Icetexane-type abietane diterpene derivatives, processes for their preparation and uses thereof

Abstract

This invention belongs to the field of medicinal chemistry technology, specifically relating to Icetexane-type abiane diterpenoid derivatives, their preparation methods, and applications. The Icetexane-type abiane diterpenoid derivatives have the structure shown in formula (I), exhibiting a novel structure and good therapeutic and / or preventive effects against acute lung injury.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry technology, specifically relating to Icetexane-type rosinane diterpenoid derivatives, their preparation methods, and applications. Background Technology

[0002] Acute lung injury (ALI) is a collective inflammatory response syndrome caused by damage to alveolar epithelial cells and capillary endothelial cells due to various direct or indirect injuries, resulting in progressive dyspnea and refractory hypoxemia. Severe cases can lead to acute respiratory distress syndrome (ARDS), a common cause of respiratory failure with high morbidity and mortality (F1000Res., 2019, 8, 1-9; N. Engl.J.Med., 2005, 353, 1685-1693). Currently, mechanical ventilation is the primary treatment in clinical practice; drug therapy such as glucocorticoids and anti-inflammatory drugs has limited effectiveness and can cause various adverse reactions such as coagulation disorders, gastric ulcers, and osteoporosis. Therefore, there is a lack of effective treatments for ALI, and there is an urgent need for safe and effective drugs to treat acute lung injury and improve patient survival rates.

[0003] Icetexane diterpenes are a class of natural products widely distributed in plants of the Lamiaceae, Cupressaceae, Celastraceae, Euphorbiaceae, Verbenaceae, and Taxaceae families, with the highest abundance found in plants of the genus *Salvia* in the Lamiaceae family (Nat. Prod. Rep., 2009, 26(9): 1195-1217). Icetexane diterpenes have a unique 6 / 7 / 6 tricyclic skeleton. Studies have shown that these compounds possess pharmacological activities such as antitumor, antimalarial, antibacterial, and antiviral activities (Med. Res. Rev., 2021, 41, 2971-2997), but pharmacological studies on their preventive and therapeutic effects on acute lung injury have not yet been reported. Summary of the Invention

[0004] This invention aims to at least solve one of the technical problems existing in the prior art. To this end, this invention proposes an Icetexane-type arosinane diterpenoid derivative, a method for its preparation, and its application in the preparation of medicaments for treating and / or preventing acute lung injury.

[0005] In a first aspect, the present invention provides a compound of general formula (I) or a pharmaceutically acceptable salt thereof, a tautomer, a racemic mixture, an enantiomer, or a diastereomer:

[0006]

[0007] in:

[0008] R1 is selected from H, OH, or R1 forms C=O with the carbon at position 3;

[0009] R2 is selected from H or R2 forms C=O with carbon at position 1;

[0010] R3 is absent or is OH;

[0011] R4 is selected from H, OH, or R4 forms C=O with carbon at position 20;

[0012] R5 is selected from H, or R5 forms C=O with the carbon at position 7;

[0013] R6 and R7 are independently selected from OCH3 and OH, respectively, or R6 and R7 form C=O with carbons at positions 11 and 12, respectively, or R6 and R7 together form...

[0014] X either does not exist or is C;

[0015] When X is C, and there is a carbon-carbon double bond between carbons at positions 1 and 10, there are carbon-carbon single bonds between carbons at positions 5, 6, 7, 10, and 20, and there is a carbon-carbon double bond between carbons at positions 11 and 12.

[0016] When X is C, and there is a carbon-carbon double bond between the carbons at positions 1 and 2, there is one or two carbon-carbon double bonds between the carbons at positions 5, 6, 7, 10, and 20, and there is a carbon-carbon double bond or a carbon-carbon single bond between the carbons at positions 11 and 12.

[0017] When X is absent, there are carbon-carbon single bonds between carbons at positions 1, 3, and 10, and carbon-carbon double bonds between carbons at positions 5 and 10, 6 and 7, and 11 and 12.

[0018] According to some embodiments of the present invention, the compound has the structure shown in general formula (II):

[0019]

[0020] in,

[0021] R6 and R7 are each independently selected from OCH3 and OH, or R6 and R7 can be formed together.

[0022] According to some embodiments of the present invention, the compound has a structure shown in general formula (III) or (IV):

[0023]

[0024] in,

[0025] R1 is selected from H, OH, or R1 forms C=O with the carbon at position 3;

[0026] R3 is absent or is OH;

[0027] R4 is selected from H or OH;

[0028] R5 is selected from H, or R5 forms C=O with the carbon at position 7;

[0029] R6 and R7 are each independently selected from OCH3 and OH, or R6 and R7 form C=O with carbons at positions 11 and 12, respectively, or R6,

[0030] R7 together form

[0031] Furthermore, the carbons at positions 10 and 20, and at positions 11 and 12, are not simultaneously single bonds.

[0032] According to some embodiments of the present invention, the compound has the structure shown in general formula (III).

[0033] in,

[0034] R1 forms C=O with the carbon at position 3;

[0035] R3 does not exist;

[0036] R4 is selected from H or OH;

[0037] R5 forms C=O with the carbon at position 7;

[0038] R6 and R7 are each independently selected from OCH3 and OH, or R6 and R7 can be formed together. According to some embodiments of the present invention, the compound has a structure represented by general formula (V):

[0039]

[0040] in,

[0041] R4 is selected from H or OH;

[0042] R5 is H;

[0043] R6 and R7 are each independently selected from OCH3 and OH, or R6 and R7 can be formed together.

[0044] According to some embodiments of the present invention, the compound is selected from:

[0045]

[0046] According to some preferred embodiments of the present invention, the compound is selected from:

[0047]

[0048] A second aspect of the present invention provides a pharmaceutical composition comprising the above-mentioned compound or a pharmaceutically acceptable salt thereof, a tautomer, a racemic mixture thereof, an enantiomer, a diastereomer, and a pharmaceutically acceptable carrier.

[0049] According to some embodiments of the present invention, the dosage form of the drug is any of the dosage forms conventional in the art, preferably in the form of a solid, semi-solid or liquid, and may be an aqueous solution, a non-aqueous solution or a suspension, more preferably a tablet, capsule, soft capsule, granule, pill, oral liquid, dry suspension, drop pill, dry extract, injection or infusion.

