CN113304154B - Pyrimidinediones as microsomal prostaglandin E2Use of synthase inhibitors - Google Patents
Pyrimidinediones as microsomal prostaglandin E2Use of synthase inhibitors Download PDFInfo
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- CN113304154B CN113304154B CN202110507114.5A CN202110507114A CN113304154B CN 113304154 B CN113304154 B CN 113304154B CN 202110507114 A CN202110507114 A CN 202110507114A CN 113304154 B CN113304154 B CN 113304154B
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
Landscapes
- Health & Medical Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Organic Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Rheumatology (AREA)
- Pain & Pain Management (AREA)
- Epidemiology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The application relates to the technical field of biological medicines, and provides an application of a pyrimidinedione compound as a microsomal prostaglandin E 2 synthase inhibitor, wherein the microsomal prostaglandin E 2 synthase inhibitor is a pyrimidinedione compound, and the pyrimidinedione compound is 2- { [ ((3-chlorophenyl) methyl ] sulfanyl } -5- (3, 4-dihydroxyphenyl) -3H,4H,5H,6H,7H, 8H-pyridinyl [2,3-d ] pyrimidine-4, 7-dione (called CLOS for short), which can be used for treating inflammation without causing side effects of cardiovascular diseases by remarkably reducing the generation of PGE 2 and inhibiting the activity of mPGES-1 protein, so that the mPGES-1 protein expression is regulated to inhibit the generation of PGE 2 to reflect anti-inflammatory activity, and simultaneously, the CLOS has no obvious influence on COX-1 and COX-2, so that the CLOS can be used for treating the inflammation.
Description
Technical Field
The application belongs to the technical field of biological medicines, and particularly relates to application of a pyrimidinedione compound as a microsomal prostaglandin E 2 synthase inhibitor.
Background
Inflammation is a complex biological defensive response of basal tissues to external stimuli such as pathogens, cellular injury, or other irritants, manifested as redness, swelling, heat, pain, and dysfunction. The inflammation may be infectious inflammation caused by infection or non-infectious inflammation caused by infection. Inflammation is often beneficial as an automatic defensive response in the human body, but sometimes inflammation is also detrimental, and transitional inflammation can cause damage to the basal tissue organ, such as attack on the body's own tissues, inflammation occurring in transparent tissues, and the like. More severe cases induce death from multiple organ failure, and therefore, transitional validation requires the provision of appropriate medications for control.
Currently, anti-inflammatory agents are used for the treatment of inflammation in response to tissue injury, and there are two main categories: one is a steroidal anti-inflammatory drug, and the other is a non-steroidal anti-inflammatory drug, namely antipyretic analgesic anti-inflammatory drugs such as aspirin and the like which are referred to in medical practice. The non-steroidal anti-inflammatory drugs are mainly used, are non-steroidal anti-inflammatory drugs which do not contain a steroidal structure, and play an anti-inflammatory role by inhibiting synthesis of prostaglandin, inhibiting aggregation of leucocytes, reducing formation of bradykinin, inhibiting aggregation of blood platelets and the like, and have the effects of anti-inflammation, anti-rheumatism, pain relieving, antipyretic, anticoagulation and the like, and are widely used for relieving osteoarthritis, rheumatoid arthritis, various fever and various pain symptoms clinically.
However, most of the nonsteroidal anti-inflammatory drugs have a certain therapeutic effect, but side effects are also obvious. Because most of nonsteroidal anti-inflammatory drugs are organic acids, the nonsteroidal anti-inflammatory drugs have high binding force with plasma proteins, so that the concentration of the drugs at an inflammation site is increased to play a role. Most patients are resistant to NSAIDs. However, almost none of NSAIDs are safe and have adverse effects on the cardiovascular system, central nervous system, blood system, skin and liver, in addition to the gastrointestinal and renal aspects of the main toxic response. Mild side effects are manifested by allergic reactions such as rubella, allergic rhinitis, asthma; and severe side effects are manifested by the conditions of absentmindedness, mental depression, influence on platelet aggregation, rise of transaminase level and the like. Therefore, the current use of non-steroidal anti-inflammatory drugs causes a plurality of adverse reactions, which affects the wide use of anti-inflammatory drugs.
Microsomal prostaglandin E 2 synthase 1 (mPGES-1) is considered a new target for anti-inflammatory therapy that controls the synthesis of prostaglandin E 2(PGE2, a major inflammatory cytokine that can lead to inflammation and cancer. Thus, mPGES-1 inhibitors are currently considered to be novel anti-inflammatory agents that exhibit their anti-inflammatory effects by inhibiting the synthesis of PGE 2. In addition, mPGES-1 inhibitors have substantially no effect on cyclooxygenase 2 (COX-2) expression and, thus, unlike conventional nonsteroidal anti-inflammatory drugs (NSAIDs), mPGES-1 inhibitors may have fewer adverse effects on vascular disease of interest.
