic acid amide derivative, a preparation method and application thereof. The chlorogenic acid amide derivative has a structure shown in general formula I, the active carboxylic acid part in the chlorogenic acid structure is chemically modified, different alkyl / aromatic amine groups and the like are connected, and the anti-inflammatory activity of the chlorogenic acid derivative is enhanced. Meanwhile, in order to improve the fat solubility of the chlorogenic acid derivative, the phenolic hydroxyl and alcoholic hydroxyl parts and the like in the structure are protected by etherification or acylation, and the fat solubility of the chlorogenic acid derivative is improved. The chlorogenic acid amide derivative has good anti-inflammatory activity in an acute inflammatory cell model in vitro and an acute gout rat model, and is an anti-inflammatory preparation with good development potential. The synthesis method of the chlorogenic acid amide derivative is simple and efficient, and compared with original drugs, has the characteristics of increasing fat solubility and stability, improving absorption performance, improving drug efficacy and reducing toxicity and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of pharmaceutical chemistry, in particular to a chlorogenic acid amide derivative, a preparation method and application thereof. BACKGROUND

[0002] Gout is the second largest metabolic disease in the world after diabetes, and there is a huge market demand for gout treatment drugs. The inflammatory response during the acute attack of gout is the main cause of joint pain and swelling, so anti-inflammatory drugs have become the main means for the prevention and treatment of acute gout.

[0003] The anti-inflammatory drugs commonly used in clinical treatment of acute gout at present mainly include colchicine, non-steroidal anti-inflammatory drugs, glucocorticoids or adrenocorticotropic hormone, etc. The main disadvantage of these drugs during use is that the toxic side effects are too large to allow long-term medication. The active natural product molecules in traditional Chinese medicinal materials have the advantages of unique multi-target action mechanism and low toxicity, and have been favored by domestic and foreign research experts in recent years.

[0004] Chlorogenic acid has good anti-inflammatory activity and good prevention and treatment effect on inflammation-related diseases, such as obvious therapeutic effect on cow mastitis, and can also play an anti-inflammatory role by inhibiting the activity of hyaluronidase. At the same time, chlorogenic acid can also effectively inhibit the activation of NLRP3 inflammasome and related inflammatory factors-IL-1β and TNF-α, and has good prevention and treatment potential for the inflammatory response during the acute attack of gout. The poor stability and pharmacokinetic properties of chlorogenic acid limit its clinical application, so chlorogenic acid is often structurally modified to improve stability and anti-inflammatory activity, such as the anti-inflammatory activity of the 1-carboxyl ethyl ester product being obviously improved compared with chlorogenic acid.

[0005] In order to further improve the metabolic stability of chlorogenic acid derivatives, according to the principle of electronic isostere, the ester group at the 1-position of chlorogenic acid is replaced by an amide group which is more stable to esterase, and it is expected that the introduction of amide group can simultaneously improve the anti-inflammatory activity and stability of the derivative, and lay the foundation for the study of the anti-inflammatory activity of chlorogenic acid. SUMMARY

[0006] The present application aims to provide a chlorogenic acid amide derivative, a preparation method and application thereof in the treatment of acute gout inflammation. The chlorogenic acid amide derivative in the present application has good anti-inflammatory activity and shows good therapeutic effect for acute gout. The synthesis method of the chlorogenic acid amide derivative of the present application is simple and efficient. The chlorogenic acid amide derivative provided by the present application has the characteristics of increasing fat solubility and stability, changing absorption performance, improving drug efficacy and reducing toxicity compared with the original drug.

[0007] The chlorogenic acid amide derivative of the present application has the structure shown in general formula I:

[0008]

[0009] wherein: R 1 is any one of alkyl, aryl, heterocyclic aryl; R 2 is any one of alkyl, aryl, heterocyclic aryl, C(O)-alkyl, C(O)-aryl, C(O)-heterocyclic aryl, C(O)-amine.

[0010] The application provides a preparation method of a chlorogenic acid amide derivative, and the process is as follows:

[0011]

[0012] wherein: R 1 is any one of n-propyl, pentyl, octyl, benzyl, propargyl, cyclohexyl, tetrahydrofurfuryl, furfuryl;

[0013] 1.0 eq of compound 2 and 1.0 eq of HOBT, 2.0 eq of EDCI are weighed in a reaction container, dissolved in DCM after being added, stirred at room temperature, then 2.0 eq of compound 3 is added, water is added after the reaction is completed at room temperature, then extracted with DCM, the organic layers are washed with water and saturated brine in sequence, the organic layer is dried with anhydrous sodium sulfate, and then concentrated under reduced pressure and column chromatography to obtain a white foamy solid, which is compound 4, i.e. a chlorogenic acid amide derivative.

[0014] Further preferably, the stirring reaction time at room temperature is 15 min.

[0015] Further preferably, the compound 3 is any one of n-propylamine, n-pentylamine, n-octylamine, benzylamine, propargylamine, cyclohexylamine, tetrahydrofurfurylamine and furfurylamine.

[0016] The researches involved in the application include but are not limited to the following factors: first, the active carboxylic acid site in the structure of chlorogenic acid is condensed with different alkyl primary amines and aryl primary amines to form a chlorogenic acid amide derivative, and the stability of the derivative is improved by replacing the electron isostere; second, in order to find a chlorogenic acid derivative with high activity and better stability, the phenolic hydroxyl and alcoholic hydroxyl sites in the structure of the chlorogenic acid derivative are protected by etherification or acylation to improve the liposolubility of the chlorogenic acid derivative; third, the pharmacological activity screening of the above chlorogenic acid amide mainly includes in vitro and in vivo anti-inflammatory activity tests. Through the analysis of structure-activity relationship, the structure-activity relationship research results are further used to guide chemical synthesis, and a new compound with higher activity and more stable chemical properties which can be used in clinical treatment is found.