[0050] According to some embodiments of the present invention, the administration method of the drug can be a conventional administration method in the art, including but not limited to injection or oral administration. The injection administration can be via intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection, or subcutaneous injection.

[0051] For dosage, a typical regimen is one to five doses per day, each dose providing about 0.01 to about 50 mg / kg of the compound of the invention, with preferred doses each providing about 0.1 to about 30 mg / kg, particularly about 0.5 to about 25 mg / kg.

[0052] A third aspect of the invention provides the use of the above-described compound or a pharmaceutically acceptable salt, tautomer, racemate, enantiomer, diastereomer, or pharmaceutical composition thereof in the preparation of a medicament for treating and / or preventing acute lung injury.

[0053] According to some embodiments of the present invention, the acute lung injury is LPS-induced acute lung injury.

[0054] According to some preferred embodiments of the present invention, the LPS is endotoxin LPS.

[0055] Beneficial effects:

[0056] This invention provides a series of novel icetexane diterpenoid derivatives and their novel applications as therapeutic agents for acute lung injury. Through extensive scientific research and creative work, the activity of icetexane diterpenoid derivatives in treating acute lung injury has been discovered. Specifically, this invention reveals that icetexane diterpenoid derivatives can effectively reduce the expression of LPS-induced inflammatory factors such as TNF-α, IL-1β, and IL-6 both in vivo and in vitro, and effectively protect against pathological changes in lung tissue of mice with LPS-induced acute lung injury. This confirms the protective effect of icetexane diterpenoid derivatives against acute lung injury and further expands the application scope of icetexane diterpenoid derivatives.

[0057] The term "pharmaceutically acceptable salt" includes salts formed with inorganic acids, organic acids, alkali metals, alkaline earth metals, and basic amino acids; preferably, the inorganic acid includes at least one of hydrochloric acid, nitric acid, sulfuric acid, phosphoric acid, and hydrobromic acid; preferably, the organic acid includes at least one of maleic acid, fumaric acid, tartaric acid, lactic acid, citric acid, acetic acid, methanesulfonic acid, p-toluenesulfonic acid, adipic acid, palmitic acid, and tannic acid; preferably, the alkali metal includes at least one of lithium, sodium, and potassium; preferably, the alkaline earth metal includes at least one of calcium and magnesium; preferably, the basic amino acid includes lysine.

[0058] The term "pharmaceutically acceptable carrier" includes at least one of diluents, excipients, fillers, binders, humectants, disintegrants, absorption enhancers, surfactants, adsorbents, lubricants, sweeteners, and flavorings; preferably, the excipients include water; preferably, the fillers include at least one of starch and sucrose; preferably, the binders include at least one of cellulose derivatives, alginates, gelatin, and polyvinylpyrrolidone; preferably, the humectants include glycerin; preferably, the disintegrants include at least one of agar, calcium carbonate, and sodium bicarbonate; preferably, the absorption enhancers include quaternary ammonium compounds; preferably, the surfactants include hexadecyl alcohol; preferably, the adsorbents include at least one of kaolin and soap clay; preferably, the lubricants include at least one of talc, calcium stearate, magnesium stearate, and polyethylene glycol.

[0059] Unless otherwise stated, the term “treatment” as used in this application includes effects that occur when a subject has a specific disease, disorder, or condition, which reduce the severity of the disease, disorder, or condition, or delay or slow the development of the disease, disorder, or condition (“therapeutic treatment”), and also includes effects that occur before a subject begins to have a specific disease, disorder, or condition (“preventive treatment”).

[0060] The term "dosage" refers to the amount that can alleviate or delay the progression of a disease, degenerative or damaging condition. It can vary depending on the specific disease being treated and other factors, including age, weight, health status, severity of symptoms, route of administration, frequency of treatment, and whether other medications are being used concurrently during treatment.

[0061] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Attached Figure Description

[0062] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0063] Figure 1 The following is an evaluation of the cytotoxicity of icetexane diterpenoid derivatives and their activity in inhibiting LPS-induced inflammatory factor release: (A) Evaluation of the effect of icetexane diterpenoid derivatives on the survival rate of RAW264.7 cells by MTT assay; (B) Structure of icetexane diterpenoid derivatives; (C) Effect of icetexane diterpenoid derivatives (10 μM) on the expression of inflammatory factors (IL-1β, IL-6, TNF-α) in LPS-induced RAW264.7 cells;

[0064] Figure 2 Figure 1 shows the concentration-dependent inhibition of LPS-induced inflammatory cytokine release in RAW162.7 cells by icetexane diterpenoid derivatives: (A) Effects of different concentrations of compound 3 (0, 10, 20 μM) on the expression of inflammatory cytokines (IL-1β, IL-6, TNF-α) in LPS-induced RAW264.7 cells; (B) Effects of different concentrations of compound 14 (0, 5, 10, 20 μM) on the expression of inflammatory cytokines (IL-1β, IL-6, TNF-α) in LPS-induced RAW264.7 cells.

[0065] Figure 3The results show the concentration-dependent inhibition of ROS and NO levels in LPS-induced RAW162.7 cells by compound 14: (A) Immunofluorescence assay of the effect of different concentrations of compound 14 (5, 10 μM) on ROS in LPS-induced RAW264.7 cells; (B) Flow cytometry assay of the effect of different concentrations of compound 14 (1, 5, 10, 20 μM) and the positive control drug dexamethasone (DEX) on ROS levels in LPS-induced RAW264.7 cells; (C) Different concentrations of compound 14... Effects of compound 14 (1, 5, 10, 20 μM) and the positive control drug dexamethasone (DEX) on NO levels in the supernatant of LPS-induced RAW264.7 cell culture medium (statistical results, *P<0.05, ***P<0.005); Effects of different concentrations of compound 14 (1, 5, 10, 20 μM) and the positive control drug dexamethasone (DEX) on ROS levels in LPS-induced RAW264.7 cells (statistical results, *P<0.05, ***P<0.005);

[0066] Figure 4 Figure 1 shows the concentration-dependent reduction of pPI3K and pAKT protein levels in LPS-induced RAW264.7 cells by compound 14: (A) Western blotting of the effects of different concentrations of compound 14 (1, 5, 10, 20 μM) on LPS-induced PI3K and p-PI3K protein levels in RAW264.7 cells and statistical results (*P<0.05, **P<0.01); (B) Western blotting of the effects of different concentrations of compound 14 (1, 5, 10, 20 μM) on LPS-induced AKT and p-AKT protein levels in RAW264.7 cells and statistical results (*P<0.05, **P<0.01, ***P<0.005).

