CN113200973B - Compound based on zaltoprofen parent structure and anti-inflammatory application thereof - Google Patents

Abstract

The invention discloses a binding supportIbuprofen or derivatives thereof and a compound with a structure shown as a formula (1) or a formula (2) are spliced to form a novel compound. The compound has anti-inflammatory activity aiming at double targets of COX-2 and PPAR-gamma, can reduce the water content of lung tissues, relieve inflammatory reaction caused by acute lung injury, and detect the expression levels of COX-2 and PPAR-gamma in the lung tissues by immunohistochemistry, and the result shows that the compound can obviously reduce the high expression of COX-2 induced by LPS and can obviously improve the low expression of PPAR-gamma induced by LPS, so the compound can be used for preparing anti-inflammatory drugs.

Description

Compound based on zaltoprofen parent structure and anti-inflammatory application thereof

Technical Field

The invention relates to a new compound, in particular to a new compound based on a Zaltoprofen (ZPF) parent structure, and also relates to a preparation method of the compound and application of the compound in preparing anti-inflammatory drugs.

Background

ZPF is a novel potent non-steroidal anti-inflammatory drug, mainly through selective inhibition of COX-2 and PGE ₂ The synthesis of the compound has the functions of relieving fever, easing pain and resisting inflammation. The ZPF is slightly damaged in the gastrointestinal tract at high dosage and may be closely related to the ZPF carboxyl group. Compared with single-target drugs, multi-target drugs have better effects in the treatment of inflammation, cancer and other diseases.

Both cyclooxygenase-2 (COX-2) and peroxisome proliferator-activated receptor (PPAR-gamma) play important roles in inflammatory reaction, so the development of a novel non-steroidal anti-inflammatory drug based on the dual target of COX-2 and PPAR-gamma has a great prospect. In addition, researches report that the existence of 3-thienyl can obviously show better selectivity of COX-1/COX-2 and has good anti-inflammatory activity; the existence of the amide derivative structure shows good COX-2 inhibition capability; furazan and furazan oxynitride can effectively reduce the irritation to the stomach and intestine as NO releasing groups, and has good anti-inflammatory effect.

Therefore, the invention starts from the parent structure of ZPF, takes COX-2 and PPAR-gamma as the dual target as the basis, reforms the parent structure of ZPF and carboxyl group, and adopts the combination method to combine thienyl and furazan nitrogen oxide as the substituent of carboxyl, and designs the novel non-steroidal anti-inflammatory drug with COX-2 and PPAR-gamma as the dual target potential.

Disclosure of Invention

The invention aims to provide a novel compound, which is prepared by splicing zaltoprofen and derivatives thereof with thiophene or benzenesulfonyl furazan compounds, so that the zaltoprofen and the derivatives thereof have better anti-inflammatory activity.

In order to achieve the purpose, the invention provides the following scheme:

a compound is formed by splicing zaltoprofen or derivatives thereof and a compound with a structure shown as a formula (1) or a formula (2),

wherein R1 is a C1-C5 alcohol; r2 is H or a conventional substituent on the phenyl ring; r3 is H or a conventional substituent on the thiophene ring.

Preferably, R1 is propanol and R2 is H.

Preferably, R3 is H.

Preferably, the zaltoprofen derivative comprises two compounds shown as a structural formula,

in the preparation of the compound, carboxyl on the zaltoprofen or a derivative thereof, hydroxyl on the compound shown in the formula (1) and amino on the compound shown in the formula (2) are subjected to esterification reaction, so that the two compounds are spliced.

In vitro cell experiments show that the compound synthesized by the invention can antagonize COX-2 expression of RAW264.7 cells induced by LPS, and the inhibition effect is obviously stronger than that of ZPF and the derivatives thereof. In addition, the compounds have a modulatory effect on the reduction of PPAR-gamma protein levels in LPS-induced RAW264.7 cells. Therefore, the compound is proved to have dual target potential of COX-2 and PPAR-gamma and can antagonize cell inflammatory reaction.

Furthermore, an LPS is adopted to induce a mouse acute pulmonary edema model and evaluate the treatment effect of the compound, and the result shows that the compound can reduce the water content of lung tissues so as to play a role in treating pulmonary edema; can relieve inflammatory reaction caused by acute lung injury and has little toxic effect on lung tissues; the immunohistochemical detection of COX-2 and PPAR-gamma expression levels in lung tissues shows that the compound can obviously reduce the high expression of COX-2 induced by LPS and can obviously improve the low expression of PPAR-gamma induced by LPS. The above studies indicate that the novel compounds exert anti-inflammatory effects through dual COX-2 and PPAR-gamma targets.

Therefore, the compound can be used for preparing anti-inflammatory drugs, especially anti-pneumonia drugs, and can be used as an active ingredient alone or together with other anti-inflammatory drugs when the drugs are prepared, so that a better synergistic effect is achieved.