[0017] The application provides an application of the chlorogenic acid amide derivative in the preparation of a medicine for preventing or treating acute gouty diseases, and the diseases include acute gouty arthritis, acute attack period of intermittent gouty arthritis, drug-induced liver injury, acute kidney injury or neuropathic pain and the like.

[0018] The present application provides the use of chlorogenic acid amide derivatives in the preparation of a medicament for preventing or treating inflammatory infectious diseases, including one or more of gouty arthritis, inflammatory bowel disease, meningitis, pancreatitis, peritonitis, vasculitis, glomerulonephritis, hepatitis, keratitis, cataract, senile macular degeneration and optic neuritis.

[0019] The present application provides chlorogenic acid amide derivatives with new structures by chemical modification of chlorogenic acid. The chlorogenic acid amide derivatives provided by the present application have the following characteristics: first, by replacing the carboxyl or ester group with a more stable amide group through an isosteric replacement, the stability of the chlorogenic acid derivative is enhanced; second, the liposolubility of the chlorogenic acid derivative is increased, the compound enters the body in the form of a prodrug, and after oral administration, it is metabolized into the original drug under the catalysis of enzymes in the body and the acidic environment of gastric juice to exert the drug efficacy, prolonging the half-life and enhancing the drug efficacy. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 Effect of chlorogenic acid amide derivative on NO production in LPS-induced RAW264.7 cells.

[0021] Figure 2 Effect of chlorogenic acid amide derivative on the survival rate of LPS-induced RAW264.7 cells.

[0022] Figure 3 Effect of PCR test 4c on the relative expression level of NLRP3 in RAW264.7 cells.

[0023] Figure 4 Effect of PCR test 4c on the relative expression level of IL-1β in RAW264.7 cells.

[0024] Figure 5 Effect of PCR test 4c on the relative expression level of TNF-α in RAW264.7 cells.

[0025] Figure 6 Effect of ELISA test 4c on the relative expression level of IL-1β in RAW264.7 cells.

[0026] Figure 7 Effect of ELISA test 4c on the relative expression level of TNF-α in RAW264.7 cells.

[0027] Figure 8 Effect of WB test compound 4c on the relative expression level of IL-1β in RAW264.7 cells.

[0028] Figure 9To test the effect of compound 4c on the relative expression level of caspase-1 in RAW264.7 cells by WB.

[0029] Figure 10 To test the effect of compound 4c on the relative expression level of INOS in RAW264.7 cells by WB.

[0030] Figure 11 To test the effect of compound 4c on the relative expression level of COX-2 in RAW264.7 cells by WB.

[0031] Figure 12 To test the effect of compound 4c on the ankle circumference of acute gout rats by the line method.

[0032] Figure 13 To test the effect of compound 4c on the ankle swelling level of acute gout rats by the line method.

[0033] Figure 14 To test the effect of compound 4c on the relative expression level of TNF-α in the serum of acute gout rats by ELISA.

[0034] Figure 15 To test the effect of compound 4c on the relative expression level of IL-1β in the serum of acute gout rats by ELISA.

[0035] Figure 16 To test the effect of compound 4c on the western blotting of NLRP3 protein in the serum of acute gout rats by WB.

[0036] Figure 17 To test the effect of compound 4c on the relative expression level of NLRP3 in the serum of acute gout rats by WB.

[0037] Figure 18 The hydrogen spectrum of compound 4a.

[0038] Figure 19 The carbon spectrum of compound 4a. DETAILED DESCRIPTION

[0039] For ease of reference, certain terms are now defined.

[0040] “Alkyl” refers to an unsubstituted, straight-chain, branched, or cyclic alkyl carbon chain of up to 15 carbon atoms. Straight-chain alkyl groups include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl. Cyclic alkyl groups (“cycloalkyl”) include, for example, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. The term “aryl” as used herein refers to an unsubstituted or substituted aromatic compound, a carbocyclic group. Aryl groups are either monocyclic or polycyclic fused compounds. For example, phenyl is a monocyclic aryl. Naphthyl is an example of an aryl group with polycyclic fusion. Aryl groups can be substituted with one or more substituents. Non-limiting examples of substituents include NH2, NO2, N(CH3)2, ONO2, F, Cl, Br, I, OH, OCH3, CO2H, CO2CH3, CN, aryl, and heteroaryl.

[0041] The chlorogenic acid amide derivative of the present invention has the structure shown in general formula I:

[0042]

[0043] Where: R 1 R is any one of alkyl, aryl, and heterocyclic aryl; 2 It is any one of alkyl, aryl, heterocyclic aryl, C(O)-alkyl, C(O)-aryl, C(O)-heterocyclic aryl, and C(O)-amino.

[0044] The synthetic route for chlorogenic acid amide derivatives of general formula I is as follows:

[0045]

[0046] Where: R 1 It is any one of n-Propyl, n-Amyl, n-Octyl, Benzyl, Propargyl, Cyclohexyl, Tetrahydrofuryl, and Furfuryl;

[0047] Weigh 1 eq of compound 2, 1 eq of HOBT, and 2 eq of EDCI into a reaction vessel. After dissolving in DCM, stir at room temperature for 10 min. Then add 2.0 eq of compound 3 and react completely at room temperature. Add water and extract with DCM. Combine the organic layers and wash with water and saturated brine in sequence. Dry the organic layers with anhydrous sodium sulfate and concentrate under reduced pressure through column chromatography to obtain a white foamy solid, which is compound 4, i.e., the chlorogenic acid amide derivative.

