CN118873532B - Application of trisubstituted pyrazole compound in preparation of medicines for inhibiting osteoclast differentiation - Google Patents
Application of trisubstituted pyrazole compound in preparation of medicines for inhibiting osteoclast differentiation Download PDFInfo
- Publication number
- CN118873532B CN118873532B CN202411396736.5A CN202411396736A CN118873532B CN 118873532 B CN118873532 B CN 118873532B CN 202411396736 A CN202411396736 A CN 202411396736A CN 118873532 B CN118873532 B CN 118873532B
- Authority
- CN
- China
- Prior art keywords
- pyrazole compound
- trisubstituted pyrazole
- osteoclast differentiation
- trisubstituted
- preparation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 210000002997 osteoclast Anatomy 0.000 title claims abstract description 61
- 230000004069 differentiation Effects 0.000 title claims abstract description 34
- -1 pyrazole compound Chemical class 0.000 title claims abstract description 30
- 239000003814 drug Substances 0.000 title claims abstract description 26
- 230000002401 inhibitory effect Effects 0.000 title claims abstract description 15
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- 229940079593 drug Drugs 0.000 title claims description 11
- 230000014509 gene expression Effects 0.000 claims abstract description 15
- 239000003963 antioxidant agent Substances 0.000 claims abstract description 9
- 230000003078 antioxidant effect Effects 0.000 claims abstract description 9
- 201000010099 disease Diseases 0.000 claims abstract description 9
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 9
- 239000003112 inhibitor Substances 0.000 claims abstract description 6
- 108090000623 proteins and genes Proteins 0.000 claims description 20
- 102000004169 proteins and genes Human genes 0.000 claims description 16
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 10
- 239000000843 powder Substances 0.000 claims description 7
- 230000005764 inhibitory process Effects 0.000 claims description 6
- 208000001132 Osteoporosis Diseases 0.000 claims description 5
- 238000000034 method Methods 0.000 claims description 5
- 230000000010 osteolytic effect Effects 0.000 claims description 5
- 206010028980 Neoplasm Diseases 0.000 claims description 4
- 210000000988 bone and bone Anatomy 0.000 claims description 4
- 229910052736 halogen Inorganic materials 0.000 claims description 4
- 238000002347 injection Methods 0.000 claims description 4
- 239000007924 injection Substances 0.000 claims description 4
- 125000001424 substituent group Chemical group 0.000 claims description 4
- 238000011282 treatment Methods 0.000 claims description 4
- 206010027476 Metastases Diseases 0.000 claims description 3
- 230000006378 damage Effects 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 claims description 3
- 230000009401 metastasis Effects 0.000 claims description 3
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 3
- 206010039073 rheumatoid arthritis Diseases 0.000 claims description 3
- 239000004480 active ingredient Substances 0.000 claims description 2
- 239000002775 capsule Substances 0.000 claims description 2
- 239000006071 cream Substances 0.000 claims description 2
- 230000002708 enhancing effect Effects 0.000 claims description 2
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 claims description 2
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 2
- 239000006187 pill Substances 0.000 claims description 2
- 239000003826 tablet Substances 0.000 claims description 2
- 125000005843 halogen group Chemical group 0.000 claims 2
- 239000006072 paste Substances 0.000 claims 1
- 238000011321 prophylaxis Methods 0.000 claims 1
- 150000001875 compounds Chemical class 0.000 abstract description 10
- 238000010186 staining Methods 0.000 abstract description 9
- 102000016938 Catalase Human genes 0.000 abstract description 8
- 108010053835 Catalase Proteins 0.000 abstract description 8
- 238000003753 real-time PCR Methods 0.000 abstract description 6
- 238000001262 western blot Methods 0.000 abstract description 5
- 102000013563 Acid Phosphatase Human genes 0.000 abstract description 3
- 108010051457 Acid Phosphatase Proteins 0.000 abstract description 3
- 238000010839 reverse transcription Methods 0.000 abstract description 3
- FEWJPZIEWOKRBE-UHFFFAOYSA-N Tartaric acid Natural products [H+].[H+].[O-]C(=O)C(O)C(O)C([O-])=O FEWJPZIEWOKRBE-UHFFFAOYSA-N 0.000 abstract description 2
- 235000002906 tartaric acid Nutrition 0.000 abstract description 2
- 239000011975 tartaric acid Substances 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 39
- 210000001185 bone marrow Anatomy 0.000 description 19
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 16
- 239000002609 medium Substances 0.000 description 14
- 210000002540 macrophage Anatomy 0.000 description 13
- 150000003217 pyrazoles Chemical class 0.000 description 13
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 12
- 102000014128 RANK Ligand Human genes 0.000 description 12
- 108010025832 RANK Ligand Proteins 0.000 description 12
- 102000007591 Tartrate-Resistant Acid Phosphatase Human genes 0.000 description 12
- 108010032050 Tartrate-Resistant Acid Phosphatase Proteins 0.000 description 12
- 239000003642 reactive oxygen metabolite Substances 0.000 description 10
- 230000015572 biosynthetic process Effects 0.000 description 9
- 230000000694 effects Effects 0.000 description 9
- 108010046938 Macrophage Colony-Stimulating Factor Proteins 0.000 description 8
- 102100028123 Macrophage colony-stimulating factor 1 Human genes 0.000 description 8
- 102000002737 Heme Oxygenase-1 Human genes 0.000 description 7
- 108010018924 Heme Oxygenase-1 Proteins 0.000 description 7
- 241000699666 Mus <mouse, genus> Species 0.000 description 7
- 239000001963 growth medium Substances 0.000 description 7
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 5
- 238000005160 1H NMR spectroscopy Methods 0.000 description 5
- 208000006386 Bone Resorption Diseases 0.000 description 5