See the examples for more specific technical solutions.

Drawings

FIG. 1: western blot was used to determine the expression levels of COX-2 and PPAR-gamma in RAW264.7 cells.

FIG. 2 is a schematic diagram: measurement of lung tissue Water content in mouse model of acute pulmonary edema.

FIG. 3: pathological section examination results of mouse acute pulmonary edema (blank control group and LPS group).

FIG. 4 is a schematic view of: pathological section examination of mice for acute pulmonary edema (D63 group and D63+ LPS group).

FIG. 5: pathological section examination of mice for acute pulmonary edema (group E63 and group E63+ LPS).

FIG. 6: pathological section examination results of acute pulmonary edema of mice (JMC2 group and JMC2 group + LPS group).

FIG. 7: pathological section examination results of acute pulmonary edema of mice (JMC5 group and JMC5 group + LPS group).

FIG. 8: pathological section examination of mice for acute pulmonary edema (JMC6 group and JMC6 group + LPS group).

FIG. 9: immunohistochemistry measured the expression levels of COX-2 in different groups of lung tissues.

FIG. 10: immunohistochemistry detected the expression levels of PPAR-gamma in lung tissues of different groups.

Detailed Description

In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the present application will be described in detail with reference to specific embodiments. It should be noted that the following description is intended to be illustrative, and not to limit the scope of the invention. The experimental procedures referred to in the specification, which are not specifically described herein, are all routine techniques in the art, and those skilled in the art can refer to various general tool specifications, scientific documents, or relevant specifications, manuals, etc., before the filing date of the present application.

Preparation of the Compound of example 1

1. Principal materials and reagents

Ethyl acetate, dichloromethane, petroleum ether, absolute ethanol, dimethyl sulfoxide, methanol, anhydrous sodium sulfate, sodium hydroxide, tetrahydrofuran, glacial acetic acid, 1, 3-propanediol, N-dimethylformamide, and the like, were purchased from national pharmaceutical group chemical agents ltd; ZPF, 4-dimethylaminopyridine, N-hydroxysuccinimide, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride were purchased from Shanghai-derived leaf Biotech Ltd; the double-circle qualitative filter paper is purchased from Hangzhou Wohua filter paper Co., Ltd; column chromatography silica gel is purchased from Qingdao oceanic factory division; the thin layer chromatography silica gel plate is purchased from Qingdao oceanic factory.

2. Synthesis of intermediates

Synthesis of M3: according to ZPF: the amount of sodium borohydride material was 1:2.5, dissolved in methanol, stirred at 45 ℃ for 2h and the progress of the reaction was monitored by TLC. After the reaction is completed, adjusting the pH of the reaction solution to be neutral by using dilute hydrochloric acid, performing suction filtration, collecting filtrate, and then performing reduced pressure distillation to remove methanol; and then adding ethyl acetate to dissolve, adding equal amount of water to extract, collecting an organic phase, repeatedly extracting the water phase for 3-5 times, combining the organic phases, adding anhydrous sodium sulfate to dry and remove water, carrying out reduced pressure distillation to remove the organic phase, dissolving methanol, carrying out reduced pressure distillation again to evaporate the solvent to obtain the target product M3.

Synthesis of M282: 5g M3 was dissolved in 40mL of acetic acid, 3mL of concentrated sulfuric acid was added dropwise, the reaction was stirred at room temperature for 15min, then at 45 ℃ for 15min, and the progress of the reaction was monitored by TLC. After the reaction is completed, adding a sodium hydroxide solution to adjust the pH of the reaction solution to subacidity, adding ethyl acetate to perform extraction (100mL multiplied by 3), combining organic phases, adding anhydrous sodium sulfate to dry and remove water, and performing reduced pressure distillation to remove the organic phase to obtain a product M282.

Synthesis of M6: dissolving 2g M282 in 25mL of acetic acid, slowly dropwise adding 1mL of 30% hydrogen peroxide, stirring and reacting at 45 ℃ for 5 hours after the dropwise addition is finished, and monitoring the reaction progress by TLC once per hour. After the reaction is completed, adding a sodium hydroxide solution to adjust the pH of the reaction solution to be weakly acidic, adding ethyl acetate to perform extraction (100mL multiplied by 3), combining organic phases, adding anhydrous sodium sulfate to dry and remove water, performing reduced pressure distillation to remove the organic phase to obtain a light yellow liquid, and purifying by silica gel column chromatography or silica gel plate to obtain a product M6.