[0048] Synthesis of compound 2:

[0049]

[0050] Chlorogenic acid (compound 1, 10.6 g, 30.0 mmol, 1 eq) and DMAP (732.0 mg, 6.0 mmol, 0.2 eq) were weighed into a 250 mL round-bottom flask, followed by the addition of acetic anhydride (20 mL) and pyridine (10 mL). The mixture was allowed to react overnight at room temperature. The next day, after TLC monitoring showed complete reaction, the reaction mixture was poured into 100 mL of an ice-water mixture and stirred at room temperature for 30 min. The pH was then adjusted to 2-3 with 3N dilute hydrochloric acid, resulting in the precipitation of a white solid. The solid was filtered off, washed with a suitable amount of water, and dried to obtain approximately 12 g of the target product 2 as a white solid, with a yield of 71%.

[0051] Example 1

[0052] Synthesis of compound 4a: Compound 2 (1.1 g, 2.0 mmol, 1 eq), HOBT (0.27 g, 2.0 mmol, 1 eq), and EDCI (764 mg, 4.0 mmol, 2 eq) were weighed into a 100 mL round-bottom flask, dissolved in dichloromethane (50 mL), and stirred at room temperature for 15 min. Then, n-propylamine 3a (240 mg, 4.0 mmol, 2 eq) was added, and the reaction was stirred at room temperature for 0.5–1 h. After the reaction was complete as monitored by TLC, dichloromethane (50 mL) was added for dilution, followed by washing with water (20 mL × 2) and saturated brine (20 mL) successively. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was subjected to column chromatography (DCM / MeOH = 80 / 1–20 / 1) to obtain the target product 4a as a white, foamy solid, 560 mg, in 46% yield. The 1H and 1C spectra of compound 4a are shown below. Figure 18 and Figure 19 As shown. 1 HNMR(400MHz, CDCl3)δ:7.60(d,J=16.0Hz,1H),7.44–7.34(m,2H),7.23(d,J=8.4Hz,1H),6.33(d,J= 16.0Hz,1H),5.84(t,J=5.8Hz,1H),5.63(q,J=3.4Hz,1H),5.54(td,J=10.8,4.5Hz,1H),5.10(dd,J=1 0.2,3.6Hz,1H),3.32–3.10(m,2H),2.94–2.84(m,1H),2.54(ddd,J=19.8,12.0,3.6Hz,2H),2.31(d, J=3.2Hz,6H),2.19(s,3H),2.09(s,3H),2.04–1.92(m,4H),1.55–1.43(m,2H),0.89(t,J=7.4Hz,3H); 13C NMR(101MHz, CDCl3)δ:170.1,169.9,169.7,169.4,168.1,168.0,165.7,143.9,143.8,142.5,132.9,126.6,12 4.0,122.9,118.4,81.5,71.7,68.1,67.0,41.4,38.5,30.6,22.7,21.7,21.0,20.7,20.6,20.59,11.2; HRMS:C 29 H 35 NO 13 +Na + ,Cal 628.2006,Found,628.2012.

[0053] Example 2

[0054] Synthesis of compound 4b: Compound 2 (1.1 g, 2.0 mmol, 1 eq), HOBT (0.27 g, 2.0 mmol, 1 eq), and EDCI (764 mg, 4.0 mmol, 2 eq) were weighed into a 100 mL round-bottom flask, dissolved in dichloromethane (50 mL), and stirred at room temperature for 15 min. Then, n-pentylamine 3b (350 mg, 4.0 mmol, 2 eq) was added, and the reaction was stirred at room temperature for 0.5–1 h. After the reaction was complete as monitored by TLC, dichloromethane (50 mL) was added for dilution, followed by washing with water (20 mL × 2) and saturated brine (20 mL) successively. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was subjected to column chromatography (DCM / MeOH = 80 / 1–20 / 1) to obtain the target product 4b as a white, foamy solid, 710 mg, in 58% yield. 1 H NMR(400MHz, CDCl3)δ:7.60(d,J=16.0Hz,1H),7.45–7.34(m,2H),7.23(d,J=8.4Hz,1H),6.33(d,J=1 6.0Hz,1H),5.88(t,J=5.8Hz,1H),5.63(q,J=3.2Hz,1H),5.53(td,J=10.8,4.6Hz,1H),5.10(dd,J=10 .2,3.6Hz,1H),3.33–3.12(m,2H),2.94–2.83(m,1H),2.61–2.46(m,2H),2.31(d,J=3.0Hz,6H),2.18 (s,3H),2.09(s,3H),2.02–1.94(m,4H),1.52–1.41(m,2H),1.35–1.21(m,4H),0.88(t,J=7.0Hz,3H); 13C NMR (101MHz, CDCl3): δ170.1,169.9,169.7,169.3,168.1,167.9,165.7,143.9,143.8,142.5,132.9,126.6,124.0,1 22.9,118.4,81.5,71.8,68.1,67.0,39.7,38.5,30.6,29.0,28.9,22.2,21.7,21.0,20.7,20.6,20.59,13.9; HRMS:C 31 H 39 NO 13 +H + ,Cal:634.2494found,634.2504.

