CN113773295A - Synthesis method of mono-substituted dihydrochromone and application of mono-substituted dihydrochromone in treating lung inflammation such as COPD (chronic obstructive pulmonary disease) - Google Patents
Synthesis method of mono-substituted dihydrochromone and application of mono-substituted dihydrochromone in treating lung inflammation such as COPD (chronic obstructive pulmonary disease) Download PDFInfo
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Abstract
Hair brushA process for synthesizing the monosubstituted dihydrochromone and its application in treating the inflammation of lung such as COPD are disclosed. The chemical structure of the 7-monosubstituted-4', 5-dihydroxydihydrochromone derivative is shown as the following formula (a), wherein in the formula (a), R1The group is Boc-Ala, Boc-Val-Gly, Boc-D-Phe, Boc-L-Phe-Phe, Boc-D-Trp, Boc-O-Bn-Tyr or Boc-D-Pro. The invention synthesizes the 7-monosubstituted-4', 5-dihydroxy dihydrochromone derivative, and experiments show that the derivative can effectively inhibit the lung inflammatory reaction. Therefore, the compound can be used for inhibiting the lung inflammatory reaction and has wide application.
Description
The technical field is as follows:
the invention belongs to the field of biological medicine, and particularly relates to a 7-monosubstituted-4', 5-dihydroxy dihydrochromone compound, a synthesis method thereof and application thereof in preparing a medicament for treating pulmonary inflammation such as COPD (chronic obstructive pulmonary disease).
Background art:
pneumonia refers to inflammation occurring in the distal lung, i.e., the pulmonary interstitium, alveolar spaces, and terminal airways. The factors causing inflammation are mainly pathogenic microorganisms such as bacteria, parasites, fungi and viruses, or chemical allergy, radiation and the like. The clinical symptoms of pneumonia are cough, fever, blood in sputum or expectoration, which is often accompanied by dyspnea or chest distress. Pneumonia can be classified into viral pneumonia, fungal pneumonia, bacterial pneumonia, mycoplasma pneumonia, physicochemical pneumonia, allergic pneumonia, pneumonia caused by other pathogens, immune pneumonia, etc. according to etiology, the most common pneumonia is bacterial pneumonia, and accounts for more than 70% of adult pneumonia. When pathogens invade lung tissues, signaling pathways such as Mitogen Activated Protein Kinase (MAPK), nuclear transcription factor- κ B, etc. are activated, triggering an inflammatory response for immunization. In the inflammatory reaction, immune cells such as alveolar macrophages, neutrophils, lymphocytes and the like are activated by a signal transduction pathway, and inflammatory mediators such as tumor necrosis factors, interleukins, interferons, growth factors, chemokines and the like are released, such as TNF-alpha, IL-6, IFN-gamma, IL-1 beta, IL-12, IL-18 and the like, so that the inflammatory reaction and the signal transduction pathway are subjected to feedback regulation. Pulmonary inflammation is an important component of various acute and chronic respiratory diseases such as COPD, asthma, ARDS and the like, which can cause patients to have symptoms such as mucus hypersecretion, airway obstruction, emphysema, pulmonary edema and the like, and complicated diseases of other organ tissues and even systemic diseases, and even the life is delayed in severe cases.
The flavonoid compounds are widely used for treating pulmonary inflammation due to the special structural characteristics of the flavonoid compounds. Corresponding to the pathological process characteristics of the occurrence and development of the inflammatory reaction of the lung tissue, the mechanism of resisting pneumonia by the flavonoid compounds mainly comprises the following ways: the compound preparation regulates signaling pathways such as MAPK, NF-kB and the like, influences the accumulation of inflammatory cells in the lung, inhibits the release of cytokines, reduces the generation of proinflammatory mediators and inhibits the generation and development of inflammatory reaction.
The invention content is as follows:
the invention aims to provide a 7-monosubstituted-4', 5-dihydroxy dihydrochromone derivative, a synthesis method thereof and application thereof in preparing a medicament for treating pulmonary inflammation such as COPD (chronic obstructive pulmonary disease).
The first purpose of the invention is to provide a 7-mono-substituted-4', 5-dihydroxy dihydrochromone derivative, the chemical structure of which is shown as the following formula (a),
in the formula (a), R1The group is Boc-Ala, Boc-Val-Gly, Boc-D-Phe, Boc-L-Phe-Phe, Boc-D-Trp, Boc-O-Bn-Tyr or Boc-D-Pro.