The synthetic route is as follows:

synthesis of ZPF derivatives

Synthesis of 3-benzenesulfonyl-4-propanol-furazan: 2g of 3, 4-diphenylsulfonyl-furazan was dissolved in 20mL of tetrahydrofuran, followed by the addition of 3g of 1, 3-propanediol and the batchwise addition of 1mL of NaOH (25%) solution as a catalyst for the reaction, the reaction was stirred at 40 ℃ and the progress of the reaction was monitored by TLC once an hour. And after the reaction is completed, distilling under reduced pressure to remove the solvent, then adding ethyl acetate to dissolve, adding an equal amount of water to extract, collecting an organic phase, repeatedly extracting the water phase for 3-5 times, combining the organic phases, adding anhydrous sodium sulfate to dry and remove water, distilling under reduced pressure to remove the organic phase to concentrate, and purifying by silica gel column chromatography after concentration to obtain the target compound. The synthetic route is as follows:

synthesis of 3-benzenesulfonyl-4-propanol-furazan

2-synthesis of thiophene formaldehyde hydrazone: putting 1.5mL of 2-thiophenecarboxaldehyde into 7mL of tetrahydrofuran, slowly dropwise adding 1mL of hydrazine hydrate for reaction, carrying out the reaction at room temperature, monitoring the reaction process by TLC, and after the reaction is completed, carrying out reduced pressure distillation to remove the solvent to obtain the target compound 2-thiophenecarboxaldehyde hydrazone, wherein the target compound 2-thiophenecarboxaldehyde hydrazone is not required to be purified and can be directly used for subsequent reaction. The synthetic route is as follows:

synthesis of 2-thiophenecarboxaldehyde hydrazone

3. Synthesis of target Compound

Synthesis of JMC 2: accurately weighing 400mg of M6, dissolving in N, N-Dimethylformamide (DMF), adding 491mg of 4-Dimethylaminopyridine (DMAP), 308mg of N-hydroxysuccinimide (NHS) and 512mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC & HCl), stirring at 35 ℃ for reaction for 2 hours, activating carboxyl on the M6 structure, adding 430mg of 3-benzenesulfonyl-4-propanol-furazan for reaction, monitoring the reaction process by TLC, distilling under reduced pressure to remove the solvent after the reaction is completed, adding ethyl acetate for dissolution, adding equal amount of water for extraction, collecting an organic phase, repeatedly extracting the aqueous phase for 3 times, combining the organic phases, adding anhydrous sodium sulfate for drying and removing water, distilling under reduced pressure to remove the organic phase for concentration, concentrating, and purifying by silica gel column chromatography to obtain the target compound. The synthetic route is as follows:

synthesis of JMC2

Synthesis of JMC 5: accurately weighing 400mg of ZPF and dissolving in DMF, adding 491mg of DMAP, 308mg of NHS and 512mg of EDC & HCl, stirring and reacting for 2h at 35 ℃, activating carboxyl on the ZPF structure, adding 430mg of 3-benzenesulfonyl-4-propanol-furazan for reaction after activation, monitoring the reaction process by TLC, distilling under reduced pressure to remove the solvent after the reaction is completed, adding ethyl acetate for dissolving, adding equal amount of water for extraction, collecting the organic phase, repeatedly extracting the water phase for 3 times, combining the organic phases, adding anhydrous sodium sulfate for drying and removing water, distilling under reduced pressure to remove the organic phase for concentration, purifying by silica gel column chromatography after concentration to obtain the target compound, wherein the synthetic route is as follows:

synthesis of JMC5

Synthesis of JMC 6: accurately weighing 420mg of M2 and dissolving in DMF, adding 491mg of DMAP, 308mg of NHS and 512mg of EDC & HCl, stirring and reacting for 2h at 35 ℃, activating carboxyl on an M2 structure, adding 430mg of 3-benzenesulfonyl-4-propanol-furazan for reaction after activation, monitoring the reaction process by TLC, removing the solvent by reduced pressure distillation after the reaction is completed, adding ethyl acetate for dissolution, adding equal amount of water for extraction, collecting the organic phase, repeatedly extracting the water phase for 3 times, combining the organic phases, adding anhydrous sodium sulfate for drying and dewatering, removing the organic phase by reduced pressure distillation for concentration, purifying by silica gel column chromatography after concentration to obtain the target compound, wherein the synthetic route is as follows:

synthesis of JMC6

Synthesis of D63: accurately weighing 400mg of ZPF (dimethyl formamide) and dissolving in DMF (dimethyl formamide), then adding 491mg of DMAP, 308mg of NHS and 512mg of EDC & HCl, stirring at 35 ℃ for reaction for 2 hours, activating carboxyl on the ZPF structure, adding 169mg of 2-thiophene formaldehyde hydrazone for reaction after activation, monitoring the reaction process by TLC (thin layer chromatography), pouring the reaction solution into ice water after the reaction is completed, separating out solids, performing suction filtration, and recrystallizing a filter cake to obtain D63, wherein the synthetic route is as follows:

synthesis of D63

Synthesis of E63: accurately weighing 400mg of M6, dissolving in DMF, then adding 491mg of DMAP, 308mg of NHS and 512mg of EDC & HCl, stirring at 35 ℃ for reaction for 2 hours, activating carboxyl on an M6 structure, adding 169mg of 2-thiophene formaldehyde hydrazone for reaction after activation, monitoring the reaction process by TLC, pouring the reaction solution into ice water after the reaction is completed, separating out solids, performing suction filtration, and recrystallizing a filter cake to obtain E63, wherein the synthetic route is as follows:

synthesis of E63

4. Structural characterization of the Compounds

Mass spectrometric identification of compounds: weighing about 1mg of each compound, dissolving with methanol, diluting to obtain 0.5 μ g/mL solution, filtering with 0.22 μm microporous membrane, detecting with LC/MS-IT-TOF, and determining molecular weight of the compound.

Nuclear magnetic resonance hydrogen spectrum of compound (a) ((b)) ¹ H-NMR) identification: weighing each compound about 5mg dissolved in DMSO-D ₆ (deuterated dimethyl sulfoxide) or CDCl ₃ (deuterated chloroform), the solution was placed in a nuclear magnetic tube, and the measurement was performed using TMS as a reference reagent.

All compounds were identified by mass spectrometry as having detected molecular weights consistent with the theoretically calculated molecular weight, D63 (C) ₂₂ H ₁₈ N ₂ O ₂ S ₂ Theoretical molecular weight 406.52, molecular weight determination [ M + H]+407.0832)、E63(C ₂₂ H ₁₈ N ₂ O ₂ S ₂ Theoretical molecular weight 406.52, molecular weight determination [ M + H ]]+407.0837)、JMC2(C ₂₈ H ₂₄ N ₂ O ₈ S ₂ Theoretical molecular weight 580.63, molecular weight determination [ M + H]+581.1061)、JMC5(C ₂₈ H ₂₄ N ₂ O ₈ S ₂ Theoretical molecular weight 580.63, molecular weight determination [ M + Na]+603.0828) and JMC6 (C) ₂₈ H ₂₄ N ₂ O ₉ S ₂ Theoretical molecular weight 596.63, molecular weight for detection [ M + Na ]]+519.0783)。

JMC2:Yellow oil. ¹ H NMR(600MHz,CDCl ₃ )δ8.03(d,J＝8.3Hz,2H),7.73(td,J＝7.6,0.9Hz,1H),7.63–7.40(m,8H),7.35(t,J＝7.5Hz,1H),7.18(dd,J＝12.0,4.3Hz,1H),7.10(dt,J＝8.1,2.1Hz,1H),4.35–4.19(m,4H),3.71(dd,J＝15.8,8.6Hz,1H),2.12(p,J＝6.1Hz,2H),1.50(d,J＝7.2Hz,3H).JMC5:White solid. ¹ H NMR(600MHz,CDCl ₃ )δ8.15(dd,J＝8.0,1.5Hz,1H),8.07–8.00(m,2H),7.75–7.71(m,1H),7.58(ddd,J＝17.5,8.1,6.0Hz,4H),7.45–7.40(m,1H),7.36(d,J＝1.8Hz,1H),7.30(td,J＝7.8,1.2Hz,1H),7.12(ddd,J＝12.4,8.0,1.9Hz,1H),4.37–4.28(m,4H),4.24(qt,J＝11.4,5.7Hz,2H),3.73(q,J＝7.1Hz,1H),2.18–2.11(m,2H),1.49(d,J＝7.2Hz,3H).JMC6:White solid. ¹ H NMR(600MHz,CDCl ₃ )δ8.19–8.14(m,1H),8.06(ddd,J＝14.2,12.0,8.2Hz,4H),7.64–7.59(m,3H),7.58–7.51(m,2H),7.39–7.34(m,1H),7.30(d,J＝0.7Hz,1H),4.62–4.44(m,2H),4.38–4.31(m,2H),4.25(ddd,J＝6.2,3.9,1.7Hz,2H),3.89(t,J＝5.8Hz,1H),2.14(dd,J＝11.8,5.9Hz,2H),1.49(d,J＝7.2Hz,3H).D63:White solid. ¹ H NMR(600MHz,CDCl ₃ )δ9.13(s,1H),7.81(s,1H),7.61–7.55(m,3H),7.53(d,J＝1.7Hz,1H),7.46–7.38(m,4H),7.18(dd,J＝3.6,0.7Hz,1H),7.04(dd,J＝5.1,3.6Hz,1H),4.39–4.36(m,2H),4.05(q,J＝7.2Hz,1H),1.50(d,J＝7.1Hz,3H).E63:White solid. ¹ H NMR(600MHz,CDCl ₃ )δ9.44(s,1H),8.00–7.75(m,3H),7.75–7.63(m,1H),7.58(dd,J＝10.7,7.2Hz,1H),7.48–7.31(m,4H),7.23–7.07(m,3H),7.07–7.00(m,1H),4.04(q,J＝7.2Hz,1H),1.51(t,J＝7.0Hz,3H).

