CN109734707B - Andrographolide decalin structure modified derivative series II and preparation method and application thereof - Google Patents

Abstract

The invention belongs to the technical field of medicines, discloses an application of andrographolide derivatives in preparing medicines for preventing and treating fibrosis of various tissues and organs of a human body, and relates to andrographolide decalin structure modified derivatives. It has a structure of a general formula II. Experiments prove that the compounds obviously inhibit the migration and activation of hepatic stellate cell LX-2; significantly inhibits the TGF-beta 1 induced mesenchymal transformation (EMT) of human alveolar II type-like cells A549 and renal epithelial cells HK-2. Remarkably inhibits the migration of primary human myocardial fibroblast HCFB induced by angiotensin II (Ang II). The compound is used as an active ingredient for preparing a medicament for resisting human tissue and organ fibrosis, has high efficiency and low toxicity, and provides a new medicament approach for treating and preventing diseases related to the fibrosis, so that the selectable range of clinical medicaments is expanded, and the medicament has good application and development prospects.

Description

Andrographolide decalin structure modified derivative series II and preparation method and application thereof

Technical Field

The invention relates to an andrographolide derivative, a synthetic method and application thereof as an anti-hepatic fibrosis drug, in particular to an andrographolide decalin structure modified derivative, belonging to the technical field of medicines.

Background

Fibrosis of human tissues and organs is a chronic disease, and is frequently found in the liver, lung, heart, kidney and other parts. 1/3 worldwide succumb to tissue fibrosis and the resultant organ failure fibrosis due to sustained injury to the epithelial tissue of the human body, which stimulates excessive proliferation of associated effector cells, deposits a large amount of extracellular matrix (ECM), and fibrosis of the tissue occurs, eventually leading to gradual loss of biological function of the tissue. Since organs with a large composition of epithelial tissues are more susceptible to the influence, fibrosis is often found in organs such as liver, lung, kidney, heart, etc.

Liver fibrosis is a pathological process in which connective tissues in the liver abnormally proliferate due to various chronic injuries, resulting in excessive precipitation of diffuse extracellular matrix in the liver, and severe cases develop into liver cirrhosis, finally resulting in liver failure. The activation of fibroblast and the production of a large amount of extracellular matrix are important links leading to hepatic fibrosis. Research has shown that hepatic epithelial-derived cells (hepatic stellate cells (HSC), hepatocytes, cholangiocytes) can all be converted into fibroblasts by epithelial-mesenchymal transition (EMT). When the liver is injured, the HSCs in a quiescent state are activated by inflammation and cytokine action, and then secrete a large amount of extracellular matrix, and express fibrosis promoting factors such as alpha-smooth muscle actin (alpha-SMA), type I collagen, interstitial metalloproteinase-2 (MMP-2) and metalloproteinase tissue inhibitor-1 (tissue inhibitor of metalloproteinase-1, TIMP-1), and the like, thereby playing an important role in the occurrence and development of hepatic fibrosis.

Pulmonary Fibrosis (PF) is a serious interstitial lung disease, and the main pathological features of the occurrence of the disease are diffuse alveolitis in the early stage, pathological proliferation and transformation of a large number of fibroblasts in the later stage, abnormal accumulation of extracellular matrix (ECM) and replacement of normal lung tissue structure, and the essence is the process of alveolar injury, excessive repair of lung tissue and abnormal remodeling. Statistics show that the 5-year survival rate is lower than 50%, the 10-year survival rate is about 30%, and an effective prevention and control means is not available at present. The etiology of the chronic pulmonary diseases is various, and a plurality of chronic pulmonary diseases, including asthma, bronchiectasis, chronic obstructive pulmonary disease, pulmonary tuberculosis, lung cancer, interstitial lung disease and the like, are accompanied with fibrosis pathological changes. The main pathological characteristics of the lung tissue include the proliferation of mesenchymal cells in lung tissue, the proliferation and deposition of extracellular matrix, the reconstruction of lung parenchyma and the like. For multiple lung diseases such as idiopathic pulmonary fibrosis, respiratory distress syndrome, eosinophilic granuloma, etc., the degree of fibrosis and fibroplasia of the lung tissue determines the clinical outcome of the disease. These diseases progress to an advanced stage, which seriously hampers the normal work and quality of life of the patient and even leads to death of the patient from respiratory failure or cardiac failure.

The impairment of renal function, whether it is a primary glomerular disease, or diabetes, ureteral obstruction, is closely related to the degree of fibrotic changes in the kidney. These diseases can cause apoptosis of renal tubular epithelial cells, infiltration of renal interstitial inflammatory cells, accumulation of myofibroblasts, and proliferation of interstitial fibroblasts, excessive deposition of extracellular matrix (ECM), renal tubular atrophy, etc. in the presence of some profibrotic factors, resulting in renal interstitial fibrosis, leading to severe impairment of renal function. Renal fibrosis is a common pathological process of chronic kidney disease, and is one of the major causes of renal failure. Renal fibrosis is a complex process involving multiple factors, including transforming growth factors, cell secretion factors, oxidative stress, inflammatory stimuli, and the like.

Myocardial Fibrosis (MF) is a pathological name proposed on the basis of modern medical research, and refers to that myocardial interstitial myocardial fibroblasts (CFs) proliferate, extracellular matrix (ECM) (mainly collagen fibers i and iii) are abnormally increased in deposition under pathological conditions, so that ventricular compliance is reduced, and normal contraction and relaxation functions of the heart are affected. MF is a common pathological change in the development of various heart diseases to a certain stage, and is also a key cause for ventricular remodeling, and a great deal of research in recent years shows that myocardial fibrosis is closely related to a plurality of heart diseases, such as atrial fibrillation, myocardial infarction, chronic heart failure, rheumatic valvular heart disease and the like. The MF can be induced by rheumatic heart disease, hypertension, myocardial infarction, heart failure and other diseases, and can also occur in the processes of age increase and coronary atherosclerosis, and the main pathological manifestations include increase of myocardial stiffness, reduction of myocardial contractility, reduction of coronary blood flow reserve, and even cause malignant arrhythmia and sudden death.