The 7-monosubstituted-4', 5-dihydroxy dihydrochromone derivative is preferably one of the following compounds:
when R is1When Boc-Ala, the 7-monosubstituted-4', 5-dihydroxydihydrochromoneThe chemical structure of the derivative is as follows:
when R is1When Boc-Val-Gly, the chemical structure of the 7-monosubstituted-4', 5-dihydroxy dihydrochromone derivative is as follows:
when R is1When Boc-D-Phe, the chemical structure of the 7-monosubstituted-4', 5-dihydroxy dihydrochromone derivative is as follows:
when R is1When Boc-L-Phe-Phe, the chemical structure of the 7-monosubstituted-4', 5-dihydroxy dihydrochromone derivative is as follows:
when R is1When Boc-D-Trp, the chemical structure of the 7-monosubstituted-4', 5-dihydroxy dihydrochromone derivative is as follows:
when R is1When Boc-O-Bn-Tyr, the chemical structure of the 7-monosubstituted-4', 5-dihydroxy dihydrochromone derivative is as follows:
when R is1When Boc-D-Pro, the 7-monosubstituted-4', 5-dihydroxyl diThe chemical structure of the hydrogen chromone derivative is as follows:
the second purpose of the invention is to provide a synthesis method of 7-mono-substituted-4', 5-dihydroxy dihydrochromone derivatives, which comprises the following steps (shown as a reaction formula i):
condensing BOC protected amino acid, dissolving with DMF, adding 1-ethyl- (3-dimethylaminopropyl) carbonyl diimine hydrochloride and 4-dimethylaminopyridine in sequence under stirring, adding naringenin, stirring at room temperature for reaction, and obtaining a 7-monosubstituted-4', 5-dihydroxydihydrochromone derivative after the reaction is stopped;
the reaction formula of the above method is as follows:
R1the group is Boc-Ala, Boc-Val-Gly, Boc-D-Phe, Boc-L-Phe-Phe, Boc-D-Trp, Boc-O-Bn-Tyr or Boc-D-Pro.
Experiments show that the 7-monosubstituted-4', 5-dihydroxy dihydrochromone derivative can inhibit the release of cytokines and reduce the production of proinflammatory mediators so as to inhibit the generation and development of inflammatory reaction.
Therefore, the third purpose of the invention is to provide the application of the 7-monosubstituted-4', 5-dihydroxy dihydrochromone derivative in preparing anti-inflammatory drugs.
The fourth object of the present invention is to provide an anti-inflammatory agent comprising the above 7-monosubstituted-4', 5-dihydroxydihydrochromone derivative as an active ingredient.
Preferably, the anti-inflammatory agent is an anti-pulmonary inflammation agent.
Further preferably, the anti-inflammatory agent is a drug for treating chronic obstructive pulmonary disease, COPD.
Preferably, the anti-inflammatory drug comprises a 7-monosubstituted-4', 5-dihydroxy dihydrochromone derivative and medically acceptable auxiliary materials.
The invention synthesizes the 7-monosubstituted-4', 5-dihydroxy dihydrochromone derivative, and experiments show that the derivative can effectively inhibit the lung inflammatory reaction. Therefore, the compound can be used for inhibiting the lung inflammatory reaction and has wide application.
Drawings
Figure 1 is a result of the anti-inflammatory activity of the compounds.
Detailed Description
The following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Example 15 Synthesis of-hydroxy-2- (4-hydroxyphenyl) -4-oxochroman-7-yl- (tert-butoxycarbonyl) phenylalanine (e 1):
BOC-phenylalanine (1mmol) was condensed in a 25mL round-bottomed flask, dried DMF (6mL) was added to dissolve it, 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride (EDCI,1.2mmol), 4-dimethylaminopyridine (DMAP,2mmol) and naringenin (naringenin,1mmol) were added sequentially with stirring, and then the reaction was stirred at room temperature for 5 hours, and stopped according to the plate count. And adding an ethyl acetate solution for many times, extracting to obtain a crude product, and performing column chromatography to obtain the target compound e 1. Yield: 45.8 percent.1H NMR(400MHz，MeOH-d4)δ7.51(dd,J＝20.2,6.2Hz,1H),7.45–7.18(m,3H),7.06(d,J＝8.5Hz,1H),5.94(dd,J＝13.6,2.2Hz,1H),5.55–5.38(m,1H),4.66–4.52(m,1H),3.30–3.18(m,1H),3.18–2.99(m,1H),2.88–2.69(m,1H),1.52–1.36(m,6H).13C NMR(100MHz，MeOH-d4)δ171.11,167.03,164.10,163.15,156.54,150.74,136.88,136.76,129.06,128.21,127.18,126.62,121.44,101.97,95.87,94.87,79.46,78.46,55.52,42.72,37.16,27.29.