[0055] Example 3

[0056] Synthesis of compound 4c: Compound 2 (1.1 g, 2.0 mmol, 1 eq), HOBT (0.27 g, 2.0 mmol, 1 eq), and EDCI (764 mg, 4.0 mmol, 2 eq) were weighed into a 100 mL round-bottom flask, dissolved in dichloromethane (50 mL), and stirred at room temperature for 15 min. Then, n-octylamine 3c (520 mg, 4.0 mmol, 2 eq) was added, and the reaction was stirred at room temperature for 0.5–1 h. After the reaction was complete as monitored by TLC, dichloromethane (50 mL) was added for dilution, followed by washing with water (20 mL × 2) and saturated brine (20 mL) successively. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was subjected to column chromatography (DCM / MeOH = 80 / 1–20 / 1) to obtain 650 mg of the target product 4c as a pale yellow oil, in 48% yield. 11H NMR (400 MHz, CDCl3) δ: 7.60 (d, J = 16.0 Hz, 1H), 7.40 (dd, J = 14.0, 5.6 Hz, 2H), 7.23 (d, J = 8.4 Hz, 1H), 6.33 (d, J = 16.0 Hz, 1H), 5.85 (t, J = 5.6 Hz, 1H), 5.63 (d, J = 3.4 Hz, 1H), 5.53 (td, J = 10.8, 4.6 Hz, 1H), 5.10 (dd, J = 10.2, 3.6 Hz, 1H), 3.22 (dt, J = 13.0, 7.2 Hz, 2H), 2.88 (d, J = 16.4 Hz, 1H), 2.62–2.45 (m, 2H), 2.31 (d, J = 3.0 Hz, 6H), 2.18 (s, 3H), 2.09 (s, 3H), 2.02–1.93 (m, 4H), 1.51–1.39 (m, 2H), 1.27 (d, J = 10.9 Hz, 10H), 0.87 (t, J = 6.7 Hz, 3H); 13 13C NMR (101 MHz, CDCl3): δ 170.1, 169.8, 169.7, 169.3, 168.1, 167.9, 165.7, 143.9, 143.8, 142.5, 132.9, 126.6, 124.0, 122.9, 118.4, 81.5, 71.8, 68.1, 67.0, 39.8, 38.5, 31.7, 30.6, 29.4, 29.1, 26.8, 22.6, 21.8, 21.0, 20.7, 20.6, 20.6, 14.1; HRMS: C 34 H 45 N 13 O + + H + , Cal 676.2969 Found, 676.2963; + Na

[0057] Example 4

[0058] Synthesis of compound 4d: Compound 2 (1.1 g, 2.0 mmol, 1 eq), HOBT (0.27 g, 2.0 mmol, 1 eq), and EDCI (764 mg, 4.0 mmol, 2 eq) were weighed into a 100 mL round-bottom flask, dissolved in dichloromethane (50 mL), and stirred at room temperature for 15 min. Then, benzylamine 3d (430 mg, 4.0 mmol, 2 eq) was added, and the reaction was stirred at room temperature for 0.5–1 h. After the reaction was complete as monitored by TLC, dichloromethane (50 mL) was added for dilution, followed by washing with water (20 mL × 2) and saturated brine (20 mL) successively. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was subjected to column chromatography (DCM / MeOH = 80 / 1–20 / 1) to obtain the target product 4d as a white, foamy solid, 470 mg, in 36% yield. 1 H NMR(400MHz, CDCl3)δ:7.59(d,J=16.0Hz,1H),7.41–7.21(m,8H),6.32(d,J=16.0H z,1H),6.15(s,1H),5.64(d,J=3.6Hz,1H),5.58–5.49(m,1H),5.11(dd,J=10.2,3.6 Hz,1H),4.42(qd,J=14.8,5.6Hz,2H),2.92(d,J=16.4Hz,1H),2.57(ddd,J=19.6,11 .8,3.6Hz,2H),2.31(d,J=3.2Hz,6H),2.15(s,3H),2.08(s,3H),2.05–1.93(m,4H); 13 C NMR (101MHz, CDCl3): δ170.1,170.0,169.7,169.5,168.1,168.0,165.6,144.0,143.8,142.5,137.6,132.9,128.8(2C),127 .7(2C),127.6,126.6,124.0,122.9,118.3,81.4,71.7,68.1,67.0,43.8,38.4,30.7,21.7,21.0,20.7,20.6,20.59; HRMS:C 33 H 35 NO 13 +H + ,Cal 654.2187Found,654.2189.

[0059] Example 5

[0060] Synthesis of compound 4e: Compound 2 (1.1 g, 2.0 mmol, 1 eq), HOBT (0.27 g, 2.0 mmol, 1 eq), and EDCI (764 mg, 4.0 mmol, 2 eq) were weighed into a 100 mL round-bottom flask, dissolved in dichloromethane (50 mL), and stirred at room temperature for 15 min. Then, propargylamine 3e (220 mg, 4.0 mmol, 2 eq) was added, and the reaction was stirred at room temperature for 0.5–1 h. After the reaction was complete as monitored by TLC, dichloromethane (50 mL) was added for dilution, followed by washing with water (20 mL × 2) and saturated brine (20 mL) successively. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was subjected to column chromatography (DCM / MeOH = 80 / 1–20 / 1) to obtain the target product 4e as a white, foamy solid, 817 mg, in 68% yield. 1 H NMR(400MHz, CDCl3)δ:7.61(d,J=16.0Hz,1H),7.46–7.34(m,2H),7.24(d,J=8.4Hz,1H),6.34 (d,J=16.0Hz,1H),6.20(t,J=5.2Hz,1H),5.64(q,J=3.2Hz,1H),5.54(td,J=10.7,4.6Hz,1H), 5.11(dd,J=10.2,3.6Hz,1H),4.11–3.94(m,2H),2.87(dt,J=16.3,3.0Hz,1H),2.65–2.44(m,2 H),2.31(d,J=2.8Hz,6H),2.25(t,J=2.4Hz,1H),2.20(s,3H),2.09(s,3H),2.04–1.92(m,4H); 13 C NMR(101MHz, CDCl3)δ:170.1,169.8,169.7,169.5,168.1,167.9,165.6,144.0,143.8,142.5,132.9,126.6,12 4.0,122.9,118.3,81.1,78.8,72.2,71.6,68.0,66.9,38.1,30.6,29.6,21.7,21.0,20.7,20.6,20.59; HRMS:C 29 H 31 NO 13 +H + ,Cal 602.1874Found,602.1880;C 29 H 31 NO 13 +Na + ,Cal624.1693,Found,624.1708.

