CN117069627A - 4- (3- (3-sulfamoyl phenyl) ureido) butyric acid compound, fluorescent marker, preparation method and application thereof - Google Patents
4- (3- (3-sulfamoyl phenyl) ureido) butyric acid compound, fluorescent marker, preparation method and application thereof Download PDFInfo
- Publication number
- CN117069627A CN117069627A CN202211255810.2A CN202211255810A CN117069627A CN 117069627 A CN117069627 A CN 117069627A CN 202211255810 A CN202211255810 A CN 202211255810A CN 117069627 A CN117069627 A CN 117069627A
- Authority
- CN
- China
- Prior art keywords
- ureido
- sulfamoylphenyl
- added
- chemical formula
- represented
- 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.)
- Pending
Links
- -1 4- (3- (3-sulfamoyl phenyl) ureido) butyric acid compound Chemical class 0.000 title claims abstract description 48
- 239000003550 marker Substances 0.000 title claims abstract description 18
- 238000002360 preparation method Methods 0.000 title claims abstract description 14
- 239000000126 substance Substances 0.000 claims abstract description 38
- 238000000034 method Methods 0.000 claims abstract description 29
- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical compound NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 claims abstract description 18
- 229960003692 gamma aminobutyric acid Drugs 0.000 claims abstract description 9
- AHWALFGBDFAJAI-UHFFFAOYSA-N phenyl carbonochloridate Chemical compound ClC(=O)OC1=CC=CC=C1 AHWALFGBDFAJAI-UHFFFAOYSA-N 0.000 claims abstract description 8
- JPVKCHIPRSQDKL-UHFFFAOYSA-N 3-aminobenzenesulfonamide Chemical compound NC1=CC=CC(S(N)(=O)=O)=C1 JPVKCHIPRSQDKL-UHFFFAOYSA-N 0.000 claims abstract description 7
- 230000003213 activating effect Effects 0.000 claims abstract description 3
- 101001043564 Homo sapiens Prolow-density lipoprotein receptor-related protein 1 Proteins 0.000 claims description 23
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 23
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 22
- 102100021923 Prolow-density lipoprotein receptor-related protein 1 Human genes 0.000 claims description 21
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 17
- ZFQWJXFJJZUVPI-UHFFFAOYSA-N tert-butyl n-(4-aminobutyl)carbamate Chemical compound CC(C)(C)OC(=O)NCCCCN ZFQWJXFJJZUVPI-UHFFFAOYSA-N 0.000 claims description 10
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 9
- 238000009169 immunotherapy Methods 0.000 claims description 9
- 230000001413 cellular effect Effects 0.000 claims description 8
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 claims description 6
- 230000008569 process Effects 0.000 claims description 6
- FYSNRJHAOHDILO-UHFFFAOYSA-N thionyl chloride Chemical compound ClS(Cl)=O FYSNRJHAOHDILO-UHFFFAOYSA-N 0.000 claims description 6
- 238000001816 cooling Methods 0.000 claims description 5
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Substances CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 claims description 4
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 claims description 4
- 150000001875 compounds Chemical class 0.000 claims description 4
- 230000002194 synthesizing effect Effects 0.000 claims description 4
- 229960000549 4-dimethylaminophenol Drugs 0.000 claims description 3
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 3
- 238000010992 reflux Methods 0.000 claims description 3
- ZAJAQTYSTDTMCU-UHFFFAOYSA-N 3-aminobenzenesulfonic acid Chemical compound NC1=CC=CC(S(O)(=O)=O)=C1 ZAJAQTYSTDTMCU-UHFFFAOYSA-N 0.000 claims description 2
- 238000004090 dissolution Methods 0.000 claims description 2
- 210000004027 cell Anatomy 0.000 description 48
- 239000006228 supernatant Substances 0.000 description 14
- 108090000623 proteins and genes Proteins 0.000 description 9
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 8
- 108700018351 Major Histocompatibility Complex Proteins 0.000 description 8
- 239000000427 antigen Substances 0.000 description 8
- 108091007433 antigens Proteins 0.000 description 8
- 102000036639 antigens Human genes 0.000 description 8
- 230000020382 suppression by virus of host antigen processing and presentation of peptide antigen via MHC class I Effects 0.000 description 8
- 239000003795 chemical substances by application Substances 0.000 description 7
- 238000002390 rotary evaporation Methods 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 238000004809 thin layer chromatography Methods 0.000 description 7
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 6
- 108010004889 Heat-Shock Proteins Proteins 0.000 description 6
- 102000002812 Heat-Shock Proteins Human genes 0.000 description 6
- 210000001744 T-lymphocyte Anatomy 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 6
- 239000000872 buffer Substances 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 238000001228 spectrum Methods 0.000 description 6
- 241000701161 unidentified adenovirus Species 0.000 description 6
- 238000005481 NMR spectroscopy Methods 0.000 description 5
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 5
- 239000007787 solid Substances 0.000 description 5
- 239000002904 solvent Substances 0.000 description 5
- 230000003833 cell viability Effects 0.000 description 4
- 210000004443 dendritic cell Anatomy 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 108090000765 processed proteins & peptides Proteins 0.000 description 4
- 238000003753 real-time PCR Methods 0.000 description 4
- 230000002441 reversible effect Effects 0.000 description 4
- 238000010186 staining Methods 0.000 description 4
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 3
- 102000008949 Histocompatibility Antigens Class I Human genes 0.000 description 3
- 238000011529 RT qPCR Methods 0.000 description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000004440 column chromatography Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 3
- 230000001939 inductive effect Effects 0.000 description 3
- 210000001778 pluripotent stem cell Anatomy 0.000 description 3
- 238000004321 preservation Methods 0.000 description 3
- 102000004196 processed proteins & peptides Human genes 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 239000000741 silica gel Substances 0.000 description 3
- 229910002027 silica gel Inorganic materials 0.000 description 3
- 102000004082 Calreticulin Human genes 0.000 description 2
- 108090000549 Calreticulin Proteins 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 208000035473 Communicable disease Diseases 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 108091054437 MHC class I family Proteins 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 238000002123 RNA extraction Methods 0.000 description 2
- 239000012980 RPMI-1640 medium Substances 0.000 description 2
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 2
- 230000030741 antigen processing and presentation Effects 0.000 description 2
