CN115636807B - Preparation method and application of two 2-arylbenzofuran derivatives - Google Patents

Abstract

The invention discloses a preparation method of two 2-arylbenzofuran derivatives and application thereof in preparing medicines for treating inflammatory diseases. The 2-arylbenzofuran derivatives are two novel compounds named as corollary honey B and corollary honey C from the genus corollary honey Artocarpus heterophyllus lam of the family Moraceae, and have the following structures:the compound has strong inhibitory activity on respiratory burst of neutrophils in rats, and IC thereof ₅₀ 0.27.+ -. 0.10. Mu.M and 1.53.+ -. 0.58, respectively; the two compounds can be further used for preparing medicines for treating inflammatory diseases, and can be used for clinically treating various oxidative damages caused by neutrophil overactivation, such as rheumatoid arthritis, compensatory anti-inflammatory reaction syndrome, systemic inflammatory reaction syndrome and the like.

Description

Preparation methods and applications of two 2-arylbenzofuran derivatives

Technical field

The invention belongs to the field of medicine and relates to a preparation method of two 2-aryl benzofuran derivatives, in particular to two 2-aryl benzofuran derivatives, jackfruit B and jackfruit, extracted and separated from jackfruit. Application of C in the preparation of drugs for the treatment of inflammatory diseases.

Background technique

Neutrophils (PMNs) are the body's first line of defense against the invasion of foreign pathogenic bacteria; when PMNs recognize the small peptides secreted by the receptors or the complex formed by bacteria and antibodies in the serum, they cause cell inflammation through receptors on the membrane. Activated, a large amount of superoxide anions (O ₂ ^·- ) are rapidly generated; O ₂ ^·-, catalyzed by superoxide dismutase and myeloperoxidase, undergoes a series of free radical chain reactions, producing various Reactive oxygen species (ROS), including hydroxyl radicals (HO·), hydrogen peroxide (H ₂ O ₂ ), and hypochlorous acid (HOCl); these ROS can efficiently destroy invading pathogenic microorganisms, a phenomenon called respiratory burst ; Under normal physiological conditions, the respiratory burst of PMNs is precisely regulated, forming the body's most effective defense mechanism against pathogenic bacteria; however, while ROS kills invading bacteria, it can also cause damage to surrounding normal tissues, causing obstruction of microcirculation, Damages vascular endothelial cells and extravascular tissue cells, releases and promotes the release of inflammatory mediators, and becomes a "destroyer"; for example, in patients with rheumatoid arthritis, neutrophils are improperly activated and produce a large amount of ROS, causing damage to articular cartilage tissue. Corrosion; in sepsis or surgical injury, trauma, burns, ischemia-reperfusion injury, over-activated PMNs can cause tissue damage, and in severe cases, lead to uncontrolled inflammatory responses, including compensatory anti-inflammatory response synthesis syndrome (cARS), systemic inflammatory response syndrome (SIRS), etc.; therefore, discovering substances that inhibit the respiratory burst of PMNs is of great significance for the treatment of various oxidative injuries caused by the respiratory burst of PMNs.

Isolating and identifying natural products with novel structures and significant activity from natural drugs has always been one of the main ways to discover new drugs; Jackfruit (Artocarpus styracifolius Pierre) is a tree species of the genus Jackfruit in the Moraceae family; "Compendium of Materia Medica" records that jackfruit is "sweet" "Slightly acidic, flat, non-toxic" and has the effects of "quenching thirst, relieving annoyance, sobering up, and replenishing qi"; modern chemical and pharmacological research shows that jackfruit is rich in a large number of isopentenyl phenolic components, which have a wide range of pharmacological properties. Activity; In the early activity screening of the study, it was found that the chloroform extraction part of the 95% ethanol extract of jackfruit root showed strong inhibitory activity on the respiratory burst of rat PMNs. Therefore, the present invention conducted in-depth research on this active site.

Contents of the invention

In view of the above problems, the object of the present invention is to provide a preparation method and application of two 2-arylbenzofuran derivatives (jackonin B and jackonectin C).