- 101150011252 CTSK gene Proteins 0.000 description 5
- 102000007568 Proto-Oncogene Proteins c-fos Human genes 0.000 description 5
- 108010071563 Proto-Oncogene Proteins c-fos Proteins 0.000 description 5
- 230000006698 induction Effects 0.000 description 5
- 238000002844 melting Methods 0.000 description 5
- 230000008018 melting Effects 0.000 description 5
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 4
- 101150001535 SRC gene Proteins 0.000 description 4
- RWCCWEUUXYIKHB-UHFFFAOYSA-N benzophenone Chemical compound C=1C=CC=CC=1C(=O)C1=CC=CC=C1 RWCCWEUUXYIKHB-UHFFFAOYSA-N 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- 241000699670 Mus sp. Species 0.000 description 3
- 108010087230 Sincalide Proteins 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 230000001464 adherent effect Effects 0.000 description 3
- 238000010609 cell counting kit-8 assay Methods 0.000 description 3
- 230000003013 cytotoxicity Effects 0.000 description 3
- 231100000135 cytotoxicity Toxicity 0.000 description 3
- 229940088598 enzyme Drugs 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000002243 precursor Substances 0.000 description 3
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- GZCWLCBFPRFLKL-UHFFFAOYSA-N 1-prop-2-ynoxypropan-2-ol Chemical compound CC(O)COCC#C GZCWLCBFPRFLKL-UHFFFAOYSA-N 0.000 description 2
- 229940122361 Bisphosphonate Drugs 0.000 description 2
- 102000055006 Calcitonin Human genes 0.000 description 2
- 108060001064 Calcitonin Proteins 0.000 description 2
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- PXGOKWXKJXAPGV-UHFFFAOYSA-N Fluorine Chemical compound FF PXGOKWXKJXAPGV-UHFFFAOYSA-N 0.000 description 2
- PWKSKIMOESPYIA-BYPYZUCNSA-N L-N-acetyl-Cysteine Chemical compound CC(=O)N[C@@H](CS)C(O)=O PWKSKIMOESPYIA-BYPYZUCNSA-N 0.000 description 2
- 108010019160 Pancreatin Proteins 0.000 description 2
- 229960004308 acetylcysteine Drugs 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 150000004663 bisphosphonates Chemical class 0.000 description 2
- 238000007664 blowing Methods 0.000 description 2
- 230000024279 bone resorption Effects 0.000 description 2
- BBBFJLBPOGFECG-VJVYQDLKSA-N calcitonin Chemical compound N([C@H](C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(N)=O)C(C)C)C(=O)[C@@H]1CSSC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1 BBBFJLBPOGFECG-VJVYQDLKSA-N 0.000 description 2
- 229960004015 calcitonin Drugs 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 229910052801 chlorine Inorganic materials 0.000 description 2
- 239000000460 chlorine Substances 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 230000002222 downregulating effect Effects 0.000 description 2
- 229910052731 fluorine Inorganic materials 0.000 description 2
- 239000011737 fluorine Substances 0.000 description 2
- 150000002367 halogens Chemical group 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 230000020395 negative regulation of osteoclast differentiation Effects 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 229940055695 pancreatin Drugs 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 229960004622 raloxifene Drugs 0.000 description 2
- GZUITABIAKMVPG-UHFFFAOYSA-N raloxifene Chemical compound C1=CC(O)=CC=C1C1=C(C(=O)C=2C=CC(OCCN3CCCCC3)=CC=2)C2=CC=C(O)C=C2S1 GZUITABIAKMVPG-UHFFFAOYSA-N 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 241000501754 Astronotus ocellatus Species 0.000 description 1
- 206010070884 Atypical femur fracture Diseases 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- BHUDDFRMRUKTAQ-UHFFFAOYSA-N C1(=CC=CC=C1)N1N=C(C(=C1)C(=O)C1=CC=C(C=C1)F)C(=O)C1=CC=C(C=C1)F Chemical compound C1(=CC=CC=C1)N1N=C(C(=C1)C(=O)C1=CC=C(C=C1)F)C(=O)C1=CC=C(C=C1)F BHUDDFRMRUKTAQ-UHFFFAOYSA-N 0.000 description 1
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 description 1
- 208000005189 Embolism Diseases 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 101710088172 HTH-type transcriptional regulator RipA Proteins 0.000 description 1
- 101001001810 Homo sapiens Pleckstrin homology domain-containing family M member 3 Proteins 0.000 description 1
- 102000043136 MAP kinase family Human genes 0.000 description 1
- 108091054455 MAP kinase family Proteins 0.000 description 1
- 101100116205 Mus musculus Dcstamp gene Proteins 0.000 description 1
- 101001001809 Mus musculus Pleckstrin homology domain-containing family M member 3 Proteins 0.000 description 1
- 102100034404 Nuclear factor of activated T-cells, cytoplasmic 1 Human genes 0.000 description 1
- 101710151542 Nuclear factor of activated T-cells, cytoplasmic 1 Proteins 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 206010052306 Periprosthetic osteolysis Diseases 0.000 description 1
- 229940122907 Phosphatase inhibitor Drugs 0.000 description 1
- 102100036332 Pleckstrin homology domain-containing family M member 3 Human genes 0.000 description 1
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 206010042434 Sudden death Diseases 0.000 description 1
- 239000006180 TBST buffer Substances 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 229940124532 absorption promoter Drugs 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 230000014461 bone development Effects 0.000 description 1
- 210000002798 bone marrow cell Anatomy 0.000 description 1
- 230000010072 bone remodeling Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 210000000845 cartilage Anatomy 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 239000012043 crude product Substances 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 229960001251 denosumab Drugs 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 239000012954 diazonium Substances 0.000 description 1
- IJGRMHOSHXDMSA-UHFFFAOYSA-O diazynium Chemical compound [NH+]#N IJGRMHOSHXDMSA-UHFFFAOYSA-O 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- ZUOUZKKEUPVFJK-UHFFFAOYSA-N diphenyl Chemical group C1=CC=CC=C1C1=CC=CC=C1 ZUOUZKKEUPVFJK-UHFFFAOYSA-N 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 210000000629 knee joint Anatomy 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 210000003141 lower extremity Anatomy 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 230000030991 negative regulation of bone resorption Effects 0.000 description 1
