CN109939109B - Application of N-thiazole-acetamide derivative as osteoclast differentiation inhibitor in preparation of anti-osteoporosis drugs - Google Patents
Application of N-thiazole-acetamide derivative as osteoclast differentiation inhibitor in preparation of anti-osteoporosis drugs Download PDFInfo
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- CN109939109B CN109939109B CN201811625045.2A CN201811625045A CN109939109B CN 109939109 B CN109939109 B CN 109939109B CN 201811625045 A CN201811625045 A CN 201811625045A CN 109939109 B CN109939109 B CN 109939109B
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Abstract
The invention provides an application of an N-thiazole-acetamide derivative in preparing an anti-osteoporosis medicine, wherein the structural formula of the N-thiazole-acetamide derivative is shown as a formula (I), the N-thiazole-acetamide derivative is different from the molecular skeleton of the current clinically used medicine for preventing and treating osteoporosis, and the compound does not belong to a phosphate compound, is not an estrogen medicine and is a brand new compound with an anti-osteoporosis effect. Meanwhile, the N-thiazole-acetamide derivative provided by the invention has high osteoclast differentiation inhibition activity and low toxicity. Therefore, the compound can be used as a new compound type for preventing and treating related diseases caused by abnormal differentiation of osteoclast, especially osteoporosis or osteopenia.
Description
Technical Field
The invention relates to the technical field of new application of medicaments, in particular to application of an N-thiazole-acetamide derivative as an osteoclast differentiation inhibitor in preparation of an anti-osteoporosis medicament.
Background
Osteoporosis (OP for short) is a systemic skeletal disease characterized by a decrease in bone mass, an increase in bone fragility due to destruction of the microstructure of bone tissue, and an increase in the risk of fracture. The clinical manifestations and signs are mainly pain, followed by short body length, humpback, fractures and respiratory disorders. Epidemiological investigations have shown that approximately 50% of women and 20% of men are at risk of fracture in people over the age of 50 (Rachner, Khosla et al.2011). And (3) displaying data of the national statistical office: by 2050, nearly half (49%) of the population in China over 50 years old is about 6.36 hundred million, and the number of osteoporosis patients in China is estimated to be about 3 hundred million by 2050.
An imbalance in the ratio between osteoclasts (osteoplasts) and osteoblasts (osteoplasts) is the pathological basis for the development of osteoporosis (Rachner, Khosla et al 2011). Relatively increased osteoclast differentiation or relatively decreased osteoblast differentiation can cause bone loss and osteoporosis. The main treatment means for clinically treating the osteoporosis at present is to reduce bone resorption and promote bone synthesis. The bone absorption inhibitor mainly comprises estrogen receptor modulators such as tamoxifen, toremifene, droloxifene, raloxifene, azoxifene, bazedoxifene, ipriflavone and the like, bisphosphates/salts such as glycolic acid phosphonate, clodronate, pamidronic acid, halogen phosphate, eslun phosphate, risedronate sodium, zoledron phosphate, ibandronate sodium and the like, calcitonin and the like. The medicines for promoting bone synthesis mainly comprise Wnt signal regulator (AMG785, BHQ880), Parathyroid hormone (PTH), calcium-sensitive receptor antagonist (such as ATF936) and statins. Also, agents which inhibit bone resorption and promote bone formation, such as Alfacalcidol, Calcitriol (Alfacalcidol, Calcitriol, RO-26-9228, ED-71), and the like. However, the above drugs, although preventing the decrease of bone density to some extent, do not significantly reduce the risk of atypical fractures, and have various degrees of side effects, and have not yet met the requirements for anti-osteoporosis therapy (Siris, Selby et al 2009). Therefore, the development of a new specific medicine for resisting osteoporosis is urgently needed at present to solve the problem that the current clinical medicine cannot meet the treatment requirement.
Osteoclasts are undifferentiated cells derived from the macrophage lineage of bone marrow, and are the only cells known to have a bone resorption effect. Nuclear Receptor activator of nuclear factor B ligand (RANKL) is a soluble transmembrane protein essential for osteoclasts to maintain their structure, function and survival. RANK binds to its ligand, activates downstream NF- κ B, Akt, mitogen-activated protein kinase (MAPK), activates nuclear receptor for T (NFAT), calcium ion channels, and calcium/calmodulin-dependent kinase signaling pathways, allowing undifferentiated bone marrow macrophages to differentiate into osteoclasts, thereby causing osteoporosis (Boyle, Simonet et al 2003). Numerous studies have demonstrated that interference with the RANKL signaling pathway inhibits osteoclast differentiation and results in pharmacological effects against osteoporosis (Kim and Kim 2016). In recent years, the development of novel anti-osteoporosis drugs by interfering with the RANKL signaling pathway has become a hotspot. The search for a drug with inhibitory activity on the osteoclast induced by RANKL will hopefully solve the problems of the current osteoporosis treatment drugs.