The technical solution of the present invention is:

The two 2-aryl benzofuran derivatives described in the present invention have substances that inhibit the respiratory burst activity of PMNs and provide medicine for the clinical treatment of inflammation related to PMNs respiratory burst; the invention specifically relates to the extract from jackfruit root. Two 2-aryl benzofuran derivatives, namely jackfruit B and jackfruit C; the chemical structural formula of the compound is as follows:

Furthermore, the compounds 1 and 2 are two 2-arylbenzofuran derivatives, both of which are new compounds that have not been reported in the literature, namely jackfruit B and jackfruit C.

Further, the preparation method of the two 2-arylbenzofuran derivatives jackfruit B and jackfruit C;

Specifically, the compound of the present invention is prepared by the following method:

Take 17.0kg of jackfruit root and extract it three times with 95% ethanol at room temperature (medicinal material and solvent 1:10) to obtain the leachate, which is filtered and combined, and concentrated under reduced pressure to obtain a total extract of 1.5kg;

Suspend the total extract with water at a ratio of 1:1, and extract with petroleum ether, chloroform, ethyl acetate, and n-butanol in sequence to obtain 213.3g of petroleum ether, 574.0g of chloroform, 382.0g of ethyl acetate, and n-butanol. Part 152.4g;

Take 532.0g of the chloroform extraction site extract, mix it with HP-20 macroporous adsorption resin (weight ratio 1:1), put it on an HP-20 macroporous adsorption resin column (column size: 10*45cm), and mix it with ethanol-water ( 0～95%) gradient elution to obtain 11 fractions Frs.H1-H11;

The fraction Fr.H6 (57.3g) was subjected to ODS column chromatography (column size: 4*22cm), MeOH-H ₂ O (volume ratio 4:6, 5:5, 6:4, 7:3, 8:2, 9:1,10:0) gradient elution to obtain 6 fractions Frs.H6O1-H6O6;

The fraction Fr.H6O3 (13.7g) was subjected to MCI CHP-20P resin column chromatography (column size: 4*45cm), MeOH-H ₂ O (volume ratio 6:4, 7:3, 8:2, 9:1, 10:0) gradient elution to obtain 7 fractions Frs.H6O3M1-H6O3M7;

The fraction Fr.H6O3M4 (1.6g)) was further subjected to Sephadex LH-20 gel column chromatography (column specification: 2*200cm)), and 7 fractions Fr.H6O3M4L1-H6O3M4L7 were eluted with methanol;

The fraction H6O3M4L4 (82.0mg)) was subjected to preparative high performance liquid phase column chromatography (column specification: 2*25cm) and eluted with 50% acetonitrile to obtain the compound jackfruit B (1) of the present invention;

The fraction Fr.H6O3M4L6 (74.0 mg) was subjected to preparative high performance liquid phase column chromatography (column specification: 2*25cm) and eluted with 50% acetonitrile to obtain the compound jackfruit C (2) of the present invention.

Further, the object of the present invention is to provide the above-mentioned compounds (two 2-arylbenzofuran derivatives, jackfruit B and jackfruit C, and their physiologically acceptable salts), in the preparation and treatment of inflammatory diseases (respiratory outbreaks with PMNs). Inflammation caused by uncontrolled inflammation) new uses in drugs.

Furthermore, the inflammatory disease refers to diseases related to or similar to rheumatoid arthritis, compensatory anti-inflammatory response syndrome, systemic inflammatory response syndrome, etc.

The beneficial effects of the present invention are: it is confirmed through activity screening experiments that the jackfruit B and jackfruit C of the present invention have a significant inhibitory effect on the burst of neutrophils in rats stimulated by phorbol myristate (PMA). The half inhibitory concentrations (IC ₅₀ ) are 0.27 ± 0.10 and 1.53 ± 0.58 μM respectively, and the activity is better than the positive control V _c (IC ₅₀ = 23.84 ± 2.20 μM); the jackfruit B and jackfruit C of the present invention can be further prepared It has become a drug to treat inflammation caused by uncontrolled respiratory bursts in PMNs.