- 239000002674 ointment Substances 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000012113 quantitative test Methods 0.000 description 1
- 238000010814 radioimmunoprecipitation assay Methods 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000007634 remodeling Methods 0.000 description 1
- 229940095743 selective estrogen receptor modulator Drugs 0.000 description 1
- 239000000333 selective estrogen receptor modulator Substances 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 229940095064 tartrate Drugs 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000004809 thin layer chromatography Methods 0.000 description 1
- 210000002303 tibia Anatomy 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 210000000689 upper leg Anatomy 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
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
- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
- A61K31/415—1,2-Diazoles
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
- A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
-
- 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]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Physical Education & Sports Medicine (AREA)
- Veterinary Medicine (AREA)
- Chemical & Material Sciences (AREA)
- Public Health (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- General Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Rheumatology (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Immunology (AREA)
- Pain & Pain Management (AREA)
- Epidemiology (AREA)
- Plural Heterocyclic Compounds (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses an application of a trisubstituted pyrazole compound in preparation of a medicament for inhibiting osteoclast differentiation, and belongs to the field of medicaments. The invention utilizes a plurality of experimental means such as tartaric acid phosphatase (TRAP) staining, reverse transcription real-time fluorescence quantitative PCR (qPCR), western immunoblotting (Western blot) and the like, and proves that the trisubstituted pyrazole compound of the following formula can obviously promote the expression of antioxidant proteins HO-1 and Catalase, thereby effectively inhibiting the differentiation and the generation of osteoclasts. Therefore, the trisubstituted pyrazole compound provided by the invention can be used as a candidate compound for developing a novel osteoclast differentiation inhibitor and treating diseases caused by overactivation of osteoclasts.
Description
Technical Field
The invention belongs to the field of medicines, and particularly relates to an application of a trisubstituted pyrazole compound in preparation of a medicament for inhibiting osteoclast differentiation.
Background
Osteoclasts (osteoclasts, ocs) are the only known cells responsible for bone resorption in vivo, and are derived from the differentiation and maturation of osteoclast precursor cells (e.g. bone marrow mononuclear/macrophages) derived from hematopoietic stem cells under the action of various factors, with RANKL action being particularly critical. RANKL activates downstream signal pathways such as NF- κ B, MAPK through binding to the receptor RANK on the osteoclast precursor cells, thereby up-regulating transcription factors such as NFATc1, c-Fos, and thereby regulating transcription of osteoclast specific genes, and finally promoting osteoclast formation and activation. Osteoclasts play a vital role in normal bone development and remodeling. However, abnormal increases in osteoclast number and/or activity can lead to various osteolytic diseases such as osteoporosis, rheumatoid arthritis, peri-prosthetic osteolysis, and cancer metastasis bone destruction. Inhibition of osteoclast differentiation and bone resorption is one of the currently accepted primary means of treating such diseases.
Currently, the most widely used anti-bone resorption drugs in clinic include bisphosphonates, the rank L monoclonal antibody diels (Denosumab), calcitonin, selective estrogen receptor modulators (raloxifene), and the like. The application of bisphosphonates and dieldrake has the risks of mandibular necrosis and atypical femur fracture, the rebound of dieldrake when it is deactivated, the possibility of potential increase of tumor risk when calcitonin is used for a long period of time, and the risk of venous embolism caused by raloxifene. Therefore, the existing anti-bone resorption drugs have obvious defects. Therefore, there is an urgent need for a novel safe and effective medicament for treating osteolytic diseases such as osteoporosis by inhibiting osteoclast differentiation and function.
Intracellular Reactive Oxygen Species (ROS) play a vital role in the process of osteoclast formation and bone resorption. While osteoclast precursor cells produce endogenous ROS upon RANKL stimulation, the use of ROS scavengers such as N-acetylcysteine (NAC) can inhibit RANKL-mediated ROS production, thereby inhibiting osteoclastogenesis, indicating that ROS are necessary for osteoclast differentiation. Within the cell are a number of antioxidant enzymes that antagonize ROS, such as heme oxygenase-1 (HO-1) and catalase (Catalase), and enhancing expression of these antioxidant enzymes scavenges ROS, thereby inhibiting the formation of osteoclasts. Therefore, targeted inhibition of ROS can be used as a novel strategy for inhibiting osteoclast differentiation, thereby treating bone-soluble diseases such as osteoporosis.
Disclosure of Invention
In view of the above, the embodiment of the application provides an application of a trisubstituted pyrazole compound in preparation of a medicament for inhibiting osteoclast differentiation.