EXAMPLE 2 Effect of Compounds on LPS-induced COX-2 and PPAR- γ expression in RAW264.7 cells

1. Principal materials and reagents

RAW264.7 mouse monocyte macrophage cell line purchased from the university of Wuhan collection.

ZPF was purchased from shanghai source leaf biotechnology limited; the major metabolite M2 of ZPF is provided by the laboratory; LPS (Ultrapure, TLR4 activator) was purchased from shanghai bi yunnan biotechnology limited; DMEM high-glucose medium was purchased from siemer heishel technologies (china) ltd; fetal bovine serum from south America was purchased from PAN ^TM Seratech corporation; penicillin-streptomycin double antibody, trypsin-EDTA digest was purchased from Hyclone; DMSO (cell grade) was purchased from Sigma, usa; serum-free cell freezing medium was purchased from Suzhou New Saimei Biotech limited; PBS buffer (dry powder) was purchased from wuhan seiver biotechnology ltd; PAGE gel Rapid preparation kit (10%) was purchased from Hippon Biotech (Shanghai) Inc.; the CCK-8 kit was purchased from santa seine biotechnology (shanghai) ltd; RIPA lysate (strong), protease phosphatase inhibitor cocktail (for mammalian sample extraction, 50 ×), BCA protein concentration assay kit (enhanced), SDA-PAGE protein loading buffer (5 ×) was purchased from shanghai bi yunnan biotechnology limited; PVDF membranes are available from Millipore, USA; protein prestainer, ECL chemiluminescent substrate (enhanced), COX-2Rabbit pAb, PPAR gamma Rabbit pAb, GAPDH Mouse mAb, HRP Goat Anti-Mouse IgG (H + L), HRP Goat Anti-Rabbit IgG (H + L) were purchased from Botaike Biotech, Inc., Wuhan Eitake.

2. Method of producing a composite material

2.1 cell culture

RAW264.7 cells were mouse mononuclear macrophages using DMEM complete medium (containing 10% FBS, 1% glutamine and diabody) at 37 deg.C, 5% CO ₂ Culturing in an incubator.

2.2Western blot

1. RAW264.7 cells were cultured according to the protocol of section 3.1 and used for the assay after the cells had entered the logarithmic growth phase. The cell density is adjusted to 0.5 to lx 10 ⁶ PermL, seeded in 6-well plates at 37 ℃ 5% CO ₂ The incubator continues to culture the cells for 12h, and then serum-free DMEM basal medium is used to add JMC2, JMC6, JMC M D63, E63, JMC5, ZPF and M2 to the final concentration for pretreatmentThe treatment was continued for 2h with 0.1. mu.g/mL LPS for 22h, with three replicates per treatment group.

2. The treated cells were collected and centrifuged at 800rpm for 5min in a 1.5mL enzyme-free EP tube, washed twice with PBS, 100. mu.L of RIPA cell lysate, 1. mu.L of PMSF (100mM) and 1. mu.L of a protein phosphatase inhibitor cocktail were added to each tube, lysed on ice for 15min, and the lysed cells were carefully hung with a cell scraper and transferred to a 1.5mL EP tube. Cells were disrupted by sonication with an ultrasonic cell disrupter (40% power, 30s sonication, 3 times sonication for 10s each, 15s intervals). Protein concentration was measured by BCA, and 20. mu.g of cell protein per group was denatured, separated on a 10% SDS-PAGE gel, and transferred to a PVDF membrane. The membranes were then blocked with 5% BSA blocking buffer for 1h, washed, and then incubated with primary antibody at 4 ℃ overnight. The membrane was washed 3 times with TBST and then incubated with horseradish peroxidase-conjugated secondary IgG antibody for 1.5h at room temperature. And (4) carrying out exposure development by using a DAB horseradish peroxidase developing kit.

3. Results of the experiment

Western blot tests show that each compound has different degrees of effects of antagonizing COX-2 expression of RAW264.7 cells induced by LPS, and the inhibition effect is remarkably stronger than that of ZPF and M2. In addition, each compound had a relieving effect on the decrease in PPAR- γ protein levels of RAW264.7 cells induced by LPS (fig. 1). The test results show that the novel derivative has double target potential of COX-2 and PPAR-gamma, further plays a role in antagonizing inflammatory reaction of RAW264.7 cells induced by LPS, and has an anti-inflammatory effect remarkably stronger than that of ZPF and M2.