Description of the drawings

Figure 1 is the chemical structural formula of jackfruit B (1) and jackfruit C (2) in the present invention;

Figure 2 is a high-resolution mass spectrum of the new compound jackamide B (1) of the present invention;

Figure 3 is a hydrogen nuclear magnetic resonance spectrum ( ¹ H NMR) diagram of the new compound jackfruit B (1) in the present invention;

Figure 4 is a nuclear magnetic resonance carbon spectrum ( ¹³ C NMR) chart of the new compound jackfruit B (1) of the present invention;

Figure 5 is the nuclear magnetic resonance DEPT 135 spectrum of the new compound jackfruit B (1) in the present invention.

Figure 6 is the nuclear magnetic resonance HSQC spectrum of the new compound jackamide B (1) of the present invention.

Figure 7 is the nuclear magnetic resonance HMBC spectrum of the new compound jackamide B (1) of the present invention;

Figure 8 is a high-resolution mass spectrum of the new compound jackamidin C (2) of the present invention;

Figure 9 is a hydrogen nuclear magnetic resonance spectrum ( ¹ H NMR) chart of the new compound jackfruit C (2) in the present invention;

Figure 10 is a nuclear magnetic resonance carbon spectrum ( ¹³ C NMR) diagram of the new compound jackfruit C (2) in the present invention;

Figure 11 is the nuclear magnetic resonance DEPT 135 spectrum of the new compound jackfruit C (2) in the present invention;

Figure 12 is the nuclear magnetic resonance HSQC spectrum of the new compound jackfruit C (2) in the present invention;

Figure 13 is the nuclear magnetic resonance HMBC spectrum of the new compound jackfruit C (2) in the present invention;

Figure 14 is a schematic diagram showing the main HMBC (H→C) correlations of the new compounds jackfruit B (1) and jackfruit C (2) in the present invention.

Detailed ways

In order to explain the technical solution of the present invention more clearly, the technical solution of the present invention will be further described in detail below with reference to the accompanying drawings:

Example 1

Preparation of jackfruit B and jackfruit C:

Take 17.0kg of jackfruit root and extract it three times with 95% ethanol at room temperature (medicinal material and solvent 1:10) to obtain the leachate, which is filtered and combined, and concentrated under reduced pressure to obtain a total extract of 1.5kg;

Suspend the total extract with water at a ratio of 1:1, and extract with petroleum ether, chloroform, ethyl acetate, and n-butanol in sequence to obtain 213.3g of petroleum ether, 574.0g of chloroform, 382.0g of ethyl acetate, and n-butanol. Part 152.4g;

Take 532.0g of the chloroform extraction site extract, mix it with HP-20 macroporous adsorption resin (weight ratio 1:1), put it on an HP-20 macroporous adsorption resin column (column size: 10*45cm), and mix it with ethanol-water ( 0～95%) gradient elution to obtain 11 fractions Frs.H1-H11;

The fraction Fr.H6 (57.3g) was subjected to ODS column chromatography (column size: 4*22cm), MeOH-H ₂ O (volume ratio 4:6, 5:5, 6:4, 7:3, 8:2, 9:1,10:0) gradient elution to obtain 6 fractions Frs.H6O1-H6O6;

The fraction Fr.H6O3 (13.7g) was subjected to MCI CHP-20P resin column chromatography (column size: 4*45cm), MeOH-H ₂ O (volume ratio 6:4, 7:3, 8:2, 9:1, 10:0) gradient elution to obtain 7 fractions Frs.H6O3M1-H6O3M7;

The fraction Fr.H6O3M4 (1.6g) was further subjected to Sephadex LH-20 gel column chromatography (column specification: 2*200cm), and 7 fractions Frs.H6O3M4L1-H6O3M4L7 were eluted with methanol;

The fraction H6O3M4L4 (82.0mg) was subjected to preparative high performance liquid phase column chromatography (column specification: 2*25cm) and eluted with 50% acetonitrile to obtain the compound jackfruit B (4.0mg, t _R 45min) of the present invention;

The fraction Fr.H6O3M4L6 (74.0mg) was subjected to preparative high performance liquid phase column chromatography (column specification: 2*25cm) and eluted with 50% acetonitrile to obtain the compound jackfruit C (15.6mg, t _R 35min) of the present invention. .

