CN114404425A

CN114404425A - Inhibitor of phosphodiesterase and application thereof

Info

Publication number: CN114404425A
Application number: CN202210100834.4A
Authority: CN
Inventors: 马宏跃; 周婧
Original assignee: Nanjing University of Chinese Medicine
Current assignee: Nanjing University of Chinese Medicine
Priority date: 2022-01-27
Filing date: 2022-01-27
Publication date: 2022-04-29

Abstract

The invention discloses an inhibitor of phosphodiesterase, and application of bufadienolide, bufanolide or bufotoxin compounds in preparation of the inhibitor of phosphodiesterase. The research of the invention finds that the compound has high binding force with PDE4D and can raise cAMP level in cells. Thus can be used for preparing anti-inflammatory, immunoregulation, neuroprotection and anti-tumor effects.

Description

Inhibitor of phosphodiesterase and application thereof

Technical Field

The application relates to the field of application of pharmaceutical compounds, in particular to a bufadienolide compound serving as a PDE4D enzyme inhibitor. In essence, the bufadienolide compound is found to be a novel PDE4D inhibitor, so that the bufadienolide compound has wide pharmacological effects.

Background

PDEs are collectively known as phosphodiesterases (phosphodiesterases), and comprise 11 families of super enzymes (PDE 1-PDE 11), which catalyze the hydrolysis of second messenger cAMP and/or cGMP, playing an important role in cellular metabolism. PDE4 is a member of the PDE family and exerts a range of actions by specifically hydrolyzing cAMP to regulate the production of pro-inflammatory, anti-inflammatory cytokines. Inhibition of PDE4 activity in downstream pathways may increase intracellular cAMP levels and activate protein kinase a (pka), thereby inhibiting signaling pathways such as nfkb and NFAT and reducing downstream cytokine and chemokine release. These factors control the expression of inflammatory mediators such as IL-2, IL-4, IL-6, IL-31 and TNF-alpha, which in turn may regulate inflammatory responses of cells such as T cells, Th2 cells, such as neutrophil degranulation, chemotaxis and adhesion to endothelial cells. In addition, because PDE4 is distributed in immune cells, epithelial cells, and brain cells, inhibition of PDE4 produces a very broad anti-inflammatory effect. In addition to T cells and Th2 cells, inhibition of PDE4 may also inhibit the inflammatory response of macrophages, Dendritic Cells (DCs), Th1, Th17 cells, and interfere with the phenotype and function of B cells. In addition, inhibition of inflammatory mediators can also enhance the barrier function of keratinocytes and epithelial cells.

PDE4 contains at least four subtypes A, B, C and D. Their respective functions are not clearly studied at present. PDE4D is quite complex, with 9 different isoforms, whose functions are also widely involved in inflammation and neurological and psychiatric disorders. One broad class of indications for PDE4D inhibitors are inflammatory diseases such as laryngopharyngitis, pneumonia, Chronic Obstructive Pulmonary Disease (COPD), asthma and rheumatoid arthritis, psoriasis, systemic lupus erythematosus, dermatitis. Since PDE4D inhibitors have the effects of improving long-term memory, promoting wakefulness, neuroprotection, and the like, PDE4 inhibitors have a therapeutic pharmacological effect on various central nervous system diseases, such as major depressive disorder, anxiety disorder, schizophrenia, parkinson's disease, alzheimer's disease, multiple sclerosis, attention deficit hyperactivity disorder, huntington's disease, stroke, autism, and the like.

Most of the existing PDE4D medicines are in the clinical development stage, and part of the compounds are not successfully marketed due to the problems of patent medicine property and toxic and side effects. The search for novel PDE4D inhibitors is of great value.