According to the embodiment of the application, the structural formula of the trisubstituted pyrazole compound is shown as the formula (1):
wherein R 1 is phenyl or substituted phenyl, substituent is selected from halogen such as fluorine, chlorine and the like, methoxy, R 2 is phenyl or substituted phenyl, substituent is selected from halogen such as fluorine, chlorine and the like, methyl, ethyl and phenyl.
More preferably, the trisubstituted pyrazole compound may have any of the following specific structural formulas Y-1, Y-2, Y-3, Y-4 and Y-5:
。
the application of the trisubstituted pyrazole compound in preparation of osteoclast differentiation inhibitors.
The trisubstituted pyrazole compound can be used for preparing medicines for treating osteolytic diseases by inhibiting osteoclast differentiation, so as to inhibit overactivation of osteoclasts in osteolytic diseases, and can also be used for preparing medicines for treating osteoporosis, tumor metastasis bone destruction or rheumatoid arthritis.
The medicament can be in various forms such as tablets, pills, powder, capsules, injection, oral liquid, ointment, cream and the like, and the medicaments in various forms can be prepared according to a conventional method in the pharmaceutical field. The administration mode can be oral administration, injection or external application.
The medicine comprises an active ingredient trisubstituted pyrazole compound and pharmaceutically acceptable pharmaceutic adjuvant. The auxiliary materials comprise diluents, excipients, fillers, adhesives, wetting agents, disintegrating agents, absorption promoters, surfactants, adsorption carriers, lubricants and the like which are conventional in the pharmaceutical field.
Use of a trisubstituted pyrazole compound as described above for the manufacture of a medicament for inhibiting the expression of an osteoclast differentiation-critical protein, the osteoclast differentiation-critical protein being a c-Fos, nfatt 1, src or Ctsk protein.
The trisubstituted pyrazole compound is applied to preparing medicaments for inhibiting ROS and medicaments for improving the expression of antioxidant proteins such as HO-1, catalase and the like.
According to the invention, an RANKL-induced mouse bone marrow macrophage osteoclast differentiation model is adopted, and experiments prove that the trisubstituted pyrazole compound can directly and effectively inhibit bone marrow macrophage differentiation to osteoclast without obvious cytotoxicity by improving antioxidant proteins such as HO-1, catalase and the like and down-regulating the expression of osteoclast differentiation key proteins such as c-Fos, nfatc1 and the like, so that the trisubstituted pyrazole compound can be used as a candidate compound for developing novel osteoclast differentiation inhibitors and treating diseases caused by overactivation of osteoclasts. Therefore, the invention has great clinical significance and application prospect.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the application and together with the description, serve to explain the principles of the application.
FIG. 1 is a graph depicting inhibition of RANKL-induced osteoclast differentiation by evaluation of 5. Mu.M trisubstituted pyrazole compounds Y-1, Y-2, Y-3, Y-4 and Y-5 by tartrate-resistant acid phosphatase (TRAP) staining.
FIG. 2 shows the result of the test for cytotoxicity of trisubstituted pyrazole compound Y-3 against bone marrow macrophages by the CCK-8 method. The data are expressed as mean.+ -. Standard deviation, and there is no significant difference in OD 450 values for each Y-3 treated group compared to the control group without Y-3 added.
FIG. 3 is a graph showing evaluation of the dose effect of trisubstituted pyrazole compound Y-3 on inhibition of osteoclast differentiation by tartaric acid-resistant acid phosphatase (TRAP) staining. And A. TRAP staining result, B. Quantitative analysis. Data are expressed as mean ± standard deviation of p <0.05, p <0.01, p <0.001, p <0.0001 compared to RANKL-treated group without Y-3.
FIG. 4 shows the effect of reverse transcription real-time fluorescent quantitative PCR (qPCR) detection of trisubstituted pyrazole compound Y-3 on RANKL induced osteoclast differentiation related gene expression. P <0.0001 compared to RANKL-treated group without Y-3.
FIG. 5 shows the effect of Western blot (Western blot) detection of trisubstituted pyrazole compound Y-3 on the expression of osteoclast differentiation-related proteins and antioxidant proteins during osteoclast differentiation. A. 5. Mu.M of Y-3 effects on the expression of Nfatc, c-Fos, ctsk and Src, etc., and B.5. Mu.M of Y-3 effects on the expression of antioxidant proteins Catalase and HO-1.
Detailed Description
The invention will be further described with reference to specific embodiments. The following examples are merely illustrative of the invention and are not intended to limit the invention in any way. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified. Unless otherwise indicated, the quantitative tests in the examples below were all performed in triplicate, and the results averaged.
EXAMPLE 1 preparation of trisubstituted pyrazole Compounds and identification thereof
A clean 25mL schlenk tube was taken, 1.0 mmol,2.0 equivalents of S-ylide 1, 0.5 mmol,1.0 equivalents of diazonium tetrafluoroborate 2 and 1.0 mmol,1.5 mmol,3.0 equivalents of NaOH were added and dissolved in 10 mL acetonitrile and stirred at room temperature for 48 hours. After the reaction is completed by TLC (thin layer chromatography) plate, the reaction solution is decompressed and concentrated, and the crude product is directly stirred with silica gel and then is subjected to column chromatography (ethyl acetate: petroleum ether=12.5%), thus obtaining the target product 3, which can be specifically:
(1- (p-tolyl) -1H-pyrazole-3, 4-diyl) bis (phenylketone)
White powder (98 mg), 80% yield, melting point ：99.3−101.0 °C;1H NMR (400 MHz, CDCl3):δ8.30 (s, 1H), 8.14−7.99 (m, 2H), 7.89−7.76 (m, 2H), 7.67 (d,J= 8.4 Hz, 2H), 7.60−7.48 (m, 2H), 7.48−7.35 (m, 4H), 7.31 (d,J= 8.0 Hz, 2H), 2.42 (s, 3H)13C NMR (100 MHz, CDCl3):δ189.2, 188.1, 151.2, 138.3, 138.2, 136.7, 136.7, 133.2, 132.9, 130.2, 130.2, 130.1, 129.1, 128.5, 128.3, 125.0, 119.9, 21.0..HRMS (ESI): m/z calcd for [M+H]+: 367.1441, found: 367.1438.