- 210000000612 antigen-presenting cell Anatomy 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000007405 data analysis Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000005206 flow analysis Methods 0.000 description 2
- 238000000684 flow cytometry Methods 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000002452 interceptive effect Effects 0.000 description 2
- 229920002521 macromolecule Polymers 0.000 description 2
- 210000002540 macrophage Anatomy 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 239000011148 porous material Substances 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 238000010898 silica gel chromatography Methods 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 1
- FRPHFZCDPYBUAU-UHFFFAOYSA-N Bromocresolgreen Chemical compound CC1=C(Br)C(O)=C(Br)C=C1C1(C=2C(=C(Br)C(O)=C(Br)C=2)C)C2=CC=CC=C2S(=O)(=O)O1 FRPHFZCDPYBUAU-UHFFFAOYSA-N 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 101710163595 Chaperone protein DnaK Proteins 0.000 description 1
- 101100016370 Danio rerio hsp90a.1 gene Proteins 0.000 description 1
- 101100193633 Danio rerio rag2 gene Proteins 0.000 description 1
- 101100285708 Dictyostelium discoideum hspD gene Proteins 0.000 description 1
- 101100125027 Dictyostelium discoideum mhsp70 gene Proteins 0.000 description 1
- 101150031823 HSP70 gene Proteins 0.000 description 1
- 101710178376 Heat shock 70 kDa protein Proteins 0.000 description 1
- 101710152018 Heat shock cognate 70 kDa protein Proteins 0.000 description 1
- 101710113864 Heat shock protein 90 Proteins 0.000 description 1
- 102100034051 Heat shock protein HSP 90-alpha Human genes 0.000 description 1
- 108010088652 Histocompatibility Antigens Class I Proteins 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- 101710172064 Low-density lipoprotein receptor-related protein Proteins 0.000 description 1
- 108010006519 Molecular Chaperones Proteins 0.000 description 1
- 101100193635 Mus musculus Rag2 gene Proteins 0.000 description 1
- 108010071384 Peptide T Proteins 0.000 description 1
- 102000004245 Proteasome Endopeptidase Complex Human genes 0.000 description 1
- 108090000708 Proteasome Endopeptidase Complex Proteins 0.000 description 1
- 238000012228 RNA interference-mediated gene silencing Methods 0.000 description 1
- 101100071627 Schizosaccharomyces pombe (strain 972 / ATCC 24843) swo1 gene Proteins 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical class [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 1
- 230000006044 T cell activation Effects 0.000 description 1
- 108091008874 T cell receptors Proteins 0.000 description 1
- 102000016266 T-Cell Antigen Receptors Human genes 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000001594 aberrant effect Effects 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 239000012043 crude product Substances 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 101150052825 dnaK gene Proteins 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 239000012645 endogenous antigen Substances 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 230000009368 gene silencing by RNA Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000011201 multiple comparisons test Methods 0.000 description 1
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 1
- 239000012044 organic layer Substances 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 239000012286 potassium permanganate Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical class O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 230000003827 upregulation Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C311/00—Amides of sulfonic acids, i.e. compounds having singly-bound oxygen atoms of sulfo groups replaced by nitrogen atoms, not being part of nitro or nitroso groups
- C07C311/30—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound nitrogen atoms, not being part of nitro or nitroso groups
- C07C311/45—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound nitrogen atoms, not being part of nitro or nitroso groups at least one of the singly-bound nitrogen atoms being part of any of the groups, X being a hetero atom, Y being any atom, e.g. N-acylaminosulfonamides
- C07C311/47—Y being a hetero atom
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/0005—Vertebrate antigens
- A61K39/0011—Cancer antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic Table
- C07F5/02—Boron compounds
- C07F5/022—Boron compounds without C-boron linkages
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0639—Dendritic cells, e.g. Langherhans cells in the epidermis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/515—Animal cells
- A61K2039/5154—Antigen presenting cells [APCs], e.g. dendritic cells or macrophages
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/515—Animal cells
- A61K2039/5158—Antigen-pulsed cells, e.g. T-cells
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1003—Carbocyclic compounds
- C09K2211/1007—Non-condensed systems
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1044—Heterocyclic compounds characterised by ligands containing two nitrogen atoms as heteroatoms
- C09K2211/1055—Heterocyclic compounds characterised by ligands containing two nitrogen atoms as heteroatoms with other heteroatoms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/50—Cell markers; Cell surface determinants
- C12N2501/599—Cell markers; Cell surface determinants with CD designations not provided for elsewhere
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Pharmacology & Pharmacy (AREA)
- Medicinal Chemistry (AREA)
- Immunology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Zoology (AREA)
- Oncology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Epidemiology (AREA)
- Materials Engineering (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Mycology (AREA)
- Hematology (AREA)
- General Engineering & Computer Science (AREA)
- Communicable Diseases (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
The application discloses a 4- (3- (3-sulfamoyl phenyl) ureido) butyric acid compound shown in a formula 1The object, a fluorescent marker, a preparation method and application thereof belong to the technical field of pharmaceutical chemistry. The method comprises the steps of: obtaining a first intermediate represented by chemical formula 2 from m-aminobenzenesulfonamide and phenyl chloroformate; obtaining a second intermediate represented by chemical formula 3 from 4-aminobutyric acid; obtaining a third intermediate represented by chemical formula 4 from the first intermediate and the second intermediate; 4- (3- (3-sulfamoylphenyl) ureido) butanoic acid is obtained from the third intermediate. The small molecule compound prepared by the application can enter DC cells through activating CD 91.[ chemical formula 1 ][ chemical formula 2 ][ chemical formula 3 ][ formula 4 ].