Bufadienolide is a natural cardiac glycoside substance, and is separated from secretion of Bufo animal. The method comprises the following steps: resibufogenin (resibufogenin), bufalin (bufalin), cinobufagin (cinobufagin), bufotalin (bufotalin), desacetylbufotalin (desacet-cinobufagin), south america bufagin (marinobafacin), cinobufotalin (cinobufotalin), arenobufagin (arenobufagin), bufotalin (bufotalin), phlorogin (helbregnenin), gamabufotalin (gamabutatalin), telebufagin (telocinobufagin), and the like. The bufogenin compounds usually form esters with arginine fatty diacid, and become bufotoxins. Toad toxins can be classified into three categories according to the difference of the acids to which they are linked, namely fatty acid amino acid esters (such as suberic acid arginine ester, pimelic acid arginine ester, etc.), fatty acid esters (C-3 position linked fatty acid esters such as hemisuberic acid ester, hemisuccinate ester) and sulfate esters (C-3 position linked with sulfate ester). Such as resibufogenin-3-sulfate-arginine ester, bufalinin-3-sulfate-arginine ester, resibufogenin-3-sulfate-arginine ester, resibufagin-3-sulfate-arginine ester, bufalinin-3-sulfate-arginine ester, cinobufagin-3-sulfate-arginine ester, desacetyllipobacterium-3-sulfate-arginine ester, arenobutain-3-sulfate-arginine ester, telogen-3-sulfate-arginine ester, and bulganin-3-sulfate-arginine ester. Bufadienolide derived from plant has multiple linked sugar chains at C-3 position, including monosaccharide, disaccharide and polysaccharide. The bufadienolide obtained by means of microbial catalysis has hydroxyl group substitution on steroid parent nucleus. Chemically modified bufadienolide generally has various substituents at the C-3 position. But the basic parent nucleus of the compound is completely the same, so the compound has high approximate pharmacological activity.

Disclosure of Invention

In order to find a novel PDE4D inhibitor, the invention provides a parent nucleus compound with a cardiac glycoside structure as a PDE4D enzyme inhibitor and application thereof in preparing medicaments.

In order to achieve the technical purpose, the invention adopts the following technical scheme:

an inhibitor of phosphodiesterase, which has a structural mother nucleus of cardiac glycoside compounds.

Preferably, the phosphodiesterase inhibitor is characterized in that the cardiac glycosides comprise bufadienolide or bufanolide or bufotoxin compounds.

Preferably, the cardiac glycoside component is a compound which is connected with alpha-pyrone ring (six-membered unsaturated lactone ring) at C-17 position, cis or trans fusion of A/B ring, trans fusion of B/C ring, and cis or trans fusion of C/D.

Preferably, the phosphodiesterase inhibitor is a cardiac glycoside in which the hydroxyl group at the C-3 position of the mother nucleus is present in a free state or in a plurality of derivative states, including but not limited to arginine dicarbonate, fatty acid ester, sulfate, diacid ester, glycosyl, disaccharide, trisaccharide, methoxy group, and nitrogen-containing group.

Preferably, the phosphodiesterase inhibitor is prepared by substituting hydroxyl, methoxy, methyl, carbonyl or acetyl at C-5, C-11, C-12, C-13 and C-16 positions of a bufadienolide parent nucleus.

Preferably, the C-14 position of the parent nucleus of bufadienolide is substituted by hydroxyl, epoxy, methyl, carbonyl or acetyl.

Experiments show that compounds with cardiac glycoside structure mother nucleus such as bufadienolide, bufanolide or bufotoxin can be used for preparing phosphodiesterase inhibitor, especially PDE4D enzyme inhibiting and cAMP increasing activities. Thus can be used for preparing anti-inflammatory and treating central nervous system diseases, including pharyngolaryngitis, pneumonia, chronic obstructive pulmonary disease, asthma, rheumatoid arthritis, psoriasis, systemic lupus erythematosus, and dermatitis; central nervous system disorders include major depressive disorder, anxiety disorder, schizophrenia, parkinson's disease, alzheimer's disease, multiple sclerosis, attention deficit hyperactivity disorder, huntington's disease, stroke, or autism.

Drawings

FIG. 1 shows that 10 bufadienols were significantly different from the control group in binding to PDE4D by ultrafiltration-mass spectrometry (P <0.001)

FIG. 2 shows that bufadienolide inhibits PDE4D activity; bufadienolide inhibits PDE4D activity. Inhibitory activity is reflected in cAMP content. (x P)<0.001, compared with a blank control group, the administration group has significant difference;^###P<0.001, there was a significant difference after administration of the drug plus Roflumilast compared to the corresponding administration group. )

FIG. 3 shows analgesic, anti-infective and anti-inflammatory activities of bufadienolide film; the bufadienolide has analgesic, anti-infectious and anti-inflammatory effects. (A) von Frey fibromyalgia threshold assay. (B) Bacterial numbers in skin tissue. (C) And observing the surface of the skin wound position. (D) HE staining of the skin showed inflammatory cell infiltration. MG: a model group; LT: low dose BF (0.05%); MT: medium dose BF (0.1%); HT: high dose BF (0.2%); PG: erythromycin ointment group. Data are expressed as Mean ± SEM, with n ═ 5.^#P<0.05，^##P<0.01, comparing the model group with the control group;^*P<0.05，^**P<0.01, treatment group compared to model group. (Red color)The arrow points to the stratum corneum; green arrows point to local fibroblasts; blue arrows point to bleeding points; yellow arrows point to inflammatory cells).