Example 2

Structural identification of jackfruit B and jackfruit C:

The isolated monomers were identified as two 2-arylbenzofuran compounds by high-resolution mass spectrometry (HR-ESI-MS) and nuclear magnetic resonance spectroscopy (1D NMR and 2D NMR). Compounds 1 and 2 are new compounds that have not been reported in the literature.

Compound 1, light yellow oil (methanol): High-resolution mass spectrometry HR-ESI-MS gave a quasi-molecular ion peak m/z 407.1853 [MH] ^- (C ₂₅ H ₂₇ O ₅ , calculated value 407.1864)), confirmed Its molecular formula is C ₂₅ H ₂₈ O ₅ ;

The infrared spectrum IR shows that there are characteristic signal peaks such as hydroxyl (3357cm ^-1 ), methyl (2925cm ^-1 ) and benzene ring (1616, 1481cm ^-1 ) in its structure;

^{The 1} H NMR spectrum (Table 1) shows three active hydrogen proton signals δ _H 9.43 (2H, s), 8.44 (1H, s)); an A ₂ B spin coupling system signal δ _H 6.71 (2H, d, J=2.2Hz), 6.23(1H,t,J=2.2Hz); a methoxy group signal δ _H 3.94(3H,s); combined with 13C NMR spectrum, two sets of typical 3-methyl-2 can be observed -Proton and carbon signals of butene substituent δ _H 5.27(1H,br t,J=7.2Hz), 3.51(2H,d,J=7.2Hz), 1.83(3H,s), 1.64(3H,s) , 5.13(1H,br t,J＝6.9Hz), 3.33(2H,d,J＝6.9Hz), 1.73(3H,s) and 1.62(3H,s); δ _c 130.7, 129.6, 124.0, 122.6, 25.6, 22.9, 22.7, 17.9, 17.8;

The ¹ H NMR data of compound 1 is very similar to the known isopentenyl 2-arylbenzofuran derivative regiafuran A ^[15] ; the difference in the hydrogen spectrum between the two is mainly reflected in the absence of regiafuran A in compound 1. The proton signal of H-2 is δ _H 6.85, and there is an additional set of proton signals belonging to the 3-methyl-2-butene substituent; these data suggest that 1 is a 2-isopentenyl substituted derivative of regiafuran A thing;

Through DEPT 135, HSQC and HMBC NMR experiments, the positions of the substituents were determined and all hydrogen and carbon signals were fully assigned (Table 1);

The two isopentenyl groups substituted at C-2 and C-4, respectively, can be determined by the following HMBC correlation observed (Figure 2): H ₂ -15 (δ _H 3.51) and C-1 (δ _C 152.9), C-2 (δ _C 106.8) is related to C-3 (δ _C 150.4); H-16 (δ _H 5.27) is related to C-2; H-20 (δ _H 3.33) is related to C-3, C-4 ( δ _C 116.1) is related to C-5 (δ _C 148.5); H-21 (δ _H 5.13) is related to C-4;

In addition, the HMBC correlation of the methoxy proton signal δ _H 3.94 (MeO-5) and C-5 (δc 148.5) confirmed its substitution at C-5;

Comprehensive analysis of HMBC related peak data determined the structure of 1 as 5-[6-hydroxy-4-methoxy-5,7-bis(3-methyl-2-butene)benzofuran-2-yl]- 1,3-Benzodiphenol (Figure 1), named jackfruit B.