Description
Technical Field
The application relates to the technical field of pharmaceutical chemistry, in particular to a 4- (3- (3-sulfamoyl phenyl) ureido) butyric acid compound, a fluorescent marker thereof, a preparation method and application.
Background
Cellular immunotherapy is of increasing interest in the treatment of infectious diseases or cancers because of its better therapeutic effect. The method achieves the aim of killing viruses by effectively presenting and stimulating immune T cells by antigen presenting cells (also called Dendritic Cells (DCs)).
For example, patent CN 107075484B discloses a method for inducing T cells for cellular immunotherapy from pluripotent stem cells, which provides a method for inducing T cells for cellular immunotherapy, comprising the steps of: (1) Providing a human pluripotent stem cell having a gene encoding a desired antigen-specific T cell receptor and knocked out Rag1 and/or Rag2 genes, and (2) inducing T cells from the pluripotent stem cell of step (1). Also provided are a cellular immunotherapy method using T cells for cellular immunotherapy and an iPS cell bank for cellular immunotherapy.
The complex of heat shock protein gp96 and antigen peptide is taken up by antigen presenting cells and presented by MHC class I molecules. To explain the aberrant efficiency of this process, it is assumed that gp96 uptake is via a receptor, which has recently been identified as CD91 (low density lipoprotein receptor-related protein). Complexes of peptides with heat shock proteins hsp90, calreticulin and hsp70 are also taken up by macrophages and dendritic cells and re-presented by MHC class I molecules. All heat shock proteins utilize the CD91 receptor even though some proteins have no homology to each other. Post-uptake processing of gp96 chaperone peptides requires a proteasome and a transporter associated with antigen processing, utilizing the classical endogenous antigen presentation pathway.
Heat Shock Proteins (HSP) gp96, HSP70, HSP90 and calreticulin have been used to elicit immune responses for the immunotherapy of cancer and infectious diseases. These immune responses, mainly against peptide T cells, mediate their effects through cytotoxicity of antigen expression. In several antigen systems tested in vitro and in vivo, dendritic cells and macrophages acquire extracellular HSPs via the receptor CD91 and cross-present peptides naturally accompanying these HSPs on MHC molecules, providing signals for T cell activation.
The molecular weight of heat shock proteins is generally large, the volume is large, and certain difficulties are caused to the immune cell uptake process.
In summary, in the prior art, the CD91 is a protein macromolecule, which has a large molecular weight, a large volume, inconvenient preservation and relatively high cost, so designing a small molecule compound capable of binding to CD91 to replace the protein macromolecule to bring antigen into DC cells is a problem to be solved.
Disclosure of Invention
The application aims to provide a 4- (3- (3-sulfamoyl phenyl) ureido) butyric acid compound, a fluorescent marker, a preparation method and application thereof, wherein the fluorescent marker proves that the compound can effectively bind and activate CD91 molecules on DC cells, enter the DC cells rapidly and can up-regulate the expression of MHC-I molecules of presentation antigens.
In order to achieve the above object, the present application provides the following technical solutions:
the application provides a 4- (3- (3-sulfamoyl phenyl) ureido) butyric acid compound shown in a formula 1.
The present application provides a method for synthesizing a 4- (3- (3-sulfamoylphenyl) ureido) butanoic acid compound, the method comprising the steps of:
obtaining a first intermediate represented by chemical formula 2 from m-aminobenzenesulfonamide and phenyl chloroformate;
obtaining a second intermediate represented by chemical formula 3 from 4-aminobutyric acid;
obtaining a third intermediate represented by chemical formula 4 from the first intermediate and the second intermediate;
4- (3- (3-sulfamoylphenyl) ureido) butanoic acid is obtained from the third intermediate.
[ chemical formula 2 ]
[ chemical formula 3 ]
[ chemical formula 4 ]
Further, a first intermediate represented by chemical formula 2 is obtained from metanilic acid amide and phenyl chloroformate, comprising the steps of:
adding m-aminobenzenesulfonamide and DMAP into a dry reaction vessel, dissolving with THF, and cooling in ice bath; phenyl chloroformate is added dropwise under ice bath, stirred at a constant temperature, and then moved to room temperature for reaction.
Further, a second intermediate represented by chemical formula 3 is obtained from 4-aminobutyric acid, comprising the steps of:
adding 4-aminobutyric acid into a dry reaction container, adding methanol for dissolution, and cooling in an ice bath; thionyl chloride is slowly added dropwise in ice bath, stirred at a constant temperature, and then moved to room temperature for reaction.
Further, a third intermediate represented by chemical formula 4 is obtained from the first intermediate and the second intermediate, comprising the steps of:
the first intermediate and the second intermediate are added into a dry reaction vessel, dissolved in methanol, added with triethylamine, and then heated for reflux reaction.
Further, 4- (3- (3-sulfamoylphenyl) ureido) butyric acid is obtained from the third intermediate, comprising the steps of:
and adding the third intermediate into a dry reaction container, adding sodium hydroxide solution, and reacting at normal temperature.
The present application provides the use of a 4- (3- (3-sulfamoylphenyl) ureido) butanoic acid compound in cellular immunotherapy, said compound being capable of binding to fluorescent molecules for the purpose of entering DC cells by activating CD91 molecules.
The application provides a BODIPY marker of 4- (3- (3-sulfamoyl phenyl) ureido) butyric acid shown in a formula 5;
[ chemical formula 5 ]
The application provides a method for synthesizing a BODIPY marker of 4- (3- (3-sulfamoyl phenyl) ureido) butyric acid; the method comprises the following steps:
preparation of 4- (3- (3-sulfamoylphenyl) ureido) butanoic acid, obtainable by the process according to any one of claims 1 to 6;
obtaining a fourth intermediate represented by formula 6 from 4- (3- (3-sulfamoylphenyl) ureido) butanoic acid and N-BOC-1, 4-butanediamine;
obtaining a fifth intermediate represented by formula 7 from the fourth intermediate;
a BODIPY marker of 4- (3- (3-sulfamoylphenyl) ureido) butyric acid is obtained from said fifth intermediate and BODIPY-CO 2H.