Figure 4 bufadienolide inhibits inflammatory mediators; (A) bufadienolide inhibits inflammatory mediators. (B) COX pathway-associated lipid metabolism changes. Each set of data is expressed as Mean ± SEM, with n ═ 4.^#P<0.05，^##P<0.01, comparing the model group with the control group;^*P<0.05，^**P<0.01, treatment group compared to model group.

FIG. 5 is a structural diagram of free bufadienolide compounds.

FIG. 6 is a structural diagram of free bufadienolide compounds.

FIG. 7 is a structural diagram of conjugated bufadienolide compounds.

FIG. 8 is a structural diagram of conjugated bufadienolide compounds.

Detailed Description

The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. The materials, reagents and the like used are all commercially available reagents and materials unless otherwise specified.

Example 1

1. Binding experiments of bufadienolide component to PDE4D

1.1 sample preparation

Control group: 1 mu g/mL bufadienolide mixed standard solution (including ester bufogenin, cinobufagin, bufalin, cinobufotalin, deacetyl cinobufotalin, bufotalin, telocinobufagin, bufotalidin, concentration is 1 mu g/mL).

Negative control group: 1 mu g/mL bufadienolide mixed standard solution +10 mu g pancreatin protein solution.

Experimental groups: 1 mu g/mL bufadienolide mixed standard solution +10 mu g PDE4D protein solution.

1.2 sample Ultrafiltration

And (3) placing the control group, the negative control group and the experimental group sample at 4 ℃ for incubation for 30min, and incubating at normal temperature for 30 min. After incubation, the samples were transferred to the upper layer of the ultrafiltration tube and centrifuged at 14000rpm for 30min at 4 ℃. Adding buffer 500 μ L into the upper layer, centrifuging at 14000rpm and 4 deg.C for 30min, transferring the residual solution in the upper layer into a new ultrafiltration tube, centrifuging at 14000rpm and 4 deg.C for 30min, and collecting the upper solution in the ultrafiltration tube. Collecting the upper layer solution, adding 100 μ L pure methanol solution into a new tube, centrifuging at 14000rpm at 4 deg.C for 20min, collecting the supernatant, centrifuging, concentrating, adding 50 μ L50% methanol solution (containing 0.1% formic acid) into each upper layer sample, and redissolving to obtain the final product.

1.3 LC-MS/MS detection conditions

Liquid phase conditions: shimadzu LC 30AD liquid chromatograph (Shimadzu, japan); waters Xbridge C18 column (4.6X 100mm, 3.5 μm); phase A is 0.1% formic acid water, phase B is acetonitrile; liquid phase gradient: 0-3 min, 10-90% of B, 3-4.5 min, 90-90% of B, 4.5-6 min, 90-10% of B, 6-7 min, 10-10% of B, 7-8.1 min and 10% of B; the sample injection amount is 5 mu L; flow rate: 0.5 mL/min.

Mass spectrum conditions: using QTRAP 4500 mass spectrometer (AB SCIEX, usa), ESI ion source, MRM positive ion mode, spray voltage: 5500V; atomization temperature 500 ℃, gas curtain gas: 30 psi; atomizing: 50psi, assist gas: 50psi, collisional gas: 10psi, Q1 scan speed: 10Da/s, Q3 scanning speed: 10 Da/s.

1.4. Experimental results of binding of bufogenin and PDE4D

The binding of bufogenin to PDE4D was determined using ultrafiltration-mass spectrometry. And evaluating the strength of the binding force according to the residual bufadienolide on the upper layer of the ultrafiltration tube. The results show that compared with the control group, the interaction with negative control Tyrisin ultrafiltration does not change significantly in the upper 10 bufadienols, the interaction with PDE4D shows that the content of the ultrafiltration in the upper 10 bufadienols is obviously improved, and the 10 bufadienols are possibly combined with PDE4D to cause the content to be improved.