Example 25 Synthesis of hydroxy-2- (4-hydroxyphenyl) -4-oxochroman-7-yl- (tert-butoxycarbonyl) phenylalanyl-phenylalanine ester (e 2):
a compound was prepared in the same manner as in example 1 except that BOC-phenylalanine in example 1 was replaced with BOC-phenylalanyl-phenylalanine. Yield: 11.3 percent. HRMS-ESI calcd for C38H38N2O9[M-H]-665.2577，found 665.2578
Example synthesis of 35-hydroxy-2- (4-hydroxyphenyl) -4-oxochroman-7-yl- (tert-butoxycarbonyl) tryptophan (e 3):
the compound was prepared in the same manner as in example 1 except that BOC-phenylalanine in example 1 was replaced with BOC-tryptophan. Yield: 18.3%. HRMS-ESI calcd for C31H30N2O8[M-H]-557.2002，found 557.1999.IR(neat,cm-1):3795，2089，1996，1929，1729，1467，1435，1390，1343，1323，1217，1109，915.659
Example synthesis of 45-hydroxy-2- (4-hydroxyphenyl) -4-oxochroman-7-yl-3- (4- (benzyloxy) phenyl) -2- ((tert-butoxycarbonyl) amino) propionate (e 4):
the compound was prepared in the same manner as in example 1 except that BOC-phenylalanine in example 1 was replaced with BOC-tyrosine. Yield: 43.6 percent.1H NMR(500MHz,MeOD)δ7.49(d,J＝8.2Hz,1H),7.43(d,J＝7.4Hz,1H),7.36(t,J＝7.5Hz,1H),7.29(dd,J＝15.3,8.0Hz,1H),7.21(d,J＝8.4Hz,1H),7.03(d,J＝8.3Hz,1H),6.96(d,J＝8.5Hz,1H),5.94(dd,J＝15.3,1.9Hz,1H),5.43(dd,J＝12.7,2.1Hz,1H),5.07(s,1H),4.54(t,J＝7.3Hz,1H),3.16(dd,J＝13.8,6.5Hz,1H),3.11–2.98(m,1H),2.76(dd,J＝17.1,2.8Hz,1H),1.44(s,4H).13C NMR(101MHz,MeOD)δ195.70,171.16,167.06,164.08,163.12,157.92,156.49,150.70,137.38,136.81,130.11,128.86,128.11,127.44,127.17,127.13,121.43,114.69,101.97,95.91,94.91,79.46,78.42,69.60,55.66,42.70,36.42,27.32.HRMS-ESI calcd for C13H25NO8[M-H]-624.2312，found 624.2312
Example 55 Synthesis of hydroxy-2- (4-hydroxyphenyl) -4-oxochroman-7-yl (tert-butoxycarbonyl) -alanine (e 5):
the compound was prepared in the same manner as in example 1 except that BOC-phenylalanine in example 1 was replaced with BOC-alanine. Yield: 36.7%. 1H NMR (400MHz, MeOD) δ 7.65-7.46 (m,1H),7.19(d, J ═ 8.0Hz,1H),5.95(d, J ═ 13.9Hz,1H),5.50(d, J ═ 11.5Hz,1H),4.37(dd, J ═ 14.0,6.9Hz,1H),3.12(dd, J ═ 17.0,12.8Hz,1H),2.81(d, J ═ 15.3Hz,1H),1.53(d, J ═ 7.3Hz,1H),1.48(d, J ═ 4.9Hz,5H),1.48(d, J ═ 4.9, 5H), 1.29.29.19, 1H),1.35 (dd, 1H), 1.23.35H).13CNMR(100MHz，MeOH－d4)δ195.78,172.29,167.02,164.10,163.16,156.64,150.95,136.86,127.44,121.45,101.96,95.85,94.85,79.33,78.48,49.54,42.74,27.32,15.97.