During the development of tissue fibrosis, fibroblasts and myofibroblasts are the key effector cells of tissue fibrosis, and these effector cells can release in large quantities the collagen components that make up the ECM, such as type i and type iii collagen. A variety of cytokines are also involved in the process of fibrosis, the most critical of which is transforming growth factor-beta (TGF-. beta.). TGF- β is a multifunctional cell growth factor that regulates cell proliferation, differentiation, and can stimulate myofibroblast massive proliferation and ECM overproduction by directly stimulating the activation of fibroblasts in situ or by the processes of endothelial-mesenchymal transition (EnMT), epithelial-metaplasia (EMT). When TGF- β continues to be activated due to injury, MAPK, EGF, Wnt/β -catenin signals are cross-activated, leading to the development of fibrosis. In addition to TGF- β, modulation of platelet derived factor (PDGF), basic fibroblast growth factor (bFGF), Connective Tissue Growth Factor (CTGF), insulin-like growth factor (IGF), angiogenesis-related cytokines, integrins, Metal Matrix Proteases (MMPs) and inhibitors Thereof (TIMPs), renin angiotensin-related proteins, natriuretic peptides, and the like, will also affect the occurrence of fibrosis in human organ tissues.

The existing medicines for treating tissue fibrosis, such as polypeptide medicines, have remarkable development, for example, the medicine LSKL for treating hepatic fibrosis inhibits TSP-1 from activating TGF-beta 1 to regulate TGF beta signal pathway by competitively combining Lys-Arg-Phe-Lys (KRFK) sequences at positions 1412-415 of TSP-and enters the preclinical research stage. The marketed medicine Carperitide for treating myocardial fibrosis can obviously reduce the expression of TGF-beta, CTGF, PAI-1 and Collagen III, and achieves the purpose of treating peritoneal fibrosis caused by peritoneal dialysis. AcSDKP is a polypeptide drug, is derived from tetrapeptide segments at the amino terminal of human thymosin beta 4(thymosin beta 4), and is a substrate of ACE. Is involved in the regulation of the activity of the renin-angiotensin-aldosterone system, and has remarkable cardiovascular protection effect. Among a number of animal models, such as the interleukin-1 β (IL-1 β) induced cardiac fibrosis model, bleomycin induced pulmonary fibrosis model, carbon tetrachloride induced hepatic fibrosis model, diabetes-related renal fibrosis and Unilateral Ureteral Obstruction (UUO) induced renal interstitial fibrosis model, AcSDKP all showed its anti-fibrotic effects: the AcSDKP weakens inflammatory reaction, inhibits the proliferation and differentiation of fibrosis effector cells, inhibits the deposition of collagen, slows down the pathogenesis process of fibrosis, and enters a preclinical research stage.

Many of the existing patents relate to drugs for preventing or treating various fibrotic diseases of human tissues and organs, including pulmonary fibrosis, hepatic fibrosis, renal fibrosis and cardiac fibrosis. Thermoly pharmaceutical company (patent No. CN106573049A), found a composition useful for selectively removing the origin of liver fibrosis and cirrhosis (i.e., hepatic stellate cell HSC) and the origin of pancreatic fibrosis and pancreatitis (activated pancreatic stellate cell PSC), and can effectively reduce or prevent further chronic fibrosis of human tissue organs by simultaneously reducing a plurality of fibrosis-associated molecules secreted or induced by these activated stellate cells. Promicik biosciences (patent No: CN105143170A) discovered a substituted aromatic compound that can treat pulmonary fibrosis, hepatic fibrosis, skin fibrosis and cardiac fibrosis. By quantifying the expression of factors such as CTGF and collagen I in real time, the results show that when the compound is orally administered to treat the bleomycin-induced pulmonary fibrosis model, the expression of the inflammatory and fibrosis markers in the lung is remarkably reduced by the treatment group.

Andrographolide (Andrographolide) is a diterpene lactone compound extracted from Andrographis paniculata (Burm.f) Nees) belonging to Acanthaceae, is one of the main effective components of Andrographis paniculata, and has effects of clearing away heat and toxic materials, cooling blood and relieving swelling. Modern pharmacological research shows that andrographolide has antiinflammatory, antibacterial, antiviral, antitumor, immunity regulating, cardiovascular disease and cerebrovascular disease treating, liver protecting, and gallbladder promoting effects. Kapil A [ Biochem Pharmacol,1993,46(1): 182-. Meanwhile, andrographolide has the effects of resisting myocardial ischemia and ischemia-reperfusion injury, protecting vascular endothelial cells, regulating lipid, lowering blood pressure, resisting atherosclerosis, preventing restenosis after angioplasty, improving hemorheology and the like, and Liu Guoli and the like [ medical guidance, 2006,25(1):48-50] discuss that andrographolide can inhibit the expression of certain pro-inflammatory proteins, the proteins play a role in nuclear factor NF-kB connection sites in genes, and andrographolide plays an anti-inflammatory role by inhibiting connection of NF-kB and DNA, so that the expression of pro-inflammatory proteins such as COX-2 is reduced. Many researches have made progress on the structural modification of andrographolide, and Fan (Chinese university of pharmacy, 2010,41(4): 326-332), and the like, take andrographolide as a guide to synthesize a series of derivatives with the structure of 12-N-substituted-14-deoxyandrographolide, preliminarily evaluate the in vitro anti-tumor activity of the derivatives, screen out compounds with the activity remarkably higher than that of andrographolide, and the compounds can increase the expression of p53 and Bax in HepG2 cells, reduce the expression of Bcl-2, and have remarkable in vivo and in vitro anti-tumor effect at 4 d. Meanwhile, the patent (patent number: CN106974906A) applied by Guangzhou Chinese medicine university in 2017 mentions that andrographolide and bleomycin are matched for use, so that the survival cycle, the weight, the abdominal circumference diameter and the activity of a tumor model mouse can be obviously improved, the pulmonary fibrosis degree of the tumor model mouse is obviously reduced, and the pharmaceutical composition can enhance the anti-tumor effect of the bleomycin and simultaneously reduce the pulmonary fibrosis caused by the bleomycin.

The inventor obtains a large number of andrographolide derivatives with novel structures in earlier researches (CN200510107247.4, CN200710053807.1, CN200710053806.7 and CN200610017357.6), applies patent protection to the application fields of part of the derivatives in the protection effects of tumor resistance, anti-inflammation, anti-HBV, HCV, acute liver injury and the like, further synthesizes decalin structure modified andrographolide derivatives, and conducts activity test research in the aspect of resisting tissue (organ) fibrosis.