(1- (3-Fluorophenyl) -1H-pyrazole-3, 4-diyl) bis (phenylketone)
White powder (79 mg), 64% yield, melting point ：151.0−151.9 °C;1H NMR (400 MHz, DMSO-d6):δ9.24 (s, 1H), 8.02 (d,J= 7.6 Hz, 2H), 7.97−7.85 (m, 4H), 7.73−7.66 (m, 2H), 7.64−7.50 (m, 5H), 7.31 (t,J= 8.2 Hz, 1H).13C NMR (100 MHz, DMSO-d6):δ193.4, 193.1, 167.7 (d,J= 244.4 Hz), 156.6, 145.3 (d,J= 10.6 Hz), 142.5, 141.3, 139.1, 138.5, 136.8 (d,J= 9.7 Hz), 135.0, 134.4, 134.0 (d,J= 3.5 Hz), 129.2, 120.9, 119.9 (d,J= 21.0 Hz), 112.5 (d,J= 26.3 Hz), 112.3 (d,J= 3.5 Hz).HRMS (ESI): m/z calcd for [M+H]+: 371.1190, found: 371.1192.
(1- (4-Fluorophenyl) -1H-pyrazole-3, 4-diyl) bis (phenylketone)
White powder (80 mg), 65% yield, melting point ：92.3−94.1 °C;1H NMR (400 MHz, CDCl3):δ8.29 (s, 1H), 8.06 (d,J= 7.2 Hz, 2H), 7.81 (m, 4H), 7.64−7.48 (m, 2H), 7.46−7.32 (m, 4H), 7.30−6.98 (m, 2H).13C NMR (100 MHz, CDCl3):δ189.0, 188.0, 162.1 (d, J= 248.6 Hz), 151.6, 138.1, 136.6, 135.3 (d,J= 2.8 Hz), 133.4, 133.0, 130.2, 129.1, 128.5, 128.4, 125.3, 121.9 (d,J= 8.5 Hz), 116.7 (d,J= 23.3 Hz).HRMS (ESI): m/z calcd for [M+H]+: 371.1190, found: 371.1196.
(1-Phenyl-1H-pyrazole-3, 4-diyl) bis ((4-fluorophenyl) methanone)
White powder (84 mg), 65% yield, melting point ：134.5−136.5 °C;1H NMR (400 MHz, CDCl3):δ8.32 (s, 1H), 8.26−8.18 (m, 2H), 7.92−7.85 (m, 2H), 7.78 (d,J= 7.6 Hz, 2H), 7.54 (t,J= 7.6 Hz, 2H), 7.46−7.41 (m, 1H), 7.14 (t,J = 8.6 Hz, 2H), 7.09 (t,J= 8.6 Hz, 2H).13C NMR (100 MHz, CDCl3):δ187.9, 186.1, 167.16 (d,J= 254.1 Hz), 164.6 (d,J= 253.5 Hz), 151.1, 139.0, 134.5 (d,J= 2.9 Hz), 133.3 (d,J= 9.4 Hz), 132.9 (d,J= 2.9 Hz), 131.9 (d,J= 9.4 Hz), 130.1, 130.0, 128.5, 125.3, 120.1, 115.9 (d,J= 21.9 Hz), 115.7 (d,J= 21.7 Hz).HRMS (ESI): m/z calcd for [2M + Na]+: 799.1939, found: 799.1946.
(1- [1,1' -Diphenyl ] -2-yl) -1H-pyrazole-3, 4-diethyl) -bis (phenylketone)
White powder (101 mg), 71% yield, melting point ：115.3−116.5 °C;1H NMR (400 MHz, CDCl3):δ7.90 (d,J= 4.4 Hz, 2H), 7.72 (s, 1H), 7.59−7.50 (m, 7H), 7.49−7.46 (m, 4H), 7.43−7.38 (m, 2H), 7.35−7.27 (m, 2H), 7.26−7.22 (m, 2H).13C NMR (100 MHz, CDCl3):δ188.4, 188.0, 151.1, 138.3, 137.9, 137.4, 137.3, 136.6, 135.0, 133.2, 132.8, 131.3, 130.3, 129.6, 129.2, 129.0, 128.8, 128.7, 128.4, 128.3, 128.0, 126.4, 123.9.HRMS (ESI): m/z calcd for [M+H]+: 429.1598, found: 429.1603.