[0067] Figure 5 Figure 1 shows the concentration-dependent inhibition of LPS-induced p-NF-κB levels and nuclear translocation in RAW264.7 cells by compound 14: (A) Western blot analysis of the effects of different concentrations of compound 14 (1, 5, 10, 20 μM) on LPS-induced NF-κB and p-NF-κB protein levels in RAW264.7 cells and statistical results (*P<0.05, **P<0.01); (B) Immunofluorescence assay of the effects of different concentrations of compound 14 (0, 5, 10 μM) on LPS-induced NF-κB protein levels in RAW264.7 cells; (C) Western blot analysis of the effects of different concentrations of compound 14 (1, 5, 10, 20 μM) on LPS-induced NF-κB protein nuclear translocation in RAW264.7 cells and statistical results (*P<0.05, **P<0.01).

[0068] Figure 6The following figures illustrate the effect of compound 3 on the dose-dependent inhibition of inflammatory cytokine expression in the lungs of mice with LPS-induced acute lung injury, and its significant lung-protective effect: (A) Effects of different doses of compound 3 (5 mg / kg, 25 mg / kg) on ​​the expression of inflammatory cytokines (IL-1β, IL-6, TNF-α) in alveolar cells of LPS-induced acute lung injury in mice and their statistical results (*P<0.05, ***P<0.005, ****P<0.001); (B) HE staining evaluation of the effects of different doses of compound 3 (5 mg / kg, 25 mg / kg) on ​​LPS-induced alveolar lesions in mice and the structure of compound 3 (*P<0.05, **P<0.01); (C) Statistical results of the effects of different doses of compound 3 (0, 5 mg / kg, 25 mg / kg) on ​​LPS (5 mg / kg)-induced lung injury in mice (***P<0.005, no significant difference in ns).

[0069] Figure 7 The following figures illustrate the effect of compound 14 in dose-dependently inhibiting the expression of inflammatory factors in the lungs of mice with LPS-induced acute lung injury and exerting a significant lung-protective effect: (A) Effects of different doses of compound 14 (5 mg / kg, 25 mg / kg) on ​​the expression of inflammatory factors (IL-1β, IL-6, TNF-α) in alveolar cells of LPS-induced acute lung injury in mice and their statistical results (*P<0.05, ***P<0.005, ****P<0.001); (B) HE staining to evaluate the effects of different doses of compound 14 (5 mg / kg, 25 mg / kg) on ​​LPS-induced alveolar lesions in mice and the structure of compound 14; (C) Statistical results of the effects of different doses of compound 14 (0, 5 mg / kg, 25 mg / kg) on ​​LPS (5 mg / kg)-induced lung injury in mice (***P<0.005). Detailed Implementation

[0070] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0071] To better understand the essence of this invention, the following description, in conjunction with the accompanying drawings, uses embodiments of the invention to further illustrate its essence, but this does not limit the scope of the invention. Any modifications made to the invention based on its essence fall within the scope of this invention.

[0072] In the embodiments of the present invention, icetexane-type rosinane diterpenoid derivatives were prepared according to the following reaction process:

[0073]

[0074] The reagents and reaction conditions involved in the above reaction process are as follows:

[0075] (Ⅰ) 2,2'-DMP, p-TsOH, 60℃, 3h, yield 67%;

[0076] (II) SeO2, Dioxane, 80-100℃, yield of main product compound 5 43%;

[0077] (Ⅲ) ① HCl, MeOH, 80℃, 30h, yield 56%; ② Ag2O, DCM, rt, 20min, yield 86%;

[0078] (Ⅳ) CH3I, K2CO3, acetone, 50℃, 28h, yield 78%;

[0079] (V)SeO2, Dioxane, 100℃, yield of main product compound 14 31%;

[0080] (VI)SeO2, Dioxane, 100℃, 30h, total yield of compounds 16 and 17 41%;

[0081] (VII) L-selectride, 70℃, 30h, yield 51%;

[0082] (VIII)SeO2, Dioxane, 100℃, 30h, yield of main product compound 15 12%.

[0083] Example 1: Preparation of Compound 2

[0084]

[0085] Compound (1) (110 mg, 0.37 mmol) was weighed into a 25 mL round-bottom flask, and 2,2'-DMP (4 mL) and p-TsOH (7 mg, 0.037 mmol) were added respectively. After purging with N2, the mixture was stirred at 60 °C and monitored by TLC. The reaction was completed in 3 hours. 20 mL of water was added to quench the reaction, and the mixture was extracted with pure petroleum ether (3 × 20 mL). The organic layers were combined, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and subjected to silica gel column chromatography (eluting with pure petroleum ether) to obtain 84 mg of compound (2), with a yield of 67%.

[0086] 1H NMR (500MHz, CDCl3) δ6.43(s,1H),5.48(s,1H),3.54(d,J=14.8Hz,1H),3.05(d,J=14.8Hz,1H),2.95(p,J= 6.9Hz,1H),2.82(ddd,J=14.2,8.2,2.6Hz,1H),2.77–2.70(m,1H),2.08–1.98(m,2H),1.94(d,J=18.0Hz,1H ),1.80(dd,J=11.7,3.9Hz,1H),1.69(s,3H),1.68(s,3H),1.39(dd,J=14.0,5.1Hz,1H),1.25(d,J=2.4Hz, 3H), 1.24 (d, J = 2.4Hz, 3H), 1.20 (d, J = 12.1Hz, 1H), 1.11 (dd, J = 13.0, 5.6Hz, 2H), 0.94 (s, 2H), 0.91 (s, 3H).