Disclosure of Invention

On the basis of earlier research results, the inventor discovers that the andrographolide derivative with the structure of the general formula II has obvious effects of preventing and treating diseases related to human tissue organ fibrosis, has high efficiency and low toxicity and has the potential of being developed into a medicament for resisting human tissue organ fibrosis by screening the anti-liver fibrosis, lung fibrosis and kidney fibrosis activity of a synthesized compound. Therefore, the invention aims to provide the andrographolide decalin structure modified derivative shown in the general formula II and a synthetic method thereof; another purpose is to provide the application of the andrographolide decalin structure modified derivative shown in the general formula II in preparing the medicine for resisting human tissue organ fibrosis.

The andrographolide decalin structure modified derivative has a structure shown in a general formula II:

wherein R is₁，R₂Is hydrogen, COR₆Wherein R is₆Is one of C1-5 alkyl, pyridyl and phenyl radicals; or R₁，R₂Forming a hemiacetal structure; r₃Is one of hydrogen, hydroxyl or amino; r₄Is CH₂、CHO、C＝NOH、C＝NHNHR₇One of the groups; r7 is a C1-5 alkyl group, a phenyl group, or a carboxamide or thiocarboxamide group; r₅Is hydrogen or hydroxy; the dotted double bond is present or absent, but not present at the same time; (ii) a But R is₁，R₂，R₃，R₅Not hydrogen at the same time.

Preferably: r₁，R₂Is hydrogen, or R₁，R₂Forming a hemiacetal structure; r₃Is hydrogen, hydroxy; r₄Is CH₂、CHO、C＝NOH、C＝NHNHR₇One of the groups; r₇Being a carboxamide or a thio groupA carboxamide group; r₅Is hydrogen or hydroxy; the dotted double bond is present but not simultaneously; (ii) a But R is₁，R₂，R₃，R₅Not hydrogen at the same time.

Among them, the following compounds are preferred:

as described above, the target compounds represented by the preferred compounds have the following synthetic routes.

In order to research the application effect of the compound in preparing anti-fibrosis drugs, the invention utilizes human hepatic stellate cell LX-2 to determine the inhibitory activity of the compound on cell migration and activation and evaluate the anti-hepatic fibrosis activity of the compound; evaluating the inhibitory activity of the compound on the transformation from A549 cells induced by TGF-beta 1 to mesenchymal cells by using human alveolar II type sample cells A549 cells, and evaluating the anti-pulmonary fibrosis activity of the compound; evaluating the inhibitory activity of the compound on the TGF-beta 1-induced mesenchymal transformation of HK-2 cells by using the HK-2 of the proximal tubular epithelial cells, and evaluating the anti-renal fibrosis activity of the compound; after primary human myocardial fibroblast HCFB is stimulated by Ang II, the influence of the compound on cell migration is detected, and the effect of the compound on myocardial fibrosis resistance is evaluated.

The cis-structure and the trans-structure of the compound have the activity of resisting human organ or tissue fibrosis, and the compound is taken as an effective medicinal component, or various prodrug forms of the compound are independently or combined with other medicaments, and are mixed with auxiliary and/or additive components acceptable in pharmacy according to various conventional pharmacy methods and process requirements to prepare various medicament dosage forms such as anti-fibrosis oral preparations, injection preparations and the like. Preferably, the compound is used for preparing medicaments for treating or preventing fibrotic diseases of various organs or tissues such as liver, lung, kidney, heart and the like. The oral preparation is tablet, pill, capsule, granule or syrup; the injection preparation comprises injection or freeze-dried powder injection and the like.

The invention has the advantages and innovation points that: the compound is determined to have specific anti-organ and/or tissue fibrosis activity by activity screening. Experiments prove that compared with the parent compound Andrographolide (AD), the compound of the invention has obviously improved effect of resisting liver, lung, kidney and/or myocardial fibrosis. Therefore, the compound is used as an active ingredient for preparing medicaments for resisting fibrosis of various tissues and organs of a human body, and provides a new medicament approach for treating and preventing fibrosis-related diseases, so that the selectable range of clinical medicaments is expanded, and the compound has good application and development prospects.

Drawings

FIG. 1 is a graph showing the effect of AD and a compound represented by the present invention (30.00. mu.M) on the activity of human hepatic stellate cells LX-2, in which: 1, AD; 2, II-44; 3, II-48; II-50; 5, II-51;

FIG. 2 shows the results of inhibition of migration of human hepatic stellate cell LX-2 by AD and the compounds represented by the present invention (statistical results) at concentrations of 1.00. mu.M and 5.00. mu.M, in which: in the figure: 1, AD; 2, II-44; 3, II-48; II-50; 5, II-51;

FIG. 3 is a graph of the effect of AD and a compound represented by the invention (30.00. mu.M) on the viability of human alveolar type II cells A549, in which: 1, AD; 2, II-44; 3, II-47; II-48; 5, II-50; II-51; 7, II-52;

FIG. 4 is a graph showing the inhibition of TGF- β 1-induced transformation of human alveolar type II cells A549 into mesenchymal cells by AD and the compounds represented by the present invention (statistical results), with low and high concentrations of compounds AD, II-44, II-47, II-48 of 0.63. mu.M and 1.25. mu.M, respectively, and low and high concentrations of the remaining compounds of 0.31. mu.M and 0.63. mu.M, respectively; in the figure: 1. comparison; TGF-beta 1; TGF-beta 1+ AD; TGF-beta 1+ II-44; TGF-beta 1+ II-47; TGF-beta 1+ II-48; TGF-beta 1+ II-50; TGF-beta 1+ II-51; TGF-beta 1+ II-52;

FIG. 5 is a graph of the effect of AD and a compound represented by the present invention (30.00. mu.M) on the viability of human proximal tubular epithelial cells HK-2, in which: 1, AD; 2, II-44; 3, II-48; II-50; 5, II-52;

FIG. 6 is a partial micrograph:. times.100 of AD in which 1. normal, and a compound represented by the present invention inhibits TGF- β 1-induced mesenchymal transition of human proximal tubular epithelial cells HK-2; TGF-beta 1; TGF-. beta.1 + II-48 (0.08. mu.M); TGF-. beta.1 + II-50 (0.08. mu.M); TGF-. beta.1 + II-52 (0.08. mu.M); TGF-. beta.1 + II-44 (0.16. mu.M); TGF-. beta.1 + AD (1.25. mu.M);

FIG. 7 shows the results of inhibition of angiotensin II (Ang II) -induced migration of HCFB from primary human cardiac fibroblasts by AD at low and high concentrations of 0.32. mu.M and 0, 63. mu.M, respectively, and the remainder at 0.16. mu.M and 0.32. mu.M, respectively, and by compounds represented by the present invention, tested concentrations were well below the respective toxic concentrations; in the figure: 1, AD; 2, II-44; 3, II-48; II-50; 5, II-52.