[ chemical formula 6 ]
[ chemical formula 7 ]
Further, from 4- (3- (3-sulfamoylphenyl) ureido) butanoic acid and N-BOC-1, 4-butanediamine, a fourth intermediate represented by formula 6 is obtained, comprising the steps of: 4- (3- (3-sulfamoyl phenyl) ureido) butyric acid and BtOH are added into a dry reaction vessel, dissolved in DMF, added with N-BOC-1, 4-butanediamine and cooled in an ice bath.
Further, a fifth intermediate represented by formula 7 is obtained from the fourth intermediate, comprising the steps of: the fourth intermediate was added to a dry reaction vessel, dissolved in dichloromethane, TFA was added and stirred at room temperature.
Further, a BODIPY marker of 4- (3- (3-sulfamoylphenyl) ureido) butyric acid is obtained from said fifth intermediate and BODIPY-CO2H, comprising the steps of: adding the fifth intermediate, BODIPY-CO2H and BtOH into a dry reaction container, dissolving with THF, and cooling in ice bath; EDCI and TEA are added, stirred at a constant temperature and then moved to room temperature for reaction.
Compared with the prior art, the application adopts the technical proposal, and has the following advantages and positive effects by way of example and not limitation:
the compound provided by the application has the characteristics of small molecular weight, easiness in combination with CD91 and rapid entry into DC cells.
Drawings
FIG. 1 is a nuclear magnetic resonance spectrum of a first intermediate according to an embodiment of the present application.
FIG. 2 is a nuclear magnetic resonance spectrum of a third intermediate according to an embodiment of the present application.
FIG. 3 is a nuclear magnetic spectrum of 4- (3- (3-sulfamoylphenyl) ureido) butyric acid provided in the example of the present application.
FIG. 4 is a nuclear magnetic resonance spectrum of a fourth intermediate according to an embodiment of the present application.
FIG. 5 is a nuclear magnetic resonance spectrum of a fluorescent marker of 4- (3- (3-sulfamoylphenyl) ureido) butyric acid provided by the embodiment of the application.
FIG. 6 is a graph showing RNA transcription levels of each CD91 group provided in the examples of the present application.
FIG. 7 is a chart showing the result of confirming the interference of pAVE3578 sequences to CD91 RNA by flow analysis in accordance with the present application.
FIG. 8 is a fluorescence photograph of DC cells after treatment with a small molecule compound according to an embodiment of the present application.
FIG. 9 is a graph showing the change in fluorescence intensity of flow cytometry analysis according to an embodiment of the present application.
FIG. 10 is a graph showing the MHC expression level of qPCR assay according to an embodiment of the present application.
Detailed Description
The following description of the embodiments of the present application will be made apparent and fully in view of the accompanying drawings, in which some, but not all embodiments of the application are shown. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.
Wherein the drawings are for illustrative purposes only and are schematic representations, not physical figures, and are not to be construed as limiting the present patent.
Example 1
Preparation of the first intermediate represented by chemical formula 2
[ chemical formula 2 ]
M-aminobenzenesulfonamide, DMAP were added to a dry 25-mL round bottom flask, dissolved in THF, and cooled to 0-5 ℃ in an ice bath.
Phenyl chloroformate is added dropwise under ice bath, stirred for 30min under heat preservation, and then moved to room temperature for reaction for 12h.
The reaction was monitored by thin layer chromatography until the complete consumption of m-aminobenzenesulfonamide.
Developing agent: PE ea=1:1, the product was visible under uv light.
The solvent was removed by rotary evaporation, and the mixture was separated by column chromatography on silica gel (PE: ea=1:1) to give a second intermediate (38 mg, 75% yield as white solid).
The nuclear magnetic spectrum is shown in figure 1 of the specification, and can be seen as follows: 1 H NMR(600MHz,DMSO-d6)10.60(bs,1H),8.10(s,1H),7.66(bd,J＝7.2Hz,1H),7.56-7.50(m,2H),7.47-7.40(m,4H),7.30-7.23(m,3H)。
example 2
Preparation of the second intermediate represented by chemical formula 3
[ chemical formula 3 ]
4-aminobutyric acid was added to a dry 50-ml round bottom flask, dissolved in methanol, and cooled to 0-5 ℃ in an ice bath.
Thionyl chloride is slowly added dropwise under ice bath, stirred for 30min under heat preservation, and then moved to room temperature for reaction for 3h.
The reaction was monitored by thin layer chromatography until complete consumption of 4-aminobutyric acid. Developing agent: DCM, meoh=5:1, the product was developed as ninhydrin.
The reaction solution was removed by rotary evaporation, and methylene chloride and saturated sodium bicarbonate solution were added to separate phases. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated by rotary evaporation, and then separated by silica gel column chromatography (DCM: meoh=5:1) to give a second intermediate (colorless liquid 91mg, yield 80%).
Example 3
Preparation of the third intermediate represented by chemical formula 1
[ chemical formula 1 ]
The second intermediate obtained in example 1, the third intermediate obtained in example 2, was added to a dry 50-ml round bottom flask, dissolved in methanol, triethylamine was added, and then heated to 70 ℃ and reacted under reflux for 12h.
The reaction was monitored by thin layer chromatography until the second intermediate was completely consumed. Developing agent: PE ea=1:4, the product was visible under uv light.
The solvent was removed by rotary evaporation, and column chromatography on silica gel (PE: ea=1:4) was used to give a fourth intermediate (103 mg, 95% yield as a white solid).