[0061] Example 6

[0062] Synthesis of compound 4f: Compound 2 (1.1 g, 2.0 mmol, 1 eq), HOBT (0.27 g, 2.0 mmol, 1 eq), and EDCI (764 mg, 4.0 mmol, 2 eq) were weighed into a 100 mL round-bottom flask, dissolved in dichloromethane (50 mL), and stirred at room temperature for 15 min. Cyclohexylamine 3f (400 mg, 4.0 mmol, 2 eq) was then added, and the reaction was stirred at room temperature for 0.5–1 h. After the reaction was complete as monitored by TLC, dichloromethane (50 mL) was added for dilution, followed by washing with water (20 mL × 2) and saturated brine (20 mL) successively. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was subjected to column chromatography (DCM / MeOH = 80 / 1–20 / 1) to obtain the target product 4f as a white, foamy solid, 720 mg, in 56% yield. 1 H NMR (400MHz, CDCl3) δ: 7.60 (d, J = 16.0Hz, 1H), 7.45–7.35 (m, 2H), 7.23 (d, J = 8.4Hz, 1H), 6.33 (d, J = 16. 0Hz,1H),5.64(dt,J=6.8,5.8Hz,2H),5.53(td,J=10.8,4.4Hz,1H),5.10(dd,J=10.2,3.6Hz,1H),3.78– 3.64(m,1H),2.91–2.80(m,1H),2.59–2.45(m,2H),2.32(dd,J=12.6,4.2Hz,6H),2.17(s,3H),2.09(s, 3H),1.99(s,4H),1.86(d,J=10.6Hz,2H),1.67(d,J=13.2Hz,3H),1.41–1.23(m,2H),1.19–1.00(m,3H); 13 C NMR (101MHz, CDCl3) δ: 170.1, 169.7, 169.2, 169.0, 168.1, 167.9, 165.7, 143.9, 143.8, 142.5, 132.9, 126.6, 124.0, 122. 9,118.4,81.4,71.7,68.1,67.1,48.4,38.4,32.9,32.8,30.6,25.4,24.7,24.65,21.7,21.0,20.7,20.6,20.59; HRMS:C 32 H 39 NO 13 +H +,Cal 646.2500Found,646.2496;C 32 H 39 NO 13 +Na + ,Cal 668.2319,Found,668.2318.

[0063] Example 7

[0064] Synthesis of compound 4g: Compound 2 (1.1g, 2.0mmol, 1eq), HOBT (0.27g, 2.0mmol, 1eq), and EDCI (764mg, 4.0mmol, 2eq) were weighed into a 100mL round-bottom flask, dissolved in dichloromethane (50mL), and stirred at room temperature for 15min. Then, 3g of tetrahydrofurfurylamide (404mg, 4.0mmol, 2eq) was added, and the reaction was stirred at room temperature for 0.5-1h. After the reaction was complete as monitored by TLC, dichloromethane (50mL) was added for dilution, followed by washing with water (20mL × 2) and saturated brine (20mL) successively. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was subjected to column chromatography (DCM / MeOH = 80 / 1-20 / 1) to obtain 4g of the target product as a white, foamy solid (840mg), 65% yield. 1 H NMR(400MHz, CDCl3)δ:7.60(d,J=16.0Hz,1H),7.45–7.34(m,2H),7.23(d,J=8.3Hz,1H),6.33(d,J=16.0Hz,1H),6 .22(d,J=4.4Hz,1H),5.63(s,1H),5.60–5.50(m,1H),5.10(dd,J=10.2,3.6Hz,1H),3.81(dddd,J=26.6,21.6,10.8 ,5.6Hz,3H),3.53(dddd,J=13.8,9.2,6.2,3.2Hz,1H),3.30–3.00(m,1H),2.94–2.81(m,1H),2.63–2.44(m,2H),2. 31(d,J=3.2Hz,6H),2.18(s,3H),2.09(s,3H),2.02–1.92(m,4H),1.88(dd,J=12.6,6.2Hz,2H),1.56–1.43(m,1H); 13C NMR(101MHz, CDCl3)δ:170.2,170.1,169.7,169.4,168.1,167.9,165.6,143.9,143.8,142.5,132.9,126.6,124.0,1 22.9,118.4,81.3,77.6,71.8,68.1,67.0,43.0,38.3,30.7,28.3,25.8,21.7,21.6,21.0,20.7,20.6,20.59; HRMS:C 31 H 37 NO 14 +H + ,Cal 648.2287Found,648.2299;C 31 H 37 NO 14 +Na + ,Cal 670.2106,Found,670.2118.

[0065] Example 8

[0066] Synthesis of compound 4h: Compound 2 (1.1 g, 2.0 mmol, 1 eq), HOBT (0.27 g, 2.0 mmol, 1 eq), and EDCI (764 mg, 4.0 mmol, 2 eq) were weighed into a 100 mL round-bottom flask, dissolved in dichloromethane (50 mL), and stirred at room temperature for 15 min. Then, furfurylamine 3h (400 mg, 4.0 mmol, 2 eq) was added, and the reaction was stirred at room temperature for 0.5–1 h. After the reaction was complete as monitored by TLC, dichloromethane (50 mL) was added for dilution, followed by washing with water (20 mL × 2) and saturated brine (20 mL) successively. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was subjected to column chromatography (DCM / MeOH = 80 / 1–20 / 1) to obtain the target product 4h as a white, foamy solid, 930 mg, in 72% yield. 1H NMR(400MHz, CDCl3)δ:7.60(d,J=16.0Hz,1H),7.44–7.32(m,3H),7.23(d,J=8.4Hz,1H), 6.32(d,J=16.0Hz,2H),6.21(d,J=3.2Hz,2H),5.63(q,J=3.2Hz,1H),5.53(td,J=10.8,4 .6Hz,1H),5.10(dd,J=10.2,3.6Hz,1H),4.41(qd,J=15.6,5.6Hz,2H),2.95–2.82(m,1H) ,2.62–2.47(m,2H),2.31(d,J=2.8Hz,6H),2.17(s,3H),2.08(s,3H),2.02–1.91(m,4H); 13 C NMR(101MHz, CDCl3)δ:170.1,169.9,169.7,169.4,168.1,167.9,165.6,150.5,143.9,143.8,142.5,142.3,132.9,126 .6,124.0,122.9,118.3,110.5,107.6,81.3,71.7,68.0,66.9,38.3,36.8,30.7,21.7,21.0,20.7,20.6,20.59; HRMS:C 31 H 33 NO 14 +H + ,Cal644.1979Found,644.1984;C 31 H 33 NO 14 +Na+,Cal666.1799,Found,666.1805.

