阅读说明：本技术 一种α-倒捻子素衍生物及其制备方法和应用 (Alpha-mangostin derivative and preparation method and application thereof ) 是由 何细新 黄仪有 邓金辉 梁津豪 于 2021-07-31 设计创作，主要内容包括：本发明属于药物化学技术领域,具体涉及一种α-倒捻子素衍生物及其制备方法和应用。本发明提供的α-倒捻子素衍生物对PDE4具有良好的选择性抑制作用,效果明显强于阳性对照药物,同时还有较好的药代动力学和类药性,口服无呕吐等不良反应,安全性高,可应用于制备治疗PDE4相关疾病的药物中；并且在抗肺纤维化实验中,α-倒捻子素衍生物显示出显著的改善肺功能参数,减少肺纤维化病变的效果,具有较好的开发潜力。(The invention belongs to the technical field of pharmaceutical chemistry, and particularly relates to an alpha-mangostin derivative and a preparation method and application thereof. The alpha-mangostin derivative provided by the invention has good selective inhibition effect on PDE4, the effect is obviously stronger than that of a positive control medicament, and meanwhile, the alpha-mangostin derivative has better pharmacokinetics and drug-like property, has no adverse reaction such as vomit when being taken orally, has high safety, and can be applied to the preparation of medicaments for treating PDE4 related diseases; in anti-pulmonary fibrosis experiments, the alpha-mangostin derivative shows remarkable effects of improving lung function parameters and reducing pulmonary fibrosis pathological changes, and has good development potential.)

1. An alpha-mangostin derivative having the structure of formula (I):

wherein R is₁Is hydrogen, alkyl, cycloalkyl, alkanoyl, benzyl or substituted benzyl;

R₂is hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, benzyl or substituted benzyl;

the substituent group of the substituted alkyl, the substituted alkenyl and the substituted benzyl is halogen, alkoxy, methylsulfonyl, ester group or carboxyl.

2. The alpha-mangostin derivative according to claim 1, wherein R is₁Is hydrogen, C_1～5Alkyl radical, C_1～5Alkanoyl, benzyl or substituted benzyl;

the R is₂Is hydrogen, C_1～5Alkyl, substituted C_1～5Alkyl radical, C_1～5Alkylene or substituted C_1～5An alkylene group.

3. The alpha-mangostin derivative according to claim 2, wherein R is₁Is hydrogen, methyl, ethyl, propyl, butyl, isopropyl, acetyl, benzyl, 3-fluoro-benzyl, 4-fluoro-benzyl, 3-methoxy-benzyl, 3-methyl-benzyl, 4-methylsulfonyl-benzyl;

the R is₂Is hydrogen, - (CH)₂)_n-COO-CH₂CH₃、-(CH₂)_m-COOH、-CH₂CH＝CH-COO-CH₂CH₃、-CH₂CH＝CH-COOH；

Wherein n is 1-4, and m is 1-4.

4. The alpha-mangostin derivative according to claim 3, wherein R is₁Is H, methyl, ethyl, isopropyl, benzyl, 3-fluoro-benzyl, 3-methoxy-benzyl, 3-methyl-benzyl, 4-methylsulfonyl-benzyl;

the R is₂Is- (CH)₂)₃-COOH、-(CH₂)₄-COOH or-CH₂CH＝CH-COOH。

5. The method for producing an α -mangostin derivative according to any one of claims 1 to 4, comprising the steps of:

taking the alpha-mangostin derivative compound 1 as a raw material, reacting the raw material with acetic anhydride or di-tert-butyl dicarbonate in a solvent, reacting the reaction product with bromoalkyl carboxylate at the temperature of 20-60 ℃ in the presence of a catalyst, and removing a protecting group and an ester group in long-chain carboxylate to obtain the alpha-mangostin derivative compound;

or, the alpha-mangostin derivative compound 1 is used as a raw material and is reacted with bromo R in a solvent₁And reacting, namely reacting with alkyl bromocarboxylate at the temperature of 20-60 ℃ in the presence of a catalyst to remove ester groups in the long-chain carboxylate.

6. Use of the α -mangostin derivative according to any one of claims 1 to 4 for the preparation of a PDE4 inhibitor.

7. A PDE4 inhibitor, comprising an effective amount of the alpha-mangostin derivative according to any one of claims 1 to 4.

8. Use of the alpha-mangostin derivative of any one of claims 1 to 4 in the preparation of a medicament for the prevention and treatment of diseases associated with PDE 4.

9. The use according to claim 8, wherein the diseases associated with PDE4 include tissue fibrosis, psoriasis, senile dementia, chronic obstructive pulmonary disease, inflammatory bowel disease and asthma.

10. An anti-fibrotic drug, comprising an effective amount of the α -mangostin derivative according to any one of claims 1 to 4.

Technical Field

The invention belongs to the technical field of pharmaceutical chemistry. More particularly, relates to an alpha-mangostin derivative, a preparation method and application thereof.

Background

Idiopathic Pulmonary Fibrosis (IPF) is a chronic progressive disease that causes a decline in lung function, resulting in respiratory failure and ultimately death. New data from the pandemic of new coronary pneumonia show that a large number of pulmonary fibrosis symptoms appear after SARS-CoV-2 infection, and similar symptoms also appear after SARS, MERS and other coronavirus infections. Although the pathogenic mechanism of IPF has been widely studied, there is still a lack of effective treatment for IPF; the median survival time of the IPF patient from diagnosis to treatment is only 2-4 years, and lung transplantation is the only surgical treatment method capable of prolonging the expected life, so that the method is high in cost, difficult to match and high in risk. On the aspect of medicine, at present, only two anti-fibrosis medicines, namely Pirfenidone (Pirfenidone) and Nintedanib (Nintedanib), exist, and adverse reactions of the two medicines occur to different degrees. Therefore, there is an urgent need to develop more novel therapeutic drugs for treating IPF diseases.

It was found that the second messenger cyclic adenosine monophosphate (cAMP) inhibits fibroblast proliferation or differentiation into myofibroblasts during the development of IPF. Phosphodiesterase 4(PDE4), the major cAMP degrading enzyme in lung fibroblasts, is up-regulated in the progression of fibrosis, and experiments with the selective PDE4 inhibitor roflumilast on the IPF model demonstrate that PDE4 inhibitors have anti-fibrotic effects in vivo and in vitro; on the other hand, PDE4 is widely involved in the inflammatory process, which also plays a role in the pathogenesis of IPF; these lines of evidence indicate that PDE4 is a potential therapeutic target for IPF. For example, chinese patent application CN103748073A discloses a new soft PDE4 inhibitor, but it has dose-dependent side effects in practical clinical applications, such as emesis, nausea, etc. Therefore, there is an urgent need to develop a novel PDE4 inhibitor with less side effects and high safety for treating diseases associated with PDE4, such as IPF disease.

Disclosure of Invention

The invention aims to solve the technical problems that the existing medicines for treating IPF are few, and the PDE4 inhibitor has the defects of dependence side effect and deficiency, and provides the alpha-mangostin derivative with the effect of obviously inhibiting PDE 4. The inventor finds that the alpha-mangostin derivative with a specific structure has good PDE4 inhibition activity and can be used for treating diseases related to PDE4 metabolism, particularly anti-pulmonary fibrosis diseases.

Therefore, the present invention aims to provide an α -mangostin derivative.

The invention also aims to provide a preparation method of the alpha-mangostin derivative.

The invention also aims to provide application of the alpha-mangostin derivative in preparing a PDE4 inhibitor and a PDE4 inhibitor containing the alpha-mangostin derivative.

The invention also aims to provide application of the alpha-mangostin derivative in preparing anti-fibrosis drugs, and an anti-fibrosis drug containing the alpha-mangostin derivative.

The above purpose of the invention is realized by the following technical scheme:

an alpha-mangostin derivative having the structure of formula (I):

wherein R is₁Is hydrogen, alkyl, cycloalkyl, alkanoyl, benzyl or substituted benzyl;

R₂hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, benzyl, substituted benzyl;

the substituent group of the substituted alkyl, the substituted alkenyl and the substituted benzyl is halogen, alkoxy, methylsulfonyl, ester group or carboxyl.

Preferably, the alkyl group comprises methyl, ethyl, propyl, butyl, isopropyl; the cycloalkyl group comprises cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl; the carboxyl is formic acid, acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, trans-crotonic acid, cis-crotonic acid, etc.

Further, said R₁Is hydrogen, C_1～5Alkyl radical, C_1～5Alkanoyl, benzyl or substituted benzyl;

the R is₂Is hydrogen, C_1～5Alkyl, substituted C_1～5Alkyl radical, C_1～5Alkylene or substituted C_1～5An alkylene group.

Preferably, said R is₁Is hydrogen, methyl, ethyl, propyl, butyl, isopropyl, acetyl, tert-butoxycarbonyl, benzyl, 3-fluoro-benzyl, 4-fluoro-benzyl, 3-methoxy-benzyl, 3-methyl-benzyl, 4-methylsulfonyl-benzyl;

the R is₂Is hydrogen, - (CH)₂)_n-COO-CH₂CH₃、-(CH₂)_m-COOH、-CH₂CH＝CH-COO-CH₂CH₃、-CH₂CH＝CH-COOH；

Wherein n is 1-4, and m is 1-4.

More preferably, said R₁Is H, methyl, ethyl, isopropyl, benzyl, 3-fluoro-benzyl, 3-methoxy-benzyl, 3-methyl-benzyl, 4-methylsulfonyl-benzyl;

the R is₂Is- (CH)₂)₃-COOH、-(CH₂)₄-COOH or-CH₂CH＝CH-COOH。

In addition, the invention also provides a preparation method of the alpha-mangostin derivative, which comprises the following steps:

taking the alpha-mangostin derivative compound 1 as a raw material, reacting the raw material with acetic anhydride or di-tert-butyl dicarbonate in a solvent, reacting the reaction product with bromoalkyl carboxylate at the temperature of 20-60 ℃ in the presence of a catalyst, and removing a protecting group and an ester group in long-chain carboxylate to obtain the alpha-mangostin derivative compound;

or, the alpha-mangostin derivative compound 1 is used as a raw material and is reacted with bromo R in a solvent₁And reacting, namely reacting with alkyl bromocarboxylate at the temperature of 20-60 ℃ in the presence of a catalyst to remove ester groups in the long-chain carboxylate.

Further, the solvent is acetone or dicarboxamide.

Still further, the catalyst is potassium carbonate, cesium carbonate or sodium hydride.

In addition, the invention also provides application of the alpha-mangostin derivative in preparing a PDE4 inhibitor.