The nuclear magnetic spectrum is shown in figure 2 of the specification, and can be seen as follows: 1 H NMR(600MHz,DMSO-d6)8.81(s,1H),7.98(t,J＝1.8Hz,1H),7.51(bd,J＝7.8Hz,1H),7.40(t,J＝7.8Hz,1H),7.35-7.31(m,3H),6.27(t,J＝6.0Hz,1H),3.59(s,3H),3.10(td,J＝7.2,6.0Hz,2H),2.34(t,J＝7.2Hz,2H),1.70(quintet,J＝7.2Hz,2H)。
example 4
Preparation of 4- (3- (3-sulfamoylphenyl) ureido) butanoic acid compound represented by chemical formula 1
[ chemical formula 1 ]
The third intermediate obtained in example 3 was added to a dry 50-mL round bottom flask, sodium hydroxide solution was added, and the reaction was carried out at room temperature for 12 hours.
Monitored by thin layer chromatography until the third intermediate is completely consumed. Developing agent: PE ea=1:4, the product was not visible under uv light and was developed using bromocresol green.
The reaction was washed with ethyl acetate (5 ml x 2), the aqueous phase was adjusted to pH 1 with dilute hydrochloric acid (1.0M), extracted with ethyl acetate (10 ml x 3), the organic phases combined and concentrated under reduced pressure to give 4- (3- (3-sulfamylphenyl) ureido) butanoic acid compound (88 mg, 92% yield as white solid).
The nuclear magnetic spectrum is shown in figure 3 of the specification, and can be seen as follows: 1 H NMR(600MHz,DMSO-d6)δ12.11(bs,1H),8.82(s,1H),7.99(t,J＝1.8Hz,1H),7.51(bd,J＝7.8Hz,1H),7.39(t,J＝7.8Hz,1H),7.35-7.30(m,3H),6.29(t,J＝6.0Hz,1H),3.10(td,J＝7.2,6.0Hz),2.25(t,J＝7.2Hz,2H),1.66(quintet,J＝7.2Hz,2H)。
example 5
Preparation of fourth intermediate represented by chemical formula 6
[ chemical formula 6 ]
4- (3- (3-sulfamoylphenyl) ureido) butanoic acid and BtOH obtained in example 4 were added to a dry 5-mL round bottom flask, dissolved in DMF, N-BOC-1, 4-butanediamine (5) was added and cooled to 0-5℃in an ice bath.
EDCI and TEA were added, stirred for 30min at room temperature, and then allowed to react for 12h.
The reaction was monitored by thin layer chromatography until complete consumption of 4- (3- (3-sulfamoylphenyl) ureido) butanoic acid. Developing agent: DCM: meoh=10:1, the product was developed as an alkaline potassium permanganate solution.
The solvent was removed by rotary evaporation and column chromatography on silica gel (DCM: meoh=10:1) gave the fourth intermediate (16.3 mg, 52% yield) as a white solid.
The nuclear magnetic spectrum is shown in figure 4 of the specification, and can be seen as follows: 1 H NMR(600MHz,DMSO-d6)δ8.84(s,1H),8.33(s,1H),7.99(bs,1H),7.84(t,J＝6.0Hz,1H),7.51(bd,J＝8.4Hz,1H),7.39(t,J＝8.4Hz,1H),7.35-7.30(m,3H),6.81(t,J＝6.0Hz,1H),6.26(t,J＝6.0Hz,1H),3.07(td,J＝7.2,6.0Hz,2H),3.03-2.98(m,2H),2.91-2.86(m,2H),2.08(t,J＝7.2Hz,2H),1.65(quintet,J＝7.2Hz,2H),1.37(s,9H),1.36-1.32(m,4H)。
example 5
Preparation of fifth intermediate represented by chemical formula 7
[ chemical formula 7 ]
The fourth intermediate obtained in example 4 was added to a dry 5-mL round bottom flask, dissolved in dichloromethane, TFA was added and stirred at room temperature.
The reaction was monitored by thin layer chromatography until the fourth intermediate was completely consumed and the reaction was completed. Developing agent: DCM: meoh=5:1.
The solvent was removed by rotary evaporation to give a crude product of the fifth intermediate (8.6 mg of white solid, 94% crude yield), which was used in the next step without isolation.
Example 6
Preparation of BODIPY marker of 4- (3- (3-sulfamoylphenyl) ureido) butanoic acid represented by chemical formula 5
[ chemical formula 5 ]
The fifth intermediate obtained in example 5, BODIPY-CO 2 H and BtOH were added to a dry 5-mL round bottom flask, dissolved in THF, and cooled to 0-5℃in an ice bath.
EDCI and TEA were added, stirred for 30min at room temperature, and then allowed to react for 12h.
The reaction was monitored by thin layer chromatography until the fifth intermediate was completely consumed and the reaction was completed. Developing agent: PE ea=1:3, the product has stronger fluorescence (rf=0.1).
The solvent was removed by rotary evaporation, and the mixture was separated by silica gel column chromatography (PE: ea=1:3) to give a BODIPY marker of 4- (3- (3-sulfamoylphenyl) ureido) butyric acid (1.7 mg, yield 51%).