[0067] Determination of the anti-inflammatory activity of chlorogenic acid amide derivatives: RAW264.7 cells were seeded in 96-well plates at a concentration of 100 μL / well (5 × 10⁻⁶). 4Cells were cultured overnight at 37°C under constant temperature and humidity. The test compound (20 μg / mL, 50 μL / well) was added for 2 h, followed by LPS (100 ng / mL, 50 μL / well) for 24 h. The experiment was divided into three groups: a control group, a 1% DMSO negative control group, and an experimental group (4a-4h). The control group consisted of normally cultured cells, without any compound or LPS stimulation. The DMSO negative control group (model group) received no compound, followed by LPS stimulation. The experimental group received the compound for 4a-4h followed by LPS stimulation. Collect 100 μL of supernatant into a new 96-well plate. Add 100 μL of the prepared urinary nitrite qualitative test (griess) A / B (v / v = 1:1) reagent to each well, shake at room temperature for 10 min, and measure the absorbance at 540 nm. Analyze the NO production level according to the regression equation of the standard curve. Add MTT (5 mg / mL, 10 μL / well) to the remaining wells and incubate at 37°C in the dark for 4 h. Discard the supernatant, add 200 μL of DMSO to each well, shake at room temperature for 10 min, and measure the absorbance at 492 nm (A).

[0068] The cell survival rate was calculated as follows: Survival rate (%) = (Experimental group - Control group) / (Model group - Control group) * 100%; In order to evaluate the anti-inflammatory activity of the synthesized chlorogenic acid amide derivative, the effects of compound 4a-4h on NO production and cell survival rate of RAW264.7 cells were tested.

[0069] The effect of compound 4c on the relative expression levels of inflammatory factors was determined by RT-PCR: RAW264.7 cells pretreated with different concentrations of compound 4c were used to extract total RNA using the TRIZOL method, followed by reverse transcription using a GeneStar one-tube genomic de-transcription kit to obtain cDNA. Quantitative real-time PCR was performed using a Roche-480 instrument with 2XM5 HiPer SYBR Premix Es Taq reagent (MF787-01), using β-actin as an internal control to detect the expression of the target genes NLRP3, IL-1β, and TNF-α.

[0070] Western blot analysis (WB) was used to detect inflammatory factor levels: RAW264.7 cells in the logarithmic growth phase were digested with trypsin, counted, and then analyzed at a ratio of 2 × 10⁻⁶ cells / mL. 6The culture medium was seeded into 6-well plates at a density of 1 / 2. After overnight incubation and adhesion, samples were added according to the experimental groups. The plates were divided into four groups: control group, model group, isoglycyrrhizin group (25 μM), and 4c (30 μM, 90 μM). The control group received 1000 μL / well of complete culture medium, the model group received 1000 μL / well of 0.11 μg / mL LPS working solution, and incubated for 24 h. The sample groups were first incubated with the corresponding concentration of sample for 2 h, and then added according to the sample addition method of the model group. The protease inhibitor and phosphatase inhibitor were added to the RIPA lysis buffer at a ratio of 1:1:100 (i.e., RIPA:protease inhibitor:phosphatase inhibitor = 100:1:1) and mixed thoroughly. Cells in a six-well plate were washed twice with pre-chilled PBS, then RIPA lysis buffer was added. Cells were scraped off and collected into centrifuge tubes and incubated on ice for 30 min. The tubes were then centrifuged at 12000 rpm for 10 min at 4°C. The supernatant was used for BCA protein concentration analysis, and 40 μg of protein was collected. Electrophoresis was performed using 12% SDS-PAGE, and the protein was transferred to a PVDF membrane. After blocking with 5% BSA, the membrane was incubated overnight at 4°C with primary antibody. Cells were washed three times with TBST for 10 min each time, followed by incubation with the corresponding HRP-labeled secondary antibody. The membrane was then developed using a high-sensitivity chemiluminescence imaging system (Cytiva, Amersham Imager 600).

[0071] Acute gout rat modeling and related index detection: 1) Adaptive feeding: In an SPF-grade animal room, rats were housed in PSU plastic independent ventilated cages, placed in the Suzhou-Hangzhou Technology G4-type IVC system, with no more than 5 rats per cage. Animals had free access to food and water. The cages were lined with sterilized corn cob bedding. The temperature was controlled at 20-26℃, the relative humidity at 40-70%, and automatic lighting was used, alternating between light and dark every 12 hours, with lights on from 7:00 am to 7:00 pm. The rats were acclimatized for 7 days; 2) Experimental components: Control group (gavage with the same volume of physiological saline), model group ( The groups were divided into four groups: (1) Gavage administration of the same volume of excipient), colchicine group (10 mg / kg colchicine), low-dose group (5 mg / kg compound 4c), medium-dose group (10 mg / kg compound 4c), and high-dose group (20 mg / kg compound 4c); 3) Rat model establishment: Rats were general anesthetized with tribromoethanol and kept in a prone position. The injection site was disinfected with alcohol. 0.2 mL of the pre-prepared MSU crystal suspension was injected into the ankle joint using a sterile syringe. The injection speed should be appropriate to avoid the increase of intracavitary pressure and crystal overflow. PS: The modeling time point is 5 days after drug administration, 1 hour after drug administration on the 5th day; 4) Experimental observation indicators: a. General detection: Ankle joint circumference is measured before modeling and at 2, 4, 8, 12, and 24 hours after modeling. Detection method: suture method. b. Sampling: After 24 hours of testing, rats are sacrificed, blood is collected, and the blood is allowed to stand at room temperature for 2 hours. Serum is obtained by centrifugation at 3000 rpm for 10 minutes and stored at -20℃. c. Western blot (WB) detection: The synovial membrane of 3 rats in each group is used for WB detection, and the indicators are NLRP3, Caspas-1, and ASC. d. ELISA kit detection: Serum from 8 rats in each group is used for IL-1β and TNF-α detection.