The application aims to solve the problem that part of anti-inflammatory drugs in the prior art can cause adverse reactions such as vascular diseases and the like, and provides a novel compound which can be developed into anti-inflammatory drugs.
Disclosure of Invention
The application aims to provide an application of a pyrimidine diketone compound as a microsomal prostaglandin E 2 synthase inhibitor, and aims to solve the problem that partial nonsteroidal anti-inflammatory drugs in the prior art can produce side effects related to vascular diseases.
In order to achieve the purposes of the application, the technical scheme adopted by the application is as follows:
in a first aspect, the present application provides a microsomal prostaglandin E 2 synthase inhibitor, wherein the microsomal prostaglandin E 2 synthase inhibitor is a pyrimidinedione compound, and the pyrimidinedione compound is 2- { [ ((3-chlorophenyl) methyl ] sulfanyl } -5- (3, 4-dihydroxyphenyl) -3H,4H,5H,6H,7H, 8H-pyridinyl [2,3-d ] pyrimidine-4, 7-dione, having the structural formula shown in formula I,
In a second aspect, the present application provides the use of a microsomal prostaglandin E 2 synthase inhibitor in the manufacture of a medicament for inhibiting inflammation.
In a third aspect, the present application provides the use of a microsomal prostaglandin E 2 synthase inhibitor in the manufacture of a medicament for the prevention and/or treatment of inflammatory diseases and/or diseases associated with microsomal prostaglandin E 2 synthase 1.
In a fourth aspect, the present application provides the use of a microsomal prostaglandin E 2 synthase inhibitor in the manufacture of a medicament for the prevention and/or treatment of complications of inflammatory diseases and/or complications of diseases associated with microsomal prostaglandin E 2 synthase 1.
In a fifth aspect, the present application provides a pharmaceutical composition for treating inflammatory diseases, which comprises a stereoisomer, a tautomer, a hydrate or a pharmaceutically acceptable salt of a microsomal prostaglandin E 2 synthase inhibitor shown in formula I, and pharmaceutically acceptable excipients.
The first aspect of the present application provides a microsomal prostaglandin E 2 synthase inhibitor, which is a pyrimidinedione compound, wherein the pyrimidinedione compound is 2- { [ ((3-chlorophenyl) methyl ] sulfanyl } -5- (3, 4-dihydroxyphenyl) -3H,4H,5H,6H,7H, 8H-pyridinyl [2,3-d ] pyrimidine-4, 7-dione, the structure of which is shown in formula I, 2- { [ ((3-chlorophenyl) methyl ] sulfanyl } -5- (3, 4-dihydroxyphenyl) -3H,4H,5H,6H,7H, 8H-pyridinyl [2,3-d ] pyrimidine-4, 7-dione (CLOS) is characterized by significantly reducing the production of PGE 2 and inhibiting the activity of the mPGES-1 protein, thereby modulating PGE-1 protein expression to inhibit PGE 2 production, and simultaneously, the 2- { [ ((3-chlorophenyl) methyl ] sulfanyl } -5- (3, 4-dihydroxyphenyl) -3H,4H,5H,6H, 8H-pyridinyl [2,3-d ] pyrimidine-4, 7-dione (CLOS) is a potent anti-inflammatory agent which has no significant effect on cardiovascular side effects than the 2,3,4, 3-dihydroxyphenyl) -3H,4H, 7-dione, meanwhile, the side effect of cardiovascular diseases is not caused, the effect is better, the adverse reaction is less, and the preparation method is suitable for wide application.
The application of the microsomal prostaglandin E 2 synthase inhibitor in preparing medicines for inhibiting inflammation is based on the fact that the microsomal prostaglandin E 2 synthase inhibitor can obviously reduce the generation of PGE 2 and inhibit the activity of mPGES-1 protein, and then the mPGES-1 protein expression is regulated to inhibit the generation of PGE 2 so as to achieve the anti-inflammatory effect, and has no obvious influence on COX-1 and COX-2, so that the microsomal prostaglandin E 2 synthase inhibitor can be widely applied to preparing medicines for inhibiting inflammation.
The use of the microsomal prostaglandin E 2 synthase inhibitor provided in the third aspect of the present application in the preparation of a medicament for preventing and/or treating inflammatory diseases and/or diseases related to microsomal prostaglandin E 2 synthase 1, based on the fact that the microsomal prostaglandin E 2 synthase inhibitor significantly reduces the production of PGE 2 and inhibits the activity of mPGES-1 protein, thereby regulating the expression of mPGES-1 protein to inhibit the production of PGE 2, and has no obvious influence on COX-1, COX-2, the microsomal prostaglandin E 2 synthase inhibitor can be widely used in the preparation of a medicament for preventing and/or treating inflammatory diseases and/or diseases related to microsomal prostaglandin E 2 synthase 1.