[0087] Example 2 Preparation of Compound 3

[0088]

[0089] Compound (1) (1.64 g, 5.5 mmol) was weighed into a 100 mL round-bottom flask, and anhydrous potassium carbonate (2.28 g, 16.5 mmol) was added. The flask was purged with N2, and 55 mL of acetone and 2.38 g (16.5 mmol) of potassium iodide were added successively. The mixture was stirred at 50 °C and monitored by TLC. The reaction was completed after 28 hours (3 eq CH3I and 3 eq K2CO3 were added three times during the reaction). The reaction was quenched by slowly adding dilute hydrochloric acid under ice bath conditions. The mixture was extracted with petroleum ether (3 × 100 mL), washed with saturated brine, dried over anhydrous sodium sulfate, evaporated to dryness, and subjected to silica gel column chromatography (PE:EA = 10:1) to obtain 1.4 g of compound (3), with a yield of 78%.

[0090] 1H NMR (500MHz, CDCl3) δ6.63(s,1H),5.41(t,J=3.6Hz,1H),3.76(s,3H),3.75(s,3H),3.71(d ,1H),3.24–3.14(m,1H),2.95(d,J=14.6Hz,1H),2.78–2.72(m,1H),2.72–2.65(m,1H),1.9 9–1.90(m,2H),1.87–1.79(m,1H),1.74–1.68(m,1H),1.30–1.22(m,1H),1.13(d,J=2.4Hz, 3H), 1.12 (d, J = 2.3Hz, 3H), 1.11–1.07 (m, 1H), 1.04–0.99 (m, 1H), 0.84 (s, 3H), 0.80 (s, 3H).

[0091] Example 3 Preparation of Compound 4

[0092]

[0093] Weigh out 863 mg (2.63 mmol) of compound (3) into a 100 mL three-necked flask. In the three-necked flask, N... 2 Under an ambient atmosphere, a tetrahydrofuran solution of tri-sec-butylborohydride (1.0 M, 20 mL) was added, and the mixture was refluxed at 70 °C for 30 h. The reaction was monitored by TLC until complete. The reaction solution was diluted with tetrahydrofuran under ice bath conditions, and the reaction was quenched by slowly adding sodium sulfate decahydrate. The pH was adjusted to weakly acidic with hydrochloric acid, and the mixture was extracted three times with ethyl acetate. The organic layers were combined, washed with saturated brine, dried with anhydrous sodium sulfate, and subjected to silica gel column chromatography (eluting with pure petroleum ether) to remove small polar impurities. The mixture was then eluted with petroleum ether:ethyl acetate = 30:1 to obtain 420 mg of compound (4), with a yield of 51%.

[0094] 1H NMR (500MHz, CDCl3) δ6.54 (s, 1H), 5.72 (s, 1H), 5.55 (t, 1H), 3.83 (d, J = 15.3Hz, 1H), 3.80 (s, 2H),3.29–3.17(m,1H),3.10(d,J=15.0Hz,1H),2.90–2.81(m,1H),2.82–2.73(m,1H),2.10–2. 00(m,3H),1.99–1.91(m,1H),1.84(dd,J=11.6,4.0Hz,1H),1.43–1.33(m,1H),1.26(d,J=2.7H z, 3H), 1.25 (d, J = 2.7Hz, 3H), 1.24–1.18 (m, 1H), 1.16–1.09 (m, 1H), 0.95 (s, 3H), 0.91 (s, 3H).

[0095] Example 4 Preparation of Compound 5

[0096]

[0097] Compound (2) (169 mg, 0.5 mmol) was weighed into a reaction tube, and selenium dioxide powder (555 mg, 5 mmol) and 1,4-dioxane (5 mL) were added successively. The tube was evacuated and heated to 80 °C for 48 h. The reaction was monitored by TLC until it was complete. The tube was filtered, and the filtrate was dried with anhydrous sodium sulfate and evaporated to dryness. The filtrate was then subjected to silica gel column chromatography (petroleum ether: ethyl acetate = 20:1) to obtain 78 mg of compound (5), with a yield of 43%.

[0098] 1 H NMR (500MHz, CDCl3) δ7.82 (s, 1H), 7.52 (s, 1H), 7.27 (d, J = 9.9Hz, 1H), 6.97 (s, 1H), 6.11 (d, J = 9.8 Hz, 1H), 3.13 (m, J = 6.9 Hz, 1H), 1.81 (s, 6H), 1.47 (s, 6H), 1.33 (d, J = 6.9 Hz, 6H).

[0099] Example 5 Preparation of compounds 6 and 7

[0100]

[0101] Compound (5) (110 mg, 0.3 mmol) was weighed into a reaction tube, 2 mL of methanol and 2 mL of concentrated hydrochloric acid were added, and the mixture was heated to 80 °C and reacted for 30 h. The reaction was monitored by TLC until it was complete. The mixture was quenched with water, extracted three times with ethyl acetate, and the ethyl acetate layers were combined. The mixture was washed with saturated brine, dried over anhydrous sodium sulfate, and evaporated to dryness. The mixture was then passed through a silica gel column in dichloromethane:methanol = 80:1 to obtain 55 mg of the acetone-removed product (compound 6), with a yield of 56%. After vacuum drying, 21 mg of the acetone-removed product was taken into a reaction tube, and silver oxide (48 mg, 0.207 mmol) and dichloromethane (1 mL) were added. The mixture was reacted at room temperature and monitored by TLC until it was complete in 20 mins. The mixture was then passed through a short silica gel column in pure dichloromethane to obtain 18 mg of compound (7), with a yield of 86%.

[0102] Compound (6): 1 H NMR(400MHz,DMSO-d6)δ8.01(s,1H),7.59(d,J=10.0Hz,1H),7.53(s,1H),6.74(s,1 H), 6.06 (d, J = 9.8Hz, 1H), 3.38–3.35 (m, 1H), 1.34 (s, 6H), 1.22 (s, 3H), 1.21 (s, 3H).

[0103] Compound (7): 1 H NMR (500MHz, CDCl3) δ7.75 (s, 1H), 7.70 (s, 1H), 7.35 (d, J = 10.0Hz, 1H), 7.25 (s, 1H) ), 6.29(d,J=9.9Hz,1H),3.15–3.05(m,1H),1.52(s,6H),1.24(s,3H),1.22(s,3H).