EXAMPLE 3 Effect of Compounds on ameliorating LPS-induced acute Lung injury in C57BL/6 mice

1. Principal materials and reagents

The SPF male C57BL/6 mice are 6-8 weeks old and 18-22 g in weight, and are used in the Experimental animal center of Huazhong university of agriculture.

Paraformaldehyde fixing solution (neutral) was purchased from wuhan seiver biotechnology ltd; LPS was purchased from wuhan seiver biotechnology limited; running buffer (dry powder) was purchased from Wuhan Severe Biotech, Inc.; TBS buffer (Dry powder) purchased from Wuhan Seville BiotechLimit company; the transmembrane buffer (dry powder) was purchased from Wuhan Seville Biotech Ltd; BSA was purchased from Wuhan Severe Biotech Ltd; tween-20 was purchased from Chemicals, Inc., national drug group; xylene was purchased from national chemical group, chemical agents, ltd; hematoxylin-eosin dye was purchased from Wuhan Bai Qiandio Biotechnology Ltd; PBS solution (0.01M) was purchased from Wuhan Bai Qiandon Biotechnology Ltd; the 10 × EDTA repair liquid is purchased from Wuhan Bai Qian degree biotechnology, Inc.; hematoxylin dye was purchased from Wuhan Biotechnology GmbH of hundred kilo degrees; h ₂ O ₂ Purchased from Wuhan Bai Qiadu Biotech Co., Ltd; DAB color kits were purchased from DAKO; the glass slide and the cover glass are purchased from Jiangsu Shitai experimental equipment Co., Ltd; hydrochloric acid, ammonia water, neutral gum were purchased from the national pharmaceutical group chemicals, ltd.

2. Method of producing a composite material

2.1 LPS-induced pulmonary edema model in mice

SPF male C57BL/6 mice, 6-8 weeks old, 18-22 g in weight, were provided by the university of agriculture laboratory animal center in Huazhong. The animals are bred in an SPF animal room with good environment, the temperature is 20-26 ℃, the relative humidity is 40-70%, and the illumination/dark cycle is 12 hours. During the acclimation phase of one week of maintenance, all animals had free access to basal feed and sterile water.

Male C57BL/6 mice were randomly divided into 12 groups of n-8: blank control group (i.p. equal volume of PBS-DMSO solution + equal volume of normal saline); LPS group (i.p. equal volume PBS-DMSO solution + i.p. 10mg/kg b.w.LPS); group D63 (intraperitoneal injection 30mg/kg b.w.D63+ equal volume of normal saline); group E63 (intraperitoneal injection 30mg/kg b.w.E63+ equal volume of normal saline); JMC2 group (intraperitoneal injection 30mg/kg b.w.JMC2+ equal volume of normal saline); JMC5 group (intraperitoneal injection 30mg/kg b.w.JMC5+ equal volume of normal saline); JMC6 group (30 mg/kg b.w. JMC6+ equal volume of normal saline for intraperitoneal injection); d63+ LPS group (i.p. 30mg/kg b.w.D63+ 10mg/kg b.w.LPS); e63+ LPS group (i.p. 30mg/kg b.w.e63+ i.p. 10mg/kg b.w.lps); JMC2+ LPS group (30 mg/kg b.w.JMC2+ 10mg/kg b.w.LPS i.p.); JMC5+ LPS group (30 mg/kg b.w.JMC5+ 10mg/kg b.w.LPS i.p.); JMC6+ LPS group (i.p. 30mg/kg b.w.jmc6+ 10mg/kg b.w.lps). All groups were intraperitoneally injected with compound/PBS-DMSO for 2h followed by LPS/saline for 6 h. After 6h of intraperitoneal injection of LSP/normal saline, all mice were sacrificed by intraperitoneal injection of sodium pentobarbital (100mg/kg b.w.).

2.2 measurement of Water content of Lung tissue

After anesthesia and exsanguination, the left lung is rapidly stripped by opening the chest, surface water and blood are sucked dry by absorbent paper, and wet weight is weighed by an electronic balance. The left lung was baked in an oven at 80 ℃ for 72h to constant weight and weighed again, this being the dry weight. The lung tissue water content (%) [ (wet-dry weight)/wet weight ] × 100% was calculated according to the formula, reflecting the severity of pulmonary edema.

2.3 pathological HE staining of Lung tissue

1. Material taking: the fresh upper lobe tissue of the right lung is fixed in 4 percent paraformaldehyde for more than 24 hours. Taking out the tissue from the fixing solution, flattening the tissue of the target part in a fume hood by using a scalpel, and placing the trimmed tissue and the corresponding label in a dehydration box.