Disclosure of Invention
Aiming at the defects of the prior art, the invention discovers a new anti-osteoporosis medicine based on a new target RANKL signal path, so as to overcome the problem that the existing osteoporosis treatment medicine can not reduce the risk of atypical fracture, and provides an application of an N-thiazole-acetamide derivative in preparing the anti-osteoporosis medicine.
The invention adopts the following technical scheme to realize the technical purpose:
the application of the N-thiazole-acetamide derivative in preparing the anti-osteoporosis medicine is disclosed, wherein the structural formula of the N-thiazole-acetamide derivative is shown as the formula (I):
wherein R is1Hydrogen, straight-chain alkyl or substituted straight-chain alkyl, cycloalkyl, alkenyl or substituted alkenyl, carbonyl or substituted carbonyl, five-membered or six-membered heterocyclic ring, benzene ring or benzene ring containing substituent, cyano, nitro, alkoxy, alkylamino, hydroxyl and amino; r2Hydrogen, straight-chain alkyl or substituted straight-chain alkyl, cycloalkyl, alkenyl or substituted alkenyl, carbonyl or substituted carbonyl, five-membered or six-membered heterocyclic ring, benzene ring or benzene ring containing substituent, cyano, nitro, alkoxy, alkylamino, hydroxyl, amino or amino containing substituent; r1And R2Can be mutually bonded to form a ring;
the ring B is a five-membered or six-membered aromatic heterocyclic group or a substituted five-membered or six-membered aromatic heterocyclic group;
R3is a benzene ring or a benzene ring containing a substituent group, a five-membered or six-membered aromatic heterocycle or a substituted five-membered or six-membered aromatic heterocycle;
R1、R2and R3In the group definition, the substituent group is hydrogen, halogen, C1-C5 alkoxy, C1-C5 alkyl, ester group, nitro, amino, cyano, alkynyl, five-membered or six-membered heterocyclic group.
The design idea of the invention is that according to a method for screening three-dimensional structure similarity, a series of compounds possibly having potential activity compounds with anti-osteoporosis effect are obtained by screening the existing compounds in the laboratory by using the known inhibitor for resisting osteoporosis or osteoclast differentiation as a template, and then an osteoclast differentiation inhibition experiment is carried out on the screened compounds at a cellular level, so that the N-thiazole-acetamide derivatives with high inhibition activity and low cytotoxicity can effectively inhibit osteoclast differentiation. Therefore, the derivatives can be safely used for preparing medicaments for treating and/or preventing osteoporosis.
The compounds selected by the invention are all existing compounds. Computer screening refers to screening an existing compound library by using computer-aided drug design related software and adopting technical means such as similarity retrieval and the like, and performing biological activity test on the screened compounds.
As a preferred embodiment, R3Is a benzene ring or a benzene ring containing a substituent, a five-membered aromatic heterocycle or a substituted five-membered aromatic heterocycle, a pyridine ring or a pyridine ring containing a substituent, a pyrimidine ring or a pyrimidine ring containing a substituent, a pyrazine ring or a pyrazine ring containing a substituent.
As a preferred embodiment, R3Is a five-membered aromatic heterocycle, a benzene ring or a substituted benzene ring, a pyridine ring or a pyrazine ring.
As a preferred embodiment, R1Hydrogen, C1-C4 alkyl, benzene ring or substituted benzene ring, five-membered or six-membered heterocycle; r2Hydrogen, ester group or substituted ester group, phenyl or substituted phenyl, cyano-group and nitro-group, and the B ring is 1, 2-substituted or 1, 3-substituted five-membered aromatic heterocycle.
As a preferred embodiment, R1Is hydrogen, straight chain alkyl of C1-C4, benzene ring or substituted benzene ring, R2Is hydrogen, an ester group or a substituted ester group, a six-membered heterocyclic ring, R1，R2The mutual bond synthesizes six-membered ring, the B ring is thiazole ring, thiophene ring, triazole ring, pyrazole ring or oxazole ring.
As a preferred embodiment, R1，R2Are bonded to each other to form benzene rings.
As a preferred embodiment, R1Is hydrogen, C1-C3 straight-chain alkyl, phenyl, 4-toluene, 4-chlorobenzene, 4-methoxybenzene, R2Is hydrogen, ethyl formate, methyl formate, formyloxy, morpholine, azomethylpiperazine, R1And R2To form a benzene ring, ring B is a 1, 3-substituted triazole ring, R3Thiophene, thiazole, benzene ring, 4-toluene, 2-chlorobenzene, 3-chlorobenzene, 4-chlorobenzene, 2-fluorobenzene, 3-fluorobenzene, 4-cyanobenzene, 4-methyl formate benzene, 4-carboxybenzene, 4-nitrobenzene, 4-alkynyl benzene, pyridine, 3-aminobenzene, 4-methoxybenzene and 4-hydroxybenzene.