The nuclear magnetic spectrum is shown in the attached figure 5 of the specification, and can be seen as follows: 1 H NMR(600MHz,CDCl3)δ8.03(s,1H),7.65(bs,1H),7.55-7.52(m,1H),7.47(bs,1H),7.37(s,1H),7.33-7.25(m,2H),7.01(s,1H),6.89(bs,1H),6.82(d,J＝3.6Hz,1H),6.27(d,J＝3.6Hz,1H),6.04(s,1H),4.26-4.12(m,2H),3.83-3.70(m,2H),(3.22,2.71)(t,J＝7.2Hz,2H),(2.91,2.49)(s,3H),(2.88,2.18)(s,3H),2.29(t,J＝7.2Hz,2H),1.70-1.30(m,6H)。
example 7
Cell culture:
DC cells were normally cultured in 10% FBS+RPMI-1640 medium and when grown to 80% or more, passaged. The cell culture supernatant was discarded, and the cells were washed with 2mL of PBS and discarded. 700uL of 0.25% trypsin was added, and the mixture was placed in a CO2 incubator for about 1.5min, and the morphology of the cells was observed under a microscope to round, and the flask was gently tapped to detach the cells. Cells were completely detached by adding 2mL of 10% FBS+RPMI-1640 medium. The cell mixture was pipetted into a sterilized 15mL centrifuge tube and centrifuged at 1000rpm for 3min at room temperature. The supernatant was discarded, resuspended in 1mL of complete medium, and 10uL of the cell suspension was added to 1uL of trypan blue, mixed well, and counted in a cell counting plate.
Example 8
qPCR analysis of DC cell CD91 RNA interference effect
On the basis of example 5, cell viability was guaranteed to be above 90%, 6 well plates were plated, 6×105 cells per well. Cell wall-attached supernatant was changed to complete medium, adenovirus in which the following 3 CD91 interference sequences pAVE-3576, pAVE-3577 and pAVE-3578 were respectively packed and Control plasmid pAVE-Control adenovirus supernatant in which the Control plasmid pAVE-Control adenovirus was packed were added, the samples were collected after 24 hours of culture, total RNA was extracted, cDNA was reverse transcribed, and qPCR analysis was performed using the CD91 primer sequences shown in the following table and GAPDH inner primer sequences, to verify the interference effect of the 3 CD91 interference sequences on CD 91.
The 3 CD91 interference sequences are shown in the following table
| Gene | 5’-3’ | 3’-5’ |
| 1.pAVE-3576 | GAUCCGUGUGAACCGCUUUAATT | UUAAAGCGGUUCACACGGAUCTT |
| 2.pAVE-3577 | GCGAACAAACACACUGGCUAATT | UUAGCCAGUGUGUUUGUUCGCTT |
| 3.pAVE-3578 | GUCCAACUACACGUUACUUAATT | UUAAGUAACGUGUAGUUGGACTT |
The primer sequences of the genes are shown in the following table
| Gene | Forward(5’-3’) | Reverse(5’-3’) |
| CD91 | CTGGCCTATCACCGTGGCTG | GACGGTCTCACGCTCGAAGG |
| MHC-I | CTACAACCAGAGCGAGGCCG | AATCCTTGCCGTCGTAGGCG |
| GAPDH | GCGGGGCTCTCCAGAACATC | TCCACCACTGACACGTTGGC |
As shown in FIG. 6, the RNA transcription level of each group of CD91, the experimental result shows that the 3 rd sequence pAVE3578 has the best interference effect, and can be used in the subsequent experiments.
Example 9
Flow analysis confirms the interference effect of pAVE3578 plasmid on CD91 RNA
On the basis of example 6, after 24 hours of culture, the pAVE3578 group with the best interference effect is collected, and the expression quantity of CD91 is detected in a flow mode:
the method comprises the following steps:
the experimental steps are as follows:
1. cells were harvested, centrifuged at 1000rpm for 5 minutes at 4℃and the supernatant discarded.
2. Cells were resuspended in 50mL staining Buffer and counted and assayed for cell viability using Trypan Blue (Trypan Blue).
3. Centrifuging the cell fluid and discarding the supernatant; cells were resuspended with a starting Buffer and the cell concentration was adjusted to 2X 107 cells/mL.
4. 50. Mu.L of diluted primary antibody (diluted to the appropriate concentration with a starting Buffer) was added to each flow tube; 50 μ L Staining Buffer was added to the blank tube.
5. To each tube, 50. Mu.L of the cell suspension was added and gently mixed.
6. Incubate for 20 min at 4℃in the dark.
7. After the incubation was completed, centrifugation was carried out at 1000rpm for 5 minutes at 4℃and the supernatant was discarded.
8. The wash was repeated 3 times with 100 μ L Staining Buffer.
9. The cells were resuspended with 100. Mu. L Staining Buffer and then detected by an upflow instrument.
Data analysis:
data analysis was performed by graphpad prism 8, significance analysis was performed with Bonferroni's multiple comparisons test, alpha=5.000%, and then statistical analysis was performed.
As shown in fig. 7, after treatment of DC cells with interfering adenoviruses, the expression level of pAVE3578 was significantly reduced compared to the non-interfering group CD 91.
Example 10
Total RNA extraction experiments
The method comprises the following steps:
the culture solution in the 6-well plate was aspirated, 1ml Trizol Reagent was added to each well, and the cells were completely lysed by blowing with a gun head.
The lysate was transferred to a 1.5ml EP tube and left at room temperature for 10 minutes.
200. Mu.L of chloroform was added thereto, mixed with vigorous shaking, and left at room temperature for 10 minutes.
Centrifuge at 12000rpm at 4℃for 10 min, aspirate the supernatant into a new centrifuge tube, add an equal volume of isopropanol and pellet at room temperature for 10 min.
Centrifuge at 12000rpm at 4℃for 15 min, discard supernatant.
The precipitate was washed once with 500 μl of 75% ethanol. Centrifugation was carried out at 12000rpm at 4℃for 5 minutes, the precipitate was recovered, and the supernatant was discarded. And (5) at normal temperature, inversely airing for 10 minutes.
The pellet was dissolved with 20. Mu. LDEPC-H2O, OD260 and OD280 were measured, and the RNA concentration was calculated.
Agarose electrophoresis checked the integrity of the RNA.
Example 11
Real-time PCR analysis
The primer design is shown below
| Gene | Forward(5’-3’) | Reverse(5’-3’) |
| CD91 | CTGGCCTATCACCGTGGCTG | GACGGTCTCACGCTCGAAGG |
| MHC-I | CTACAACCAGAGCGAGGCCG | AATCCTTGCCGTCGTAGGCG |
| GAPDH | GCGGGGCTCTCCAGAACATC | TCCACCACTGACACGTTGGC |
Reagents and apparatus
Real-time fluorescent quantitative universal reagent (Shanghai worker)
Real-time fluorescence quantitative PCR instrument (Applied Biosystems,7900, U.S.)