[0104] Example 6 Preparation of Compound 8

[0105]

[0106] Compound (2) (127 mg, 0.37 mmol) was weighed into a reaction tube, and selenium dioxide powder (131 mg, 1.2 mmol) and 1,4-dioxane (4 mL) were added successively. The tube was evacuated and refluxed at 100 °C for 1 h until the reaction was complete. The mixture was filtered, and the filtrate was dried with anhydrous sodium sulfate. The mixture was then stirred and subjected to silica gel column chromatography (petroleum ether: ethyl acetate = 20:1) to obtain 22 mg of compound (8), with a yield of 18%.

[0107] 1H NMR (400MHz, CDCl3) δ7.25(s,1H),7.24(d,J=9.8Hz,1H),6.50(s,1H),5.93(d,J=9.8Hz,1H), 5.87(t,J=7.1Hz,1H),3.07–2.95(m,4H),1.74(s,5H),1.29(s,3H),1.27(s,3H),1.26(s,6H).

[0108] Example 7 Preparation of Compound 9

[0109]

[0110] During the preparation of compound (5) from compound (2), TLC monitoring showed that after the reaction was complete, there was a clear round "dot" below the product spot that was covered by a trailing band. Therefore, compound (5) was prepared multiple times, concentrated to obtain crude product, and then separated by PE:EA = 2:1 silica gel column to obtain 27 mg of compound (9).

[0111] 1 H NMR (400MHz, CDCl3) δ7.49(s,1H),7.11(dd,J=9.8,0.8Hz,1H),7.04(s,1H),6.03(d,J=9.5Hz,1H),5.08(d,J=5.5Hz,1H), 4.54(d,J=5.5Hz,1H),3.03(m,J=14.1,7.0Hz,1H),1.78(d,J=9.0Hz,6H),1.58(s,3H),1.32(s,3H),1.27(d,J=7.0Hz,7H).

[0112] Example 8 Preparation of Compound 10

[0113]

[0114] During the preparation of compound (5), TLC monitoring showed that after the reaction was complete, there were a few obvious round "dots" above the product spot. Therefore, compound (5) was prepared multiple times, concentrated to obtain crude product, and 23 mg of compound (10) was obtained by silica gel column chromatography (PE:EA = 30:1).

[0115] 1 H NMR(600MHz,DMSO-d6)δ7.55(d,J=11.8Hz,1H),6.96(s,1H),6.65(d,J=11.7Hz, 1H), 3.49 (s, 2H), 2.93 (p, J = 6.9Hz, 1H), 1.70 (s, 6H), 1.21 (s, 3H), 1.20 (s, 9H).

[0116] Example 9 Preparation of compounds 12 and 13

[0117]

[0118] Compound (3) (165 mg, 0.5 mmol) was weighed into a reaction tube, and selenium dioxide powder (444 mg, 4 mmol) and 1,4-dioxane (5 mL) were added successively. The mixture was evacuated and refluxed at 100 °C for 1.5 h until the reaction was complete. The mixture was filtered, and the filtrate was dried with anhydrous sodium sulfate. The mixture was then stirred and subjected to silica gel column chromatography (PE:EA = 20:1) to obtain 42 mg of compound (12) with a yield of 25% and 48 mg of compound (13) with a yield of 28%.

[0119] Compound (12): 1 H NMR (400MHz, CDCl3) δ7.12 (s, 1H), 6.78 (s, 1H), 6.39 (d, J = 9.6Hz, 1H), 5.85 (dd ,J=9.7,4.2Hz,1H),5.75(t,J=7.0Hz,1H),3.90–3.88(m,1H),3.87(s,4H),3.84 (s,3H),3.38–3.27(m,1H),3.02(dd,J=13.3,7.2Hz,1H),2.94(dd,J=13.2,6.6 Hz, 1H), 1.26 (d, J = 1.7Hz, 3H), 1.25 (d, J = 1.7Hz, 3H), 1.10 (s, 3H), 1.06 (s, 3H).

[0120] Compound (13): 1 H NMR (400MHz, CDCl3) δ7.52(s,1H),7.29(d,J=10.0Hz,1H),6.81(s,1H),5.95(d,J=9.6Hz,1H),5.86(t,J=7.0Hz, 1H),3.88(s,3H),3.88(s,3H),3.40–3.29(m,1H),2.99(d,J=7.0Hz,2H),1.27(s,3H),1.25(s,3H),1.25(s,6H).

[0121] Example 10 Preparation of Compound 14

[0122]

[0123] Compound (3) (167 mg, 0.5 mmol) was weighed into a reaction tube, and selenium dioxide powder (782 mg, 7.5 mmol) and 1,4-dioxane (5 mL) were added successively. The mixture was evacuated and refluxed at 100 °C for 24 h. The mixture was filtered, and the filtrate was dried with anhydrous sodium sulfate. The mixture was then stirred and subjected to silica gel column chromatography (petroleum ether: ethyl acetate = 20:1) to obtain 55 mg of compound (14), with a yield of 31%.

[0124] 1 H NMR (500MHz, CDCl3) δ7.94(s,1H),7.87(s,1H),7.32(d,J=9.8Hz,1H),6.95(s,1H),6.13(d,J =9.8Hz, 1H), 3.98 (d, J = 1.1Hz, 7H), 3.42 (m, J = 6.9Hz, 1H), 1.47 (s, 6H), 1.31 (d, J = 7.0Hz, 6H).

[0125] Example 11 Preparation of Compound 11

[0126]

[0127] During the preparation of compounds (12, 13, 14), TLC monitoring showed that compound (11) was present both during and at the end of the reaction. By preparing compounds (12, 13, 14) multiple times, the fraction containing compound (11) was concentrated, and finally compound (11) was obtained by separation using a semi-preparative liquid phase (acetonitrile:water = 45:55; 4 mL / min).

[0128] 1 H NMR(500MHz, CDCl3)δ7.05(s,1H),6.77(s,1H),6.33(dt,J=9.6,2.0Hz,1H),5.79(dt,1H),5.63(t,J=7.0Hz,1H),3 .88(s,3H),3.84(s,3H),3.39–3.20(m,1H),2.98–2.90(m,3H),2.17(s,2H),1.27(s,3H),1.25(s,3H),1.07(s,5H).