The microsomal prostaglandin E 2 synthase inhibitor provided in the fourth aspect of the present application is used for preparing a medicament for preventing and/or treating complications of inflammatory diseases and/or complications of diseases related to microsomal prostaglandin E 2 synthase 1, and is based on the fact that the microsomal prostaglandin E 2 synthase inhibitor can achieve anti-inflammatory effects by remarkably reducing the production of PGE 2 and inhibiting the activity of mPGES-1 protein, and further regulating the expression of mPGES-1 protein to inhibit the production of PGE 2, and has no obvious influence on COX-1 and COX-2, so that the microsomal prostaglandin E 2 synthase inhibitor can be widely used in the preparation of medicaments for preventing and/or treating complications of inflammatory diseases and/or diseases related to the microsomal prostaglandin E 2 synthase 1.
The application provides a pharmaceutical composition for treating inflammatory diseases, which comprises a stereoisomer, a tautomer, a hydrate or a pharmaceutically acceptable salt of a microsomal prostaglandin E 2 synthase inhibitor shown as a formula I and pharmaceutically acceptable auxiliary materials. Because the pharmaceutical composition comprises the microsomal prostaglandin E 2 synthase inhibitor shown in the formula I, the microsomal prostaglandin E 2 synthase inhibitor can obviously reduce the generation of PGE 2 and inhibit the activity of mPGES-1 protein, and further regulate and control the expression of mPGES-1 protein to inhibit the generation of PGE 2 so as to achieve the anti-inflammatory effect, and has no obvious influence on COX-1 and COX-2, the obtained pharmaceutical composition has the advantages of being applicable to the treatment of inflammation, simultaneously having no side effect of causing cardiovascular diseases, having better effect and less adverse reaction, and being suitable for wide application.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the embodiments or the description of the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and that other drawings can be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph of cytotoxicity analysis of interleukin-1 beta mediated A549 cells by CLOS provided in the examples of the present application.
FIG. 2 is a graph of cytotoxicity analysis of lipopolysaccharide-mediated rat primary peritoneal macrophages provided by an embodiment of the present application.
FIG. 3 is a graph showing the protein activity of the CLOS and the positive control drug MK886 according to the embodiment of the present application on PGE 2 in a cell-free ELISA test.
FIG. 4 is a graph showing the protein activity analysis of mPGES-1 protein by CLOS and MK886 as a positive control agent in a cell-free ELISA assay.
FIG. 5 is a graph showing the effect of CLOS on the affinity of mPGES-1 protein provided in the examples of the application.
FIG. 6 shows the effect of CLOS and a positive control drug MK886 provided by the example of the present application on PGE 2 products on interleukin-1β (IL-1β) -mediated A549 cells.
FIG. 7 shows the effect of CLOS and a positive control drug MK886 on PGE 2 on Lipopolysaccharide (LPS) -mediated primary macrophages of the abdominal cavity of rats, as provided by the examples of the present application.
FIG. 8 shows the effect of CLOS and a positive control drug MK886 on PGI 2 on Lipopolysaccharide (LPS) -mediated primary macrophages in the abdominal cavity of rats, as provided by the examples of the present application.
FIG. 9 shows the effect of CLOS and a positive control drug MK886 on TXA 2 on Lipopolysaccharide (LPS) -mediated primary macrophages of the abdominal cavity of rats, as provided by the examples of the present application.
FIG. 10 shows the effects of CLOS and a positive control drug MK886 on PGI 2 and TXA 2 on Lipopolysaccharide (LPS) -mediated primary macrophages of the abdominal cavity of rats, as provided by the examples of the present application.
FIG. 11 shows the effect of CLOS on the expression levels of mPGES-1, COX-1 and COX-2 proteins on interleukin-1 beta (IL-1 beta) mediated A549 cells provided by the examples of the application.
FIG. 12 shows the effect of CLOS on the expression levels of mPGES-1 and COX-2 proteins on Lipopolysaccharide (LPS) -mediated primary peritoneal macrophages of rats as provided by the examples of the present application.
Detailed Description
In order to make the technical problems, technical schemes and beneficial effects to be solved more clear, the application is further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the application.
In the present application, the term "and/or" describes an association relationship of an association object, which means that three relationships may exist, for example, a and/or B may mean: a alone, a and B together, and B alone. Wherein A, B may be singular or plural. The character "/" generally indicates that the context-dependent object is an "or" relationship.
In the present application, "at least one" means one or more, and "a plurality" means two or more. "at least one of" or the like means any combination of these items, including any combination of single item(s) or plural items(s). For example, "at least one (individual) of a, b, or c," or "at least one (individual) of a, b, and c," may each represent: a, b, c, a-b (i.e., a and b), a-c, b-c, or a-b-c, wherein a, b, c may be single or multiple, respectively.
It should be understood that, in various embodiments of the present application, the sequence number of each process described above does not mean that the execution sequence of some or all of the steps may be executed in parallel or executed sequentially, and the execution sequence of each process should be determined by its functions and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.