Example 65 Synthesis of-hydroxy-2- (4-hydroxyphenyl) -4-oxochroman-7-yl (tert-butoxycarbonyl) valyl-glycine (e 6):
the compound was prepared in the same manner as in example 1 except that BOC-phenylalanine in example 1 was replaced with BOC-valyl-glycine. Yield: 34.6 percent. NMR (500MHz, MEOH-d4) δ 12.00(s,1H),8.05(s,1H),7.43(d, J ═ 7.5Hz,2H),7.15(d, J ═ 8.1Hz,2H),6.01(d, J ═ 15.1Hz,2H),5.36(d, J ═ 12.4Hz,1H),5.12(d, J ═ 8.0Hz,1H),4.54(s,1H), 3.07-2.88 (m,1H),2.78(d, J ═ 17.0Hz,1H),2.15(s,1H),1.83(d, J ═ 5.3H)Hz,2H),1.70(d,J＝8.5Hz,1H),1.49(s,9H),1.03(d,J＝3.6Hz,6H).13CNMR(126MHz,MeOD)δ195.39,172.44,166.95,163.86,162.93,156.11,150.68,136.40,127.36,121.74,102.32,96.58,95.61,80.23,78.38,77.50,77.25,76.99,52.36,43.17,40.85,28.14,24.86,22.70,21.56.HRMS-ESI calcd for C27H32N2O9[M-H]-527.2108，found 527.2105
Example synthesis of- (tert-butyl) 2- (5-hydroxy-2- (4-hydroxyphenyl) -4-oxochroman-7-yl) -pyrrolidine-1, 2-dicarboxylate (e 7):
the compound was prepared in the same manner as in example 1 except that BOC-phenylalanine in example 1 was replaced with BOC-proline. Yield: 28.5 percent. NMR (400MHz, MEOH-d4) δ 7.56(t, J ═ 9.1Hz,1H),7.20(s,1H),5.95(d, J ═ 13.3Hz,1H),5.48(d, J ═ 12.6Hz,1H),4.51(s,1H),3.51(dd, J ═ 16.9,8.7Hz,1H), 3.19-3.02 (m,1H),2.79(d, J ═ 17.1Hz,1H), 2.55-2.32 (m,1H),2.21(dd, J ═ 11.4,5.7Hz,1H),2.01(d, J ═ 9.6Hz,1H),1.49(d, J ═ 9.4Hz,5H).13C NMR(100MHz,MeOD)δ171.69,167.02,164.10,163.46,163.13,154.89,154.28,150.86,150.68,136.98,136.87,127.37,127.24,121.47,121.21,101.96,95.88,94.89,80.41,80.19,78.46,78.42,59.15,59.05,42.73,30.45,29.59,27.32,24.06,23.23.HRMS-ESI calcd for C25H27NO8[M-H]-468.1737，found 468.1736。
Example 8: research on anti-inflammatory activity of 7-monosubstituted-4', 5-dihydroxydihydrochromone compound
The derivatives of the invention were tested for inhibition of pneumococcal cytokine expression levels using a conventional qPCR method.
First, culture of cells
Human lung epithelial cells BEAS-2B were cultured in a DMEM high-sugar medium (complete medium) supplemented with 10% (V/V) FBS, 100U/mL penicillin and 100. mu.g/mL streptomycin. The culture conditions were 37 ℃ and 5% CO2And carrying out passage when the cells grow to 80% -90% of the fusion degree.
Second, cell administration and induction of cell inflammation
Experiments were performed using cells in logarithmic growth phase, 1 x 10 cells5Inoculating to 24-well plate at 37 deg.C and 5% CO2Culturing to 90% for use. Setting grouping: blank stimulation group (|), LPS stimulation group (+), positive control group (naringenin) and dosing group (e 1-7). The medium was carefully removed, and fresh complete medium containing 100. mu.M of the compound was added to the positive control group and the administered group, respectively, and an equal volume of DMSO was added to the blank stimulation group and the LPS stimulation group. After 1 hour, 1. mu.g/mL LPS was added for 2 hours to induce cell inflammation, respectively, except for the blank stimulation group. Each treatment was 3 replicates.