Detailed description of the preferred embodiments

The invention is illustrated below with reference to specific embodiments. It should be understood that these embodiments are illustrative of the invention only and are not limiting upon the scope of the invention. The compound related to the invention is not limited to the representative structure used in the examples, and different substituents can be replaced to obtain the compound with anti-fibrosis activity; the compound of the invention can be obtained by taking various causes causing fibrosis as research objects and has the function of resisting human tissue organ fibrosis; other various in vivo and in vitro research models (methods) can also be utilized to obtain the compound of the invention which has the effect of resisting human tissue organ fibrosis.

EXAMPLE 1 inhibition of migration of human hepatic stellate cell LX-2 by Compounds of the invention

Under the stimulation of cytokines such as various inflammation mediators, growth factors and the like, hepatic stellate cells migrate to an inflammation part of damaged hepatic tissues, and then proliferate, activate, synthesize ECM components such as collagen and the like, which are the key points for the development of hepatic fibrosis. Therefore, the scratch damage method is adopted to evaluate the hepatic fibrosis resistance of the compound of the invention.

1) Cell culture and drug treatment

Human hepatic stellate cell LX-2 (provided by Beijing Beinanna Chuanglian union Biotechnology research institute) is compared with andrographolide to research the in vitro anti-hepatic fibrosis effect of the compound. Culturing LX-2 cellsIn a culture solution containing 10% (V/V) fetal calf serum, 100 mug/mL streptomycin and 100IU/mL penicillin RPMI1640, the volume fraction of the culture solution is 5% CO₂Culturing in an incubator at 37 deg.C under saturated humidity. Andrographolide, produced by Sichuan Erythrosin 37025, Jinxin Biotech, Inc. (batch No. 120822), has a purity of greater than 99%; the compound of the invention is synthesized by the laboratory of the inventor, and the purity is more than 99 percent, as follows.

2) MTT method for determining cytotoxicity

LX-2 cells in log phase of growth were digested with 0.25% (W/V) trypsin and diluted to 3.5X 10 in RPMI1640 medium containing 10% (V/V) fetal bovine serum⁵The cell suspension was plated in 96-well plates at 200. mu.L/well with a volume fraction of 5% CO at 37 ℃₂The culture was carried out in an incubator for 24h, and media containing different concentrations of drug were added, up to a final concentration of 30.00. mu.M, and repeated for 4 wells per treatment. The culture was continued for 48h, MTT (5mg/mL) was added at 20. mu.L/well, the culture was continued for 4h, the supernatant was discarded, 150. mu.L of DMSO was added, shaking L0 min was performed, and the absorbance was measured using a microplate reader. The measurement wavelength was 570nm and the reference wavelength was 450 nm. The cell viability after the compound was applied was calculated as the ratio of the survival (%) to the a value of the drug group/a value of the cell control group x 100%, and the results were averaged as shown in fig. 1.

3) Method for observing influence of drug on LX-2 cell migration by scratch damage method

LX-2 cells in log phase of growth were digested with 0.25% (W/V) trypsin and diluted to 1.0X 10 in RPMI1640 medium containing 10% (V/V) fetal bovine serum⁶Cell suspension/mL, plated in 96-well plates at 200. mu.L per well. After culturing for 12h, the cells are grown into a fused state, the original culture medium is discarded, a culture medium containing 0.5% (V/V) serum is added for re-synchronization culture for 12h, then streaking is carried out, the streaking is carried out twice by PBS, and 200 mu L of RPMI1640 culture medium containing the compound to be detected is added and immediately photographed under a microscope. Replicate 3 wells and set controls. After 24h incubation, the measurements were taken by pictures under a microscope. Mobility inhibition was calculated as [ 1- (dosing group 0h scratch distance-24 h scratch distance)/(blank group 0h scratch distance-24 h scratch distance)]X 100%, the results are averaged, see figure 2.

4) Results of the experiment

FIG. 1 the results show that: at a concentration of 30 μ M, the inhibition of LX-2 proliferation by the compounds of the present invention was significantly reduced compared to AD.

The results show that when the attached drawings 1 and 2 are combined: under the condition of non-toxic concentration, the compound can obviously inhibit the migration of human hepatic stellate cell LX-2, and has stronger migration inhibition effect on LX-2 and higher safety index compared with AD.

Example 2 inhibition of TGF-. beta.1-induced transformation of human type II alveolar epithelial cells A549 into mesenchymal cells by Compounds of the present invention

The II type alveolus epithelial cells in the alveolus are stimulated by cytokines such as inflammatory mediators, growth factors and the like, the cell morphology is changed from cobblestone shape to fusiform shape, the Epithelial Mesenchymal Transition (EMT) is completed, the function of interstitial cells is achieved, then collagen fibers are synthesized, and the disease course of interstitial pulmonary fibrosis can be aggravated by the deposition of a large amount of collagen fibers. Therefore, the anti-pulmonary fibrosis effect of the compound of the present invention was evaluated by morphological observation.

1) Cell culture and drug treatment

Human type II alveolar epithelial cells A549 are adopted, and compared with andrographolide, the in-vitro anti-pulmonary fibrosis effect of the compound is researched. A549 cells were cultured in RPMI1640 medium containing 10% (V/V) fetal bovine serum, 100. mu.g/mL streptomycin, and 100IU/mL penicillin, with volume fraction of 5% CO₂Culturing in an incubator at 37 deg.C under saturated humidity.

2) MTT method for determining cytotoxicity

A549 cells in logarithmic phase of growth were digested with 0.25% (W/V) trypsin and diluted to 2.5X 10 in RPMI1640 medium containing 10% (V/V) fetal bovine serum⁴The cell suspension was plated in 96-well plates at 200. mu.L/well with a volume fraction of 5% CO at 37 ℃₂Culturing in an incubator for 24h, adding a drug-containing culture medium with the final concentration of the drug of 30.00 mu mol/L at most, repeating each treatment for 4 wells, and continuing culturing for 48 h. Other examples are also the same as example 1. The results were averaged as shown in FIG. 3.