Example 2

2. Evaluation of inhibition of bufadienolide on PDE4D

2.1 Experimental methods

Cellular cAMP levels were used to reflect PDE4D enzyme activity. The normal liver cancer HepG2 cells were seeded in 96-well plates at 5X 104 cells/well at 100. mu.L/well. The cells are cultured overnight, and after the cells are attached to the wall, the drugs are added, and the cells are cultured for 24 h. After 24h, 200. mu.L of LPBS was added to wash the cells, 100. mu.L of 0.6mol/L perchloric acid was added, the cells were incubated on ice for 30min, and 33. mu.L of 2.5mol/L sodium hydroxide was added to neutralize the cells. Centrifugation was carried out at 12000rpm for 30min at 4 ℃ to collect the supernatant. ELISA kit determination of cAMP. Adding 50 mu L of sample and standard substance to the bottom of the well of the enzyme-labeled plate, immediately adding 50 mu L of prepared biotinylation antibody working solution, and incubating for 45min at 37 ℃ after sealing the plate by a sealing membrane. Removing the sealing plate film, discarding the liquid, adding 350 μ L of washing solution into each hole, soaking for 1-2 min, discarding, and repeating for 3 times. Adding 100 μ L of the working solution of enzyme conjugate into each well, mixing, incubating at 37 deg.C for 30min after sealing with a plate membrane, removing the plate membrane, discarding the solution, adding 350 μ L of washing solution into each well, soaking for 1-2 min, discarding, and repeating for 5 times. Adding 90 μ L of substrate solution (TMB) into each well, mixing, sealing plate, and developing at 37 deg.C in dark for 15 min. The reaction was terminated by adding 50. mu.L of a terminator solution, and the absorbance at 450nm was measured immediately.

2.2 inhibition of the PDE4D enzyme by Bufonis venenum sterol

After 300ng/mL Roflumilast administration, the cAMP content is remarkably reduced to (2.54 +/-1.11) ng/mL, after 100ng/mL and after 300ng/mL of Roflumilast administration, the cAMP content is remarkably increased to (24.69 +/-1.32) ng/mL and (37.06 +/-1.52) ng/mL (P <0.001), and after Arenofagin (100ng/mL and 300ng/mL) is administered in combination with Roflumilast (300ng/mL), the cAMP content is remarkably reduced to (17.6 +/-1.06) ng/mL and (27.44 +/-1.39) ng/mL, and the difference is remarkable compared with the corresponding administration group (P < 0.001).

After Bufotalin administration at 100ng/mL and 300ng/mL, the cAMP content was significantly increased to (21.73. + -. 1.57) ng/mL and (63.03. + -. 4.99) ng/mL (P <0.001) compared to the blank control group, while Bufotalin (100ng/mL and 300ng/mL) was administered in combination with Roflumilast (300ng/mL), with a significant decrease in cAMP to (1.62. + -. 0.61) ng/mL and (7.01. + -. 0.95) ng/mL, with significant difference (P <0.001) compared to the corresponding administration group.

After administration of 100ng/mL and 300ng/mL Telocinobufagin, the cAMP content was significantly increased to (20.62 + -1.86) ng/mL and (30.08 + -2.7) ng/mL (P <0.001) compared to the blank control group, while administration of Telocinobufagin (100ng/mL and 300ng/mL) in combination with Roflumilast (300ng/mL) had a significant decrease in cAMP to (11.41 + -1.74) ng/mL and (18.01 + -1.36) ng/mL, with significant difference (P <0.001) compared to the corresponding administration group.

After administration of 100ng/mL and 300ng/mL Bufotalidin, the cAMP content was significantly increased to (26.86. + -. 1.82) ng/mL and (41.64. + -. 1.25) ng/mL (P <0.001) compared to the blank control group, while administration of Bufotalidin (100ng/mL and 300ng/mL) in combination with Roflumilast (300ng/mL) had a significant decrease in cAMP to (5.95. + -. 1.42) ng/mL and (12.85. + -. 0.97) ng/mL, with a significant difference (P <0.001) compared to the corresponding administration group.

Thus, Arenobufagin, Bufotalin, Teloxicobufagin and Bufotalidin (100ng/mL and 300ng/mL) increased the intracellular cAMP levels, presumably by acting in conjunction with PDE 4D.