[0072] * indicates a significant difference compared to the control group, # indicates a significant difference compared to the model group, and the number of p values ​​represents the degree of significance; **p<0.01, ***p<0.001, ****p<0.0001, compared to the control group; #p<0.05, ##p<0.01, ###p<0.001, ####p<0.001, ####p<0.0001, compared to the model group.

[0073] The effect of compounds on NO formation: such as Figure 1 As shown, compounds 4c, 4b, and 4d at a concentration of 20 μg / mL exhibited good inhibitory activity against lipopolysaccharide-induced NO production in RAW264.7 cells, with compound 4c showing the strongest inhibitory effect. Other compounds had relatively little effect on lipopolysaccharide-induced NO production in RAW264.7 cells.

[0074] Effects of compounds on cell viability: such as Figure 2As shown, compounds 4h and 4e at a concentration of 20 μg / mL had little effect on the lipopolysaccharide-induced decrease in RAW264.7 cell survival; while compounds 4c, 4d, 4a, and 4g could protect cells from the lipopolysaccharide-induced decrease in cell survival; and compound 4f had a promoting effect on the lipopolysaccharide-induced decrease in RAW264.7 cell survival.

[0075] Based on the preliminary anti-inflammatory activity test results, compound 4c was selected as the preferred compound, and its anti-inflammatory activity in the LPS+ATP-induced RAW264.7 inflammatory cell model was further tested.

[0076] The effects of compound 4c on the levels of NLRP3 and related inflammatory factors in RAW264.7 cells were investigated. Compound 4c at concentrations of 30 and 90 μM, and a positive control (isoglycyrrhizin) at 25 μM, were prepared. First, RT-PCR was used to examine the effect of compound 4c on the relative expression levels of mRNAs of inflammatory genes NLRP3, IL-1β, and TNF-α in RAW264.7 cells. Then, ELISA was used to examine the effect of compound 4c on the relative expression levels of inflammatory factors IL-1β and TNF-α in RAW264.7 cells. Next, Western blotting (WB) was used to evaluate the effect of compound 4c on the relative expression levels of inflammatory factors IL-1β and capase-1, and inflammatory proteins iNOS and COX2. Immunofluorescence was used to confirm the effect of compound 4c on the NF-κB signaling pathway. The results showed that compound 4c at final concentrations of 30 and 90 μM could inhibit the production of mRNAs of inflammatory factors NLRP3, IL-1β, and TNF-α. Figures 3-9 It can reduce the relative expression levels of the inflammatory proteins iNOS and COX2. Figure 10 and Figure 11 It can regulate the nuclear transport of NF-κB transcription factors and is a negative regulator of NF-κB nuclear transcription.

[0077] 1) PCR test: In the LPS+ATP-induced acute RAW264.7 cell inflammation model, isoliquiritigenin was used as a positive control. PCR tests revealed that LPS stimulation significantly increased the relative expression levels of the inflammasome NLRP3 and inflammatory factors IL-1β and TNF-α. Pretreatment with compound 4c significantly reversed the increase in the relative expression levels of NLRP3 and inflammatory factors in RAW264.7 cells, especially at high concentrations, exhibiting an anti-inflammatory effect comparable to isoliquiritigenin. (See [link to article]). Figures 3-5 .

[0078] 2) ELISA Assay: The effect of compound 4c on the relative expression levels of inflammatory factors IL-1β and TNF-α in an LPS+ATP-induced acute inflammation model of RAW264.7 cells was subsequently investigated using ELISA. LPS stimulation significantly increased the relative expression levels of inflammatory factors, while pretreatment with compound 4c significantly reduced their relative expression levels. Furthermore, compound 4c showed a superior inhibitory effect compared to isoliquiritin. (See [link to ELISA test]). Figure 6 and Figure 7 .

[0079] 3) Western blot (WB) test: The effect of compound 4c on the relative expression levels of inflammatory factors IL-1β and capas-1 in an LPS+ATP-induced acute inflammation model of RAW264.7 cells was subsequently investigated using a Western blot (WB) test. It was found that LPS treatment significantly increased the relative expression levels of IL-1β and capas-1 in the model group, while pretreatment with compound 4c effectively reduced the relative expression levels of IL-1β and capas-1. Even at low concentrations, the inhibitory effect of 4c was slightly better than that of isoliquiritigenin. (See [link to article]). Figure 8 and Figure 9 .

[0080] In an LPS+ATP-induced acute inflammation model of RAW264.7 cells, INOS and COX-2 are key enzymes in the synthesis of NO and PEG-2. To further evaluate the anti-inflammatory activity of compound 4c, we investigated the effect of 4c on the relative expression levels of INOS and COX-2 in RAW264.7 cells using Western blotting. Figure 10 and Figure 11 As shown, after LPS treatment, the relative expression levels of INOS and COX-2 increased significantly, while pretreatment with compound 4c could effectively reduce their relative expression levels, with a slightly better inhibitory effect than isoliquiritin.

[0081] 4) NF-κB Assay: LPS-induced inflammatory responses can mediate the translocation of NF-κB to the nucleus, where it acts as a regulator of inflammatory proteins such as INOS and COX2. To investigate the potential role of compound 4c in NF-κB translocation, we used immunofluorescence analysis. Six hours after LPS treatment, NF-κB (red) translocated from the cytoplasm to the nucleus. Pretreatment with different concentrations of 4c significantly inhibited this translocation, indicating that 4c can significantly suppress LPS-induced NF-κB nuclear translocation in RAW264.7 cells and acts as a negative regulator of NF-κB transport.