Further, the invention also provides a PDE4 inhibitor, which comprises an effective amount of the alpha-mangostin derivative.

In addition, the invention also provides application of the alpha-mangostin derivative in preparing medicines for preventing and treating diseases related to PDE 4.

Still further, the diseases associated with PDE4 include tissue fibrosis, psoriasis, senile dementia, chronic obstructive pulmonary disease, inflammatory bowel disease and asthma. The research in the prior art finds that PDE4 is closely related to various diseases, and PDE4 inhibitors can be used as targets of related diseases.

Preferably, the disease associated with PDE4 is tissue fibrosis.

In addition, the invention also provides an anti-fibrosis drug which comprises an effective amount of the alpha-mangostin derivative.

The invention has the following beneficial effects:

experiments prove that the alpha-mangostin derivative provided by the invention has good selective inhibition effect on PDE4, the effect is obviously stronger than that of a positive control medicament, and meanwhile, the alpha-mangostin derivative has better pharmacokinetics and drug-like property, has no adverse reaction such as vomit when being taken orally, has high safety, and can be applied to the preparation of medicaments for treating PDE4 related diseases; in anti-pulmonary fibrosis experiments, the alpha-mangostin derivative shows remarkable effects of improving lung function parameters and reducing pulmonary fibrosis pathological changes, and has good development potential.

Drawings

FIG. 1 is a statistical chart of the results of acute toxicity tests of compound 18a of example 8 at an oral dose of 1.5 g/kg.

FIG. 2 is a statistical chart of the results of the anti-IPF assay of Compound 18a in example 9 of the present invention; wherein, the graph A shows the lung respiration function index measuring results of various groups of rats; panel B-hematoxylin-eosin stained sections of various groups of lung tissue: the unstained area is alveolar epithelial carcinoma, the blue is the nucleus and the red is the cytoplasm; panel C-Masson stained sections of various groups of lung tissue: the unstained area is alveolar epithelial carcinoma, the dark blue area is collagen, and the red area is a cell structure; panel D-western blot analysis of fibronectin and alpha-actin expression levels relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody.

FIG. 3 is a statistical chart of the results of compound 18a of example 10 of the present invention inhibiting epithelial-mesenchymal transition in vitro.

In the figure, p-value is calculated by a pairing test, compared with a control group,^#p<0.05，^##p<0.01，^###p<0.001，^####p<0.0001. for each of the models, compared to the model set,^*p<0.05，^**p<0.01，^***p<0.001，^****p<0.0001。

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

The synthetic route and the main products of the invention are as follows:

EXAMPLE 1 Synthesis of Compounds 12a and 12b

Dissolving the compound 1 (0.49-0.73 mmol) in 5mL of dichloromethane, and then adding acetic anhydride (0.54mmol) or di-tert-butyl dicarbonate (0.81mmol) and a catalytic amount of (DMAP) at room temperature; after reacting for 3h at room temperature, the solvent is evaporated to dryness under reduced pressure to obtain a crude product, and the crude product is subjected to silica gel column chromatography and eluted by petroleum ether/ethyl acetate to obtain yellow solids 12a and 12 b.

5-Hydroxy-8-methoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-6-oxo-2H,6H-pyra no[3,2-b]xanthen-9-yl acetate(12a).Yield:93％.¹H NMR(400MHz,CDCl₃)δ13.50(s,1H),7.10(s,1H),6.73(d,J＝10.1Hz,1H),6.25(s,1H),5.58(d,J＝10.0Hz,1H),5.27–5.18(m,1H),4.14(d,J＝6.4Hz,2H),3.77(s,3H),2.39(s,3H),1.83(s,3H),1.69(s,3H),1.47(s,6H).¹³C NMR(100MHz,CDCl₃)δ182.1,168.1,160.4,158.0,156.3,153.8,148.9,146.6,138.9,132.2,127.3,122.8,116.9,115.6,110.6,104.6,104.0,94.2,78.2,61.7,28.4,26.4,25.9,20.9,18.2.ESI-MSm/z:451.2[M+H]⁺.

Tert-butyl(5-hydroxy-8-methoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-6-oxo-2H,6H-pyrano[3,2-b]xanthen-9-yl)carbonate(12b).Yield:74％.¹HNMR(400MHz,CDCl₃)δ13.51(s,1H),7.16(s,1H),6.73(d,J＝10.1Hz,1H),6.25(s,1H),5.58(d,J＝10.0Hz,1H),5.26–5.17(m,1H),4.15(d,J＝6.5Hz,2H),3.79(s,3H),1.84(s,3H),1.68(s,3H),1.57(s,9H),1.47(s,6H).¹³CNMR(100MHz,CDCl₃)δ182.2,160.3,158.0,156.4,153.8,150.3,149.2,146.7,138.8,132.1,127.3,122.9,116.7,115.6,110.0,104.6,104.0,94.2,84.6,78.1,61.7,28.4,27.6,26.4,25.9,18.2.ESI-MSm/z:509.2[M+H]⁺.

EXAMPLE 2 Synthesis of Compounds 14a to 14i

Dissolving the compound 1 (0.34-0.73 mmol) in acetone (4-5 mL), and adding K₂CO₃(0.74-0.98 mmol) and alkyl iodide or benzyl bromide or substituted benzyl bromide (0.49-0.88 mmol), and stirring for 3-24 h at room temperature; after the reaction is finished, extracting by ethyl acetate and anhydrous Na₂SO₄Drying, and performing silica gel column chromatography (petroleum ether/ethyl acetate elution) on the crude product to obtain yellow solids 14 a-14 i.

5-Hydroxy-8,9-dimethoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-2H,6H-pyrano[3,2-b]xanthen-6-one(14a).Yield:77％.¹H NMR(400MHz,CDCl₃)δ13.75(s,1H),6.74(s,1H),6.73(d,J＝10.0Hz,1H),6.23(s,1H),5.57(d,J＝10.0Hz,1H),5.28-5.20(m,1H),4.12(d,J＝6.6Hz,2H),3.96(s,3H),3.79(s,3H),1.84(s,3H),1.68(s,3H),1.47(s,6H).¹³C NMR(100MHz,CDCl₃)δ182.2,159.8,158.3,158.1,156.4,155.5,144.2,137.4,132.0,127.2,123.3,115.9,112.0,104.6,104.0,98.5,94.1,78.1,61.1,56.2,28.5,26.3,26.1,18.3.ESI-MS m/z:423.3[M+H]⁺.

9-Ethoxy-5-hydroxy-8-methoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-2H,6H-pyrano[3,2-b]xanthen-6-one(14b).Yield:90％.¹H NMR(400MHz,CDCl₃)δ13.77(s,1H),6.73(d,J＝10.0Hz,1H),6.71(s,1H),6.22(s,1H),5.56(d,J＝10.0Hz,1H),5.28-5.21(m,1H),4.16(q,J＝7.0Hz,2H),4.13-4.09(m,2H),3.80(s,3H),1.85(s,3H),1.68(s,3H),1.52(t,J＝7.0Hz,3H),1.46(s,6H).¹³C NMR(100MHz,CDCl₃)δ182.2,159.8,158.1,157.6,156.4,155.5,144.3,137.2,131.9,127.2,123.3,115.9,111.8,104.6,104.0,99.0,94.1,78.0,64.7,60.9,28.4,26.3,26.1,18.3,14.7.ESI-MS m/z:437.1[M+H]⁺.

5-Hydroxy-9-isopropoxy-8-methoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-2H,6H-pyrano[3,2-b]xanthen-6-one(14c).Yield:86％.¹H NMR(400MHz,CDCl₃)δ13.80(s,1H),6.74(d,J＝10.0Hz,1H),6.72(s,1H),6.23(s,1H),5.56(d,J＝10.0Hz,1H),5.29-5.21(m,1H),4.68(hept,J＝6.1Hz,1H),4.12(d,J＝6.6Hz,2H),3.79(s,3H),1.85(s,3H),1.68(s,3H),1.47(s,6H),1.46(d,J＝6.1Hz,6H).¹³C NMR(100MHz,CDCl₃)δ182.2,159.8,158.2,156.6,156.4,155.5,144.8,137.4,131.9,127.2,123.3,115.9,111.6,104.6,104.0,99.8,94.1,78.0,71.3,60.8,28.5,26.4,26.1,22.0,18.3.ESI-MS m/z:473.1[M+Na]⁺ _.

5-Hydroxy-8-methoxy-9-(benzyloxy)-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-2H,6H-pyrano[3,2-b]xanthen-6-one(14d).Yield:99％.¹H NMR(400MHz,CDCl₃)δ13.75(s,1H),7.51-7.47(m,1H),7.47-7.45(m,1H),7.45-7.42(m,1H),7.42-7.39(m,1H),7.39-7.34(m,1H),6.80(s,1H),6.73(d,J＝10.5Hz,1H),6.22(s,1H),5.57(d,J＝10.0Hz,1H),5.29-5.23(m,1H),5.19(s,2H),4.14(d,J＝6.6Hz,2H),3.83(s,3H),1.85(s,3H),1.69(s,3H),1.47(s,6H).¹³C NMR(100MHz,CDCl₃)δ182.2,159.9,158.1,157.2,156.4,155.3,144.4,137.5,135.8,132.0,128.9,128.5,127.5,127.2,123.3,115.9,112.2,104.6,104.0,99.7,94.1,78.1,70.8,61.1,28.5,26.4,26.1,18.3.ESI-MS m/z:521.1[M+Na]⁺.

5-Hydroxy-8-methoxy-9-((3-fluorobenzyl)oxy)-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-2H,6H-pyrano[3,2-b]xanthen-6-one(14e).Yield:86％.¹H NMR(400MHz,CDCl₃)δ13.71(s,1H),7.39(td,J＝7.9,5.7Hz,1H),7.26–7.18(m,2H),7.06(tdd,J＝8.4,2.6,1.0Hz,1H),6.77(s,1H),6.73(d,J＝10.0Hz,1H),6.23(d,J＝0.7Hz,1H),5.57(d,J＝10.0Hz,1H),5.29–5.21(m,1H),5.19(s,2H),4.14(d,J＝6.7Hz,2H),3.84(s,3H),1.85(s,3H),1.69(s,3H),1.46(s,6H).¹³C NMR(100MHz,CDCl₃)δ182.0,163.0(d,¹J_C-F＝246.9Hz),159.8,158.0,156.8,156.2,155.2,144.3,138.2(d,³J_C-F＝7.4Hz),137.6,132.0,130.4(d,³J_C-F＝8.3Hz),127.1,123.1,122.6(d,⁴J_C-F＝2.9Hz),115.7,115.3(d,²J_C-F＝21.2Hz),114.1(d,²J_C-F＝22.3Hz),112.3,104.5,103.9,99.6,94.0,78.0,69.9(d,⁴J_C-F＝2.1Hz),61.0,28.3,26.2,25.9,18.2.¹⁹F NMR(376MHz,CDCl₃)δ-112.2.ESI-MS m/z:517.1[M+H]⁺.