EXAMPLE 2 TRAP staining evaluation of the inhibition of RANKL-induced osteoclast differentiation by trisubstituted pyrazole compounds Y-1, Y-2, Y-3, Y-4 and Y-5
(1) Mouse bone marrow macrophage isolated culture and digestion plate
Removing sudden death from 6-8 week old mice, sterilizing the whole body of the mice with alcohol, taking out femur and tibia of hind limbs of the mice, removing meat on bones, removing cartilage on knee joints, rapidly centrifuging (stopping when the revolution rises to 10000 rpm) in a centrifuge to obtain bone marrow, blowing the bone marrow uniformly with alpha-MEM culture solution (abbreviated as complete culture medium) containing 10% fetal calf serum and 1% penicillin/streptomycin double antibody, spreading the bone marrow in a cell culture dish of 10 cm for 1 day, taking supernatant, centrifuging at 1000 rpm for 4 min, discarding supernatant, culturing the obtained precipitate uniformly with complete culture medium containing 15 ng/mL M-CSF for 2 days, and obtaining adherent cells as bone marrow macrophages. When bone marrow macrophages grew to about 90%, they were washed 2 times with pre-warmed Dulbecco Phosphate Buffer Solution (DPBS), 2 mL pancreatin was added to the petri dish and left to stand 2 min in an incubator at 37 ℃ in the absence of light. After most of the cell morphology is rounded, the cell dish is tapped to loosen the adherent cells. Immediately adding 2 mL complete culture medium, stopping pancreatin reaction, and repeatedly blowing off adherent cells with gun tip to make all cells fall off from the bottom of the dish. The cells were collected in a centrifuge tube, 1000 rpm, centrifuged 4, min, the supernatant discarded, and the pellet was blown up with an appropriate amount of complete medium to obtain a bone marrow macrophage suspension. mu.L was aspirated with a pipette and counted under a 10-fold microscope using a cell counting plate. Cell densities of 4×10 5/well in 6-well plates, 1×10 5/well in 24-well plates, 5000/well in 96-well plates were plated;
(2) Osteoclast differentiation Induction and TRAP staining experiments
The bone marrow macrophages described above were plated into 24-well plates at 1X 10 5 cells per well. The cells were cultured in complete medium containing 15 ng/mL M-CSF for 1 day. Cells were replaced with osteoclast differentiation induction medium (complete medium containing 15 ng/mL M-CSF and 100 ng/mL RANKL) containing 5. Mu.M of different trisubstituted pyrazole compounds (Y-1, Y-2, Y-3, Y-4 or Y-5) dissolved in dimethyl sulfoxide (DMSO) or an equal volume of dimethyl sulfoxide (DMSO) solvent after cell attachment, the solution was changed every 2 days, cells in 24 well plates were washed 2 times with DPBS, fixed 10min times with 300. Mu.L of 4% PFA at room temperature, and washed 1 time with DPBS when a large number of osteoclasts (about 5 days) were present in the control group (i.e., DMSO-only). After DPBS removal, cells were stained with tartrate-resistant acid phosphatase (TRAP) to assess the formation of each group of osteoclasts (i.e., cells with trap+ and more than 3 nuclei). As shown in FIG. 1, 5. Mu.M trisubstituted pyrazole compounds Y-1, Y-2, Y-3, Y-4 and Y-5 all had a significant inhibitory effect on osteoclast formation.
Experimental example 3 CCK-8 detection of cytotoxicity of trisubstituted pyrazole Compound Y-3 on bone marrow macrophages
The mouse bone marrow macrophages were isolated from the mouse bone marrow and cultured as described above, spread into 96-well plates at 5000 cells per well, and cultured in complete medium containing 15 ng/mL M-CSF for 1 day. Dividing cells into 7 groups of 0, 0.3125, 0.625, 1.25, 2.5, 5 or 10 mu M Y-3, setting 6 repeats each, culturing the cells after the cells are attached, changing the culture medium into a complete culture medium containing 15 ng/mL M-CSF and the Y-3 with different concentrations, removing the complete culture medium respectively at the time of 24 hours, 48 hours and 72h, washing the cells with a proper amount of DPBS for 1-2 times, adding 100 mu L of 10% CCK-8 reagent into each hole, culturing in a 37 ℃ incubator in dark for 30 min, and finally detecting the OD value at 450 nm by an enzyme-labeled instrument. As shown in FIG. 2, the OD values obtained in the Y-3 treated group were not significantly different from those obtained in the non-Y-3 treated group at 24h,48h and 72h, indicating that the concentration of Y-3 at 10. Mu.M and below was not cytotoxic to bone marrow macrophages.
Experimental example 4 TRAP staining to detect dose Effect of trisubstituted pyrazole Compound Y-3 on inhibition of osteoclast formation
Mouse bone marrow macrophages were isolated from mouse bone marrow and cultured as described above, plated in 24 well plates at 1X 10 5 cells per well, and the cells were divided into 8 groups (including NM group and 0, 0.3125, 0.625, 1.25, 2.5, 5 or 10. Mu.M Y-3 treated groups, 3 replicates each) and cultured for 1 day in complete medium containing 15 ng/mL M-CSF. After the cells had attached, the cells were replaced with fresh complete medium (NM group) or osteoclast differentiation induction medium containing Y-3 at the above-mentioned different concentrations, and the liquid was changed every 2 days, and when a large number of osteoclasts (about 5 days) were present in the control group (i.e., 0. Mu.M Y-3 group), the formation of each group of osteoclasts (i.e., cells having TRAP+ and more than 3 nuclei) was evaluated by staining with anti-tartrate acid phosphatase (TRAP). TRAP staining results showed (FIG. 3) that Y-3 inhibited osteoclast formation dose-dependently, and that both the number and size of osteoclasts were significantly reduced in the 0.625. Mu.M treatment, and more significantly in the 1.25 and 2.5. Mu.M treatments, and that osteoclast formation was almost completely inhibited at concentrations of 5. Mu.M and above.