[0129] Example 12 Preparation of Compound 15

[0130]

[0131] Compound (4) (200 mg, 0.6 mmol) was weighed into a reaction tube, and selenium dioxide (666 mg, 6 mmol) and 1,4-dioxane (4 mL) were added. After purging with argon, the reaction was carried out at 100 °C for 30 h. TLC monitoring showed that the product spot remained basically stable. The product was filtered and dried, and then subjected to silica gel column chromatography (petroleum ether: ethyl acetate = 10:1) to obtain 24 mg of compound (15), yield: 12%. Compounds (15) (5 mg) and (17) (8 mg) were also separated as byproducts.

[0132] 1 H NMR (400MHz, CDCl3) δ13.55(s,1H),8.15(d,J=10.3Hz,1H),7.36(d,J=12.3Hz,1H),7.07(s,1H),6.82(d,J= 12.3Hz, 1H), 6.33 (d, J = 10.3Hz, 1H), 4.01 (s, 3H), 3.56–3.41 (m, 1H), 1.52 (s, 6H), 1.31 (s, 3H), 1.29 (s, 3H).

[0133] Example 13 Preparation of compounds 16 and 17

[0134]

[0135] Compound (1) (530 mg, 1.76 mmol) was weighed into a reaction tube, and selenium dioxide (1.95 g, 17.6 mmol) and 1,4-dioxane (18 mL) were added. After purging with argon, the reaction was carried out at 100 °C for 30 h. TLC monitoring showed that the product spot was basically stable and unchanged. The product was filtered and dried, and then subjected to silica gel column chromatography (petroleum ether: ethyl acetate = 10:1) to obtain compounds (16) and (17), with a total yield of 41%.

[0136] Compound 16: 1 H NMR (400MHz, CDCl3) δ8.98(d,J=10.5Hz,1H),7.68(d,J=8.0,0.6Hz,1H),7.38(d,J=8.0Hz,1H),7.16 (d,J=1.2Hz,1H),6.40(d,J=10.5Hz,1H),3.77–2.44(m,1H),1.49(s,6H),1.20(s,3H),1.18(s,3H).

[0137] Compound 17: 1H NMR(500MHz, CDCl3)δ8.00(s,1H),7.50(d,J=9.8Hz,1H),7.40(s,1H),7.22(s,1H) ,6.29(d,J=9.8Hz,1H),3.17–3.06(m,1H),1.53(s,6H),1.22(s,3H),1.20(s,3H).

[0138] In this invention, cell experiments and animal experiments revealed that icetexane-type rosinane diterpenoid derivatives have significant therapeutic effects on acute lung injury in vivo and in vitro. The experimental methods and results are as follows.

[0139] Lipopolysaccharide (LPS) is a major component of bacterial endotoxins. Exposure to the lungs can induce an acute inflammatory response, damaging pulmonary vascular endothelial cells and triggering acute lung injury by activating leukocytes and promoting the release of oxygen free radicals and cytokines. Endotoxins have been widely used in animal and cellular models of acute lung injury for screening anti-acute lung injury drugs (Am. J. Physiol. Lung Cell Mol. Physiol., 2008, 295, L379-399).

[0140] Example 14 Cytotoxicity of Icetexane-type rosinane diterpenoid derivatives on RAW264.7 cell line

[0141] The cytotoxicity of icetexane diterpenoid derivatives to the RAW264.7 cell line was determined using the MTT assay, which included the following steps:

[0142] RAW264.7 cells in logarithmic growth phase were seeded at a density of 3000 cells per well in 96-well plates and cultured at 37°C in a 5% CO2 incubator for 12 hours. After cell attachment, drugs were administered according to concentration gradients and in triplicate for each group, and the cells were cultured in the drug-treated groups for another 24 hours. The drug-treated culture medium in each well was discarded, and 100 μL of 10% MTT (thiazolyl blue) diluted in DMEM medium was added to each well. The cells were then cultured at 37°C in a 5% CO2 incubator for approximately 2.5 hours. After removing the supernatant, 100 μL of DMSO was added, and the absorbance of each well was measured at 490 nm using a microplate reader.

[0143] Based on the absorbance value of each well, calculate the cell viability using the following formula: Cell viability (%) = (OD value of drug administration well - OD value of blank group) / (OD value of normal well - OD value of blank group) × 100. Plot the results.

[0144] Experimental results are as follows Figure 1As shown in Figures A and B, at a concentration of 10 μM, none of the compounds showed cytotoxicity to RAW264.7 cells within 24 hours. Compounds 14 and 3 did not reduce cell viability in RAW264.7 cells at 60 μM.

[0145] Example 15 Effects of Icetexane-type abeline diterpenoid derivatives on the expression of LPS-induced inflammatory factors

[0146] The effects of icetexane diterpenoid derivatives on the expression of LPS-induced inflammatory cytokines TNF-α, IL-1β, and IL-6 were investigated in RAW264.7 cells, including the following specific steps:

[0147] Log-grown RAW264.7 cells were seeded evenly in 12-well plates at a density of 20,000 cells per well and cultured at 37°C in a 5% CO2 incubator for 12 hours. The following day, after cell attachment, cells were divided into groups and treated with LPS (Liquid Propionate) in triplicate, with each group cultured in 3 replicates for an additional 8 hours at 37°C in a 5% CO2 incubator. Cells were collected, total RNA was extracted, and the expression of TNF-α, IL-1β, and IL-6 mRNA was detected using qRT-PCR.

[0148] Table 1 qRT-PCR primer sequences

[0149]

[0150] Experimental results are as follows Figure 1 As shown in Figure C, Icetexane diterpenoid derivatives at 10 μM can reduce the expression of related inflammatory factor mRNA levels at the LPS-induced cellular level, indicating that Icetexane diterpenoid derivatives have certain anti-inflammatory effects.

[0151] Following the steps outlined in Example 15, the expression of TNF-α, IL-1β, and IL-6 mRNA was detected using qRT-PCR, with the difference being that the groups were: control group, model group, and groups with different concentrations of the drug.

[0152] Experimental results are as follows Figure 2 As shown in the figure, Figure A represents the concentration-effect relationship of compound 3, and Figure B represents the concentration-effect relationship of compound 14, indicating that the anti-inflammatory effects of these two compounds at the cellular level are concentration-dependent.

[0153] Example 16 Compound 14 reduces LPS-induced NO and ROS expression at the cellular level.