5-Hydroxy-8-methoxy-2,2-dimethyl-9-((3-methylbenzyl)oxy)-7-(3-methylbut-2-en-1-yl)-2H,6H-pyrano[3,2-b]xanthen-6-one(14f).Yield:76％.¹HNMR(400MHz,CDCl₃)δ13.75(s,1H),7.33–7.24(m,3H),7.21–7.15(m,1H),6.80(s,1H),6.73(d,J＝10.0Hz,1H),6.22(s,1H),5.56(d,J＝10.0Hz,1H),5.28–5.22(m,1H),5.16(s,2H),4.13(d,J＝6.6Hz,2H),3.82(s,3H),2.39(s,3H),1.85(s,3H),1.46(s,6H).¹³CNMR(100MHz,CDCl₃)δ182.1,159.7,158.0,157.2,156.2,155.2,144.3,138.5,137.3,135.6,131.9,129.1,128.7,128.0,127.1,124.4,123.1,115.8,112.0,104.5,103.9,99.6,94.0,77.9,70.8,60.9,28.3,26.2,25.9,21.5,18.2.ESI-MSm/z:513.1[M+H]⁺.

5-Hydroxy-8-methoxy-9-((3-methoxybenzyl)oxy)-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-2H,6H-pyrano[3,2-b]xanthen-6-one(14g).Yield:85％.¹HNMR(400MHz,CDCl₃)δ13.75(s,1H),7.33(t,J＝7.9Hz,1H),7.07–7.01(m,2H),6.90(ddd,J＝8.3,2.7,1.0Hz,1H),6.79(s,1H),6.73(d,J＝10.0Hz,1H),6.23(s,1H),5.57(d,J＝10.1Hz,1H),5.27–5.22(m,1H),5.18(s,2H),4.14(d,J＝6.7Hz,2H),3.84(s,3H),3.83(s,3H),1.85(s,3H),1.69(s,3H),1.46(s,6H).¹³CNMR(100MHz,CDCl₃)δ182.1,159.9,159.8,158.0,157.1,156.2,155.2,144.3,137.4,137.3,131.9,129.9,127.1,123.2,119.4,115.8,113.7,112.8,112.1,104.5,103.9,99.6,94.0,77.9,70.5,61.0,55.3,28.3,26.2,25.9,18.2.ESI-MS m/z:529.1[M+H]⁺.

5-Hydroxy-8-methoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-9-((4-(methylsulfonyl)benzyl)oxy)-2H,6H-pyrano[3,2-b]xanthen-6-one(14h).Yield:88％.¹HNMR(400MHz,CDCl₃)δ13.67(s,1H),8.01(d,J＝8.3Hz,2H),7.69(d,J＝8.2Hz,2H),6.76(s,1H),6.72(d,J＝10.0Hz,1H),6.22(s,1H),5.57(d,J＝10.0Hz,1H),5.28(s,2H),5.27–5.22(m,1H),4.14(d,J＝6.6Hz,2H),3.84(s,3H),3.09(s,3H),1.86(s,3H),1.69(s,3H),1.47(s,6H).¹³CNMR(100MHz,CDCl₃)δ181.9,159.9,157.9,156.5,156.2,155.1,144.2,142.0,140.5,137.9,132.1,128.0,127.8,127.2,123.0,115.7,112.5,104.6,103.9,99.6,94.0,78.0,69.5,61.1,44.5,28.3,26.2,25.9,18.2.ESI-MSm/z:577.1[M+H]⁺.

5-Hydroxy-8-methoxy-9-((4-fluorobenzyl)oxy)-2,2-dimethyl7-(3-methylbut-2-en-1-yl)-2H,6H-pyrano[3,2-b]xanthen-6-one(14i).Yield:89％.¹HNMR(400MHz,CDCl₃)δ13.72(s,1H),7.45(dd,J＝8.6,5.4Hz,2H),7.10(t,J＝8.7Hz,2H),6.78(s,1H),6.73(d,J＝10.0Hz,1H),6.22(s,1H),5.57(d,J＝10.0Hz,1H),5.29–5.20(m,1H),5.14(s,2H),4.13(d,J＝6.6Hz,2H),3.80(s,3H),1.85(s,3H),1.69(s,3H),1.46(s,6H).¹³C NMR(100MHz,CDCl₃)δ182.0,162.7(d,¹J_C-F＝247.3Hz),159.8,158.0,156.9,156.2,155.2,144.3,137.5,131.9,131.4(d,⁴J_C-F＝3.1Hz),129.3(d,³J_C-F＝8.3Hz),127.1,123.1,115.8(d,²J_C-F＝21.7Hz),115.7,112.2,104.5,103.9,99.5,94.0,78.0,70.1,60.9,28.3,26.2,25.9,18.2.¹⁹F NMR(376MHz,CDCl₃)δ-113.4.ESI-MSm/z:517.1[M+H]⁺.

EXAMPLE 3 Synthesis of Compounds 13a to 13d

Dissolving the compound 12a or 12b (0.18-0.20 mmol) in acetone (5mL), and adding Cs₂CO₃(0.36-0.40 mmol) and bromoalkylcarboxylic acid ethyl ester or bromocrotonic acid ethyl ester (0.18-0.79 mmol), and stirring at room temperature overnight; evaporating the solvent to dryness, adding water, and extracting with ethyl acetate for 3 times; collecting organic phase, anhydrous Na₂SO₄Drying and removing the solvent to obtain a crude product. Separating by silica gel column chromatography, eluting by ethyl acetate/petroleum ether to obtain white solids 13 a-13 d.

Ethyl2-((9-acetoxy-8-methoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-6-oxo-2H,6H-pyrano[3,2-b]xanthen-5-yl)oxy)acetate(13a).Yield:62％.¹H NMR(400MHz,CDCl₃)δ7.07(s,1H),7.04(d,J＝10.1Hz,1H),6.55(s,1H),5.69(d,J＝10.2Hz,1H),5.26–5.21(m,1H),4.69(s,2H),4.26(q,J＝7.2Hz,2H),4.11(d,J＝6.6Hz,2H),3.76(s,3H),2.38(s,3H),1.83(s,3H),1.67(s,3H),1.47(s,6H),1.30(t,J＝7.1Hz,3H).¹³C NMR(100MHz,CDCl₃)δ176.4,169.5,168.3,158.8,157.5,154.0,152.9,148.0,146.5,138.7,131.9,129.8,123.3,119.3,116.8,112.2,110.2,110.1,100.1,77.9,71.3,61.6,61.1,28.4,26.1,25.8,20.9,18.2,14.2.ESI-MS m/z:537.1[M+H]⁺.

Ethyl4-((9-((tert-butoxycarbonyl)oxy)-8-methoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-6-oxo-2H,6H-pyrano[3,2-b]xanthen-5-yl)oxy)butanoate(13b).Yield:39％.¹HNMR(400MHz,CDCl₃)δ7.11(s,1H),6.70(d,J＝10.1Hz,1H),6.51(s,1H),5.67(d,J＝10.1Hz,1H),5.26–5.21(m,1H),4.21–4.09(m,4H),4.01(t,J＝6.2Hz,2H),3.79(s,3H),2.63(t,J＝7.5Hz,2H),2.23–2.15(m,2H),1.84(s,3H),1.66(s,3H),1.56(s,9H),1.46(s,6H),1.27(t,J＝7.1Hz,3H).¹³CNMR(100MHz,CDCl₃)δ176.3,173.5,158.5,157.6,155.0,152.9,150.5,148.2,146.6,138.6,131.5,129.9,123.5,119.2,116.4,112.1,111.0,109.5,99.7,84.4,77.7,74.1,61.6,60.4,30.9,28.3,27.6,26.1,25.8,25.6,18.2,14.3.ESI-MSm/z:623.3[M+H]⁺.

Ethyl5-((9-((tert-butoxycarbonyl)oxy)-8-methoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-6-oxo-2H,6H-pyrano[3,2-b]xanthen-5-yl)oxy)pentanoate(13c).Yield:18％.¹HNMR(400MHz,CDCl₃)δ7.11(s,1H),6.71(d,J＝10.1Hz,1H),6.51(s,1H),5.66(d,J＝10.1Hz,1H),5.27–5.21(m,1H),4.17–4.11(m,4H),3.99(t,J＝6.1Hz,2H),3.79(s,3H),2.42(t,J＝7.0Hz,2H),1.94–1.85(m,4H),1.83(s,3H),1.65(s,3H),1.56(s,9H),1.46(s,6H),1.26(t,J＝7.1Hz,3H).¹³CNMR(100MHz,CDCl₃)δ176.3,173.6,158.5,157.7,155.2,152.9,150.5,148.2,146.5,138.6,131.5,129.8,123.5,119.3,116.5,112.1,111.0,109.5,99.6,84.4,77.7,74.9,61.6,60.3,34.1,29.7,28.3,27.6,26.1,25.8,21.6,18.2,14.3.ESI-MSm/z:637.3[M+H]⁺.

Ethyl(E)-4-((9-((tert-butoxycarbonyl)oxy)-8-methoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-6-oxo-2H,6H-pyrano[3,2-b]xanthen-5-yl)oxy)but-2-enoate(13d).Yield:70％.¹HNMR(400MHz,CDCl₃)δ7.14(dt,J＝15.7,4.8Hz,1H),7.12(s,1H),6.68(d,J＝10.1Hz,1H),6.55(s,1H),6.25(dt,J＝15.7,1.9Hz,1H),5.68(d,J＝10.1Hz,1H),5.25–5.17(m,1H),4.71(dd,J＝4.8,2.0Hz,2H),4.23(q,J＝7.1Hz,2H),4.12(d,J＝6.6Hz,2H),3.79(s,3H),1.82(s,3H),1.66(s,3H),1.57(s,9H),1.46(s,6H),1.31(t,J＝7.2Hz,3H).¹³CNMR(100MHz,CDCl₃)δ176.3,166.3,158.6,157.6,154.1,152.9,150.5,148.3,146.6,143.0,138.7,131.7,130.3,123.3,122.1,119.2,116.2,112.3,111.1,109.6,100.2,84.4,77.8,73.1,61.6,60.5,28.3,27.6,26.2,25.8,18.2,14.3.ESI-MS m/z:643.3[M+Na]⁺.