Table 1 ¹ H NMR and ¹³ C NMR spectral data of compounds 1 and 2

^a Bruker Avance 600 NMR instrument; chemical shifts referenced to solvent peak correction of deuterated dimethyl sulfoxide (δ _H 2.50; δ _C 39.5)

^b Bruker Avance 600 NMR instrument; chemical shifts referenced to solvent peak correction of deuterated methanol (δ _H 3.31; δ _C 49.0)

Compound 2, light yellow oil (methanol); high-resolution mass spectrometry HR-ESI-MS gave a quasi-molecular ion peak m/z389.1402[MH] ^- (C ₂₄ H ₂₁ O ₅ , calculated value is 389.1394), which was determined The molecular formula is C ₂₄ H ₂₂ O ₅ ;

The ¹ H and ¹³ C spectra of compound 2 are very similar to the known isopentenyl 2-arylbenzofuran derivative artoidonesianinA-1 ^[16] ;

The main difference between the two is that 2 has an additional set of typical 2,2-dimethylchromene groups fused with benzene rings (cyclized isopentenyl groups) signal δ _H 6.63 (1H,d,J =10.0Hz), 5.52(1H,d,J=10.0Hz), 1.45(3H×2,s); δ _C 118.0, 127.6, 77.9, 28.2;

At the same time, 2 is missing the 4-OMe signals δ _H 3.93 and δ _C 56.0 in the NMR data of the known compound artoidonesianin A-1; this information suggests that 2 is a compound containing two 2,2-dimethylchromene groups. 2-arylbenzofuran derivatives; by analyzing the DEPT 135, HSQC and HMBC NMR spectra of 2, the fused position of the two 2,2-dimethylchromene groups on the benzene ring was determined, and all All hydrogen and carbon signals were assigned (Table 1);

Two 2,2-dimethylchromene groups are fused at positions C-2/C-3 and C-4/C-5 respectively, which can be confirmed by the following HMBC related signals: H-15 (δ _H 6.73) Related to C-1 (δ _C 152.8), C-2 (δ _C 100.9) and C-3 (δ _C 148.0); H-16 (δ _H 5.61) related to C-1;

H-20 (δ _H 6.63) is related to C-3, C-4 (δ _C 106.7), C-5 (δ _C 147.4) and H-21 (δ _H 5.52) is related to C-4; comprehensive analysis of HMBC data The structure of 2 was determined to be 5-[2H,9H-2,2,9,9-tetramethyl-furo[2,3-f]pyrano[2,3-h][1]benzopyra Pyran-6-yl]-1,3-benzenediol (Figure 1), named jackfruit C.

Example 3

Cytotoxicity evaluation experiment of jackfruit B and jackfruit C:

Use the following experimental steps to isolate and purify rat PMNs; take clean-grade SD rats (Experimental Animal Center of Jiangxi University of Traditional Chinese Medicine, Animal Qualification Certificate No.: JZDW2011304), take 9 mL of blood from the orbit, and instill 1 mL of 1% heparin sodium for anticoagulation vertically. into a good glass centrifuge tube; add 4.5% dextran T-500 physiological saline solution in a ratio of 5:1, mix well, and let stand at 4°C for about 1 hour; take the supernatant and add it in a ratio of 3:1. Centrifuge the centrifuge tube pre-filled with lymphocyte separation liquid at 800 rpm (275g) for 15 minutes. Take out the centrifuge tube. The tube is divided into three layers. The upper layer is light yellow serum, and the white mist area in the middle is monocytes and lymphocytes. The lower layer that settles to the bottom of the tube is PMNs; discard the supernatant, add 2 mL of special separation liquid to rinse once, oscillate and centrifuge at 2500 rpm (531g) for 5 minutes; discard the supernatant, and add 2 mL of double distilled water to each centrifuge tube. water, pipette and shake for 20 seconds (to swell the red blood cells), immediately add 2 mL of 1.8% NaCl solution and mix well, centrifuge at 2500 rpm (531g) for 5 minutes, discard the supernatant, and repeat this operation until no blood cells remain; Rinse 1-2 times with HBSS-FCS buffer, 2 mL of HBSS-FCS solution each time, centrifuge at 2500 rpm (531g) for 3 minutes, discard the supernatant; separate PMNs, and add HBSS-FCS buffer again Add 2 mL of solution, mix well, measure the viability by trypan blue staining (the viability of PMNs within 3 hours is >95%), and store it at 4°C as a cell mother suspension for later use.