Experimental method
Reverse transcription reaction system
Reverse transcription procedure
42℃ 30min；85℃ 10min.
Real-time fluorescent quantitative reaction system
Quantitative PCR reaction procedure
Denaturation at 95℃for 5min
95 ℃ for 12 seconds; 60 ℃,40 seconds, 40 cycles
95 ℃ for 10 seconds; 60 ℃ for 10 seconds
40 ℃ for 30 seconds
Example 12
Treatment of DC cells with fluorescently labeled small molecule compounds
Ensuring the cell viability to be above 90 percent, paving 6 pore plates, and 6 times 105 cells per pore. Removing the supernatant after cell adherence, changing into a complete culture medium, respectively adding a Control plasmid pAVE-Control adenovirus supernatant and a 3 rd sequence pAVE3578 adenovirus supernatant, and uniformly mixing; after incubation for 24h, cells (40. Mu.M) were treated with fluorescent-labeled small molecule compounds and photographed at 3, 6, 12, 24, 48 h.
FIG. 8 shows photographs of the same field of view under white light and fluorescence at each time point, showing that the difference between 24 and 48h after treatment of DC cells with small molecule compounds is most pronounced.
Example 13
Flow detection of cell fluorescence
On the basis of example 8, DC cell samples were collected at various time points and the fluorescence intensity of cells was measured by flow cytometry (FIG. 4).
The method comprises the following steps:
1. cells were harvested, centrifuged at 1000rpm for 5 minutes at 4℃and the supernatant discarded.
2. Cells were resuspended in 100. Mu.L PBS and counted and assayed for cell viability using Trypan Blue (Trypan Blue).
3. Centrifuging the cell fluid and discarding the supernatant; cells were resuspended in PBS and the cell concentration was adjusted to 2X 107 cells/mL.
4. The cells were detected by flow up meter. Significance analysis was performed at α=0.05.
As shown in fig. 9, after treatment of DC cells with small molecule compounds for 24 and 48h, CD91 was RNA-interfered (pAVE-3587 group) with significantly lower fluorescence intensity than the RNA non-interfered group of CD91 (pAVE-Control group) (p < 0.05), and there was no significant difference in fluorescence intensity between the experimental and Control groups at the remaining time points (p > 0.05).
Example 14
Detection of MHC expression level
MHC (major histocompatibility complex) is a tool for presenting antigens to DC cells; upregulation of expression of MHC in DC cells is an important marker for enhanced antigen presenting capacity.
DC cells were treated with fluorescent-labeled small molecule compounds, and DC cell samples were collected at 6,16,24 hours on the basis of example 8, total RNA extraction was performed as described in example 6, real-time PCR analysis was performed as described in example 7, and MHC-I primer sequences are shown in the following Table.
| Gene | Forward(5’-3’) | Reverse(5’-3’) |
| MHC-I | CTACAACCAGAGCGAGGCCG | AATCCTTGCCGTCGTAGGCG |
As shown in FIG. 10, the MHC expression level on DC cells was examined at 6,16,24 hours, and it was found that after the application of a small molecule compound, the MHC expression on DC cells was up-regulated at 16 hours.
Although the exemplary aspects of the present disclosure have been described for illustrative purposes, those skilled in the art will appreciate that the foregoing description is merely illustrative of preferred embodiments of the application and is not intended to limit the scope of the application in any way, including additional implementations in which functions may be performed out of the order of presentation or discussion. Any alterations and modifications of the present application, which are made by those of ordinary skill in the art based on the above disclosure, are intended to be within the scope of the appended claims.
Claims (12)
1. A4- (3- (3-sulfamoylphenyl) ureido) butanoic acid compound represented by formula 1.
2. A method of synthesizing a 4- (3- (3-sulfamoylphenyl) ureido) butanoic acid compound, the method comprising the steps of:
obtaining a first intermediate represented by chemical formula 2 from m-aminobenzenesulfonamide and phenyl chloroformate;
obtaining a second intermediate represented by chemical formula 3 from 4-aminobutyric acid;
obtaining a third intermediate represented by chemical formula 4 from the first intermediate and the second intermediate;
4- (3- (3-sulfamoylphenyl) ureido) butanoic acid is obtained from the third intermediate.
3. The method according to claim 2, wherein the first intermediate represented by chemical formula 2 is obtained from metanilic acid amide and phenyl chloroformate, comprising the steps of:
adding m-aminobenzenesulfonamide and DMAP into a dry reaction vessel, dissolving with THF, and cooling in ice bath; phenyl chloroformate is added dropwise under ice bath, stirred at a constant temperature, and then moved to room temperature for reaction.
4. The method according to claim 2, wherein the second intermediate represented by chemical formula 3 is obtained from 4-aminobutyric acid, comprising the steps of:
adding 4-aminobutyric acid into a dry reaction container, adding methanol for dissolution, and cooling in an ice bath; thionyl chloride is slowly added dropwise in ice bath, stirred at a constant temperature, and then moved to room temperature for reaction.
5. The method according to claim 2, wherein a third intermediate represented by chemical formula 4 is obtained from the first intermediate and the second intermediate, comprising the steps of:
the first intermediate and the second intermediate are added into a dry reaction vessel, dissolved in methanol, added with triethylamine, and then heated for reflux reaction.
6. The process according to claim 2, characterized in that 4- (3- (3-sulfamoylphenyl) ureido) butyric acid is obtained from said third intermediate, comprising the following steps:
and adding the third intermediate into a dry reaction container, adding sodium hydroxide solution, and reacting at normal temperature.