EXAMPLE 4 Synthesis of Compounds 15 a-15 g and 19b

Dissolving the compounds 14 a-14 i (0.14-0.71 mmol) in acetone (5mL) or DMF (3mL), respectively, and adding K₂CO₃(0.28-1.07 mmol) and (E) -4-bromocrotonate ethyl ester (0.28-1.86 mmol), refluxing the reaction overnight, and transferring under reduced pressureRemoving solvent, adding water, and extracting with ethyl acetate of the same volume for 3 times; the organic phase is passed through anhydrous Na₂SO₄Drying and evaporating the solvent to obtain a crude product; separating by silica gel column chromatography, eluting with petroleum ether/ethyl acetate to obtain white solid 15 a-15 g or 19 b.

Ethyl(E)-4-((8,9-dimethoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-6-oxo-2H,6H-pyrano[3,2-b]xanthen-5-yl)oxy)but-2-enoate(15a).Yield:79％.¹H NMR(400MHz,CDCl₃)δ7.13(dt,J＝15.7,4.8Hz,1H),6.68(s,1H),6.66(d,J＝10.5Hz,1H),6.51(s,1H),6.23(dt,J＝15.7,1.8Hz,1H),5.65(d,J＝10.1Hz,1H),5.26–5.19(m,1H),4.70(dd,J＝4.7,1.8Hz,2H),4.20(q,J＝7.1Hz,2H),4.08(d,J＝6.7Hz,2H),3.91(s,3H),3.76(s,3H),1.81(s,3H),1.63(s,3H),1.43(s,6H),1.28(t,J＝7.1Hz,3H).¹³C NMR(100MHz,CDCl₃)δ176.2,166.3,158.1,157.5,157.2,154.3,154.1,144.0,143.2,137.2,131.4,130.2,123.7,122.0,116.4,114.3,112.2,111.2,100.2,98.0,77.6,73.1,60.9,60.4,56.0,28.3,26.0,25.9,18.2,14.3.ESI-MS m/z:535.2[M+H]⁺.

Ethyl(E)-4-((9-ethoxy-8-methoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-6-oxo-2H,6H-pyrano[3,2-b]xanthen-5-yl)oxy)but-2-enoate(15b).Yield:86％.¹H NMR(400MHz,CDCl₃)δ7.14(dt,J＝15.7,4.8Hz,1H),6.70(s,1H),6.69(d,J＝10.1Hz,1H),6.54(s,1H),6.24(dt,J＝15.7,1.9Hz,1H),5.67(d,J＝10.1Hz,1H),5.28-5.22(m,1H),4.72(dd,J＝4.8,1.9Hz,2H),4.23(q,J＝7.1Hz,2H),4.15(q,J＝7.0Hz,2H),4.10(d,J＝6.7Hz,2H),3.80(s,3H),1.84(s,3H),1.65(s,3H),1.52(t,J＝7.0Hz,3H),1.46(s,6H),1.31(t,J＝7.1Hz,3H).¹³C NMR(100MHz,CDCl₃)δ176.4,166.4,158.2,157.7,156.6,154.4,154.2,144.2,143.4,137.3,131.5,130.2,123.8,122.1,116.5,114.3,112.3,111.3,100.3,98.6,77.8,73.2,64.5,60.9,60.6,28.4,26.1,26.1,18.4,14.8,14.4.ESI-MS m/z:549.2[M+H]⁺.

Ethyl(E)-4-((9-isopropoxy-8-methoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-6-oxo-2H,6H-pyrano[3,2-b]xanthen-5-yl)oxy)but-2-enoate(15c).Yield:78％.¹H NMR(400MHz,CDCl₃)δ7.14(dt,J＝15.7,4.8Hz,1H),6.68(d,J＝10.1Hz,1H),6.66(s,1H),6.52(s,1H),6.24(dt,J＝15.6,1.9Hz,1H),5.65(d,J＝10.1Hz,1H),5.31-5.20(m,1H),4.72(dd,J＝4.8,1.9Hz,2H),4.65(p,J＝6.1Hz,1H),4.21(q,J＝7.1Hz,2H),4.09(d,J＝6.8Hz,2H),3.77(s,3H),1.83(s,3H),1.64(s,3H),1.44(s,6H),1.43(d,J＝6.1Hz,6H),1.29(t,J＝7.1Hz,3H).¹³C NMR(100MHz,CDCl₃)δ176.3,166.3,158.1,157.6,155.5,154.3,154.2,144.7,143.3,137.4,131.3,130.2,123.8,122.1,116.5,114.1,112.2,111.2,100.2,99.4,77.7,73.1,71.0,60.7,60.5,28.3,26.1,26.0,21.9,18.3,14.3.ESI-MS m/z:563.2[M+H]⁺.

Ethyl(E)-4-((9-(benzyloxy)-8-methoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-6-oxo-2H,6H-pyrano[3,2-b]xanthen-5-yl)oxy)but-2-enoate(15d).Yield:81％.¹H NMR(400MHz,CDCl₃)δ7.50-7.47(m,1H),7.47-7.45(m,1H),7.45-7.41(m,1H),7.41-7.38(m,1H),7.38-7.33(m,1H),7.15(dt,J＝15.7,4.8Hz,1H),6.78(s,1H),6.69(d,J＝10.1,1H),6.53(s,1H),6.25(dt,J＝15.7,1.9Hz,1H),5.67(d,J＝10.1Hz,1H),5.29-5.23(m,1H),5.20(s,2H),4.72(dd,J＝4.8,1.9Hz,2H),4.23(q,J＝7.1Hz,2H),4.12(d,J＝6.6Hz,2H),3.83(s,3H),1.84(s,3H),1.66(s,3H),1.46(s,6H),1.31(t,J＝7.1Hz,3H).¹³C NMR(100MHz,CDCl₃)δ176.4,166.4,158.2,157.6,156.3,154.2,144.4,143.3,137.5,136.0,131.5,130.3,128.9,128.4,127.4,123.8,122.2,116.5,114.7,112.3,111.3,100.3,99.3,77.8,73.2,70.7,61.0,60.6,28.4,26.2,26.1,18.4,14.4.ESI-MS m/z:611.2[M+H]⁺.

Ethyl(E)-4-((8-methoxy-2,2-dimethyl-9-((3-methylbenzyl)oxy)-7-(3-methylbut-2-en-1-yl)-6-oxo-2H,6H-pyrano[3,2-b]xanthen-5-yl)oxy)but-2-enoate(15e).Yield:80％.¹HNMR(400MHz,CDCl₃)δ7.34-7.23(m,3H),7.20-7.09(m,2H),6.77(s,1H),6.69(d,J＝10.1Hz,1H),6.53(d,J＝0.7Hz,1H),6.24(dt,J＝15.7,1.9Hz,1H),5.67(d,J＝10.1Hz,1H),5.29-5.21(m,1H),5.16(s,2H),4.72(dd,J＝4.8,1.9Hz,2H),4.23(q,J＝7.1Hz,2H),4.12(d,J＝6.7Hz,2H),3.83(s,3H),2.39(s,3H),1.84(s,3H),1.66(s,3H),1.46(s,6H),1.31(t,J＝7.1Hz,3H).¹³CNMR(100MHz,CDCl₃)δ176.3,166.3,158.1,157.5,156.3,154.1,144.3,143.2,138.5,137.4,135.8,131.4,130.1,129.0,128.6,128.0,124.4,123.7,122.1,116.4,114.6,112.2,111.2,100.2,99.2,77.6,73.1,70.7,60.9,60.4,28.3,26.0,25.9,21.5,18.2,14.3.ESI-MSm/z:625.2[M+H]⁺.

Ethyl(E)-4-((8-methoxy-9-((3-methoxybenzyl)oxy)-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-6-oxo-2H,6H-pyrano[3,2-b]xanthen-5-yl)oxy)but-2-enoate(15f).Yield:84％.¹HNMR(400MHz,CDCl₃)δ7.33(t,J＝7.9Hz,1H),7.14(dt,J＝15.7,4.8Hz,1H),7.07–7.01(m,2H),6.89(ddd,J＝8.3,2.7,0.9Hz,1H),6.77(s,1H),6.69(d,J＝10.1Hz,1H),6.25(dt,J＝15.7,1.9Hz,1H),5.67(d,J＝10.1Hz,1H),5.28–5.22(m,1H),5.18(s,2H),4.72(dd,J＝4.8,1.9Hz,2H),4.23(q,J＝7.1Hz,2H),4.12(d,J＝6.7Hz,2H),3.84(s,3H),3.83(s,3H),1.46(s,6H),1.31(t,J＝7.1Hz,3H).¹³CNMR(100MHz,CDCl₃)δ176.3,166.3,159.9,158.1,157.5,156.1,154.1,144.3,143.2,137.5,137.5,131.5,130.2,129.8,123.6,122.0,119.4,116.4,114.6,113.6,112.8,112.2,111.2,100.2,99.3,77.6,73.1,70.5,60.9,60.5,55.3,28.3,26.0,25.9,18.2,14.3.ESI-MSm/z:641.2[M+H]⁺.

Ethyl(E)-4-((8-methoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-9-((4-(methyl-sulfonyl)benzyl)oxy)-6-oxo-2H,6H-pyrano[3,2-b]xanthen-5-yl)oxy)but-2-enoate(15g).Yield:71％.¹HNMR(400MHz,CDCl₃)δ8.01(d,J＝8.3Hz,2H),7.70(d,J＝8.3Hz,2H),7.14(dt,J＝15.7,4.8Hz,1H),6.74(s,1H),6.68(d,J＝10.1Hz,1H),6.53(s,1H),6.25(dt,J＝15.7,1.8Hz,1H),5.68(d,J＝10.1Hz,1H),5.29(s,2H),5.27–5.21(m,1H),4.72(dd,J＝4.8,1.8Hz,2H),4.23(q,J＝7.1Hz,2H),4.13(d,J＝6.7Hz,2H),3.84(s,3H),3.08(s,3H),1.85(s,3H),1.67(s,3H),1.46(s,6H),1.31(t,J＝7.1Hz,3H).¹³CNMR(100MHz,CDCl₃)δ176.1,166.3,158.2,157.4,155.5,154.1,154.0,144.2,143.1,142.2,140.4,137.9,131.6,130.3,127.9,127.7,123.4,122.1,116.3,115.1,112.3,111.2,100.1,99.3,77.7,73.1,69.5,61.1,60.5,44.5,28.3,26.0,25.9,18.2,14.3.ESI-MSm/z:689.1[M+H]⁺.