Example 3 pharmacological testing of bufadienolide against inflammation, pain, and infection

1. Experimental methods

1.1 preparation of bufadienolide coating agent

PVA is used as a main film forming matrix, and sodium carboxymethylcellulose (CMC-Na) is used for improving the film texture in an auxiliary way. Glycerin as plasticizer, azone as penetration enhancer, and ethanol as solvent and antiseptic of bufadienolide. The PVA was dissolved in water at 80 ℃ under sealed stirring and heating. Bufadienoline was then dissolved in ethanol (20%, w/w) and appropriate amount of water. Adding the cooled PVA matrix and related auxiliary materials, ultrasonically mixing uniformly, preparing a film coating agent, and sealing and storing.

1.2 animal grouping and modeling

Negative control (skin damage, n ═ 10)

Positive control (skin damage + bacterial infection, n ═ 10)

Yang medicine (skin damage + bacterial liquid infection, daubing erythromycin ointment, n ═ 10)

Bufadienolide plastics has high content of (skin damage + bacterial liquid infection, smearing venenum Bufonis)0.2% (w/w) of film coating agent, 50mg/cm²，n＝10)

Toad steroid alkene coating agent (skin breakage + bacterial liquid infection, coating toad venom coating agent 0.1% (w/w),50 mg/cm)²，n＝10)

The toad steroid alkene plastics has low content (skin breakage + bacterial liquid infection, toad venom plastics 0.05% (w/w),50 mg/cm)²，n＝10)

Selecting healthy rabbits, disinfecting with 75% alcohol, cutting the skin of the area with a syringe needle, cutting # characters at intervals of about 3mm, only damaging the epidermis without reaching the dermis, and smearing 0.1ml of bacterial liquid. After 4h, 0.1mL of the bacterial suspension was applied again to increase the infection level. The following day, localized redness and swelling of the skin with an increase in temperature was seen. The reaction indexes are that the rabbit skin shakes, the back bends or avoids. The administration was once daily, 0.3 mL/dose, and the pain threshold was measured before administration. The mental state and the diet condition of the rabbits were observed every day, and the general condition of the skin surface of the rabbits was observed and recorded. At D3, 5 rabbits per group were sacrificed and skin tissue was harvested for determination of skin charge and lipidomics. At D7, skin tissue was taken for HE staining.

1.3 skin lipidomics assay

Skin tissue was weighed in advance, then cut into pieces and ground into a homogenate with a mortar, and 2ml of a 4 ℃ pre-cooled physiological saline was added while grinding, to disperse the tissue sufficiently. Then 2ml of a mixed solution of n-hexane and ethyl acetate precooled at the temperature of 80 ℃ below zero (v: v ═ 1:1) is added, vortex mixing is carried out, ice bath ultrasound is carried out for 20min after vortex mixing, centrifugation is carried out for 15min under the condition of 3000rpm after standing for 5min, and supernate is collected. Then extracting again, combining the two extracting solutions, concentrating and volatilizing. Samples were analyzed using UPLC-QTRAP-MS (AB 5500).

2 anti-inflammatory, analgesic and anti-infective test results

2.1 analgesic, anti-infective and anti-inflammatory Activity of bufadienolide coating Agents

As shown in FIG. 3 below, the present invention evaluated the analgesic activity of toad venom film-coating agents using von Frey cellosilk. The results in fig. 3A show that toad venom film-coats (0.2% and 0.1%, 50mg/cm2) significantly increased the pain threshold of rabbit skin in a dose-dependent manner (P < 0.01). Exhibit significant anti-infective activity.

High and medium dose film coatings (0.2%, 0.1% and 0.05%) all significantly reduced bacterial numbers at D3, with a significance level of P <0.01 (fig. 3B). Bufadienolide exhibited significant anti-infective activity.

The results of gross observation of rabbit skin (fig. 3C) show that at D7, the toad venom film-coating agent high dose group had intact skin and the wound healed significantly compared to the model group. Histopathological observation shows that the inflammatory cell infiltration of the coating agent in the high-dose group is reduced. The skin lesion sites in the model group and the low dose group were still seen to have more inflammatory cell infiltration and local bleeding. Bufadienolide exhibited significant anti-inflammatory activity.