Experimental example 5 qPCR Effect of trisubstituted pyrazole Compound Y-3 on the expression of osteoclast differentiation-related Gene
P2-generation BMM cells isolated from mouse bone marrow cells were plated in 6-well plates according to the number of 4X 10 5 cells per well, the cells were divided into 6 groups (including NM group and 0, 1.25, 2.5, 5 or 10. Mu.M Y-3 treated groups, 3 multiplex wells per group), cultured in complete medium containing 15 ng/mL M-CSF for 1 day, replaced with fresh complete medium (NM group) or osteoclast differentiation induction medium containing the above-mentioned different concentrations of Y-3 after cell attachment, changed for every other day until the control group (i.e., 0. Mu.M Y-3 group) was terminated when a large number of osteoclasts (about 5-6 days) were present, DPBS was washed 2 times, total RNA was extracted using Trizol kit, after measuring the concentration, was obtained by reverse transcription into cDNA in 500. Mu.L in a reaction system, followed by real-time fluorescent quantitative PCR on a Bio-CFX 96 instrument, 2. Mu.L cDNA template, 0.5. Mu.L upstream primer BR, 10. Mu.37. Mu.L primer, and 35H gene expression was calculated for each target gene of 24. Mu.24. Mu.L. As a result, as shown in FIG. 4, the mRNA expression levels of the osteoclast differentiation-related genes Nfatc, ctsk, acp5, oscar, dcstamp, mmp9 and Atp v0d2 decreased in a concentration-dependent manner with an increase in the concentration of Y-3, further indicating that Y-3 inhibits osteoclast differentiation.
Experimental example 6 Western blot detection of the Effect of trisubstituted pyrazole Compound Y-3 on the expression of osteoclast differentiation-associated protein and antioxidant protein
Bone marrow macrophages were plated into 6-well plates at 4X 10 5 cells per well, divided into a control group and a drug-treated group, each group was repeated 3 times, and after culturing with complete medium containing 15 ng/mL M-CSF for 1 day, the medium was changed to osteoclast differentiation-inducing medium, and 5. Mu.M Y-3 was added to the drug-treated group, and the control group was added with an equal amount of dimethyl sulfoxide solvent, and protein samples were collected at 0,1, 3, and 5 days, respectively, every other day. The specific sample collection steps are that the culture medium in a 6-hole plate is discarded, DPBS is cleaned for 2 times, 100 mu L of RIPA lysate containing protease inhibitor and phosphatase inhibitor is added into each hole, cells in the hole plate are scraped off, the cells are collected into a centrifuge tube, after 10min of the cells are cracked on ice, 13000 and rpm are centrifuged for 15 and min, and the supernatant is obtained as a protein sample. After the concentration of the protein samples was determined by BCA kit, an equal amount of protein samples was taken for electrophoresis on 10% SDS-PAGE gel, transferred to NC membrane after protein bands were separated, NC membrane was blocked with blocking solution (TBST containing 5% skimmed milk) at room temperature for 1 hour, then antibodies against Ctsk, src, C-Fos, nfatc1, catalase or HO-1 were added, respectively, and incubated overnight at 4℃and the next day, after washing 3 times on a shaker with 1 XTBE, the corresponding secondary antibodies were added for incubation 1h at room temperature and washing 3 times with 1 XTBE, and developed. As shown in FIG. 5, the induction of RANKL on osteoclast differentiation related proteins Ctsk and c-Fos, src, nfatc1 and the inhibition of oxidation resisting proteins Catalase and HO-1 by 5 mu M Y-3 can be remarkably reversed, which shows that Y-3 inhibits osteoclast differentiation by up-regulating oxidation resisting proteins and down-regulating key factors of osteoclast differentiation.
Claims (8)
1. Use of a trisubstituted pyrazole compound having a structural formula as shown in formula (1) in the preparation of an osteoclast differentiation inhibitor:
;
R 1 is phenyl or substituted phenyl, the substituent is selected from halogen, R 2 is phenyl or substituted phenyl, and the substituent is selected from halogen, methyl, ethyl and phenyl.
2. The use according to claim 1, wherein the trisubstituted pyrazole compound is used for the preparation of a medicament for the treatment of osteolytic diseases.
3. The use according to claim 1, wherein the trisubstituted pyrazole compound is used for the preparation of a medicament for the prophylaxis or treatment of osteoporosis, tumor metastasis bone destruction or rheumatoid arthritis.
4. The use according to claim 1, wherein the trisubstituted pyrazole compound is used for the preparation of a medicament for inhibiting the expression of an osteoclast differentiation-critical protein.
5. The use of claim 1, wherein the trisubstituted pyrazole compound is used in the manufacture of a medicament for enhancing antioxidant protein expression for targeted inhibition of ROS.
6. The use according to any one of claims 1 to 5, wherein the trisubstituted pyrazole compound is of any one of the following formulae:
。
7. The method according to claim 1to 5, wherein the inhibitor or the drug is administered orally, by injection or topically in the form of a tablet, pill, powder, capsule, injection, oral liquid, paste or cream.