Ethyl(E)-4-((9-((3-fluorobenzyl)oxy)-8-methoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-6-oxo-2H,6H-pyrano[3,2-b]xanthen-5-yl)oxy)but-2-enoate(19b).Yield:89％.¹HNMR(400MHz,CDCl₃)δ7.38(td,J＝7.9,5.7Hz,1H),7.26–7.11(m,3H),7.05(td,J＝8.6,2.7Hz,1H),6.74(s,1H),6.69(d,J＝10.1Hz,1H),6.53(s,1H),6.25(dt,J＝15.7,2.0Hz,1H),5.67(d,J＝10.1Hz,1H),5.28–5.22(m,1H),5.18(s,2H),4.72(dd,J＝4.8,1.9Hz,2H),4.23(q,J＝7.1Hz,2H),4.13(d,J＝6.7Hz,2H),3.84(s,3H),1.85(s,3H),1.66(s,3H),1.46(s,6H),1.31(t,J＝7.1Hz,3H).¹³CNMR(100MHz,CDCl₃)δ176.2,166.3,163.0(d,¹J_C-F＝246.8Hz),158.2,157.5,155.9,154.1,154.1,144.3,143.2,138.4(d,³J_C-F＝7.2Hz),137.6,131.5,130.4(d,³J_C-F＝8.3Hz),130.2,123.6,122.6(d,⁴J_C-F＝2.9Hz),122.1,116.4,115.2(d,²J_C-F＝21.0Hz),114.8,114.1(d,²J_C-F＝22.2Hz),112.2,111.2,100.2,99.2,77.7,73.1,69.8(d,⁴J_C-F＝2.0Hz),61.0,60.4,28.3,26.0,25.9,18.2,14.3.¹⁹FNMR(376MHz,CDCl₃)δ-112.3.ESI-MSm/z:629.1[M+H]⁺.

EXAMPLE 5 Synthesis of Compounds 15h, 15i and 18d

Dissolving a compound 14i (0.19-4.3 mmol) in acetone (5-20 mL), and adding Cs₂CO₃(0.38-8.5 mmol), (E) -4-bromocrotonic acid ethyl ester (8.6mmol) or 4-bromobutyric acid ethyl ester (0.39mmol), refluxing overnight, removing the solvent under reduced pressure, adding water, and extracting with ethyl acetate of equal volume for 3 times; collecting organic phase, passing through anhydrous Na₂SO₄Drying, performing silica gel column chromatography, and eluting with petroleum ether/ethyl acetate to obtain white solids 15h, 15i and 18 d.

Ethyl4-((9-((4-fluorobenzyl)oxy)-8-methoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-6-oxo-2H,6H-pyrano[3,2-b]xanthen-5-yl)oxy)butanoate(15h).Yield:88％.¹HNMR(400MHz,CDCl₃)δ7.45(dd,J＝8.5,5.5Hz,2H),7.10(t,J＝8.6Hz,2H),6.76(s,1H),6.71(d,J＝10.1Hz,1H),6.50(s,1H),5.66(d,J＝10.1Hz,1H),5.29–5.24(m,1H),5.14(s,2H),4.19–4.10(m,4H),4.02(t,J＝6.2Hz,2H),3.81(s,3H),2.63(t,J＝7.5Hz,2H),2.20(p,J＝6.6Hz,2H),1.85(s,3H),1.66(s,3H),1.46(s,6H),1.27(t,J＝7.1Hz,3H).¹³CNMR(100MHz,CDCl₃)δ176.2,173.5,162.6(d,¹J_C-F＝246.9Hz),158.1,157.5,155.8,154.9,154.0,144.2,137.4,131.7(d,⁴J_C-F＝3.2Hz),131.3,129.7,129.2(d,³J_C-F＝8.2Hz),123.7,116.5,115.7(d,²J_C-F＝21.6Hz),114.8,112.1,111.1,99.7,99.2,77.5,74.1,69.9,60.9,60.4,30.9,28.3,26.0,25.9,25.6,18.2,14.3.¹⁹FNMR(376MHz,CDCl₃)δ-113.6.ESI-MSm/z:631.2[M+H]⁺.

Ethyl(Z)-4-((9-((4-fluorobenzyl)oxy)-8-methoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-6-oxo-2H,6H-pyrano[3,2-b]xanthen-5-yl)oxy)but-2-enoate(15i).Compound15i was isolated from the crude product of compound 18d.¹HNMR(400MHz,CDCl₃)δ7.45(dd,J＝8.5,5.4Hz,2H),7.11(t,J＝8.7Hz,2H),6.83(dt,J＝11.7,4.8Hz,1H),6.76(s,1H),6.71(d,J＝10.1Hz,1H),6.53(s,1H),5.88(dt,J＝11.7,2.4Hz,1H),5.66(d,J＝10.1Hz,1H),5.31–5.25(m,1H),5.15(s,2H),5.13(dd,J＝4.7,2.4Hz,2H),4.13(q,J＝7.2Hz,4H),3.80(s,3H),1.83(s,3H),1.64(s,3H),1.46(s,6H),1.25(t,J＝7.1Hz,3H).¹³CNMR(100MHz,CDCl₃)δ176.2,166.0,162.6(d,¹J_C-F＝246.8Hz),158.1,157.5,155.9,154.5,154.1,147.7,144.1,137.5,131.6(d,⁴J_C-F＝3.1Hz),131.3,129.9,129.2(d,³J_C-F＝8.2Hz),123.6,119.1,116.5,115.7(d,²J_C-F＝21.6Hz),114.7,112.2,111.1,100.0,99.2,77.6,73.5,69.9,60.9,60.2,28.3,26.1,25.8,18.2,14.2.¹⁹FNMR(376MHz,CDCl₃)δ-113.6.ESI-MSm/z:629.2[M+H]⁺.

Ethyl(E)-4-((9-((4-fluorobenzyl)oxy)-8-methoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-6-oxo-2H,6H-pyrano[3,2-b]xanthen-5-yl)oxy)but-2-enoate(18d).Yield：86％.¹HNMR(400MHz,CDCl₃)δ7.45(dd,J＝8.5,5.5Hz,2H),7.18–7.07(m,3H),6.77(s,1H),6.69(d,J＝10.1Hz,1H),6.54(s,1H),6.24(dt,J＝15.7,1.9Hz,1H),5.67(d,J＝10.1Hz,1H),5.29–5.20(m,1H),5.15(s,2H),4.72(dd,J＝4.8,1.9Hz,2H),4.23(q,J＝7.1Hz,2H),4.12(d,J＝6.7Hz,2H),3.81(s,3H),1.84(s,3H),1.66(s,3H),1.46(s,6H),1.31(t,J＝7.1Hz,3H).¹³CNMR(100MHz,CDCl₃)δ176.2,166.3,162.7(d,¹J_C-F＝247.0Hz),158.1,157.5,156.0,154.11,154.08,144.3,143.2,137.6,131.6(d,⁴J_C-F＝3.1Hz),131.5,130.2,129.2(d,³J_C-F＝8.3Hz),123.6,122.0,116.4,115.7(d,²J_C-F＝21.6Hz),114.7,112.2,111.2,100.1,99.2,77.7,73.1,70.0,60.9,60.4,28.3,26.0,25.9,18.2,14.3.¹⁹F NMR(376MHz,CDCl₃)δ-113.6.ESI-MS m/z:629.1[M+H]⁺.

EXAMPLE 6 Synthesis of Compounds 16a to 16c, 17a to 17e, 18a to 18c, 19a and 20a to 20c

Respectively dissolving compounds 13a to 13d,15a to 15i,18d and 19b (0.04 to 0.56mmol) in acetone/water (v/v,1:4), adding NaOH (0.22 to 2.81mmol), reacting at 60 ℃ for 2 to 4 hours, pouring the reaction product into 10mL of water after the reaction is finished, acidifying by 5 percent HCl to adjust the pH value to be 1, and extracting by ethyl acetate; the organic phase is passed through anhydrous Na₂SO₄Drying and concentrating to obtain a crude product; silica gel chromatography, dichloromethane/methanol elution gave the title compound (white solid).

2-((9-Hydroxy-8-methoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-6-oxo-2H,6H-pyrano[3,2-b]xanthen-5-yl)oxy)acetic acid(16a).Yield:58％.¹H NMR(400MHz,DMSO-d₆)δ6.91(d,J＝10.1Hz,1H),6.75(s,1H),6.64(s,1H),5.85(d,J＝10.1Hz,1H),5.17–5.09(m,1H),4.57(s,2H),3.97(d,J＝6.7Hz,2H),3.69(s,3H),1.77(s,3H),1.61(s,3H),1.42(s,6H).¹³C NMR(100MHz,DMSO-d₆)δ175.9,170.8,158.1,157.3,156.3,154.0,153.9,143.8,136.7,130.9,130.7,124.3,116.6,113.1,111.9,110.3,101.8,99.9,78.2,71.1,60.6,28.3,26.0,25.8,18.4.ESI-HRMS m/z:[M+H]⁺(calcd for C₂₆H₂₆O₈ 467.1700,found 467.1696).

4-((9-Hydroxy-8-methoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-6-oxo-2H,6H-pyrano[3,2-b]xanthen-5-yl)oxy)butanoic acid(16b).Yield:73％.¹H NMR(400MHz,Methanol-d₄)δ6.74–6.69(m,2H),6.51(s,1H),5.78(d,J＝10.1Hz,1H),5.24–5.17(m,1H),4.06(d,J＝6.5Hz,2H),3.97(t,J＝6.3Hz,2H),3.76(s,3H),2.59(t,J＝7.4Hz,2H),2.19–2.10(m,2H),1.82(s,3H),1.66(s,3H),1.45(s,6H).¹³C NMR(100MHz,Methanol-d₄)δ178.2,177.2,159.8,159.0,157.2,156.1,155.8,145.0,138.4,131.9,131.4,125.3,117.1,114.7,113.3,111.9,102.5,100.7,78.8,75.5,61.3,31.6,28.5,26.9,26.7,26.0,18.4.ESI-HRMS m/z:[M+H]⁺(calcd for C₂₈H₃₀O₈ 495.2013,found 495.2009).