2.2 Activity of bufadienolide anti-inflammatory mediators

47 inflammatory lipids were detected and quantified, including those from the Cyclooxygenase (COX), Lipoxygenase (LOX), docosahexaenoic acid (DHA), and Linoleic Acid (LA) pathways. From the heat map of fig. 4A, the 0.2% and 0.1% doses of toad venom film-coating agents greatly reduced lipid upregulation in the model group. Figure 4B shows some of the lipids that changed significantly. Specifically, upregulation of several metabolites produced by the COX pathway showed a decrease in the BF panel, including 2,3-Dinor-TXB2, 9(S) -HpOTrE, 13(S) -HpOTrE, 2, 3-dinitro-PGE 1, 6-keto-PGF1 alpha, 13, 14-dihydro-PGE 1, etc. (P < 0.05).

Example 3 antidepressant Activity test

Mice were randomized into normal, model, fluoxetine positive control and bufadienolide groups, with 12 mice per group. Three consecutive days, i.e. 0.8mg/kg LPS lipopolysaccharide is intraperitoneally injected, after half an hour of the last injection, fluoxetine (30mg/kg) is intraperitoneally injected, a toad venom solution (containing 15.76 mu g/mg of gamabufotalin, 8.85 mu g/mg of arenobufagin, 3.00 mu g/mg of telocinobufagin, 2.88 mu g/mg of bufotalin, 5.19 mu g/mg of cinobufotalin and 4.52 mu g/mg of bufalin) is injected, the low dose is 40mg/kg, the high dose is 80mg/kg, the tail suspension immobility time and forced swimming immobility time of the mice are measured, and the cumulative immobility time of 4min after 6min is recorded. As shown in table 2 below, bufadienolide-enriched venenum bufonis exhibited significant anti-depressant pharmacological effects.

TABLE 2 antidepressant Activity test

Taken together, the above results indicate that bufadienolide is an inhibitor of PDE4D and is capable of elevating intracellular cAMP levels. After being prepared into a film coating agent, the compound shows obvious anti-inflammatory, analgesic and anti-infective activities in a staphylococcus aureus infection model, and also has a certain antidepressant activity.

The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications or equivalents may be made to the technical solution without departing from the principle of the present invention, and these modifications or equivalents should also be regarded as the protection scope of the present invention.

Claims

1. An inhibitor of phosphodiesterase, characterized in that the inhibitor has a structural mother nucleus of cardiac glycoside compounds.

2. The phosphodiesterase inhibitor according to claim 1, wherein the cardiac glycoside comprises a bufadienolide or a bufanolide or a bufotoxin-like compound.

3. The inhibitor of phosphodiesterase according to claim 2, wherein the cardiac glycoside is a compound in which the α -pyrone ring is linked to the C-17 position, the a/B ring is fused in cis or trans, the B/C ring is fused in trans, and most of C/D is fused in cis or trans.

4. The inhibitor of phosphodiesterase according to claim 3, wherein the hydroxyl group at C-3 of the mother nucleus of the cardiac glycoside is present in free form or in several derivatives including but not limited to arginine dicarbonate, fatty acid esters, sulfates, diesters, glycosyl, disaccharide, trisaccharide, methoxy, nitrogenous groups.

5. The inhibitor of phosphodiesterase according to any one of claims 1 to 4, wherein the C-5, C-11, C-12, C-13 and C-16 positions of the parent nucleus of bufadienolide are substituted by hydroxyl, methoxy, methyl, carbonyl or acetyl.

6. The phosphodiesterase inhibitor according to claim 5, wherein the C-14 position of the bufadienolide nucleus is substituted by a hydroxyl group, an epoxy group, a methyl group, a carbonyl group or an acetyl group.

7. Application of compound with cardiac glycoside structure mother nucleus in preparing phosphodiesterase inhibitor is provided.

8. The use of claim 7, wherein the phosphodiesterase inhibitor is an inhibitor of PDE 4D.

9. Application of bufadienolide, bufanolide or bufotoxin compounds in preparing medicine for resisting inflammation and treating central nervous system diseases is provided.

10. The use of claim 9, wherein the inflammatory disease is selected from the group consisting of pharyngolaryngitis, pneumonia, chronic obstructive pulmonary disease, asthma, rheumatoid arthritis, psoriasis, systemic lupus erythematosus, dermatitis; central nervous system disorders include major depressive disorder, anxiety disorder, schizophrenia, parkinson's disease, alzheimer's disease, multiple sclerosis, attention deficit hyperactivity disorder, huntington's disease, stroke, or autism.