Thirdly, extracting RNA and measuring qPCR
1. Completely removing the culture medium from the cultured cells, and adding RA2 in 0.5mL RNA extraction kit per well for lysis (the cells can be frozen at-80 ℃ for one night, the lysis effect is better, and the cells can be lysed at 4 ℃ for 10min and then collected);
2. collecting cell lysate, extracting Total RNA according to the kit instruction, and measuring the concentration of the Total RNA by using an ultramicro ultraviolet-visible spectrophotometer;
RT-PCR (20 uL per system) including 5X BUFFER 4. mu.L, Total RNAX. mu.L (1pg-1ug, most of the time 200-2After O (16-X) microliter is placed in an 8-junction tube, serial numbers are marked, and reverse transcription is carried out in a PCR instrument set at 50 ℃ for 15min,85 ℃ for 5s and 16 ℃ for a period of time which is in the range of oc;
4. addition of 80. mu.L DEPC-H to the transcribed cDNA2O to 100 mu L, and centrifuging and mixing evenly, and keeping at-20 ℃ for later use;
5. according to (SYBR 10. mu.L + DEPC-H)2O10 muL + primer 0.5 muL) and the number of samples, taking 18 muL/well to a 96-well plate, adding 2 muL cDNA to form a 20 muL system, centrifuging at 1200rpm for 1min, and mixing uniformly;
6. placing the sample plate into a CFX Connect read-Time System (Real-Time fluorescence quantitative PCR instrument) for detection, and performing detection according to the procedures of (95 ℃ for 2min, 95 ℃ for 20s,57 ℃ for 20s, and 72 ℃ for 20s) for 39 times, 95 ℃ for 1min, 55 ℃ for 30s, and 95 ℃ for 30s, wherein the total Time of the instrument is 2.5 h;
after 7.2h, the data were saved and analyzed, using 2-ΔΔCtAnalyzing the experimental result by a method, wherein the calculation formula is as follows: the delta Ct target gene is Ct target gene-Ct reference gene, and the delta Ct target gene is delta Ct experimental target gene-delta Ct control group target gene. 2-ΔΔCtThe expression fold of the target gene in the experimental group relative to the control group is shown.
Designing an amplification primer: the invention aims at IL-6 gene, and takes GAPDH as reference gene. The gene library in NCBI is searched for the target gene, the target gene sequence in GeneBank is checked, and the design of primer parameters is performed according to the detection requirements, as shown in Table 1 below.
TABLE 1
Synthesis of cDNA: and (3) synthesizing cDNA by using the total RNA as a template, adopting 5x HiScript II Q Select RT SuperMix, mixing each solution by vortex oscillation before use, quickly centrifuging, and collecting liquid remained on the tube wall. The preparation of the reaction system was completed in an ice bath, and after thawing the template RNA on ice, the reverse transcription reaction system was prepared as shown in table 2:
TABLE 2
Genome removal and reverse transcription reaction are carried out in a Veriti 96well Thermal Cycler PCR instrument, and the reaction program is as follows: the reverse transcription product is stored in a refrigerator at the temperature of minus 20 ℃ for standby after being stored for 15min at the temperature of 50 ℃ and 5s at the temperature of 85 ℃.
Real-time qPCR: the real-time qPCR reaction system was prepared as shown in Table 3, the sequences of primers used in this experiment are shown in Table 1, the GAPDH gene was selected as a reference, and the primers were synthesized by Biotechnology engineering (Shanghai) GmbH.
TABLE 3
Performing Real-Time qPCR reaction in a CFX Connect Real-Time System Real-Time fluorescent quantitative PCR instrument, wherein the amplification program comprises the following steps: 95 ℃ for 2min, then 40 cycles were performed: 95 ℃ for 20s,57 ℃ for 20s and 72 ℃ for 20 s. The temperature was raised from 55 ℃ to 95 ℃ to obtain a melting curve. The results were automatically analyzed by analysis software to generate an amplification curve and calculate the Ct value. By using 2-ΔΔctAnalyzing the experimental result by a method, wherein the calculation formula is as follows: delta CtTarget gene＝CtTarget gene-CtInternal reference gene，△△CtTarget gene＝△CtGenes of interest in the experimental group-△CtControl group target gene。2-ΔΔctThe expression fold of the target gene in the experimental group relative to the control group was expressed, and normalization treatment was performed based on the LPS-stimulated group.