5-((9-Hydroxy-8-methoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-6-oxo-2H,6H-pyrano[3,2-b]xanthen-5-yl)oxy)pentanoic acid(16c).Yield:77％.¹H NMR(400MHz,CDCl₃)δ6.80(s,1H),6.71(d,J＝10.1Hz,1H),6.52(s,1H),5.66(d,J＝10.1Hz,1H),5.30–5.21(m,1H),4.08(d,J＝6.4Hz,2H),3.99(t,J＝5.8Hz,2H),3.80(s,3H),2.50(t,J＝6.8Hz,2H),1.98–1.87(m,4H),1.82(s,3H),1.65(s,3H),1.45(s,6H).¹³C NMR(100MHz,CDCl₃)δ178.8,176.4,158.2,157.6,155.1,154.7,153.5,142.5,136.9,131.6,129.8,123.6,116.6,114.7,112.0,111.0,101.2,99.7,77.5,74.7,62.0,33.7,29.5,28.3,26.3,25.8,21.4,18.2.ESI-HRMS m/z:[M+H]⁺(calcd for C₂₉H₃₂O₈ 509.2170,found 509.2166).

(E)-4-((9-Hydroxy-8-methoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-6-oxo-2H,6H-pyrano[3,2-b]xanthen-5-yl)oxy)but-2-enoic acid(17a).Yield:71％.¹H NMR(400MHz,MeOD+CDCl₃)δ7.13(dt,J＝15.6,4.9Hz,1H),6.72(s,1H),6.69(d,J＝10.1Hz,1H),6.57(s,1H),6.18(dt,J＝15.7,1.9Hz,1H),5.81(d,J＝10.1Hz,1H),5.20(d,J＝6.5Hz,1H),4.66(dd,J＝4.8,1.5Hz,2H),4.06(d,J＝6.3Hz,2H),3.76(s,3H),1.82(s,3H),1.65(s,3H),1.46(s,6H).¹³C NMR(100MHz,MeOD+CDCl₃)δ178.1,169.5,159.7,158.9,157.3,155.8,155.2,145.0,144.7,138.4,132.0,131.7,125.1,123.5,116.9,114.6,113.4,111.9,102.4,101.1,78.9,74.3,61.3,28.5,26.9,26.0,18.4.ESI-HRMSm/z:[M+H]⁺(calcd for C₂₈H₂₈O₈ 493.1857,found 493.1857).

(E)-4-((8,9-Dimethoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-6-oxo-2H,6H-pyrano[3,2-b]xanthen-5-yl)oxy)but-2-enoic acid(17b).Yield:44％.¹H NMR(400MHz,CDCl₃)δ7.26(dt,J＝15.6,4.5Hz,1H),6.72(s,1H),6.68(d,J＝10.1Hz,1H),6.55(s,1H),6.30(dt,J＝15.6,1.8Hz,1H),5.69(d,J＝10.1Hz,1H),5.27–5.21(m,1H),4.77(dd,J＝4.5,1.9Hz,2H),4.11(d,J＝6.6Hz,2H),3.95(s,3H),3.79(s,3H),1.83(s,3H),1.65(s,3H),1.46(s,6H).¹³C NMR(100MHz,CDCl₃)δ176.4,171.3,158.2,157.6,157.4,154.5,154.1,146.1,144.1,137.4,131.7,130.4,123.7,121.1,116.4,114.5,112.3,111.3,100.3,98.1,77.8,73.0,61.1,56.1,28.4,26.1,26.1,18.3.ESI-HRMS m/z:[M+H]⁺(calcd for C₂₉H₃₀O₈ 507.2013,found 507.2017).

(E)-4-((9-Ethoxy-8-methoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-6-oxo-2H,6H-pyrano[3,2-b]xanthen-5-yl)oxy)but-2-enoic acid(17c).Yield:64％.¹H NMR(400MHz,CDCl₃)δ7.29-7.22(m,1H),6.69(s,1H),6.68(d,J＝10.1Hz,1H),6.54(s,1H),6.29(dt,J＝15.6,2.0Hz,1H),5.68(d,J＝10.1Hz,1H),5.28-5.21(m,1H),4.77(dd,J＝4.2,1.5Hz,2H),4.22-4.12(m,2H),4.11(d,J＝7.1Hz,2H),3.80(s,3H),1.84(s,3H),1.66(s,3H),1.52(t,J＝7.0Hz,3H),1.46(s,6H).¹³C NMR(100MHz,CDCl₃)δ176.4,171.3,158.2,157.7,156.7,154.4,154.1,146.2,144.2,137.3,131.5,130.4,123.7,121.1,116.4,114.3,112.2,111.3,100.3,98.6,77.8,73.0,64.5,60.9,28.4,26.1,26.1,18.3,14.7.ESI-HRMS m/z:[M+H]⁺(calcd for C₃₀H₃₂O₈ 521.2170,found 521.2166).

(E)-4-((9-Isopropoxy-8-methoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-6-oxo-2H,6H-pyrano[3,2-b]xanthen-5-yl)oxy)but-2-enoic acid(17d).Yield:63％.¹H NMR(400MHz,CDCl₃)δ7.29-7.21(m,2H),6.70(s,1H),6.68(d,J＝10.0Hz,1H),6.55(s,1H),6.29(dt,J＝15.7,1.9Hz,1H),5.68(d,J＝10.1Hz,1H),5.28-5.22(m,1H),4.77(dd,J＝4.5,2.0Hz,2H),4.67(hept,J＝6.1Hz,1H),4.10(d,J＝6.7Hz,2H),3.79(s,3H),1.84(s,3H),1.66(s,3H),1.46(s,6H),1.46(s,3H),1.44(s,3H).¹³C NMR(100MHz,CDCl₃)δ176.4,170.8,158.2,157.7,155.6,154.4,154.1,146.1,144.8,137.5,131.5,130.4,123.8,121.0,116.4,114.2,112.2,111.3,100.3,99.5,77.8,73.0,71.1,60.8,28.4,26.2,26.1,22.0,18.3.ESI-HRMS m/z:[M+H]⁺(calcd for C₃₁H₃₄O₈ 535.2326,found 535.2321).

(E)-4-((9-(Benzyloxy)-8-methoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-6-oxo-2H,6H-pyrano[3,2-b]xanthen-5-yl)oxy)but-2-enoic acid(17e).Yield:55％.¹H NMR(400MHz,CDCl₃)δ7.52-7.47(m,1H),7.47-7.45(m,1H),7.45-7.42(m,1H),7.42-7.38(m,1H),7.38-7.33(m,1H),7.29-7.22(m,1H),6.78(s,1H),6.68(d,J＝10.0Hz,1H),6.54(s,1H),6.36-6.24(m,1H),5.69(d,J＝10.1Hz,1H),5.25(t,J＝6.1Hz,1H),5.20(s,2H),4.82-4.70(m,2H),4.12(d,J＝6.4Hz,2H),3.83(s,3H),1.84(s,3H),1.66(s,3H),1.46(s,6H).¹³CNMR(100MHz,CDCl₃)δ176.4,171.0,158.3,157.6,156.3,154.3,154.1,146.1,144.4,137.6,136.0,131.6,130.4,128.9,128.4,127.4,123.7,121.1,116.4,114.7,112.3,111.3,100.4,99.4,77.8,73.0,70.7,61.1,28.4,26.2,26.1,18.4.ESI-HRMS m/z:[M+H]⁺(calcd for C₃₅H₃₃FO₈ 583.2326,found 583.2321).

(E)-4-((9-((4-Fluorobenzyl)oxy)-8-methoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-6-oxo-2H,6H-pyrano[3,2-b]xanthen-5-yl)oxy)but-2-enoic acid(18a).Yield:78％.¹H NMR(400MHz,CDCl₃)δ7.45(dd,J＝8.6,5.4Hz,2H),7.25(dt,J＝15.6,4.5Hz,1H),7.11(t,J＝8.7Hz,2H),6.77(s,1H),6.68(d,J＝10.2Hz,1H),6.55(s,1H),6.30(dt,J＝15.6,2.0Hz,1H),5.69(d,J＝10.1Hz,1H),5.27–5.21(m,1H),5.15(s,2H),4.77(dd,J＝4.5,2.0Hz,2H),4.12(d,J＝6.7Hz,2H),3.81(s,3H),1.84(s,3H),1.66(s,3H),1.47(s,6H).¹³C NMR(100MHz,CDCl₃)δ176.3,170.6,162.7(d,¹J_C-F＝246.9Hz),158.2,157.5,156.0,154.1,154.0,146.0,144.3,137.6,131.6(d,⁴J_C-F＝3.2Hz),131.55,130.3,129.3(d,³J_C-F＝8.2Hz),123.5,120.9,116.2,115.7(d,²J_C-F＝21.7Hz),114.7,112.2,111.2,100.2,99.2,77.7,72.8,70.0,60.9,28.3,26.0,25.9,18.2.¹⁹FNMR(376MHz,CDCl₃)δ-113.5.ESI-HRMS m/z:[M+H]⁺(calcd for C₃₅H₃₃FO₈ 601.2232,found 601.2232).

4-((9-((4-Fluorobenzyl)oxy)-8-methoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-6-oxo-2H,6H-pyrano[3,2-b]xanthen-5-yl)oxy)butanoic acid(18b).Yield:87％.¹HNMR(400MHz,CDCl₃)δ7.45(dd,J＝8.6,5.4Hz,2H),7.10(t,J＝8.7Hz,2H),6.77(s,1H),6.68(d,J＝10.1Hz,1H),6.53(s,1H),5.68(d,J＝10.1Hz,1H),5.28–5.23(m,1H),4.11(d,J＝6.6Hz,2H),4.02(t,J＝5.9Hz,2H),3.81(s,3H),2.76(t,J＝7.1Hz,2H),2.21(p,J＝6.7Hz,2H),1.85(s,3H),1.66(s,3H),1.46(s,6H).¹³CNMR(100MHz,CDCl₃)δ177.7,176.7,162.7(d,¹J_C-F＝246.9Hz),158.4,157.6,156.1,154.7,154.2,144.3,137.5,131.6(d,⁴J_C-F＝3.2Hz),131.5,130.0,129.3(d,³J_C-F＝8.3Hz),123.6,116.3,115.7(d,²J_C-F＝21.6Hz),114.6,112.2,111.0,99.9,99.2,77.7,74.1,70.0,60.9,31.4,28.3,26.1,25.9,25.7,18.2.¹⁹FNMR(376MHz,CDCl₃)δ-113.5.ESI-HRMS m/z:[M+H]⁺(calcd for C₃₅H₃₅FO₈603.2389,found 603.2373).