The terminology used in the embodiments of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used in this application and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.
The weights of the relevant components mentioned in the description of the embodiments of the present application may refer not only to the specific contents of the components, but also to the proportional relationship between the weights of the components, so long as the contents of the relevant components in the description of the embodiments of the present application are scaled up or down within the scope of the disclosure of the embodiments of the present application. Specifically, the mass described in the specification of the embodiment of the application can be a mass unit which is known in the chemical industry field such as mu g, mg, g, kg.
The terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated for distinguishing between objects such as substances from each other. For example, a first XX may also be referred to as a second XX, and similarly, a second XX may also be referred to as a first XX, without departing from the scope of embodiments of the application. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature.
In a first aspect of the present application, a microsomal prostaglandin E 2 synthase inhibitor is provided, the microsomal prostaglandin E 2 synthase inhibitor is a pyrimidinedione compound, and the pyrimidinedione compound is 2- { [ ((3-chlorophenyl) methyl ] sulfanyl } -5- (3, 4-dihydroxyphenyl) -3H,4H,5H,6H,7H, 8H-pyridinyl [2,3-d ] pyrimidine-4, 7-dione, the structural formula is shown in formula I,
The first aspect of the present application provides a microsomal prostaglandin E 2 synthase inhibitor, which is a pyrimidinedione compound, and the pyrimidinedione compound is 2- { [ ((3-chlorophenyl) methyl ] sulfanyl } -5- (3, 4-dihydroxyphenyl) -3H,4H,5H,6H,7H, 8H-pyridyl [2,3-d ] pyrimidine-4, 7-dione, the structure of which is shown in formula I, 2- { [ ((3-chlorophenyl) methyl ] sulfanyl } -5- (3, 4-dihydroxyphenyl) -3H,4H,5H,6H,7H, 8H-pyridyl [2,3-d ] pyrimidine-4, 7-dione (abbreviated as CLOS) which exhibits anti-inflammatory activity by significantly reducing the production of PGE 2 and inhibiting the activity of mPGES-1 protein, thereby modulating the expression of mPGES-1 protein to inhibit PGE 2 production, and at the same time, which has no effect on the side effect of 3-chlorophenyl) -5- (3, 4-dihydroxyphenyl) methyl ] sulfanyl } -5- (3, 4-d) pyrimidine-4H, 5H,6H,7H, 8H-pyridyl [2,3-d ] pyrimidine-4, 7-dione (abbreviated as CLOS), which has no significant effect on cardiovascular side effects on the cardiovascular side of 2, 3-H, 5H,6H, 3-d ] pyrimidine-4, 7-dione, meanwhile, the side effect of cardiovascular diseases is not caused, the effect is better, the adverse reaction is less, and the preparation method is suitable for wide application.
In some embodiments, the microsomal prostaglandin E 2 synthase inhibitor as shown in structural formula I inhibits PGE 2 production by modulating protein expression of microsomal prostaglandin E 2 synthase 1. Anti-inflammatory activity is manifested by inhibition of PGE 2 production.
In some embodiments, the microsomal prostaglandin E 2 synthase inhibitor of formula I has no effect on COX-1, COX-2, and the microsomal prostaglandin E 2 synthase inhibitor does not strike a balance between PGI 2 and TXA 2 while acting, thus, the risk of the CLOS causing cardiovascular side effects in use is less than that of conventional selective COX-2 inhibitors or other non-steroidal anti-inflammatory drugs.
In a second aspect, embodiments of the present application provide an application of a microsomal prostaglandin E 2 synthase inhibitor in the preparation of a medicament for inhibiting inflammation.
The application of the microsomal prostaglandin E 2 synthase inhibitor in preparing medicines for inhibiting inflammation is based on the fact that the microsomal prostaglandin E 2 synthase inhibitor can obviously reduce the generation of PGE 2 and inhibit the activity of mPGES-1 protein, and then the mPGES-1 protein expression is regulated to inhibit the generation of PGE 2 so as to achieve the anti-inflammatory effect, and has no obvious influence on COX-1 and COX-2, so that the microsomal prostaglandin E 2 synthase inhibitor can be widely applied to preparing medicines for inhibiting inflammation.
In a third aspect, embodiments of the present application provide the use of a microsomal prostaglandin E 2 synthase inhibitor in the manufacture of a medicament for the prevention and/or treatment of inflammatory diseases and/or diseases associated with microsomal prostaglandin E 2 synthase 1.