The results are detailed in table 4:
the test results show that the 7-monosubstituted-4', 5-dihydroxydihydrochromone derivative can effectively inhibit the expression level of IL-6 in the tested human lung-derived epithelial cell strains, particularly the compounds e2 and e7, and show better anti-inflammatory activity compared with naringenin in a positive control group (figure 1). The experimental results show that the compound has good anti-inflammatory activity and can be used for researching medicaments for resisting pulmonary inflammation.
Claims (10)
1.7-monosubstituted-4', 5-dihydroxy dihydrochromone derivative with the chemical structure as shown in the following formula (a),
in the formula (a), R1The group is Boc-Ala, Boc-Val-Gly, Boc-D-Phe, Boc-L-Phe-Phe, Boc-D-Trp, Boc-O-Bn-Tyr or Boc-D-Pro.
2. The 7-monosubstituted-4', 5-dihydroxydihydrochromone derivative according to claim 1 wherein the derivative is represented by one of the following compounds:
when R is1When Boc-Ala, the chemical structure of the 7-mono-substituted-4', 5-dihydroxy dihydrochromone derivative is as follows:
when R is1When Boc-Val-Gly, the chemical structure of the 7-monosubstituted-4', 5-dihydroxy dihydrochromone derivative is as follows:
when R is1When Boc-D-Phe, the chemical structure of the 7-monosubstituted-4', 5-dihydroxy dihydrochromone derivative is as follows:
when R is1When Boc-L-Phe-Phe, the chemical structure of the 7-monosubstituted-4', 5-dihydroxy dihydrochromone derivative is as follows:
when R is1When Boc-D-Trp, the chemical structure of the 7-monosubstituted-4', 5-dihydroxy dihydrochromone derivative is as follows:
when R is1When Boc-O-Bn-Tyr, the chemical structure of the 7-monosubstituted-4', 5-dihydroxy dihydrochromone derivative is as follows:
when R is1When Boc-D-Pro, the chemical structure of the 7-monosubstituted-4', 5-dihydroxy dihydrochromone derivative is as follows:
3. a method for synthesizing a 7-monosubstituted-4', 5-dihydroxydihydrochromone derivative according to claim 1, which is represented by the reaction formula i, comprising the following steps:
condensing BOC-amino acid, dissolving with DMF, adding 1-ethyl- (3-dimethylaminopropyl) carbonyl diimine hydrochloride and 4-dimethylaminopyridine in sequence under stirring, adding naringenin, stirring at room temperature for reaction, and obtaining a 7-monosubstituted-4', 5-dihydroxydihydrochromone derivative after the reaction is stopped;
the reaction formula of the above method is as follows:
R1the group is Boc-Ala, Boc-Val-Gly, Boc-D-Phe, Boc-L-Phe-Phe, Boc-D-Trp, Boc-O-Bn-Tyr or Boc-D-Pro.
4. Use of the 7-monosubstituted-4', 5-dihydroxydihydrochromone derivative according to claim 1 for the preparation of an anti-inflammatory medicament.
5. The use of claim 4, wherein the anti-inflammatory agent is an anti-pulmonary inflammation agent.
6. The use according to claim 5, wherein the anti-inflammatory agent is a drug for the treatment of chronic obstructive pulmonary disease, COPD.
7. An anti-inflammatory agent comprising the 7-monosubstituted-4', 5-dihydroxydihydrochromone derivative according to claim 1 as an active ingredient.
8. An anti-inflammatory agent as in claim 7, wherein said agent is an anti-pulmonary inflammatory agent.
9. An anti-inflammatory drug according to claim 8, wherein said anti-inflammatory drug is a drug for the treatment of chronic obstructive pulmonary disease, COPD.
10. The anti-inflammatory agent of claim 7, wherein the anti-inflammatory agent comprises a 7-mono-substituted-4', 5-dihydroxydihydrochromone derivative and a pharmaceutically acceptable excipient.
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