(Z)-4-((9-((4-Fluorobenzyl)oxy)-8-methoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-6-oxo-2H,6H-pyrano[3,2-b]xanthen-5-yl)oxy)but-2-enoic acid(18c).Yield:52％.¹HNMR(400MHz,Chloroform-d)δ7.45(dd,J＝8.5,5.4Hz,2H),7.10(t,J＝8.7Hz,2H),6.92(dt,J＝11.8,4.6Hz,1H),6.76(s,1H),6.67(d,J＝10.1Hz,1H),6.52(s,1H),5.89(dt,J＝11.8,2.4Hz,1H),5.66(d,J＝10.1Hz,1H),5.26–5.21(m,1H),5.14(s,2H),5.06(dd,J＝4.6,2.5Hz,2H),4.09(d,J＝6.8Hz,2H),3.79(s,3H),1.80(s,3H),1.61(s,3H),1.45(s,6H).¹³CNMR(101MHz,CDCl₃)δ176.2,170.4,162.6(d,¹J_C-F＝247.0Hz),158.2,157.5,156.0,154.2,154.1,150.2,144.2,137.6,131.6(d,⁴J_C-F＝3.3Hz),131.4,130.1,129.2(d,³J_C-F＝8.2Hz),123.5,118.3,116.3,115.7(d,²J_C-F＝21.6Hz),114.6,112.1,111.0,100.1,99.2,77.7,73.4,69.9,60.9,28.3,26.0,25.8,18.2.¹⁹FNMR(376MHz,CDCl₃)δ-113.6.ESI-HRMS m/z:[M+H]⁺(calcd for C₃₅H₃₃FO₈601.2232,found 601.2222).

(E)-4-((9-((3-Fluorobenzyl)oxy)-8-methoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-6-oxo-2H,6H-pyrano[3,2-b]xanthen-5-yl)oxy)but-2-enoic acid(19a).Yield:87％.¹HNMR(400MHz,CDCl₃)δ7.39(td,J＝8.0,5.8Hz,1H),7.29–7.18(m,3H),7.05(tdd,J＝8.4,2.6,1.0Hz,1H),6.75(s,1H),6.69(d,J＝10.1Hz,1H),6.54(s,1H),6.30(dt,J＝15.7,1.9Hz,1H),5.69(d,J＝10.1Hz,1H),5.27–5.22(m,1H),5.19(s,2H),4.77(dd,J＝4.5,2.0Hz,2H),4.13(d,J＝6.7Hz,2H),3.84(s,3H),1.85(s,3H),1.67(s,3H),1.47(s,6H).¹³CNMR(100MHz,CDCl₃)δ176.2,170.8,163.0(d,¹J_C-F＝246.7Hz),158.2,157.5,155.9,154.1,154.0,146.0,144.3,138.4(d,³J_C-F＝7.4Hz),137.7,131.6,130.4(d,³J_C-F＝8.4Hz),130.3,123.5,122.6(d,⁴J_C-F＝2.8Hz),120.9,116.2,115.2(d,²J_C-F＝21.3Hz),114.8,114.1(d,²J_C-F＝22.4Hz),112.2,111.2,100.2,99.2,77.7,72.8,69.8(d,⁴J_C-F＝1.9Hz),61.0,28.3,26.0,25.9,18.2.¹⁹FNMR(376MHz,CDCl₃)δ-112.3.ESI-HRMSm/z:[M+H]⁺(calcdforC₃₅H₃₃FO₈ 601.2232,found 601.2236).

(E)-4-((8-Methoxy-9-((3-methoxybenzyl)oxy)-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-6-oxo-2H,6H-pyrano[3,2-b]xanthen-5-yl)oxy)but-2-enoic acid(20a).Yield:79％.¹H NMR(400MHz,CDCl₃)δ7.33–7.21(m,4H),7.19–7.15(m,1H),6.78(s,1H),6.69(d,J＝10.1Hz,1H),6.55(s,1H),6.30(dt,J＝15.7,2.0Hz,1H),5.69(d,J＝10.1Hz,1H),5.29–5.22(m,1H),5.16(s,2H),4.77(dd,J＝4.6,2.0Hz,2H),4.12(d,J＝6.7Hz,2H),3.83(s,3H),2.39(s,3H),1.84(s,3H),1.66(s,3H),1.46(s,6H).¹³C NMR(100MHz,CDCl₃)δ176.3,171.0,158.1,157.5,156.3,154.2,154.0,146.0,144.3,138.5,137.4,135.8,131.5,130.3,129.0,128.6,128.0,124.4,123.6,121.0,116.3,114.5,112.2,111.2,100.3,99.2,77.7,72.9,70.7,60.9,28.3,26.0,25.9,21.5,18.2.ESI-HRMS m/z:[M+H]⁺(calcd for C₃₆H₃₆O₈ 597.2483,found 597.2473).

(E)-4-((8-Methoxy-9-((3-methoxybenzyl)oxy)-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-6-oxo-2H,6H-pyrano[3,2-b]xanthen-5-yl)oxy)but-2-enoic acid(20b).Yield:75％.¹HNMR(400MHz,CDCl₃)δ7.33(t,J＝7.9Hz,1H),7.25(dt,J＝15.7,4.5Hz,1H),7.08–7.01(m,2H),6.89(dd,J＝8.1,2.6Hz,1H),6.77(s,1H),6.68(d,J＝10.1Hz,1H),6.54(s,1H),6.30(dt,J＝15.7,2.0Hz,1H),5.69(d,J＝10.1Hz,1H),5.28–5.22(m,1H),5.18(s,2H),4.77(dd,J＝4.6,2.0Hz,2H),4.12(d,J＝6.7Hz,2H),3.84(s,3H),3.83(s,3H),1.84(s,3H),1.66(s,3H),1.46(s,6H).¹³C NMR(100MHz,CDCl₃)δ176.3,170.4,159.9,158.2,157.5,156.2,154.1,154.0,145.9,144.3,137.5,131.5,130.3,129.8,123.6,120.9,119.4,116.3,114.6,113.6,112.8,112.2,111.2,100.3,99.3,77.7,72.9,70.5,61.0,55.3,28.3,26.0,25.9,18.2.ESI-HRMS m/z:[M+H]⁺(calcd for C₃₆H₃₆O₉613.2432,found 613.2421).

(E)-4-((8-Methoxy-2,2-dimethyl-7-(3-methylbut-2-en-1-yl)-9-((4-(methylsulfonyl)benzyl)oxy)-6-oxo-2H,6H-pyrano[3,2-b]xanthen-5-yl)oxy)but-2-enoic acid(20c).Yield:67％.¹HNMR(400MHz,DMSO-d₆)δ8.00(d,J＝8.3Hz,2H),7.77(d,J＝8.3Hz,2H),7.12(s,1H),7.02(dt,J＝15.7,4.8Hz,1H),6.66(s,1H),6.61(d,J＝10.2Hz,1H),6.11(dt,J＝15.7,1.9Hz,1H),5.90(d,J＝10.1Hz,1H),5.44(s,2H),5.17–5.10(m,1H),4.65(dd,J＝4.8,1.9Hz,2H),4.01(d,J＝6.7Hz,2H),3.76(s,3H),3.24(s,3H),1.77(s,3H),1.61(s,3H),1.43(s,6H).¹³C NMR(100MHz,DMSO-d₆)δ175.6,167.3,158.2,157.3,156.1,154.0,153.9,144.2,143.6,142.6,140.9,136.5,131.5,131.0,128.5,127.8,124.1,122.8,115.8,114.2,112.3,111.1,100.3,100.1,78.3,73.3,69.7,61.0,43.9,28.3,26.1,25.8,18.5.ESI-HRMS m/z:[M+H]⁺(calcd for C₃₆H₃₆O₁₀S 661.2102,found 661.2090).

EXAMPLE 7 determination of the inhibitory Activity of Compounds on PDE4

The inhibitory activity of the compounds on PDE4D2 was determined, as represented by compound 18a, and their selectivity for the PDEs subtypes was determined, with the other compounds having similar effects to compound 18 a.

The experimental method comprises the following steps:

the PDEs expressing plasmids were first transformed into e.coli (e.coli) strain BL21(codonplus), and recombinant plasmid-carrying e.coli (e.coli) cells were grown in LB medium at 37 ℃ to a600 ═ 0.7, followed by the addition of 0.1mM isopropyl- β -D-thiogalactopyranoside (IPTG) for induction of expression and culture at 15 ℃ for 20 h. Recombinant PDEs catalytic domains were purified by Ni-NTA affinity column (Qiagen), Q column (GE Healthcare) and Superdex 100 column (GE Healthcare).

To be provided with³Guanosine H-cyclic phosphate (cGMP) or³Measuring Phosphodiesterase (PDEs) activity by taking H-cyclic adenosine monophosphate (cAMP) as a substrate; the detection buffer contained 20mM Tris-HCl (pH 7.5), 10mM MgC l₂Or 4mM MnCl₂1mM DTT and 10-30 nM³H-cGMP or³H-cAMP (20,000-30,000 CPM/assay); the reaction was carried out at room temperature for 15min, and then 0.2M zinc sulfate was added to terminate; reaction product (A)³H-cyclic guanosine monophosphate cGMP or³H-cyclic adenosine monophosphate cAMP) is precipitated with 0.2N barium hydroxide, while unreacted: (³H-cyclic guanosine monophosphate cAMP or³cGMP) was retained in the supernatant, measured by Perkinelmer 2910 liquid scintillation counter, and IC was calculated by non-linear regression₅₀The value is obtained.

The experimental results are shown in tables 1-2.

Inhibitory Activity of the Compounds of Table 1 on PDE4D2

As can be seen from the table, the present invention providesHas significant inhibitory activity on PDE4D2, and most of the compounds IC₅₀Significantly less than the positive control Rolipram (Rolipram); among them, compounds 16a, 18d and 19b had poor inhibitory effect and IC₅₀>1000。

TABLE 2 Compound 18a Selectivity for PDEs subtypes

PDEs	IC50(nM)	Index of selectivity
			PDE4D2(86-413)	4.2±0.5	-
PDE4B2(152-487)	140±18	33
			PDE1C(147-531)	440±92	104
PDE2A(580-919)	940±79	223
			PDE3A(679-1087)	>10000	>2300
PDE5A1(535-860)	93±20	22
			PDE7A1(130-482)	298±9	70
PDE8A1(480-828)	710±65	169
			PDE9A2(181-506)	>10000	>2300
PDE10A2(449-770)	260±6	61

As can be seen from the table, compound 18a showed very weak inhibitory effect on PDE3A and PDE9A, IC₅₀The value exceeds 10 mu M, and the selectivity multiple exceeds 2000; compound 18a showed a comparison of PDE4D with PDE1C, PDE2A and PDE8A against PDE4D>100-fold selectivity, compared with PDE5A, PDE7A and PDE10A>A selectivity of 22 times; compound 18a also showed 33-fold moderate selectivity over PDE 4B. The above results indicate that compound 18a is a selective PDE4D inhibitor.