2. And (3) dehydrating: and (4) putting the dehydration box into a hanging basket, and dehydrating by sequentially gradient alcohol in the dehydration machine. The method comprises the steps of 1h of 75% alcohol, 4h of 85% alcohol, 2h of 90% alcohol, 2h of 95% alcohol, 1h of absolute ethanol I, 30min of absolute ethanol II, 5-10 min of alcohol benzene, 5-10 min of xylene I, 5-10 min of xylene II, 1h of paraffin I, 1h of paraffin II and 1h of paraffin III.

3. Embedding: embedding the wax-soaked tissue in an embedding machine. Firstly, molten wax is put into an embedding frame, tissues are taken out from a dehydration box and put into the embedding frame according to the requirements of an embedding surface before the wax is solidified, and corresponding labels are attached. Cooling at-20 deg.C, solidifying wax, taking out the wax block from the embedding frame, and trimming.

4. Slicing: the trimmed wax block was sliced on a paraffin slicer to a thickness of 4 μm. The slices float on warm water at 40 ℃ of a slice spreading machine to spread the tissues, the tissues are taken out by a glass slide, the slices are baked in an oven at 60 ℃, and the slices are taken out and stored at normal temperature for later use after being baked by water, dried and waxed.

5. Paraffin section dewaxing to water: placing the slices in xylene I20 min-xylene II 20 min-absolute ethyl alcohol I10 min-absolute ethyl alcohol II 10 min-95% alcohol 5 min-90% alcohol 5 min-80% alcohol 5 min-70% alcohol 5 min-distilled water washing.

6. Hematoxylin staining nuclei: and (3) slicing the cut slices into Harris hematoxylin for dyeing for 3-8 min, washing with tap water, differentiating for several seconds by 1% hydrochloric acid alcohol, washing with tap water, returning blue by 0.6% ammonia water, and washing with running water.

7. Eosin staining of cytoplasm: and (5) dyeing the slices in eosin dye liquor for 1-3 min.

8. Dewatering and sealing: placing the slices in 95% alcohol I5 min-95% alcohol II 5 min-absolute ethanol I5 min-absolute ethanol II 5 min-xylene I5 min-xylene II 5min to dehydrate and transparent in sequence, taking out the slices from xylene, air drying, and sealing with neutral gum.

9. Microscopic examination and analysis: microscopic examination and image acquisition and analysis.

2.4 immunohistochemical detection of Lung tissue COX-2 and PPAR-gamma expression

1. Paraffin section dewaxing to water: the paraffin sections are placed in a 65 ℃ oven to be dried for 2h, and are dewaxed to water (the sections are sequentially placed in xylene I for 20 min-xylene II for 20 min-absolute ethanol I for 10 min-absolute ethanol II for 10 min-95% ethanol for 5 min-90% ethanol for 5 min-80% ethanol for 5 min-70% ethanol for 5 min), and are washed with PBS for three times, and each time is 5 min.

2. Antigen retrieval: placing the slices in citric acid pH 6.0 or EDTA pH 9.0, 8.0 buffer solution, repairing with microwave at medium fire for 8min, cooling for 8min, and cutting off power at medium and low fire for 7 min.

3. Blocking endogenous peroxidase: the sections were placed in 3% hydrogen peroxide solution, incubated for 25min at room temperature in the dark, and the slides were washed 3 times 5min each time in PBS (pH7.4) with shaking on a destaining shaker.

4. Serum blocking: 3% BSA was added dropwise to the assembled ring to cover the tissue uniformly, and the block was performed at room temperature for 30 min.

5. Primary antibody incubation: BSA solution was removed and approximately 50. mu.L of diluted primary antibody (COX-2, PPAR-. gamma.) was added to each section to cover the tissue and incubated overnight at 4 ℃.

6. And (3) secondary antibody incubation: washing with PBS for 3 times, 5min each time, removing PBS solution, adding 50-100 μ L of secondary antibody of corresponding species to each slice, and incubating at room temperature for 50 min.

DAB color development: washing with PBS for 3 times, 5min each time, removing PBS, adding 50-100 μ L of fresh DAB solution into each slice, and controlling color development with a microscope.

8. Counterstaining cell nuclei: after the color development is completed, the color is washed by distilled water or tap water, hematoxylin counterstain, 1% hydrochloric acid alcohol is differentiated (about 1s), the color is washed by tap water, ammonia water is turned to blue, and the color is washed by running water.

9. Dewatering and sealing: placing the slices in 75% alcohol for 5 min-85% alcohol for 5 min-anhydrous ethanol I for 5 min-anhydrous ethanol II for 5 min-n-butanol for 5 min-xylene I for 5min, dehydrating, removing the slices from xylene, air drying, and sealing with neutral gum.