3) Morphological observation method for detecting influence of drug on A549 cell EMT

A549 cells in the logarithmic phase of growth were treated with 0.25% (W-V) Trypsin digestion, dilution to 2.5X 10 in RPMI1640 medium containing 10% (V/V) fetal bovine serum⁴Cell suspension/mL, plated in 96-well plates at 200. mu.L per well. After 24h of incubation, the cells were grown to confluent state, the original medium was discarded, serum-free medium was added and re-synchronized for 24h, the medium was discarded, washed twice with PBS, and 200. mu.L of RPMI1640 medium containing TGF-. beta.1 (5ng/mL) and various concentrations of test compound was added and photographed under a microscope (100X). Replicate 3 wells and set controls. After 48h incubation, pictures were taken under the microscope. A total of 5 fields were selected for each compound in three wells of the same concentration and greater than 100 cells were measured. The pictures were processed using photoshopCS6 image software and their circularity was calculated (formula e-4 pi × S/C)²Where e represents circularity, S represents area, and C represents perimeter). The results were averaged and are shown in FIG. 4.

4) Results of the experiment

The results in figure 3 show that the inhibitory activity of the compounds of the present invention on human a549 cell proliferation is significantly reduced compared to AD.

The results of figures 3 and 4 show that: the compound can obviously inhibit epithelial mesenchymal transition of A549 cells under the nontoxic concentration, and has stronger inhibition effect on the mesenchymal transition of human II-type alveolar epithelial cells and higher safety index compared with AD.

Example 3 inhibition of TGF-beta 1-induced transformation of human proximal tubular epithelial cells HK-2 into mesenchymal cells by Compounds of the invention

Early studies found that tubular epithelial cells can transdifferentiate into fibroblasts and express their marker protein, fibroblast-specific protein (FSP1, fibroblastic protein 1), and that tubular epithelial cell-mesenchymal cell transdifferentiation is one of the important pathogenesis of renal interstitial fibrosis. Therefore, the anti-renal fibrosis effect of the compound of the invention is evaluated by a morphological observation method after TGF-beta 1 stimulation.

1) Cell culture and drug treatment

In vitro anti-renal fibrosis of the compounds of the present invention was studied using human proximal tubular epithelial cells HK-2 (provided by the Chinese type culture Collection) in comparison with andrographolide ADAnd (5) performing a maintenance action. HK-2 cells were cultured in DMEM-F12 medium containing 10% fetal bovine serum (V/V), 100. mu.g/mL streptomycin, 100IU/mL penicillin, with a volume fraction of 5% CO₂The cells were cultured in an incubator at 37 ℃ under saturated humidity.

2) MTT method for determining cytotoxicity

HK-2 cells in log phase of growth were digested with 0.25% (W/V) trypsin + 0.02% EDTA (W/V) and then diluted to 7.0X 10 with DMEM-F12 medium containing 10% (V/V) fetal bovine serum⁴The cell suspension was plated in 96-well plates at 200. mu.L/well with a volume fraction of 5% CO at 37 ℃₂Culturing in incubator for 24h, changing into culture medium containing different concentrations of drug with maximum final concentration of 30.00 μ M, repeating each treatment for 4 wells, and culturing for 48 h. The rest is the same as example 1. The results were averaged as shown in FIG. 5.

3) Observation of the effects of drugs on HK-2 cell morphology following TGF-1 stimulation

HK-2 cells grown to logarithmic phase were digested with 0.25% (W/V) trypsin + 0.02% EDTA and then diluted to 5.0X 10 with DMEM-F12 medium containing 10% (V/V) fetal bovine serum⁴Cell suspension/mL, plated in 96-well plates at 200. mu.L per well. After 24h of culture, the cells grow into a monolayer, the original culture medium is discarded, the cells are washed twice by using 0.01M PBS (phosphate buffer solution), the serum-free culture medium is replaced to synchronize, after 24h of culture, the serum-free culture medium is discarded, and 200 mu L of DMEM-F12 culture medium containing the compounds to be detected and the stimulation factor TGF-beta 1(5ng/mL) with different concentrations is added. Replicate 3 wells and set controls. After 48h incubation, the photographs were recorded under a microscope. The morphological changes of some of the compounds of the invention after exposure to cells are shown in FIG. 6.

4) Results of the experiment

The results in FIG. 5 show that none of the compounds of the present invention significantly increased the proliferation of human proximal tubular epithelial cells HK-2 over the parent compound AD.

The results of fig. 5, table 1 and fig. 6 show that: the compound can obviously inhibit the epithelial mesenchymal transformation of the HK-2 cells under the nontoxic concentration, and has stronger inhibiting effect on the epithelial mesenchymal transformation of the HK-2 cells and higher safety index compared with AD.

TABLE 1 Effect of AD and the inventive Compounds on mesenchymal transformation of human tubular epithelial cells HK2

Note: the test concentration is 0.08-1.25 mu M; comparison: epithelial cells have interaction, the tissue structure is compact, and the cells are in a typical paving stone shape; TGF-beta 1 treatment: the epithelial cells lose the typical state, the interaction among the cells disappears, the tissue structure is relatively loose, the cell density is reduced, and the cube is in the form of a paved stone-shaped epithelial cell converted into a spindle-shaped fiber cell; very strong (inhibitory effect): the cells are almost the same as the control, spindle shapes are rarely seen under the visual field, intercellular interaction is recovered, and the shape is recovered to be in the typical paving stone shape; strong (inhibitory effect): the invasiveness of the cells is inhibited, the cells are compact, the cell state is almost completely recovered, and spindle fibrous cells are rarely seen; moderate (inhibitory effect): the cell density is increased, and most cells are still in a cubic state

EXAMPLE 4 inhibition of angiotensin II (Ang II) -induced primary human myocardial fibroblast migration by Compounds of the invention

Research shows that the myocardial fibroblast is the main effector cell of myocardial fibrosis, and after being stimulated by active substances such as Ang II and the like, the myocardial fibroblast can generate phenotypic change, has enhanced migration capability and is converted into the myofibroblast with the function of secreting extracellular matrix. Therefore, compared with andrographolide, the MTT method is adopted to detect the influence of the compound on the proliferation activity of the HCFB (human fibroblast growth factor beta) of the primary human myocardial fibroblasts; the inhibition of Ang ii-induced migration of primary human myofibroblasts by the compounds of the invention was evaluated by a scratch injury method.