7. Use of a 4- (3- (3-sulfamoylphenyl) ureido) butanoic acid compound according to claim 1 in cellular immunotherapy, characterized in that: the compounds are capable of binding fluorescent molecules for the purpose of entering DC cells by activating CD91 molecules.
8. A BODIPY marker of 4- (3- (3-sulfamoylphenyl) ureido) butyric acid represented by formula 5.
9. A method of synthesizing a BODIPY marker of 4- (3- (3-sulfamoylphenyl) ureido) butanoic acid, the method comprising the steps of:
preparation of 4- (3- (3-sulfamoylphenyl) ureido) butanoic acid, obtainable by the process according to any one of claims 1 to 6;
obtaining a fourth intermediate represented by formula 6 from 4- (3- (3-sulfamoylphenyl) ureido) butanoic acid and N-BOC-1, 4-butanediamine;
obtaining a fifth intermediate represented by formula 7 from the fourth intermediate;
from said fifth intermediate and BODIPY-CO 2 H gives a BODIPY marker of 4- (3- (3-sulfamoylphenyl) ureido) butanoic acid.
10. The process according to claim 9, wherein a fourth intermediate of formula 6 is obtained from 4- (3- (3-sulfamoylphenyl) ureido) butanoic acid and N-BOC-1, 4-butanediamine, comprising the steps of: 4- (3- (3-sulfamoyl phenyl) ureido) butyric acid and BtOH are added into a dry reaction vessel, dissolved in DMF, added with N-BOC-1, 4-butanediamine and cooled in an ice bath.
11. The method according to claim 9, wherein a fifth intermediate represented by formula 7 is obtained from the fourth intermediate, comprising the steps of: the fourth intermediate was added to a dry reaction vessel, dissolved in dichloromethane, TFA was added and stirred at room temperature.
12. The method according to claim 9, wherein a BODIPY marker of 4- (3- (3-sulfamoylphenyl) ureido) butyric acid is obtained from said fifth intermediate and BODIPY-CO2H, comprising the steps of: the fifth intermediate, BODIPY-CO 2 H and BtOH are added into a dry reaction vessel, dissolved by THF and cooled by ice bath; EDCI and TEA are added, stirred at a constant temperature and then moved to room temperature for reaction.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211255810.2A CN117069627A (en) | 2022-10-13 | 2022-10-13 | 4- (3- (3-sulfamoyl phenyl) ureido) butyric acid compound, fluorescent marker, preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211255810.2A CN117069627A (en) | 2022-10-13 | 2022-10-13 | 4- (3- (3-sulfamoyl phenyl) ureido) butyric acid compound, fluorescent marker, preparation method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117069627A true CN117069627A (en) | 2023-11-17 |
Family
ID=88702939
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211255810.2A Pending CN117069627A (en) | 2022-10-13 | 2022-10-13 | 4- (3- (3-sulfamoyl phenyl) ureido) butyric acid compound, fluorescent marker, preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117069627A (en) |
-
2022
- 2022-10-13 CN CN202211255810.2A patent/CN117069627A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110746410B (en) | Leucine aminopeptidase and monoamine oxidase activated near-infrared fluorescent probe, synthetic method and biological application | |
| CN105624192B (en) | Preparation of breast cancer cell strain capable of stably secreting near-infrared fluorescence labeled exosomes | |
| WO2016105605A1 (en) | Comprehensive and comparative flow cytometry-based methods for identifying the state of a biological system | |
| WO2019237442A1 (en) | Anti-human b7-h4 monoclonal antibody, identification method, use and kit | |
| CN111826443B (en) | Application of serum exosome micro RNAs and liver cancer detection kit | |
| WO2012137538A1 (en) | Cytotoxic t cell inducing composition | |
| CN107400668B (en) | Extraction method of tussah microsporidian template DNA and application of tussah microsporidian template DNA in molecular diagnosis | |
| CN109820866A (en) | A kind of c-Myc gene expression inhibitor and its application | |
| CN113956342B (en) | Tumor neogenesis antigen polypeptide and application thereof | |
| CN117069627A (en) | 4- (3- (3-sulfamoyl phenyl) ureido) butyric acid compound, fluorescent marker, preparation method and application thereof | |
| CN103276060B (en) | Gene probe composition and kit for detecting epithelial ovarian cancer | |
| JP2005532034A5 (en) | ||
| CN110885366B (en) | Tumor marker heat shock factor 2binding protein of liver cancer and application thereof | |
| CN115521288B (en) | Compounds with enhanced immunogenicity for eliciting antigen-specific responses, fluorescent markers, methods of preparation and uses thereof | |
| CN109161543A (en) | For being enriched with DNA probe and its application of low frequency DNA mutation | |
| CN108949963A (en) | A kind of fluorescent quantitative PCR detection method and its detection kit of KIR2DL2 mRNA | |
| CN109593067B (en) | Tetrazole molecular probe compound or composition and application thereof | |
| CN110642861A (en) | PET/CT tracer agent with selectivity on different lung cancer cells and preparation method and application thereof | |
| CN1940074A (en) | Construction, screen and use for stomach cancer target AFP gene siRNAs expression carrier | |
| CN117105813A (en) | Compounds with enhanced immunogenicity for use as chimeric antigens, fluorescent markers thereof, preparation method and uses thereof | |
| CN113582864B (en) | PRMTI type methyltransferase inhibiting active compound and preparation and application thereof | |
| CN117105880A (en) | Urea compounds for inducing antigen-specific reaction, fluorescent markers thereof, preparation method and application | |
| CN111675644B (en) | Indole diaryl methane compound and preparation method and application thereof | |
| CN103820481B (en) | The structure of chicken peripheral blood mononuclear lymphocyte PD-L2 recombinant plasmid, gene abundance real-time detection method and application thereof | |
| CN107880029A (en) | Design, synthesis and the application of a kind of indole derivatives antitumoral compounds containing pyrazol framework |
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 |