[0154] The specific steps include the following:

[0155] 1. NO detection

[0156] Log-grown RAW264.7 cells were seeded evenly in 12-well plates at a density of 20,000 cells per well and cultured at 37°C in a 5% CO2 incubator for 12 hours. The following day, after cell attachment, control, model, and groups receiving different drug concentrations were established and cultured for another 8 hours at 37°C in a 5% CO2 incubator. The supernatant was collected, and the cells were centrifuged at 1500 rpm for 10 minutes to remove cell debris. The NO content in the supernatant of each group was measured using a NO detection kit (Beyotime).

[0157] 2. ROS testing

[0158] (1) Detection of ROS expression by fluorescence imaging: Log-grown RAW264.7 cells were seeded evenly in 6-well plates at a density of 10,000 cells per well and cultured at 37°C in a 5% CO2 cell culture incubator for 12 h. After cell adhesion the next day, control group, model group and different drug concentration groups were set up and cultured for another 8 h at 37°C in a 5% CO2 cell culture incubator. The supernatant was removed, and the cells were washed 3 times with PBS. 1 ml of basal medium diluted with the reactive oxygen species fluorescent probe was added to each well and incubated at 37°C in a 5% CO2 cell culture incubator for 30 min. The probe medium was removed, and the cells were washed 3 times with PBS. DAPI diluted with PBS was added and incubated in the dark for 20 min. The supernatant was removed, and the cells were washed 3 times with PBS. Finally, 1 ml of PBS was added to each well and the cells were photographed under a fluorescence microscope.

[0159] (2) Flow cytometry detection of ROS expression: Log-grown RAW264.7 cells were seeded evenly in 12-well plates at a density of 10,000 cells per well and cultured at 37°C in a 5% CO2 cell culture incubator for 12 h. After cell adhesion the next day, control group, model group and different drug concentration groups were set up and cultured for another 8 h at 37°C in a 5% CO2 cell culture incubator. The supernatant was collected into the corresponding EP tubes, washed twice with PBS, and 0.5 mL of reactive oxygen species fluorescent probe diluted in basal culture medium was added to each well. The cells were incubated at 37°C in a 5% CO2 cell culture incubator for 30 min. The supernatant was removed, washed twice with PBS, and EDTA-free trypsin was added. The cells were placed in a 37°C oven for 2 min. The cells were removed and the corresponding supernatant collected above was added to stop digestion. The cells were collected into the corresponding EP tubes, centrifuged at 1500 rpm / 4°C for 5 min, the supernatant was discarded, washed twice with PBS, and finally resuspended in 300 μL of PBS for flow cytometry detection.

[0160] Experimental results are as follows Figure 3As shown, Figure A shows the results of ROS immunofluorescence, Figure B shows the results of ROS flow cytometry, indicating that compound 14 significantly reduced LPS-induced ROS expression according to the concentration gradient; Figure C shows the expression of NO in the supernatant, indicating that compound 14 significantly reduced the NO content in the supernatant at 10 μmol.

[0161] Example 17 Effects of Compound 14 on P-PI3K, P-AKT, and P-NF-KB2 proteins in RAW264.7 cells

[0162] The detection was performed using Western blotting, including the following specific steps:

[0163] Log-grown RAW264.7 cells were used, and 5 × 10⁶ cells were used per well. 5 Cells were seeded at a uniform density in 6-well plates and cultured at 37°C in a 5% CO2 incubator for 12 h. The next day, after cell adhesion, control, model, and groups with different drug concentrations were established and cultured for another 8 h at 37°C in a 5% CO2 incubator. The supernatant was discarded, cells were washed with PBS, and collected. 100 μL of cell lysis buffer was added to each well, and the cells were lysed on ice for 10 min. The total protein concentration extracted from the cells was determined using a BCA kit. 100 μg of total protein was loaded onto the plate and separated using a 10% polyacrylamide gel electrophoresis (20 mA). The protein was transferred to a PVDF membrane (80V, 1.5 h), blocked with 5% skim milk powder at room temperature for 2 h, washed three times with TBST buffer, and then incubated overnight at 4°C with primary antibodies against NF-KB2, P-NF-KB2, PI3K, P-PI3K, AKT, P-AKT, and β-actin, respectively. The PVDF membrane was washed three times with TBST, then secondary antibody was added and incubated at room temperature for 2 hours. It was then washed three times with TBST again. Chemiluminescent developing solution was uniformly added to the PVDF membrane, and the band exposure was detected using an imager, followed by photographing.

[0164] Experimental results are as follows Figure 4 ,and Figure 5 As shown in Figure A, phosphorylated PI3K, AKT, and NF-KB2 were significantly downregulated as the concentration of compound 14 increased.

[0165] Example 18 Effect of Compound 14 on Nuclear Translocation of NF-KB2 Protein in RAW264.7 Cells

[0166] The assay was performed using immunofluorescence and nucleoprotein extraction detection methods, including the following specific steps:

[0167] RAW264.7 cells in logarithmic growth phase were digested with 0.25% trypsin and pipetted to form single cells. 10,000 cells per well were seeded in 6-well plates (containing cell crawling slides) and cultured overnight in an oven for cell adhesion. Control group, model group, and groups with different drug concentrations were set up. The cells were cultured for another 8 hours in a 37°C, 5% CO2 cell culture incubator. The supernatant was removed, and the cells were washed 3 times with PBS. The cells were then fixed with fixative (4% paraformaldehyde in PBS solution) at 4°C for 12 hours, washed 3 times with PBS, permeabilized with Triton-X100 in PBS solution for 10 minutes, washed 3 times with PBS, blocked with 5% BSA in PBS solution for 60 minutes, and then NF-KB2 antibody was added. The cells were then incubated overnight in a humidified chamber at 4°C. The next day, wash three times with PBS for 5 minutes each time, then add Alexa Fluor 488goat anti-rabbit IgG secondary antibody, incubate at 37°C in the dark for 2 hours, wash three more times with PBS, add 200 μL Hoechest 33342 staining solution, stain at room temperature in the dark for 5-10 minutes, wash three times with PBS, add anti-fluorescence quenching agent to the slide, cover the slide with the slide upside down, observe under a microscope, and take pictures.