The use of the microsomal prostaglandin E 2 synthase inhibitor provided in the third aspect of the present application in the preparation of a medicament for preventing and/or treating inflammatory diseases and/or diseases related to microsomal prostaglandin E 2 synthase 1, based on the fact that the microsomal prostaglandin E 2 synthase inhibitor significantly reduces the production of PGE 2 and inhibits the activity of mPGES-1 protein, thereby regulating the expression of mPGES-1 protein to inhibit the production of PGE 2, and has no obvious influence on COX-1, COX-2, the microsomal prostaglandin E 2 synthase inhibitor can be widely used in the preparation of a medicament for preventing and/or treating inflammatory diseases and/or diseases related to microsomal prostaglandin E 2 synthase 1.
In a fourth aspect, embodiments of the present application provide a microsomal prostaglandin E 2 synthase inhibitor for the manufacture of a medicament for the prevention and/or treatment of complications of inflammatory diseases and/or complications of diseases associated with microsomal prostaglandin E 2 synthase 1.
The microsomal prostaglandin E 2 synthase inhibitor provided in the fourth aspect of the present application is used for preparing a medicament for preventing and/or treating complications of inflammatory diseases and/or complications of diseases related to microsomal prostaglandin E 2 synthase 1, and is based on the fact that the microsomal prostaglandin E 2 synthase inhibitor can achieve anti-inflammatory effects by remarkably reducing the production of PGE 2 and inhibiting the activity of mPGES-1 protein, and further regulating the expression of mPGES-1 protein to inhibit the production of PGE 2, and has no obvious influence on COX-1 and COX-2, so that the microsomal prostaglandin E 2 synthase inhibitor can be widely used in the preparation of medicaments for preventing and/or treating complications of inflammatory diseases and/or diseases related to the microsomal prostaglandin E 2 synthase 1.
In a fifth aspect, the present application provides a pharmaceutical composition for treating inflammatory diseases, which comprises a stereoisomer, a tautomer, a hydrate or a pharmaceutically acceptable salt of a microsomal prostaglandin E 2 synthase inhibitor shown in formula I, and pharmaceutically acceptable excipients.
The application provides a pharmaceutical composition for treating inflammatory diseases, which comprises a stereoisomer, a tautomer, a hydrate or pharmaceutically acceptable salt of a microsomal prostaglandin E 2 synthase inhibitor shown as a formula I and pharmaceutically acceptable auxiliary materials. Because the pharmaceutical composition comprises the microsomal prostaglandin E 2 synthase inhibitor shown in the formula I, the microsomal prostaglandin E 2 synthase inhibitor can obviously reduce the generation of PGE 2 and inhibit the activity of mPGES-1 protein, and further regulate and control the expression of mPGES-1 protein to inhibit the generation of PGE 2 so as to achieve the anti-inflammatory effect, and has no obvious influence on COX-1 and COX-2, the obtained pharmaceutical composition has the advantages of being applicable to the treatment of inflammation, simultaneously having no side effect of causing cardiovascular diseases, having better effect and less adverse reaction, and being suitable for wide application.
In some embodiments, the pharmaceutical composition further comprises one or more excipients selected from the group consisting of a thickener, a filler, a diluent, and a pharmaceutical carrier.
In some embodiments, the pharmaceutical composition is formulated in any one of the following forms: syrups, suspensions, powders, granules, tablets, capsules, lozenges, aqueous solutions, creams, ointments, washes, gels, emulsions and aerosols.
The following description is made with reference to specific embodiments.
Example 1
Cell Activity assay
Test procedure
(1) A549 cell culture and treatment
A549 cell line was purchased from AMERICAN TYPE Culture Collection (ATCC, manassas, VA, USA).
Cell culture medium was RPMI 1640, and 10% fetal bovine serum (Gibco BRL Co, GRAND ISLAND, NY, USA) and 1% penicillin G (100 ml/unit), streptomycin (100 mg/ml) and L-glutamine (2 mM) (Gibco BRL Co, GRAND ISLAND, NY, USA) were added.
Cells were incubated in an environment containing 5% co 2 at 37 ℃; a549 cells were seeded at a density of 1 x 10 5 in 6-well plates and incubated for 24 hours.
(2) Isolation and culture of rat primary peritoneal macrophages
SD rats (weight 180-280 g) purchased from hong Kong university were supplied, peritoneal macrophages (RPM) isolated from the rats, and cultured in Dulbecco's Modified Eagle's Medium (DMEM); wherein, the medium was supplemented with 10% fetal bovine serum (Gibco BRL Co, GRAND ISLAND, NY, USA) and 1% penicillin G (100 ml/unit), streptomycin (100 mg/ml) and L-glutamine (2 Mm) (Gibco BRL Co, GRAND ISLAND, NY, USA).
SD rats were given standard water, and the rat abdominal cavity was rinsed with cold sterile Hank's Balanced Salt Solution (HBSS) to obtain primary RPM; centrifuging the washing solution at 1500rpm for 10 minutes, and discarding the supernatant; cells were resuspended in 15mL of complete DMEM medium and seeded in a 2mL system into 6-well plates; cells were incubated for 2 hours at 37 ℃ in an environment containing 5% co 2, then the attached cells were washed twice with pre-warmed complete DMEM medium to ensure no non-adherent suspension cells.