[0082] The results above show that 4c has good inhibitory activity on the secretion of inflammasome NLRP3 and related inflammatory factors such as Caspas-1, IL-1β, and TNF-α in RAW 264.7 cells. It also shows good inhibitory effect on the secretion of inflammatory proteins INOS and COX-2. In addition, it is also a negative regulator of NF-κB transport. Therefore, 4c has good in vitro anti-inflammatory activity.

[0083] Subsequently, the in vivo anti-inflammatory activity of the preferred compound 4c was evaluated using an acute gout rat model: first, the effects of compound 4c on the ankle circumference and swelling degree of rats were investigated, and then the effects of 4c on the relative expression levels of inflammatory factors in rat blood were investigated.

[0084] 1) Effects of compound 4c on ankle joint circumference and swelling in rats: In an acute gout rat model, the control group maintained a normal ankle joint circumference (approximately 2.6 cm) and had zero swelling. After MSU treatment, the model group showed a significant increase in ankle joint circumference (2.9 ± 0.1 cm) and swelling (12% ± 2%). Pretreatment with medium and high doses of compound 4c significantly reduced ankle joint circumference and swelling in rats, indicating that medium and high doses have good anti-inflammatory activity in the acute gout rat model. However, the low-dose group showed no significant effect. Figure 12 and Figure 13 .

[0085] 2) ELISA test of the relative expression levels of TNF-α and IL-1β: The effect of 4c on the relative expression levels of inflammatory factors TNF-α and IL-1β in rat serum was tested using an ELISA assay. After stimulation with urate crystal suspension injected into the ankle joint, the inflammation level in the model group was significantly increased. Low and medium doses of 4c administered by gavage could effectively reduce the relative expression levels of inflammatory factors TNF-α and IL-1β, especially the high-dose group, which reduced the inflammatory factor levels to the control group level, demonstrating a good anti-inflammatory effect. (See [link to study]). Figure 14 and Figure 15 .

[0086] 3) Western blot (WB) test on the effect of 4c on the level of inflammatory factors in rat blood: Subsequently, the effect of 4c on the relative expression level of the inflammasome NLRP3 in the blood of an acute gout rat model was tested by Western blot (WB). Figure 16 and Figure 17 As shown, after stimulation of the ankle joint by injection of MSU suspension, the relative expression level of NLRP3 in the model group increased significantly. The medium-dose and high-dose groups of compound 4c could reduce the relative expression level of NLRP3, while the low-dose group showed no significant effect.

[0087] Based on the above results of in vivo activity experiments in rats with acute gout, compound 4c can be seen to have a significant alleviating effect on ankle joint circumference and swelling in rats with acute gout. It also has a significant inhibitory effect on the relative expression levels of inflammasome NLRP3 and inflammatory factors IL-1β and TNF-α in the blood of model rats, and is a good anti-inflammatory compound.

[0088] As can be seen from the above, the chlorogenic acid amide derivative of the present invention can be used to prepare drugs for the prevention or treatment of acute gout diseases, including acute gouty arthritis, intermittent gouty arthritis, drug-induced liver injury, acute kidney injury, or neuropathic pain.

[0089] The chlorogenic acid amide derivatives of the present invention can be used to prepare drugs for the prevention or treatment of inflammatory infectious diseases, including: gouty arthritis, inflammatory bowel disease, meningitis, pancreatitis, peritonitis, vasculitis, glomerulonephritis, hepatitis, keratitis, cataracts, age-related macular degeneration, and optic neuritis.

[0090] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. A chlorogenic acid amide derivative, characterized in that, It has the structure shown in general formula I: ， Where: R 1 It is n-octyl or benzyl; R 2 It is an acetyl group.

2. A method for preparing the chlorogenic acid amide derivative according to claim 1, characterized in that, The process is as follows: ， Where: R 1 It is n-octyl or benzyl; Weigh 1.0 eq of compound 2, 1.0 eq of HOBT, and 2.0 eq of EDCI into a reaction vessel. Dissolve the compounds in DCM and stir at room temperature. Then add 2.0 eq of compound 3 and react completely at room temperature. Add water and extract with DCM. Combine the organic layers and wash them with water and saturated brine. Dry the organic layers with anhydrous sodium sulfate and concentrate under reduced pressure through a column to obtain a white foamy solid, which is compound 4, i.e., a chlorogenic acid amide derivative.

3. The method for preparing the chlorogenic acid amide derivative according to claim 2, characterized in that, The reaction was carried out at room temperature with stirring for 15 minutes.

4. The method for preparing the chlorogenic acid amide derivative according to claim 2, characterized in that, Compound 3 is n-octylamine or benzylamine.

5. The use of the chlorogenic acid amide derivative according to claim 1 in the preparation of a drug for the prevention or treatment of acute gout.

6. The use of the chlorogenic acid amide derivative according to claim 5 in the preparation of a drug for the prevention or treatment of acute gout, characterized in that, The diseases mentioned include one or more of the following: acute gouty arthritis, acute exacerbation of intermittent gouty arthritis, drug-induced liver injury, acute kidney injury, or neuropathic pain.

7. The use of the chlorogenic acid amide derivative according to claim 1 in the preparation of a drug for the prevention or treatment of inflammatory infectious diseases.

8. The use of the chlorogenic acid amide derivative according to claim 7 in the preparation of a drug for the prevention or treatment of inflammatory infectious diseases, characterized in that, The inflammatory infectious diseases mentioned include one or more of the following: gouty arthritis, inflammatory bowel disease, meningitis, pancreatitis, peritonitis, vasculitis, glomerulonephritis, hepatitis, keratitis, cataracts, age-related macular degeneration, and optic neuritis.

Images