As a most preferred embodiment, the structural formula of the N-thiazole-acetamide derivative is one of the following structural formulas:
more preferably, the compounds 3g, 6g, 4i, 2e and 4k in the above structural formula have a significant effect on inhibitory activity or safety.
Further, the medicament comprises pharmaceutically acceptable salts, carriers and/or excipients.
Compared with the prior art, the invention has the following advantages and effects:
the N-thiazole-acetamide derivative provided by the invention can inhibit osteoclast differentiation to a certain extent, and has better inhibitory activity and safety. The N-thiazole-acetamide derivative provided by the invention has a simple structure and is easy to synthesize; and the compound has low toxicity, and can be safely used for preparing medicaments for treating and/or preventing osteoporosis or osteopenia.
Drawings
FIG. 1 is a graph showing the inhibition of osteoclast differentiation by 3 g.
FIG. 2 is a graph showing the inhibition of osteoclast differentiation at 6 g.
FIG. 3 is a graph showing the inhibition of osteoclast differentiation by 4 i.
Fig. 4 is a graph of 2e osteoclast differentiation inhibition.
FIG. 5 is a graph of the 4k osteoclast differentiation inhibition.
Detailed Description
The present invention will be further described with reference to the following specific examples and drawings, which are not intended to limit the invention in any manner. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the present invention are commercially available.
Example 1: computer drug screening
Compound library pretreatment: the compound library is a self-contained compound database. The compound library was processed as follows: removing ions and complexing water molecules, adding charges, protonating and generating three-dimensional conformation. These procedures were all completed in the drug design software package Discovery Studio 2.5. Wherein the protonation is carried out under the condition of pH 6.5-8.5. And preparing a small molecule library for virtual screening.
Three-dimensional similarity search is carried out by using known anti-osteoporosis drugs and osteoclast differentiation inhibitors, and compounds with similarity higher than 80% are found. The invention predicts that the compound stock library in the laboratory has 20 compounds with potential activity by taking a computer drug screening program as a self-developed program WEGA in the laboratory. The 20 compounds were screened for activity of inhibiting osteoporosis differentiation at the cellular level, and 4 of the compounds (1a to 1d) were found to have an effect of inhibiting osteoclast differentiation. These 4 compounds are all thiazole amide derivatives.
Example 2: determination of toxicity of Compound cells
S1, cell culture.
RAW264.7 cells were cultured in vitro. Conventional maintenance culture and passage were carried out at 37 ℃ under 5% carbon dioxide concentration using a high-glucose medium containing 10% fetal bovine serum (DMEM).
S2, compound intervention.
Cells were collected in log phase and cell suspension was made to 1X 105one/mL, added to 96 well cell culture plates. In carbon dioxideAfter 24h incubation in the incubator, the medium was changed to medium containing different concentrations of the compound, and the incubation was continued for 2 days, and cytotoxicity was measured on day 3. Test compounds were formulated as solutions of different concentrations using DMSO. Duplicate wells of 3 parallel were placed at each concentration and compared to a control group without compound treatment.
And S3, testing.
The MTT [3- (4, 5-dimethylthiazole-2) -2, 5-diphenyl tetrazolium bromide ] method is adopted to determine cells after the compound is treated for 6 days. The absorbance (OD) of the wells was measured using a microplate reader (detection wavelength 570nm, reference wavelength 630 nm).
And S4, result processing.
The growth inhibition rate of each drug on RAW264.7 cells was calculated according to the following formula:
the inhibition rate of RAW264.7 cell growth is used as ordinate, the log value of compound concentration is used as abscissa to draw the inhibition curve graph of each compound on cell growth, and half toxicity concentration CC is obtained according to the inhibition rate of each compound on cell50I.e., the concentration of drug that inhibits cell growth by 50%.
According to the formula: selecting suppression constant (SI) ═ CC50/IC50The selective inhibition constant of each compound was calculated to evaluate the safety of administration of each compound. The selective inhibition constant results for all compounds are shown in table 1.
Example 3: osteoclast differentiation inhibition assay
S1, cell culture.
RAW264.7 cells were cultured in vitro. Conventional maintenance culture and passage were carried out at 37 ℃ under 5% carbon dioxide concentration using a high-glucose medium containing 10% fetal bovine serum (DMEM).
S2, compound intervention.
Cells were collected in log phase and cell suspension was made to 2X 104One/ml, added to 96 well cell culture plates. Culturing in carbon dioxide incubator for 24 hrThe culture medium was changed to a medium containing 100ng/ml RANKL and different compound concentrations, and the medium was continuously cultured for 5 days, and every 2 days, medium containing the same RANKL concentration and compound concentration was changed, and osteoclast was stained by TRAP staining method on day 5. Duplicate wells of 3 parallel were placed at each concentration and compared to a control group without compound treatment.