10. Microscopic examination and analysis: microscopic examination and image acquisition and analysis.

3. Results of the experiment

The invention adopts an acute pulmonary edema model of LPS induced male C57BL/6 to further evaluate the treatment effect of each compound. As shown in fig. 2, LPS significantly induced the water content in lung tissue, and each compound reduced the water content in lung tissue to a different extent compared to LPS group, without significant difference compared to blank group. The test result shows that each compound has good treatment effect on the lung edema of mice induced by LPS.

In order to further evaluate the therapeutic effect of each compound on LPS-induced pulmonary edema in mice, the present invention evaluated the therapeutic effect of the compound from the viewpoint of pathological lesion and immunohistochemistry. The pathological section examination results are shown in FIGS. 3-8, and compared with the blank control group, the lung tissue structure of LPS group is abnormal, part of alveolar wall is thickened, alveolar atrophy is collapsed, and lung is materialized (arrow); a large number of neutrophils (arrows) were visible in the lung parenchyma, suggesting that the lung tissue of the LPS group exhibits a high degree of inflammatory cell infiltration. The lung tissues of the D63, E63 and JMC5 groups were slightly abnormal in structure, part of alveolar walls were thickened (arrows), and no obvious inflammatory cell infiltration was observed in the tissues; the lung tissue structures of the JMC2 group and the JMC6 group are normal, the alveolar structures are clear, the alveolar walls are not thickened, a large number of inflammatory cells are not found in the tissues, and red blood cells (arrows) are filled in part of alveolar cavities, so that the compounds have little toxic effect on the lung tissues of mice. The lung tissue structure of the D63+ LPS group is slightly abnormal, the alveolar structure is unclear, a small amount of alveoli are atrophic, and capillaries on part of alveolar walls are expanded (arrows); a small number of neutrophils were visible in the tissue (arrow), and inflammatory cells were significantly less than in the LPS group; lung tissue structure of the E63+ LPS group was slightly abnormal, alveolar structure was unclear, and a small amount of alveolar atrophy (arrow); a small number of neutrophils were visible in the tissue (arrows); inflammatory cells were significantly less than LPS group; the lung tissue structure of JMC2+ LPS group was slightly abnormal, part of alveolar wall was thickened, and a small number of inflammatory cells were visible (arrows); partial alveolar fusion expansion (arrow); the lung tissue structure of the JMC5+ LPS group is abnormal, partial alveoli collapse and collapse, and the lung is materialized (arrow); and a small number of inflammatory cells are visible (arrows); the lung tissue structure of the JMC6+ LPS group is slightly abnormal, the alveolar structure is clearer, and a small amount of inflammatory cells can be seen in the tissue (arrows). The above results indicate that each compound has an inflammatory response relieving LPS-induced acute lung injury in male C57BL/6 mice to various degrees.

To further investigate whether each compound also plays a crucial role in the dual-target COX-2 and PPAR-gamma treatment of LPS-induced lung injury in mice, immunohistochemical analysis was used to detect the expression levels of COX-2 and PPAR-gamma in lung tissues of different groups, and the results are shown in FIGS. 9-10. LPS can obviously improve the expression of COX-2 and reduce the expression of PPAR-gamma, and each compound can obviously reduce the expression of COX-2 level induced by LPS to different degrees and can obviously improve the reduction phenomenon of PPAR-gamma level induced by LPS, thereby playing the role of treating pathological lung injury of mice induced by LPS. The above studies indicate that each compound can relieve the inflammatory response of LPS-induced acute lung injury in male C57BL/6 mice in a COX-2 and PPAR-gamma dependent manner.

The above-described embodiments are only intended to illustrate the preferred embodiments of the present invention, and not to limit the scope of the present invention, and various modifications and improvements made to the technical solution of the present invention by those skilled in the art without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims (6)

1. A compound is prepared by splicing zaltoprofen or derivatives thereof with a compound with a structure shown in a formula (1) or a formula (2),

wherein R1 is a C1-C5 alcohol;

the zaltoprofen derivative is two compounds with structural formulas shown as follows,

the two compounds are spliced by performing esterification reaction on carboxyl on the zaltoprofen or a derivative thereof, hydroxyl on the compound shown in the formula (1) and amino on the compound shown in the formula (2).

2. The compound of claim 1, wherein: r1 is propanol.

3. A method of synthesizing a compound according to claim 1 or 2, wherein: the two compounds are spliced by performing esterification reaction on carboxyl on the zaltoprofen or a derivative thereof, hydroxyl on the compound shown in the formula (1) and amino on the compound shown in the formula (2).

4. Use of a compound according to claim 1 or 2 for the preparation of an anti-inflammatory medicament, characterized in that: the compound plays an anti-inflammatory activity by acting on double targets of COX-2 and PPAR-gamma.

5. Use according to claim 4, characterized in that: the anti-inflammatory drug is an anti-pneumonia drug.

6. An anti-inflammatory agent comprising the compound according to claim 1 or 2 as an active ingredient.

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