1) Cell culture

Primary human cardiac fibroblast HCFB (supplied by Shanghai-Na Biotech Co., Ltd., Shang) was cultured in a culture flask containing 8% fetal bovine serum, 100. mu.g/mL streptomycin and 100IU/mL penicillin in H-DMEM medium, and cultured in a 5% volume CO2 incubator at saturated humidity and 37 ℃.

2) MTT method for determining cytotoxicity

HCFB cells in logarithmic growth phase were digested with 0.25% trypsin, and then diluted to 5.0X 10 in H-DMEM medium containing 8% fetal bovine serum⁴The cell suspension was plated in 96-well plates with 7000 cells/well at 37 ℃ with a volume fraction of 5% CO₂And culturing in an incubator with saturated humidity for 24h, adding culture media containing different concentrations of AD or the compound of the invention, and continuing culturing for 48h, wherein the rest is the same as example 1.AD and compounds of the invention are as in example 1. The results were averaged.

3) Scratch (migration) experiment to observe the growth of drug inhibition on AngII-stimulated HCFB migration

The log phase HCFB cells were digested with 0.25% (W/V) trypsin, diluted into a cell suspension in H-DMEM medium containing 8% fetal bovine serum, and plated in 96-well plates at 20000 cells per well. Culturing for 24 hr until the cells grow into fusion state, removing original culture medium, adding serum-free culture medium, synchronously culturing for 24 hr, marking with 200 μ L standard gun head, cleaning with 0.01M PBS twice, adding 200 μ L medicine containing different concentrations of compound to be tested and Ang II (10 μ L)^-7mol/L) of H-DMEM (containing 0.5% DMSO) medium, a blank with H-DMEM medium containing 0.5% DMSO, and an Ang II and 0.5% DMSO-containing H-DMEM medium as Ang II group, which were repeated in 3 wells. The measurement was performed by taking a photograph under a microscope before the culture (0h) and 24h after the culture, respectively. Migration distance is edge distance (0h) -edge distance (24 h). Inhibition rate ═ [ (Ang ii group migration distance-drug group migration distance)/(Ang ii group migration distance-blank group migration distance)]X 100%, the results are averaged, see figure 7.

4) Results of the experiment

The results show that: compared with AD treatment at the same concentration, the compound of the invention has no cytotoxicity in the experimental concentration range, and the compound of the invention has stronger inhibition effect on Ang II stimulated HCFB migration capacity than AD (see figure 7).

EXAMPLE 5 Synthesis of preferred Compounds II-44, II-47, II-48 and II-52

Will be SeO₂(0.168g, 1.5mmol) was placed in a 25mL round bottom flask, 10mL of methylene chloride was added, and 1.16g of 70% aqueous t-butanol peroxide (equivalent toSlowly dropping into reaction system at 9mmol t-BuOOH), stirring at room temperature for 30min, adding 1g dehydroandrographolide (TC, 3mmol), reacting at room temperature for 12 hr, monitoring by TLC, and adding saturated Na₂SO₃Adjusting pH of the solution to neutral, CH₂Cl₂Extracting with saturated saline, mixing organic phases, and extracting with anhydrous Na₂SO₄Drying, filtering, evaporating under reduced pressure to remove solvent, parching, separating with dry silica gel column chromatography, and eluting with pure ethyl acetate to obtain pale yellow solid 550mg, i.e. compound I-41, with yield 55%. Mp 99.6-100.5 ℃. IR (KBr)3397,2933,2873,1747,1084,1039,1001,982cm^-1。¹H NMR(400MHz,Chloroform-d)δ7.18(s,1H),6.86(dd,J＝15.7,10.2Hz,1H),6.17(d,J＝15.8Hz,1H),5.02(s,1H),4.83(s,2H),4.71(s,1H),4.41(d,J＝2.7Hz,1H),4.22(d,J＝11.1Hz,1H),3.53(dd,J＝11.3,4.8Hz,1H),3.33(d,J＝11.1Hz,1H),2.84(d,J＝10.1Hz,1H),1.96(d,J＝13.9Hz,1H),1.81–1.74(m,3H),1.61(dd,J＝13.6,3.1Hz,1H),1.52(dt,J＝13.5,3.3Hz,1H),1.27(s,3H),1.18(dd,J＝13.6,4.2Hz,1H),0.80(s,3H).¹³C NMR(101MHz,CDCl₃)δ172.34,149.46,143.45,135.12,129.12,121.66,112.60,80.71,72.67,69.69,64.10,56.13,46.86,42.39,38.82,37.97,29.69,29.63,28.03,22.36,15.00.。.

Compound I-41(3g, 8.62mmol) was placed in a 100mL round-bottom flask, 60mL tetrahydrofuran was added, and 10 drops of concentrated H were added dropwise with stirring₂SO₄Refluxing at 70 ℃, slowly adding 3mL of benzaldehyde (10.34mmol) dropwise into the reaction system, continuing to react for 4h, monitoring the reaction by TLC, and adding saturated Na₂CO₃Adjusting pH of the solution to neutral, steaming under reduced pressure to remove organic solvent, extracting with ethyl acetate/saturated saline water, mixing organic phases, and adding anhydrous Na₂SO₄Drying, filtering, distilling under reduced pressure to remove ethyl acetate, parching, and separating with dry silica gel column chromatography, wherein the ethyl acetate: petroleum ether (1: 1) is used for elution to obtain 1.97g of light yellow solid, namely the compound II-48, with the yield of 65.7 percent. Mp 202.3-203.0 ℃. IR (KBr)3503,2946,1744,1731,1457,1395,1346,1098,1068,1051,758,699cm^-1。¹H NMR(400MHz,Chloroform-d)δ7.51(d,J＝6.5Hz,2H),7.37(q,J＝8.3,7.3Hz,3H),7.20(s,1H),6.95(dd,J＝15.7,10.2Hz,1H),6.22(d,J＝15.7Hz,1H),5.79(s,1H),5.07(s,1H),4.85(s,2H),4.78(s,1H),4.45(s,1H),4.34(d,J＝11.3Hz,1H),3.73(dd,J＝12.6,4.7Hz,1H),3.62(d,J＝11.3Hz,1H),2.89(d,J＝10.2Hz,1H),2.46-2.33(m,1H),1.99(d,J＝13.9Hz,1H),1.88(dd,J＝13.5,2.0Hz,1H),1.76(dq,J＝12.8,3.8Hz,1H),1.53(s,3H),1.29-1.20(m,1H),1.00(s,3H).¹³C NMR(101MHz,Chloroform-d)δ172.22,149.51,143.61,138.91,134.93,129.06,128.88,128.34,126.23,121.95,113.12,95.29,72.49,69.63,69.57,56.28,46.50,38.92,37.19,36.53,28.77,25.69,21.63,15.28.