[0168] Log-grown RAW264.7 cells were used, and 5 × 10⁶ cells were used per well. 5 Cells were seeded at a uniform density in 6-well plates and cultured at 37°C in a 5% CO2 incubator for 12 hours. The following day, after cell adhesion, control, model, and groups with different drug concentrations were established and cultured for another 8 hours at 37°C in a 5% CO2 incubator. The supernatant was discarded, cells were washed with PBS, and collected. Nucleoproteins (NF-κB) were extracted from the cells using a nucleoprotein extraction kit (Solepro), and the expression of NF-κB2 in the nucleoproteins was detected by Western blotting.

[0169] Experimental results are as follows Figure 5 As shown in Figures B and C, the green fluorescence decreases and nuclear translocation decreases with increasing drug concentration, indicating that compound 14 can dose-dependently reduce nuclear translocation of NF-KB2 protein in RAW264.7. The Western blot results of the nuclear protein in Figure B also confirm this.

[0170] Example 19: Inhibitory effect of Icetexane-type abeline diterpenoid derivatives on LPS-induced ALI mice

[0171] The specific steps include the following:

[0172] Laboratory animals: Healthy male C57 / BL mice, weighing 18–22 g, from the Laboratory Animal Center of Central South University. Room temperature was controlled at 20–25°C and humidity at 40–70% by air conditioning; free access to water and food was provided.

[0173] Drugs and reagents: Icetexane diterpenoid derivatives were prepared into drug concentrations (2.2 mg / mL and 11 mg / mL) using 5% DMSO and 95% injection-grade oil. The dosage for mice was 5 mg / kg and 25 mg / kg, with each mouse receiving 50 μL. All reagents used were of analytical grade.

[0174] Experimental Methods: Twenty-four mice were randomly divided into four groups: a solvent control group, an LPS group, an LPS + low-dose group (5 mg / kg), and an LPS + high-dose group (25 mg / kg), with six mice in each group. The low-dose (5 mg / kg) and high-dose (25 mg / kg) groups received 50 μL of the drug via intraperitoneal injection, according to the concentration of the stock solution. The control group and LPS group received an equal volume of solvent. One hour after drug administration, the LPS group, the LPS + low-dose group, and the LPS + high-dose group established an acute lung injury mouse model by intratracheal infusion of LPS (5 mg / kg), while the control group received phosphate-buffered saline (PBS) via intratracheal infusion. Lung tissue was collected from each group of mice 12 hours after intratracheal infusion of LPS for subsequent experiments.

[0175] H&E staining of lung tissue pathological sections: The collected lung tissue is fixed, dehydrated, paraffin-impregnated, embedded, sectioned, and stained with H&E.

[0176] Lung tissue RNA extraction: A portion of lung tissue was cut from each group, placed in a grinding tube, and grinding beads were added. 1 ml of Trizol solution was added to each group, and the tissue was ground in a grinder. The grinding solution was centrifuged at 12000 rpm / 15 min / 4℃, and the supernatant was collected in another clean EP tube. The total RNA was extracted from the tissue following the subsequent steps of the Trizol RNA extraction method. The RNA was reverse transcribed, and the expression of TNF-α, IL-1β, and IL-6 mRNA was detected by qRT-PCR.

[0177] Statistical methods: Data are expressed as mean ± standard error (mean ± SE), and the sample size for each group is 6. P < 0.05 is considered statistically significant. * This indicates that the corresponding two groups have P < 0.05; ** This indicates that the corresponding two groups have P < 0.01; *** **** indicates that the corresponding two groups have P < 0.005; **** indicates that the corresponding two groups have P < 0.001.

[0178] Experimental results are as follows Figure 6 , 7 As shown, where, according to Figure 6 and Figure 7Figure A shows that the mRNA levels of TNF-α, IL-1β, and IL-6-related inflammatory factors were significantly increased in the LPS group. After drug intervention, the levels of the three inflammatory factors decreased to varying degrees, with the high-dose group showing a more significant decrease. This result further illustrates that icetexane-type rosinane diterpenoid derivatives can effectively inhibit LPS-induced inflammatory responses.

[0179] In addition, according to Figure 6 and Figure 7 Figure B in the diagram shows that the LPS group exhibited significantly increased inflammatory cell infiltration, damage and even rupture of some alveolar walls, and marked capillary dilation and congestion, directly reflecting the severity of acute lung injury. Both the LPS + low-dose and high-dose groups showed varying degrees of improvement in lung tissue pathology, with the high-dose group showing a more significant effect. These results indicate that icetexane diterpenoid derivatives can effectively alleviate the pathological changes in lung tissue of acute lung injury.

[0180] In summary, this study demonstrates that icetexane-type abelane diterpenoid derivatives effectively reduce the mRNA expression of pro-inflammatory factors TNF-α, IL-1β, and IL-6 both in vivo and in vitro. Compound 14 also reduces LPS-induced NO and ROS expression. Its mechanism of action involves downregulating the PI3K-AKT pathway, reducing NF-KB2 nuclear translocation, and thus decreasing LPS-induced inflammatory responses. In vivo, it reduces LPS-induced pathological changes in lung tissue, effectively alleviating acute lung injury. Given the current lack of effective drugs for treating acute lung injury, this research has significant application value, providing a new direction for the clinical treatment of acute lung injury and further expanding the application scope of icetexane-type abelane diterpenoid derivatives.

[0181] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. Use of the compound or a pharmaceutically acceptable salt thereof in the preparation of medicaments for the treatment and / or prevention of acute lung injury: The compound is ; Acute lung injury is a collective transitional inflammatory response syndrome caused by damage to alveolar epithelial cells and capillary endothelium due to various direct or indirect injuries, resulting in progressive dyspnea and refractory hypoxemia.

2. The use of a pharmaceutical composition in the preparation of a medicament for treating and / or preventing acute lung injury, characterized in that, The pharmaceutical composition comprises the compound of claim 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier; Acute lung injury is a collective transitional inflammatory response syndrome caused by damage to alveolar epithelial cells and capillary endothelial cells due to various direct or indirect injuries, resulting in progressive dyspnea and refractory hypoxemia.

3. The application according to any one of claims 1 or 2, characterized in that, The acute lung injury mentioned is LPS-induced acute lung injury.

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