EXAMPLE 8 pharmacokinetic and drug-like assays for Compound 18a

Taking compound 18a as a representative, the pharmacokinetics and drug-like properties of compound 18a were determined, and the effects of other compounds were similar to those of compound 18 a.

The experimental method comprises the following steps:

(1) pharmacokinetic experiments: SD rats 6 (3 intravenous injections and 3 oral injections) were selected, plasma was collected at 10 time points at different time points after administration,determining the concentration of the compound in plasma by HPLC-MS/MS method, and analyzing the pharmacokinetic parameter AUC_(0-t)、AUC_(0-∞)t_1/2、T_max、C_maxAnd the like.

(2) In vitro liver microsome stability test

Preparing 0.5mM test sample or positive control (ketanserin); 1.5 mu M of a test sample or a positive control sample is put in a liver microsome suspension of 0.75 mg/mL; 6mM NADPH in PBS; 30 μ L of 0.75mg/mL liver microsome suspension containing 1.5 μ M compound or positive control per 96-well plate at time points (0, 5, 15, 30, 45 and 60 min); adding 150 μ L of ACN containing internal standard at 0 point, adding 15 μ L of prepared NADPH (6mM), and packaging the reaction plate; preheating a 96-well reaction plate in a water bath at 37 ℃ for 5min, and adding 15 mu L of prepared NADPH (6mM) to start reaction (the final concentration of the compound and a positive control in the reaction system is 1 mu M); adding 150 μ L of ACN containing internal standard at 5, 15, 30, 45 and 60min to terminate the reaction; shaking the reaction plate for 5min, and storing the sample in a refrigerator at-80 deg.C until the sample is measured; centrifuging the sample at 4000rpm for 15min before sample measurement, taking 80 microliter of supernatant, adding 80 microliter of ultrapure water, mixing uniformly, and performing LC-MS/MS sample injection analysis.

(3) Acute toxicity test in mice: 30 adult clean-grade Kunming mice are taken, 10 mice are taken in each group, 3 groups are divided into a blank control group (solvent), a normal group and 1500mg/kg at random. The drug was suspended in 0.5% CM C-Na and administered orally at 1 ml/kg. The animals were observed for their response immediately after administration, for 12 hours, once a day, for 14 consecutive days, and the acute toxicity of the compound was evaluated.

The results are shown in tables 3-4 and FIG. 1.

TABLE 3 pharmacokinetic profiles of Compound 18a in SD rats (oral administration 5mg/kg)

As can be seen, after oral administration of 5mg/kg Compound 18a to SD rats, t_1/2、C_maxAnd AUC were 1.02h, 1067ng/mL, and 2106h ng/mL, respectively.

TABLE 4 determination of drug-like Properties of Compound 18a

As can be seen from the table, IC of compound 18a to human cytochrome P450 enzymes CYP1A2, 2B6, 2D6 and 3A4 subtype₅₀IC for CYP2C9 with a value of greater than 25 μ M₅₀The value was 1.7 μ M, indicating that compound 18a does not generally induce drug-drug interactions; and IC of Compound 18a on the hERG potassium channel₅₀Values greater than 30 μ M, with lower risk of cardiotoxicity; in addition, acute toxicity to mice at a dose of 1.5g/kg was evaluated, and the results, see fig. 1, show that compound 18a exhibits good safety without acute toxicity at an oral dose of 1.5 g/kg.

In conclusion, compound 18a has reasonable selectivity and physicochemical properties, and is suitable for subsequent pharmacodynamic studies. Other compounds also have similar properties.

EXAMPLE 9 Compound 18a anti-IPF assay

The experimental method comprises the following steps: 40 male SD rats (approved by the ethical Committee of the institute of animal research organization of Zhongshan university (SYSU-IAUC-2021-. Before the experiment, the rats had free access to food and water, then the control group was treated with normal saline, and the rest were subjected to a single intratracheal instillation with BLM at a dose of 5 mg/kg; treatment starting on the next day, with the drug vehicle (control), compound 18a (10.0mg/kg) or PFD (150mg/kg) for 4 weeks: compound 18a and PFD were dissolved in 0.5% sodium carboxymethylcellulose solution and administered orally at a dose of 0.4mL/100 g. Changes in weight, hair and respiration status were observed and recorded throughout the experimental interval. After 28 days of oral administration, the respiratory level of each group was measured.

At the end of the assay, rats were injected intraperitoneally with 4% sodium pentobarbital, 10mL of blood was drawn from the abdominal aorta, centrifuged at 2800 rpm and 4 ℃ for 10min, and serum was collected and stored in a refrigerator at-80 ℃. The lower left lung fracture was removed after euthanasia, and the tissues were immersed in 4% buffered paraformaldehyde at room temperature and embedded in paraffin to make 5 μm thick sections, mounted on slides, and hematoxylin-eosin and Masson (Masson) stained.

The lung tissue of each group was homogenized in lysis buffer supplemented with 1% protease inhibitor cocktail and 1mM phenylmethanesulfonyl fluoride, the lysates were centrifuged at 12,000 g and 4 ℃ for 10min, and the supernatants were collected for subsequent immunoblot analysis.

20 μ g of each supernatant sample described above was used for western blotting: protein samples were separated by SDS-PAGE and then transferred to polyvinylidene fluoride membranes (Millipore USA), which were blocked with 5% skimmed milk powder in Tris-buffered saline Tween 20(TBS-T) for 1.5h at room temperature and probed with the following primary antibodies continuously overnight at 4 ℃: anti-FN antibody (1:5000, Abcam, ab2413, USA), anti- α -SMA antibody (1:300, Abcam, ab7817, USA) and anti-GAPDH antibody (1 μ g/mL, Abcam, ab9484, USA); the membranes were then stained with the corresponding goat anti-mouse lgG secondary antibody (Boster, china) for 1 hour at room temperature, and finally, the signal was visualized using ECL reagent (usa) and the immunoblot bands were densitometric using Bio-Rad Quality One software.

Referring to fig. 2, it can be seen from fig. 2A that the lung function parameters including flow rate in expiration (EF50), peak flow rate in expiration (PEF), respiratory rate (n), and enhanced pause (gold border) were significantly increased in the model group (Mod) rats compared to the control group (Con), indicating successful establishment of the BLM-induced IPF model; under the same conditions, compound 18a at an oral dose of 10mg/kg had better anti-IPF effect than the commercial drug Pirfenidone (PFD) at an oral dose of 150mg/kg after 28 consecutive days. Also, lung function-related parameters such as EF50, PEF and gold-edged index were restored to levels close to those of the control group in the compound 18a group, indicating that the PDE4 inhibitor compound 18a has a significant effect in improving lung function in IPF rats.

As can be seen in FIGS. 2B and 2C, hematoxylin-eosin staining showed that the alveolar disorder of the model group, the alveolar wall was ruptured and fused, and there was significant inflammatory cell infiltration; both compound 18a (10mg/kg) and PFD (150mg/kg) were effective in reducing structural damage to the lung and fibrotic lesions. In addition, the expression of collagen deposition, one of the pathological features of IPF, was greatly increased, as shown by Masson stained sections, with a large area of collagen deposition and cell proliferation 5C around the trachea in the model group, while both compound 18a and PFD almost completely inhibited collagen deposition to normal levels.

As can be seen in fig. 2D, compound 18a and PFD significantly reduced the levels of α -SMA and Fibronectin by western blotting, up-regulating the expression levels of the marker proteins α -smooth muscle actin (α -SMA) and Fibronectin (Fibronectin) in the lung tissue of IPF relative to the expression levels in the model group.

The above results all indicate that the PDE4 inhibitor compound 18a improves Bleomycin (BLM) -induced pulmonary fibrosis.

EXAMPLE 10 Compound 18a inhibits Epithelial Mesenchymal Transition (EMT) in vitro

Epithelial-mesenchymal transition is an important pathogenesis of IPF disease, and can be induced by the pre-fibrotic cytokine TGF-beta (transforming growth factor-beta), during which the expression levels of hall markers such as Fibronectin (Fibronectin), N-cadherin (N-cadherin) and vimentin (vimentin) are changed. This example further assesses the anti-IPF potential of compound 18a in a TGF- β induced a549 cell model by immunoblot (western bolt) analysis.

The experimental method comprises the following steps: a549 cells (purchased from Shanghai cell Bank of Chinese academy of sciences) were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum and placed in a medium containing 5% CO₂The constant temperature incubator is 37 ℃; a549 cells at 5X 10⁵Inoculating the seeds/mL into a cell culture dish, and culturing overnight; dividing cells into blank control group, model group and administration group, adding 10ng/mL TGF-beta except blank group, and administering groupCompound 18a was added at final concentrations of 20 μ M and 10 μ M; after 48h incubation, the cell dish was placed at 0 ℃ and the medium was discarded, followed by three washes with 4 ℃ PBS solution; total cellular protein was extracted with RIPA lysate and protein was quantified using BCA method: protein levels in each set of samples were determined by Western blotting using antibodies to N-cadherin (1:1000, CST, 13116, usa), vimentin (1:1000, CST, 5741, usa) and FN (1:5000, Abcam, ab2413, usa). All results are given as average scanning electron microscopy. A p-value of less than 0.05 was considered statistically significant using one-way ANOVA with Ponferroni (Bonferroni) multiple comparisons. The results are shown in FIG. 3.

As can be seen, the expression levels of Fibronectin (Fibronectin), N-cadherin and vimentin in A549 cells were significantly up-regulated 48h after TGF-beta-induction; compound 18a treatment significantly reduced the high expression of N-cadherin, vimentin fibers, and fibronectin in a dose-dependent manner (10-20 μmol/L), indicating that the PDE4 inhibitor, Compound 18a, prevented TGF- β induced EMT processes.

EXAMPLE 11 Compound 18a beagle emetic assay

The experimental method comprises the following steps: the beagle dogs (8 male beagle dogs purchased from Guangzhou institute of medicine, Inc., IAC No: SYSUIAUC-2021-; animals were observed for a continuous period of 180 minutes for retching, excessive salivation, and vomiting. See table 5 for results.

TABLE 5 Compound 18a Biggee emetic test results

Medicine	Dosage (mg/kg, p.o.)	Number of vomited animals/number of test animals
			Compound 18a	10	0/4
Rolipram	1	4/4

As can be seen from the table, compound 18a of the present invention does not cause emesis and is more compliant when administered orally than rolipram.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.