Determine the cytotoxicity of jackfruit B and jackfruit C to PMNs with reference to relevant literature on the standard trypan blue exclusion method; take 50 μL of PMNs cell mother suspension and dilute it with 2% calf serum HBSS to a cell concentration of 2×10 ⁶ cells/ mL; mix 1 mL of PMNs cell dilution with 10 μL of DMSO or test compound (dissolved in DMSO, final concentration range from 1 to 1000 μM), and incubate at 37°C for 30 minutes; add 112 μL of 0.4% trypan blue to each sample Dye solution, under a high-power microscope, calculate the cytotoxic effect of the sample on PMNs by counting the absorption of trypan blue by 100 cells; the results show that jackfruit B and jackfruit C have no obvious cytotoxicity to PMNs at concentrations lower than 150 μM.

Example 4

Determination of the respiratory burst inhibition rate of jackfruit B and jackfruit C in rat PMNs:

Use the same steps as in Example 3 to prepare rat PMNs cell mother suspension; take 50 μL of PMNs cell mother suspension and dilute it with 2% calf serum HBSS to a cell concentration of 2×10 ⁶ cells/mL; store at 4°C Backup; PMNs undergo a respiratory burst after being activated by the exogenous irritant - phorbol ethyl myristate (phorbol) (PMA), producing a large amount of reactive oxygen species. The free radicals are captured by the luminescent agent luminol to produce chemical Luminescence (CL), PMN-CL intensity is positively correlated with the cell number of PMNs and the respiratory burst and phagocytosis function of PMNs; BPCL-K ultra-weak luminescence meter (Beijing Institute of Biophysics, Chinese Academy of Sciences, supporting BPCL Appl.7.2 data Processing workstation) parameters are set as follows: luminous pool temperature 37°C, voltage value 800V, maximum detection time 1800s, counting time interval 5s;

Preheat the instrument for half an hour before use and take the baseline; after the baseline is stable, take 1ml of PMNs cell dilution solution in the luminescent cup, add 200μl of luminol working solution to it, and place it in the ultra-weak luminescence measuring instrument and incubate for 10 minutes ( Parameter settings: luminescent pool temperature 37°C, voltage 800V, maximum detection time 1800s), record the self-luminescence process (counting time interval 5s); then add 10μl sample solution (using 10μl DMSO as solvent control) and continue the measurement for 5min, add 8μg ·ml ^-1 PMA stimulant 10μl, continue the measurement for 15 minutes, record the measurement results; PMN-CL intensity is expressed by the peak height of luminescence count; calculate the PMN-CL inhibition rate according to formula (1):

Taking the PMN-CL inhibition rate as the ordinate and the sample concentration as the abscissa, a dose-effect relationship curve is established. Through the dose-effect curve, the concentration of the sample (ie, IC ₅₀ value) when the luminescence inhibition rate is 50% can be calculated; taking vitamin C ( V _c ) as a positive control;

The results showed that the two stilbene compounds had a significant inhibitory effect on the burst of rat PMNs stimulated by PMA, and their IC ₅₀ were 0.27±0.10 and 1.53±0.58μM respectively, both of which were better than the positive control V _c (IC ₅₀ =23.84 ±2.20μM).

Finally, it should be understood that the embodiments described in the present invention are only used to illustrate the principles of the embodiments of the present invention; other modifications may also fall within the scope of the present invention; therefore, alternatives to the embodiments of the present invention are provided as examples rather than limitations. Configurations may be considered consistent with the teachings of the invention; accordingly, embodiments of the invention are not limited to those expressly introduced and described herein.

Claims (4)

1. Two 2-arylbenzofuran derivatives, wherein the two 2-arylbenzofuran derivatives are corollary honey B and corollary honey C; the chemical structural formula of the compound is shown as follows:

2. use of two 2-arylbenzofuran derivatives as defined in claim 1 and their physiologically acceptable salts for the preparation of a medicament for the treatment of inflammatory diseases.

3. The use according to claim 2, wherein the inflammatory disease is an inflammatory disease caused by uncontrolled breathing bursts of PMNs.

4. The use according to claim 2, wherein the inflammatory disease is rheumatoid arthritis, compensatory anti-inflammatory response syndrome and systemic inflammatory response syndrome.