(3) Both a549 cells and RPM cells were seeded in 6-well plates. After pretreatment with different concentrations of CLOS (0. Mu.M, 2.5. Mu.M, 5. Mu.M, 10. Mu.M, 20. Mu.M), A549 cells and PRM cells were stimulated with IL-1β (1 ng/ml) and LPS (1. Mu.g/ml), respectively, for 24 hours, and MTT solution was added to each well and incubated for 4 hours. Thereafter, 100 μl of 10% sds-HCL solution was added to each well and incubated for 18 hours to dissolve formazan crystals using a microplate UV/VIS spectrophotometer (Tecan, mannedorf, switzerland). Absorbance was measured at 570nm absorption wavelength and 650nm reference wavelength. The absorbance (OD) of the blank (untreated with compound and IL-1β, LPS) was set to 100%.
Analysis of results
Cell viability assay in example 1a 549 cells and PRM cells were stimulated with IL-1β and LPS, respectively, for 24 hours after pretreatment with different concentrations of CLOS. Thereafter, the cell activity was examined using the MTT method.
The results are shown in FIG. 1 and FIG. 2, where FIG. 1 is a cytotoxicity assay of CLOS on IL-1β -activated A549 cells and FIG. 2 is a cytotoxicity assay of CLOS on LPS-activated RPM cells, and the results show that at concentrations below 20 μM, CLOS has no significant cytotoxicity on IL-1β -activated A549 cells and LPS-activated RPM cells.
Example 2
Effect of CLOS on PGE 2 and mPGES-1 protein activity in cell-free ELISA assay
Test procedure
(1) Preparation of mPGES-1 protein
A549 cells were seeded at a density of 1.6x10 6 in a 175cm 2 flask and incubated for 24 hours; the cells were stimulated with 1ng/mL IL-1. Beta. For 72 hours; cells were collected again and washed 2 times with PBS; the collected cell pellet was centrifuged at 1000rpm for 5 minutes, and then the supernatant was discarded; cells were disrupted by sonicator disruption by resuspension of cell pellet with pre-chilled homogenization buffer (0.1M potassium phosphate buffer pH7.4, 1mM phenylmethanesulfonyl fluoride, 60. Mu.g/mL soybean trypsin inhibitor, 1. Mu.g/mL leupeptin, 2.5mM GSH and 250mM sucrose). The suspension was collected and centrifuged at 10000g for 10 minutes. The supernatant was collected and centrifuged at 50000rpm for 1.5 hours. After super-high speed centrifugation, the supernatant was discarded, and the pellet was collected and resuspended in homogeneous buffer and stored at-80 ℃.
(2) Microsomal membranes of A549 cells were diluted in potassium phosphate buffer (0.1M, pH 7.4) containing 2.5mM GSH (total volume 100. Mu.L). PGE 2 synthesis was initiated by addition of PGH2 (20. Mu.M final concentration). The working solution was incubated at 4℃for 1 min, and then the reaction was stopped with 100. Mu.L of stop solution (40 mM FeCl2, 80mM citric acid and 10. Mu.M 11. Beta. -PGE 2). PGE 2 levels were measured by ELISA. In the test, MK886 was selected as a positive control.
Analysis of results
Since MK886 (ic50=2.4 μm) is one of the earliest discovered mPGES-1 inhibitors, which is generally used as a reference inhibitor in mPGES-1 protein activity assays, the effect of CLOS on PGE 2 and mPGES-1 protein activity was examined by cell-free ELISA methods.
The results are shown in FIG. 3 and FIG. 4, FIG. 3 is an analytical graph for determining the protein activity of PGE 2, and FIG. 4 is an analytical graph for determining the protein activity of mPGES-1; as can be seen from FIG. 3, CLOS reduces PGE 2 protein activity, as does the positive control MK886, which also reduces PGE 2 protein activity; as can be seen from FIG. 4, CLOS decreases mPGES-1 protein activity, as does the positive control MK886, which also decreases mPGES-1 protein activity. Thus, CLOS may reduce PGE 2 and mPGES-1 protein activity simultaneously. Similarly, the positive drug MK886 can also reduce the activity of both proteins.
Example 3
Affinity detection of mPGES-1 proteins with Compounds in cell-free assays
Test procedure
The measurement was carried out using a Fort Bio Octet Red instrument, all using 96-well plates (Greiner Bio-One, PN: 655209). All final reaction volumes were controlled to 200 μl/well. Biotinylated mPGES-1 protein was immobilized on the SA tip. The measuring step comprises the following steps: baseline zeroing, compound binding, dissociation. Protein and drug affinity results analysis was performed by Forte-Bio data analysis software. To measure the interaction between compound CLOS and mPGES-1 protein, 7 concentrations of compound CLOS (3.13, 6.25, 12.5, 25, 50, 100. Mu.M) were used in this assay.
Analysis of results
Since the results of example 2 have shown that CLOS can reduce the protein activity of both PGE 2 and mPGES-1, the present study was followed to test the binding capacity of CLOS to mPGES-1 protein using molecular docking. As a result, the binding ability of CLOS to mPGES-1 protein was shown in FIG. 5.
Example 4
Effect of CLOS on IL-1 beta-induced PGE 2 protein expression in a549 cells
Test procedure
(1) A549 cell line was purchased from AMERICAN TYPE Culture Collection (ATCC, manassas, VA, USA). Cell culture medium was RPMI 1640, and 10% fetal bovine serum (Gibco BRL Co, GRAND ISLAND, NY, USA) and 1% penicillin G (100 ml/unit), streptomycin (100 mg/ml) and L-glutamine (2 Mm) (Gibco BRL Co, GRAND ISLAND, NY, USA) were added. Cells were incubated in an environment containing 5% CO 2 at 37 ℃.
(2) A549 cells were seeded in 6-well plates at a density of 1 x 10 5 and incubated for 24 hours; pretreatment with CLOS (10. Mu.M), MK886 (10. Mu.M), DEX (0.5. Mu.M) was performed for 1 hour, followed by stimulation with interleukin-1β (IL-1β) (1 ng/mL) for 24 hours.
Analysis of results
Since the experiment of example 3 has demonstrated that CLOS has affinity for mPGES-1 protein, CLOS was further tested on the IL-1β -induced A549 cell model.
The results are shown in FIG. 6, in which IL-1β -induced PGE 2 expression levels in A549 cells were significantly increased after 24 hours of stimulation. Pretreatment with CLOS effectively reduces PGE 2 expression levels. Dexamethasone (DEX) and MK886 also significantly reduced PGE 2 expression levels in this cell model.
Example 5
Detection of PGE 2、PGI2 and TXA 2 expression levels in A549 cells and RPM
Test procedure
A549 cells and RPM cells were seeded in 6-well plates for use in subsequent experiments. Both cells were pretreated with CLOS (10. Mu.M) or the positive drug MK886 (10. Mu.M) or DEX (0.5. Mu.M) for 1 hour. A549 cells were then stimulated with IL-1 β and RPM cells were stimulated with LPS for 24 hours. After stimulation, the medium was collected. Culture medium summary PGE 2 levels were measured using ELISA methods. The expression levels of 6-keto-pgf1α and TXB 2 in the medium were chosen to represent the expression levels of PGI 2 and TXA 2, as the former is a stable degradation product of the latter.
Analysis of results
Since PGI 2 and TXA 2 were enzymatically degraded to 6-keto-pgf1α and TXB 2 in a very short period of time under natural conditions, the expression level of the latter was chosen to reflect the expression level of the former two in this study.
Fig. 7 is an analytical graph of PGE 2 levels, showing that RPM cells expressed significantly increased PGE 2 levels after 24 hours of LPS stimulation. In contrast, PGE 2 expression levels decreased in a dose-dependent manner following pretreatment with CLOS, with PGE 2 expression levels tending to decrease as CLOS concentration increases; both dexamethasone and MK886 significantly reduced the expression level of PGE 2.
FIG. 8 is an analytical graph of PGI 2 expression levels, showing that PGI 2 expression levels decreased in a dose-dependent manner after pretreatment with CLOS, and PGI 2 expression levels tended to decrease as CLOS concentration increased; both dexamethasone and MK886 significantly reduced the expression level of PGI 2.
Fig. 9 is an analytical graph of TXA 2 expression levels, as can be seen, the expression levels of TXA 2 were not significantly affected after pretreatment with CLOS; both dexamethasone and MK886 significantly reduced the expression level of TXB 2, TXA 2.
Fig. 10 is an analytical plot of the ratio of TXA 2 expression levels to PGI 2 expression levels, showing that CLOS has no significant effect on the balance between PGI 2 and TXA 2; dexamethasone also breaks the balance between PGI 2 and TXA 2, which significantly alters the ratio between PGI 2 and TXA 2.
Example 6
Effects of CLOS on the expression levels of mPGES-1, COX-1 and COX-2 proteins in IL-1β -induced A549 cells and on the expression levels of mPGES-1 and COX-2 proteins in LPS-induced RPM cells
Test procedure
(1) After the pretreated and stimulated cells were collected, washed with pre-chilled PBS. Cells were then lysed with RIPA lysis buffer (CELL SIGNALING technology, boston, MA, USA) mixed with 1 x protease inhibitor (Roche APPLIED SCIENCE, germany); protein concentration was determined using Bio-Rad protein quantification reagent (Bio-Rad, hercules, calif., USA).
(2) The proteins were separated by gel electrophoresis on 13.5% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE).
(3) After electrophoretic separation, the separated proteins were transferred from the SDS-PAGE gel onto nitrocellulose membranes (NC membrane, GE HEALTHCARE LIFE SCIENCES, buckinghamshire, UK). Then, the NC film is sealed by 5% skimmed milk; thereafter, the membrane was incubated overnight with primary antibodies (. Beta. -actin, COX-1, COX-2, mPGES-1); the membrane was then incubated with a fluorescent secondary antibody IRDye 800CW goat anti-mouse IgG (H+L) or IRDye 800CW goat anti-mouse IgG (H+L) secondary antibodies (Li-COR, lincoln, NE, USA) for 1 hour at room temperature. Antigen-antibody complex bands were then obtained by Odyssey CLxImager (Li-COR, USA) and protein expression levels were quantified and analyzed using Odyssey v3.0 software (Li-COR, USA). The density ratio of COX-1, COX-2, mPGES-1 protein to beta-actin protein was analyzed by Odyssey v 3.0.
Analysis of results
(1) Effect of CLOS on IL-1 beta-induced expression levels of mPGES-1, COX-1 and COX-2 proteins in a549 cells
As CLOS reduces PGE 2 expression levels after stimulation in selected cell models. The effect of CLOS on protein expression levels of mPGES-1, COX-1 and COX-2 in these models was thus verified by the western blot method following this study.
As a result, the expression of mPGES-1 protein in the IL-1β -induced A549 cells was significantly increased as shown in FIG. 11. Whereas CLOS pretreatment has a significant inhibitory effect on cellular mPGES-1 protein expression, but no significant effect on COX-1 and COX-2 protein expression.
(2) Effect of CLOS on the expression levels of mPGES-1 and COX-2 proteins in LPS-induced RPM cells
Since CLOS reduced the expression levels of PGE 2 in RPM cells and did not significantly affect the expression levels of PGI 2 and TXA 2, the present study used the western blot method to verify the effect of CLOS on protein expression levels of mPGES-1 and COX-2 in LPS stimulated RPM.
As shown in FIG. 12, LPS stimulation significantly increased the expression levels of mPGES-1 and COX-2 proteins in RPM cells. As can be seen from FIG. 12 (A), the pretreatment with CLOS had a dose-dependent inhibitory effect on the cell-expressed mPGES-1 protein, and as can be seen from FIG. 12 (B), there was no significant effect on the COX-2 protein.
Taken together, the compound CLOS is a novel mPGES-1 inhibitor; in a cell-free assay, CLOS significantly reduced PGE 2 production and inhibited mPGES-1 protein activity, indicating that it may exhibit anti-inflammatory activity by modulating mPGES-1 protein expression to inhibit PGE 2 production. In cell experiments, CLOS down-regulates the expression of mPGES-1 protein and the production of PGE 2 in a dose-dependent (10, 5, 2.5. Mu.M) manner, which also demonstrates that it can exhibit anti-inflammatory activity by modulating mPGES-1 protein expression to inhibit PGE 2 production. At the same time, CLOS does not break the balance between PGI 2 and TXA 2, and has no obvious effect on COX-1 or COX-2, which means that CLOS has less risk of causing cardiovascular side effects in application than traditional selective COX-2 inhibitors or other non-steroidal anti-inflammatory drugs. Thus, the CLOS can be used in the preparation of a medicament for inhibiting inflammation, in the preparation of a medicament for preventing and/or treating inflammatory diseases and/or diseases related to microsomal prostaglandin E 2 synthase 1, in the preparation of a medicament for preventing and/or treating complications of inflammatory diseases and/or complications of diseases related to microsomal prostaglandin E 2 synthase 1.
The foregoing description of the preferred embodiments of the application is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the application.
Claims (2)
1. Use of a microsomal prostaglandin E 2 synthase 1 inhibitor for the manufacture of a medicament for inhibiting inflammation associated with microsomal prostaglandin E 2 synthase 1, wherein the microsomal prostaglandin E 2 synthase 1 inhibitor is a pyrimidinedione compound, and the pyrimidinedione compound is 2- { [ ((3-chlorophenyl) methyl ] sulfanyl } -5- (3, 4-dihydroxyphenyl) -3H,4H,5H,6H,7H, 8H-pyridinyl [2,3-d ] pyrimidine-4, 7-dione, having the structural formula shown in formula I,
Formula I.
2. The use of a microsomal prostaglandin E 2 synthase 1 inhibitor in the manufacture of a medicament for the treatment of inflammatory diseases associated with microsomal prostaglandin E 2 synthase 1, said microsomal prostaglandin E 2 synthase 1 inhibitor is a pyrimidinedione compound, and said pyrimidinedione compound is 2- { [ ((3-chlorophenyl) methyl ] sulfanyl } -5- (3, 4-dihydroxyphenyl) -3H,4H,5H,6H,7H, 8H-pyridinyl [2,3-d ] pyrimidine-4, 7-dione, having the structural formula shown in formula I,
Formula I.
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