And S3, testing.
And (3) staining the osteoclasts formed by differentiation by using a TRAP kit, and counting the number of the osteoclasts fused with ≧ 3 cell nuclei.
And S4, result processing.
The inhibition rate of each drug on RANKL-induced osteoclast differentiation was calculated according to the following formula:
plotting osteoclast differentiation inhibition rate as ordinate and log value of compound concentration as abscissa, and calculating half effective rate IC according to osteoclast differentiation inhibition rate of each compound50I.e. the concentration of the drug which inhibits osteoclast differentiation by 50%.
According to the formula: selecting suppression constant (SI) ═ CC50/IC50The selective inhibition constant of each compound was calculated to evaluate the safety of administration of each compound.
According to the selectivity index SI ═ CC50/IC50The effect of the compounds against the differentiation of osteoporosis was evaluated according to the following criteria. SI (Standard interface)<1.0 indicates that the compound is toxic and ineffective, 1.0 or more and SI or less than 2.0 indicates that the compound is low-efficiency toxic, namely weak positive, 2.0<SI<10.0 shows that the compound is effective and low in toxicity, namely positive, and SI is more than or equal to 10.0 shows that the compound is efficient and low in toxicity, namely strong positive.
As shown in the results in Table 1, the present inventors found that 5 compounds have different degrees of osteoclast differentiation inhibition, among which 3g activity is the best, therapeutic index is the highest, and IC is the highest500.001, SI 97200 (see FIG. 1, Table 1); it is composed ofA second time of 6g, IC500.02, SI 8310 (see FIG. 2, Table 1); the next to active is 4i, IC500.05, and 7370 SI (see fig. 3, table 1); again, the activities are 2e and 4k, IC500.06 and 0.08 respectively, and 2065 and 6940 respectively for SI (see fig. 4 and 5, table 1).
TABLE 1 Structure of compound obtained by drug screening and its inhibitory action on osteoclast differentiation
The invention discovers a thiazole amide derivative by a computer-aided drug design similarity retrieval method. Through an osteoclast differentiation inhibition experiment and a cytotoxicity (MTT) experiment, 3g, 6g, 4i, 2e and 4k of the compounds have osteoclast differentiation inhibition activity less than 0.1 mu M, low cytotoxicity and high treatment index. Half inhibitory dose (IC) of 3g for osteoclast differentiation50) At 0.001. mu.M, a cell semilethal dose (CC)50) 97.2 μ M, with an inhibition constant (SI) of 97200 (see FIG. 1, Table 1); 6g half inhibitory dose (IC) for osteoclast differentiation50) At 0.02. mu.M, a cell semilethal dose (CC)50) 166.2 μ M, with an inhibition constant (SI) of 8310 (see FIG. 2, Table 1); the half inhibitory dose (IC50) for osteoclast differentiation of 4i was 0.05. mu.M, the half lethal dose (CC50) was 368.5. mu.M, and the inhibition constant (SI) was selected to be 7370 (see FIG. 3, Table 1); 2e half inhibitory dose (IC) on osteoclast differentiation50) At 0.06. mu.M, a cell semilethal dose (CC)50) 123.9 μ M, an inhibition constant (SI) of 2065 was chosen (see FIG. 4, Table 1); half inhibitory dose (IC) of 4k on osteoclast differentiation50) At 0.08. mu.M, a cell semilethal dose (CC)50) The inhibition constant (SI) was chosen to be 6940 for 555.2. mu.M (see FIG. 5, Table 1). The results show that the compounds have high osteoclast differentiation inhibition activity and low cytotoxicity, and can be used as an osteoclast inhibitor to prepare osteoporosis or osteopenia prevention and treatment medicines.
Claims (1)
- The application of the N-thiazole-acetamide derivative in preparing the anti-osteoporosis medicine is characterized in that the structural formula of the N-thiazole-acetamide derivative is one of the following structural formulas:
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| EP1402900A1 (en) * | 2001-06-11 | 2004-03-31 | Takeda Chemical Industries, Ltd. | Medicinal compositions |
| CN107050021A (en) * | 2017-01-23 | 2017-08-18 | 广州中大南沙科技创新产业园有限公司 | Application of the thiazole amide derivatives in osteosporosis resistant medicament is prepared |
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| EP1402900A1 (en) * | 2001-06-11 | 2004-03-31 | Takeda Chemical Industries, Ltd. | Medicinal compositions |
| CN107050021A (en) * | 2017-01-23 | 2017-08-18 | 广州中大南沙科技创新产业园有限公司 | Application of the thiazole amide derivatives in osteosporosis resistant medicament is prepared |
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