Placing compound II-48(1g, 2.3mmol) in 50mL round bottom flask, adding 15mL acetone and 3g PDC, refluxing at 70 deg.C for 45min, monitoring by TLC, spreading diatomaceous earth, filtering off insoluble substance, evaporating the filtrate under reduced pressure to remove organic solvent, extracting with ethyl acetate/saturated saline water, mixing organic phases, and adding anhydrous Na₂SO₄Drying, filtering, distilling under reduced pressure to remove ethyl acetate, parching, and separating with dry silica gel column chromatography, wherein the ethyl acetate: petroleum ether (1: 2) is used for elution to obtain 0.54g of yellow solid, namely compound II-44, with the yield of 54.3%. Mp 154.9-155.8 ℃. IR (KBr)3432,2938,1754,1686,1455,1390,1101,762,766cm^-1。¹H NMR(400MHz,Chloroform-d)δ9.44(s,1H),7.53-7.43(m,3H),7.40(d,J＝6.1Hz,3H),7.22(s,1H),6.95-6.89(m,1H),6.59(dd,J＝15.8,9.9Hz,1H),6.23(d,J＝15.9Hz,1H),5.80(s,1H),4.84(s,2H),4.44-4.33(m,2H),3.78-3.67(m,3H),2.82(d,J＝9.3Hz,1H),1.78(dq,J＝13.0,3.8Hz,1H),1.53(s,3H),0.98(s,3H).¹³C NMR(101MHz,Chloroform-d)δ193.29,172.10,150.67,143.23,141.24,138.66,135.13,129.07,128.97,128.37,126.20,121.52,95.31,81.08,69.70,69.37,53.61,48.95,36.44,36.24,36.22,29.26,25.65,23.76,20.98,15.86.

Placing compound II-44(1.6g, 3.68mmol) and thiosemicarbazide (400mg, 4.38mmol) in a 50mL round-bottom flask, adding 30mL absolute ethyl alcohol and 10 drops of glacial acetic acid, refluxing at 80 ℃ for 50min, monitoring by TLC after the reaction is finished, evaporating under reduced pressure to remove the organic solvent, extracting with ethyl acetate/saturated saline, combining the organic phases, and adding anhydrous Na₂SO₄Drying, filtering, distilling under reduced pressure to remove ethyl acetate, parching, and separating with dry silica gel column chromatography, wherein the ethyl acetate: petroleum ether (1: 1) is eluted,1.3g of a yellow solid, i.e., Compound II-47, was obtained in a yield of 81.3%. Mp 128.2-129.3 ℃. IR (KBr)3423,2967,1752,1585,1519,1277,1103,1071, 762,700cm^-1。¹H NMR(400MHz,Chloroform-d)δ9.32(s,1H),7.55-7.48(m,2H),7.38(d,J＝7.5Hz,4H),7.25(s,1H),6.82(d,J＝4.1Hz,1H),6.58(dd,J＝15.9,10.0Hz,1H),6.36(d,J＝5.8Hz,2H),6.25(s,1H),6.17(d,J＝16.0Hz,1H),5.79(s,1H),4.88(s,2H),4.38(d,J＝11.4Hz,1H),3.72(d,J＝10.3Hz,2H),2.87(d,J＝10.2Hz,1H),1.78(dd,J＝13.7,4.0Hz,1H),1.52(s,3H),1.00(s,3H).¹³C NMR(101MHz,Chloroform-d)δ172.48,145.87,142.98,138.72,138.45,128.97,128.38,126.20,120.15,95.30,70.00,69.43,55.13,49.03,36.77,36.47,36.23,30.36,29.92,25.69,23.39,21.02,16.07.

Placing compound II-47(1g, 1.97mmol) in a 50mL round-bottom flask, adding a mixed solution of 15mL glacial acetic acid and 5mL water, reacting at room temperature for 24h, monitoring the reaction by TLC, evaporating under reduced pressure to remove organic solvent, extracting with ethyl acetate/saturated saline, combining organic phases, and adding anhydrous Na₂SO₄Drying, filtering, distilling under reduced pressure to remove ethyl acetate, parching, and separating with dry silica gel column chromatography, wherein the ethyl acetate: petroleum ether (1: 1) is used for eluting to obtain 385mg of light yellow solid, namely the compound II-52, and the yield is 38.5 percent. Mp 165.7-166.6 ℃. IR (KBr)3423,2935,1748,1631,1528,1407,1360,1042cm^-1。¹H NMR(400MHz,DMSO-d₆)δ11.15(s,1H),8.08(s,1H),7.60(s,1H),7.56(s,1H),6.70(d,J＝3.3Hz,1H),6.51(dd,J＝15.9,10.1Hz,1H),6.33(s,1H),6.16(d,J＝15.9Hz,1H),4.89(s,2H),3.89(d,J＝11.0Hz,1H),3.46(d,J＝11.0Hz,2H),3.24(dd,J＝10.1,4.7Hz,1H),2.78(d,J＝9.9Hz,1H),2.35-2.21(m,2H),1.08(s,3H),0.76(s,3H).¹³C NMR(101MHz,DMSO-d₆)δ177.63,172.24,146.03,145.42,137.44,133.45,126.91,119.73,78.56,70.13,62.46,54.73,48.86,41.57,37.19,35.70,27.13,24.28,22.68,15.41.

EXAMPLE 6 Synthesis of preferred Compounds II-50, II-51

Dissolving compound AD-H (5g) in 7.5mL of methanol, slowly adding 10mL of 10% diluted hydrochloric acid, heating to 75 deg.C, reacting for 5H, slowly cooling to room temperature after reaction, adding 15mL of water into the system, addingIn the process, a large amount of white solid is separated out, the filtration is carried out, the filter cake is recrystallized by methanol to obtain the white solid, and the white solid is dried and weighed to be 1.538g, namely CC, Yield 53 percent; mp 215.4-215.9 ℃; IR (KBr)3383,2922,2848,1753,1454,1381,1353,1091,1040,985,812;¹H NMR(400MHz,CDCl₃)δ7.21(s,1H),6.64(dd,J＝15.7,10.6Hz,1H),6.17(d,J＝15.8Hz,1H),5.52(s,1H),4.85(s,2H),4.30(d,J＝11.0Hz,1H),3.51(t,J＝9.5Hz,2H),2.94(d,J＝7.6Hz,1H),2.63(s,1H),2.44(d,J＝10.8Hz,1H),2.12(d,J＝17.3Hz,1H),1.90(m,1H),1.85–1.76(m,1H),1.70(m,2H),1.53(s,3H),1.36(dd,J＝12.4,4.6Hz,1H),1.27(s,3H),1.24–1.14(m,1H),0.84(s,3H).¹³C NMR(100MHz,DMSO)δ172.87,147.12,136.32,132.56,127.48,122.64,122.15,79.37,70.65,62.99,59.98,49.97,42.08,38.27,35.94,27.80,23.70,23.23,22.67,15.95.

will be SeO₂(0.168g, 1.5mmol) was placed in a 25mL round-bottom flask, 10mL of dichloromethane was added, 1.16g of 70% t-butanol peroxide aqueous solution (equivalent to 9mmol t-BuOOH) was slowly dropped into the reaction system, stirring was carried out at room temperature for 30min, 1gCC (3mmol) was added, the reaction was continued at room temperature for 12h, the completion of the reaction was monitored by TLC, and saturated Na was used₂SO₃Adjusting pH of the solution to neutral, CH₂Cl₂Extracting with saturated saline, mixing organic phases, and extracting with anhydrous Na₂SO₄Drying, filtering, evaporating under reduced pressure to remove solvent, parching, separating with dry silica gel column chromatography, eluting with pure ethyl acetate, and eluting with compound II-50 and Mp 128.5-129.4 deg.C.¹H NMR(400MHz,DMSO-d₆)δ7.67(s,1H),6.77(d,J＝15.8Hz,1H),6.42(d,J＝15.8Hz,1H),5.53(s,1H),4.89(s,2H),4.61(s,1H),3.98(d,J＝10.9Hz,1H),3.43(d,J＝10.9Hz,2H),3.17(dd,J＝10.6,4.8Hz,1H),2.04-1.87(m,2H),1.89-1.76(m,2H),1.54(dd,J＝9.0,4.6Hz,2H),1.50(s,3H),1.09(s,3H),0.86(s,3H).¹³C NMR(101MHz,DMSO-d₆)δ172.44,146.50,138.25,134.52,127.03,124.77,118.27,78.86,76.80,70.09,62.69,41.75,41.27,40.32,29.64,27.16,23.57,22.92,19.89,18.21.

Placing compound II-44(1g, 1.97mmol) in 50mL round bottom flask, adding mixed solution of 15mL glacial acetic acid and 5mL water, reacting at room temperature for 24h, monitoring by TLC, evaporating under reduced pressure to remove organic solvent, and adding ethyl acetateExtracting with ester/saturated saline, mixing organic phases, and extracting with anhydrous Na₂SO₄Drying, filtering, distilling under reduced pressure to remove ethyl acetate, parching, and separating by dry silica gel column chromatography to obtain compound II-51. Mp 108.2-109.1 ℃.¹H NMR(400MHz,Chloroform-d)δ9.39(s,1H),7.20(d,J＝2.2Hz,1H),6.89(dt,J＝5.3,2.4Hz,1H),6.51(dd,J＝15.9,9.8Hz,1H),6.16(d,J＝15.9Hz,1H),4.81(d,J＝2.0Hz,2H),4.28(d,J＝11.1Hz,1H),3.50(dd,J＝10.7,5.1Hz,3H),2.79-2.72(m,1H),1.27(s,3H),0.78(s,3H).¹³C NMR(101MHz,Chloroform-d)δ193.35,172.45,151.09,143.00,140.77,135.39,129.17,121.14,80.68,77.24,69.75,63.87,53.69,49.49,42.13,37.34,36.02,29.69,27.47,24.64,22.34,15.76。

Claims (8)

1. The andrographolide decalin structure modified derivative is characterized by being selected from the following compounds:

2. the use of the andrographolide decalin structure-modified derivative of claim 1 in the preparation of a medicament, wherein the andrographolide decalin structure-modified derivative is used as an active ingredient in the preparation of a medicament for treating or preventing liver fibrosis.

3. The use of the andrographolide decalin structure-modified derivative of claim 1 in the preparation of a medicament, wherein the andrographolide decalin structure-modified derivative is used as an active ingredient in the preparation of a medicament for treating or preventing pulmonary fibrosis.

4. The use of the andrographolide decalin structure-modified derivative of claim 1 in the preparation of a medicament, wherein the andrographolide decalin structure-modified derivative is used as an active ingredient in the preparation of a medicament for treating or preventing renal fibrosis.

5. The use of the andrographolide decalin structure modified derivative in the preparation of a medicament according to claim 1, wherein the andrographolide decalin structure modified derivative is used as an active ingredient in the preparation of a medicament for treating or preventing myocardial fibrosis.

6. The use of the andrographolide decalin structure-modified derivative of claim 1 in the preparation of a medicament, wherein the andrographolide decalin structure-modified derivative is used as an active ingredient or combined with other medicaments, and is mixed with pharmaceutically acceptable auxiliary ingredients to prepare an anti-fibrotic oral preparation or an anti-fibrotic injection preparation.

7. The use of the andrographolide decalin structure-modified derivative according to claim 6, wherein the oral formulation is tablet, pill, capsule, granule or syrup; the injection preparation is injection or freeze-dried powder injection.

8. The method for preparing the andrographolide decalin structure modified derivatives II-44, II-47, II-48, II-50, II-51 and II-52 is characterized in that the synthetic route is as follows:

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