8. The method according to claim 1 to 5, wherein the inhibitor or the drug comprises a trisubstituted pyrazole compound as an active ingredient and pharmaceutically acceptable excipients.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202411396736.5A CN118873532B (en) | 2024-10-09 | 2024-10-09 | Application of trisubstituted pyrazole compound in preparation of medicines for inhibiting osteoclast differentiation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202411396736.5A CN118873532B (en) | 2024-10-09 | 2024-10-09 | Application of trisubstituted pyrazole compound in preparation of medicines for inhibiting osteoclast differentiation |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN118873532A CN118873532A (en) | 2024-11-01 |
| CN118873532B true CN118873532B (en) | 2025-02-07 |
Family
ID=93219798
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202411396736.5A Active CN118873532B (en) | 2024-10-09 | 2024-10-09 | Application of trisubstituted pyrazole compound in preparation of medicines for inhibiting osteoclast differentiation |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN118873532B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN120267652B (en) * | 2025-06-11 | 2025-08-05 | 浙大城市学院 | Application of benzothiazinone vinylidene indene compounds in the preparation of drugs for inhibiting osteoclast differentiation |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1545415A (en) * | 2001-06-18 | 2004-11-10 | ����ŵά�� | Bone anabolic compounds and methods of use |
| CN111789833A (en) * | 2020-08-31 | 2020-10-20 | 苏州大学 | Application of 2-bromopalmitic acid in the preparation of medicines for preventing and treating bone loss related diseases |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003032989A1 (en) * | 2001-10-18 | 2003-04-24 | Boehringer Ingelheim Pharmaceuticals, Inc. | 1,4-disubstituted benzo-fused urea compounds as cytokine inhibitors |
| US20110230536A1 (en) * | 2008-10-24 | 2011-09-22 | Calcimedica, Inc. | Phenylpyrazole inhibitors of store operated calcium release |
| KR101395658B1 (en) * | 2012-09-20 | 2014-05-16 | 서울대학교산학협력단 | Compositions for preventing and treating osseous metabolic disease comprising 4-(5-(2-methoxyphenyl)-3-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)-4-oxobutanoic acid |
-
2024
- 2024-10-09 CN CN202411396736.5A patent/CN118873532B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1545415A (en) * | 2001-06-18 | 2004-11-10 | ����ŵά�� | Bone anabolic compounds and methods of use |
| CN111789833A (en) * | 2020-08-31 | 2020-10-20 | 苏州大学 | Application of 2-bromopalmitic acid in the preparation of medicines for preventing and treating bone loss related diseases |
Also Published As
| Publication number | Publication date |
|---|---|
| CN118873532A (en) | 2024-11-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Wang et al. | Quercetin suppresses apoptosis of chondrocytes induced by IL-1β via inactivation of p38 MAPK signaling pathway | |
| CN118873532B (en) | Application of trisubstituted pyrazole compound in preparation of medicines for inhibiting osteoclast differentiation | |
| Ge et al. | Protective effects of naringin on glucocorticoid-induced osteoporosis through regulating the PI3K/Akt/mTOR signaling pathway | |
| Najar et al. | A complete map of the Calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) signaling pathway | |
| US8445437B2 (en) | Treatment and prevention of cardiovascular disease using mast cell stabilizers | |
| US20120046246A1 (en) | Methods for treating osteoclast-related disease, compounds and compositions thereof | |
| Chen et al. | Oroxylin A attenuates osteoarthritis progression by dual inhibition of cell inflammation and hypertrophy | |
| Wu et al. | Dihydromyricetin inhibits inflammation of fibroblast-like synoviocytes through regulation of nuclear factor-κB signaling in rats with collagen-induced arthritis | |
| Venkateish et al. | Morin inhibits the activity of pancreatic lipase and adipogenesis | |
| Qiu et al. | Neratinib exerts dual effects on cartilage degradation and osteoclast production in Osteoarthritis by inhibiting the activation of the MAPK/NF-κB signaling pathways | |
| US20100035940A1 (en) | Cyclin Dependent Kinase Inhibitors | |
| Chen et al. | Rongjin Niantong Fang ameliorates cartilage degeneration by regulating the SDF-1/CXCR4-p38MAPK signalling pathway | |
| Yang et al. | Casticin suppresses RANKL-induced osteoclastogenesis and prevents ovariectomy-induced bone loss by regulating the AKT/ERK and NF-κB signaling pathways | |
| AU2004279260A1 (en) | Composition for treatment of osteoarthritis containing apigenin as chondroregenerative agent | |
| US10195167B2 (en) | Compounds for use in the treatment of conditions associated with NADPH oxidase | |
| JP2006515276A (en) | Furan derivative having preventive and therapeutic effects on osteoporosis and pharmaceutical composition containing the same | |
| CN120267652B (en) | Application of benzothiazinone vinylidene indene compounds in the preparation of drugs for inhibiting osteoclast differentiation | |
| Zhang et al. | Inhibition of protein disulfide isomerase mitigates steroid-induced osteonecrosis of the femoral head by suppressing osteoclast activity through the reduction of cellular oxidative stress | |
| Niu et al. | Siweixizangmaoru Decoction Ameliorated Type II Collagen-Induced Arthritis in Rats via Regulating JAK2–STAT3 and NF-κB Signaling Pathway | |
| Eun et al. | Saururus chinensis (Lour.) Baill. extract promotes skeletal muscle cell differentiation by positively regulating mitochondrial biogenesis and AKT/mTOR signaling in vitro | |
| Zhang et al. | Carpaine ameliorates synovial inflammation by promoting p65 degradation and inhibiting the NF-κB signalling pathway | |
| Huang et al. | Lactate-upregulated ARG2 expression induces cellular senescence in fibroblast-like synoviocytes of osteoarthritis via activating the mTOR/S6K1 signaling pathway | |
| Gao et al. | The active ingredient of Evodia rutaecarpa reduces inflammation in knee osteoarthritis rats through blocking calcium influx and NF‐κB pathway | |
| CN119548487A (en) | Application of 5-(tetradecyloxy)-2-furoic acid in the preparation of drugs for inhibiting osteoclast differentiation and function | |
| Xian-Hui et al. | The effect of TCM-induced HAMP on key enzymes in the hydrolysis of AD model cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |