CN115368430B - Preparation method and application of tripterine metal complex - Google Patents
Preparation method and application of tripterine metal complex Download PDFInfo
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- -1 tripterine metal complex Chemical class 0.000 title claims abstract description 19
- 238000002360 preparation method Methods 0.000 title claims abstract description 10
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims abstract description 56
- KQJSQWZMSAGSHN-JJWQIEBTSA-N celastrol Chemical class C([C@H]1[C@]2(C)CC[C@@]34C)[C@](C)(C(O)=O)CC[C@]1(C)CC[C@]2(C)C4=CC=C1C3=CC(=O)C(O)=C1C KQJSQWZMSAGSHN-JJWQIEBTSA-N 0.000 claims abstract description 36
- 229940002612 prodrug Drugs 0.000 claims abstract description 8
- 239000000651 prodrug Substances 0.000 claims abstract description 8
- KQJSQWZMSAGSHN-UHFFFAOYSA-N (9beta,13alpha,14beta,20alpha)-3-hydroxy-9,13-dimethyl-2-oxo-24,25,26-trinoroleana-1(10),3,5,7-tetraen-29-oic acid Natural products CC12CCC3(C)C4CC(C)(C(O)=O)CCC4(C)CCC3(C)C2=CC=C2C1=CC(=O)C(O)=C2C KQJSQWZMSAGSHN-UHFFFAOYSA-N 0.000 claims description 31
- AQKDBFWJOPNOKZ-UHFFFAOYSA-N Celastrol Natural products CC12CCC3(C)C4CC(C)(C(O)=O)CCC4(C)CCC3(C)C2=CC=C2C1=CC(=O)C(=O)C2C AQKDBFWJOPNOKZ-UHFFFAOYSA-N 0.000 claims description 31
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 18
- 238000000034 method Methods 0.000 claims description 18
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 15
- 239000003960 organic solvent Substances 0.000 claims description 14
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 12
- 238000001035 drying Methods 0.000 claims description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 11
- 230000008569 process Effects 0.000 claims description 11
- 238000004090 dissolution Methods 0.000 claims description 9
- 239000002904 solvent Substances 0.000 claims description 8
- 239000012154 double-distilled water Substances 0.000 claims description 7
- 229910052742 iron Inorganic materials 0.000 claims description 7
- 238000002156 mixing Methods 0.000 claims description 7
- 239000000843 powder Substances 0.000 claims description 7
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 6
- 238000003760 magnetic stirring Methods 0.000 claims description 6
- 150000003839 salts Chemical class 0.000 claims description 6
- 238000000926 separation method Methods 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 5
- VTLYFUHAOXGGBS-UHFFFAOYSA-N Fe3+ Chemical compound [Fe+3] VTLYFUHAOXGGBS-UHFFFAOYSA-N 0.000 claims description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 4
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 4
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 4
- 239000003795 chemical substances by application Substances 0.000 claims description 4
- 238000003260 vortexing Methods 0.000 claims description 4
- 230000001093 anti-cancer Effects 0.000 claims description 3
- 239000002184 metal Substances 0.000 claims description 3
- 229910052751 metal Inorganic materials 0.000 claims description 3
- 239000005416 organic matter Substances 0.000 claims description 3
- 238000002390 rotary evaporation Methods 0.000 claims description 3
- 238000010025 steaming Methods 0.000 claims description 3
- 238000002604 ultrasonography Methods 0.000 claims description 3
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 2
- CWYNVVGOOAEACU-UHFFFAOYSA-N Fe2+ Chemical compound [Fe+2] CWYNVVGOOAEACU-UHFFFAOYSA-N 0.000 claims description 2
- 239000002253 acid Substances 0.000 claims description 2
- 150000004677 hydrates Chemical class 0.000 claims description 2
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 claims description 2
- 239000002798 polar solvent Substances 0.000 claims description 2
- 239000002516 radical scavenger Substances 0.000 claims description 2
- 238000000527 sonication Methods 0.000 claims description 2
- 229910002651 NO3 Inorganic materials 0.000 claims 1
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 claims 1
- 238000012216 screening Methods 0.000 claims 1
- 239000000126 substance Substances 0.000 claims 1
- 239000000243 solution Substances 0.000 abstract description 14
- 229910021645 metal ion Inorganic materials 0.000 abstract description 5
- 210000004881 tumor cell Anatomy 0.000 abstract description 4
- 206010028980 Neoplasm Diseases 0.000 abstract description 3
- 108090000623 proteins and genes Proteins 0.000 abstract description 3
- 102000004169 proteins and genes Human genes 0.000 abstract description 3
- 230000002829 reductive effect Effects 0.000 abstract description 3
- 230000009466 transformation Effects 0.000 abstract description 3
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 230000008901 benefit Effects 0.000 abstract description 2
- 230000001988 toxicity Effects 0.000 abstract description 2
- 231100000419 toxicity Toxicity 0.000 abstract description 2
- ZKHQWZAMYRWXGA-KQYNXXCUSA-J ATP(4-) Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-J 0.000 abstract 1
- ZKHQWZAMYRWXGA-UHFFFAOYSA-N Adenosine triphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O ZKHQWZAMYRWXGA-UHFFFAOYSA-N 0.000 abstract 1
- TTWYZDPBDWHJOR-IDIVVRGQSA-L adenosine triphosphate disodium Chemical compound [Na+].[Na+].C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP([O-])([O-])=O)[C@@H](O)[C@H]1O TTWYZDPBDWHJOR-IDIVVRGQSA-L 0.000 abstract 1
- 230000015572 biosynthetic process Effects 0.000 abstract 1
- 230000000536 complexating effect Effects 0.000 abstract 1
- 230000002708 enhancing effect Effects 0.000 abstract 1
- 238000000746 purification Methods 0.000 abstract 1
- 239000012266 salt solution Substances 0.000 abstract 1
- 238000003786 synthesis reaction Methods 0.000 abstract 1
- 230000005909 tumor killing Effects 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- 238000000862 absorption spectrum Methods 0.000 description 4
- 230000003013 cytotoxicity Effects 0.000 description 4
- 231100000135 cytotoxicity Toxicity 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 3
- 230000003833 cell viability Effects 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- NQXWGWZJXJUMQB-UHFFFAOYSA-K iron trichloride hexahydrate Chemical compound O.O.O.O.O.O.[Cl-].Cl[Fe+]Cl NQXWGWZJXJUMQB-UHFFFAOYSA-K 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 238000001157 Fourier transform infrared spectrum Methods 0.000 description 2
- 238000001237 Raman spectrum Methods 0.000 description 2
- YRKCREAYFQTBPV-UHFFFAOYSA-N acetylacetone Chemical compound CC(=O)CC(C)=O YRKCREAYFQTBPV-UHFFFAOYSA-N 0.000 description 2
- 230000009918 complex formation Effects 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000027756 respiratory electron transport chain Effects 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 238000000967 suction filtration Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 206010067484 Adverse reaction Diseases 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 101100220087 Caenorhabditis elegans cdc-37 gene Proteins 0.000 description 1
- 238000005079 FT-Raman Methods 0.000 description 1
- 229910017135 Fe—O Inorganic materials 0.000 description 1
- 238000005033 Fourier transform infrared spectroscopy Methods 0.000 description 1
- 101710113864 Heat shock protein 90 Proteins 0.000 description 1
- 229910021577 Iron(II) chloride Inorganic materials 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 101710132589 Peroxidase 2 Proteins 0.000 description 1
- BELBBZDIHDAJOR-UHFFFAOYSA-N Phenolsulfonephthalein Chemical compound C1=CC(O)=CC=C1C1(C=2C=CC(O)=CC=2)C2=CC=CC=C2S(=O)(=O)O1 BELBBZDIHDAJOR-UHFFFAOYSA-N 0.000 description 1
- 108091000080 Phosphotransferase Proteins 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 239000006146 Roswell Park Memorial Institute medium Substances 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
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- 230000006838 adverse reaction Effects 0.000 description 1
- 230000009435 amidation Effects 0.000 description 1
- 238000007112 amidation reaction Methods 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 230000022131 cell cycle Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 238000010668 complexation reaction Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 230000000857 drug effect Effects 0.000 description 1
- 230000032050 esterification Effects 0.000 description 1
- 238000005886 esterification reaction Methods 0.000 description 1
- 229910001447 ferric ion Inorganic materials 0.000 description 1
- 229960002089 ferrous chloride Drugs 0.000 description 1
- 241000411851 herbal medicine Species 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- NMCUIPGRVMDVDB-UHFFFAOYSA-L iron dichloride Chemical compound Cl[Fe]Cl NMCUIPGRVMDVDB-UHFFFAOYSA-L 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 201000005202 lung cancer Diseases 0.000 description 1
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- 150000002823 nitrates Chemical class 0.000 description 1
- 208000002154 non-small cell lung carcinoma Diseases 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 229960003531 phenolsulfonphthalein Drugs 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 150000008442 polyphenolic compounds Chemical class 0.000 description 1
- 235000013824 polyphenols Nutrition 0.000 description 1
- 230000007420 reactivation Effects 0.000 description 1
- 230000004043 responsiveness Effects 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 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 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J63/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton has been modified by expansion of only one ring by one or two atoms
- C07J63/008—Expansion of ring D by one atom, e.g. D homo steroids
-
- 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
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Abstract
A preparation method and application of a tripterine metal complex belong to the field of tripterine derivative transformation. The invention adds ferric salt solution into tripterine solution, fully and uniformly mixes, and stands for 5min to obtain CEL-Fe complex. The synthesized tripterine iron complex has the advantages of dissociable ATP response, and simple and feasible synthesis and purification scheme. The metal ions occupy the binding sites of the tripterine and the target protein, so that the physiological toxicity is effectively reduced, and the complexing state can be released by the high-concentration ATP (adenosine triphosphate) competition of the tumor part, thereby being beneficial to enhancing the selective killing of tumor cells and becoming a potential tripterine prodrug strategy.
Description
Technical Field
The invention belongs to the technical field of preparation of tripterine derivatives, and particularly relates to a preparation method and application of a tripterine iron complex.
Background
Celastrol (CEL) is an active substance extracted from traditional Chinese herbal medicines, and plays an important role in anti-inflammatory, anticancer, and inhibiting autoimmune diseases and neurodegenerative disease progression. Mechanically, some protein targets have been reported, including heat shock protein 90 (Hsp 90), cell division cycle 37 (Cdc 37), ikkβ kinase beta (ikkβ), peroxidase-2, etc., and affect numerous metabolic processes through multiple signaling pathways [1] . Despite its broad application prospect, numerous preclinical research results show that the water solubility is poor, the bioavailability is low, the therapeutic window is narrow, the adverse reaction is serious, and the clinical application of the drug as anticancer drug is limited [2] 。
Structure modifications based on CEL have resulted in numerous derivatives including C-20 carboxy esterification or amidation modifications, C-3 hydroxy substitution, and C-6 substitution [3][4] . Wherein derivatives having a stronger selectivity for tumor cells can be selected to reduce toxicity to normal cells and ensure the sameAnticancer effect. Structural modification of CEL is also the starting point for the present design as a current direction of intense research.
Inspired by the rapid, simple and responsive dissociable coordination mode of the coordination compound formed by polyphenol and metal ions in recent years [5] We have found that the C-2 carbonyl and C-3 hydroxyl groups on CEL ring A are structurally similar to acetylacetone, with the ability to form coordination compounds with ferric ions. In the CEL-Fe complex, the iron element occupies the site where CEL originally interacts with protein, so that cytotoxicity is reduced, and the CEL cytotoxicity can be recovered through the action of coordination competitor ATP, so that the CEL-Fe complex becomes a prodrug strategy with good application prospect. Based on the advantages of simple preparation, high stability, controllable cytotoxicity and the like of the CEL-Fe complex, the research provides more possibility for clinical transformation of CEL.
Reference is made to:
[1]Lim H Y,Ong P S,Wang L,et al.Celastrol in cancer therapy:Recent developments,challenges and prospects[J].Cancer Letters,Elsevier B.V.,2021,521(September):252–267.
[2]Shi J,Li J,Xu Z,et al.Celastrol:A Review of Useful Strategies Overcoming its Limitation in Anticancer Application[J].Frontiers in Pharmacology,2020,11(November).
[3]Li N,Xu M,Zhang L,et al.Discovery of Novel Celastrol-Imidazole Derivatives with Anticancer Activity In Vitro and In Vivo[J].Journal of Medicinal Chemistry,2022,65(6):4578–4589.
[4]Wang G,Xiao Q,Wu Y,et al.Design and synthesis of novel celastrol derivative and its antitumor activity in hepatoma cells and antiangiogenic activity in zebrafish[J].Journal of Cellular Physiology,2019,234(9):16431–16446.
[5]Guo J,Ping Y,Ejima H,et al.Engineering multifunctional capsules through the assembly of metal-phenolic networks[J].Angewandte Chemie-International Edition,2014,53(22):5546–5551.
disclosure of Invention
The invention designs and synthesizes the celastrol iron complex, namely CEL-Fe, which is simple to operate and can effectively increase the selectivity to tumor cells.
In order to achieve the above purpose, the technical scheme of the invention comprises the following steps:
(1) Tripterine ((2 r,4as,6as,12br,14as,14 br) -10-hydroxy-2,4a,6a,9,12b,14 a-hexa-methyl-11-oxo-1, 2,3, 4a,5, 6a,11,12b,13,14 a,14 b-tetradecahydro-2-carboxic acid) is added into a reactor and dissolved with an organic solvent to obtain a solution a;
the organic solvents used include, but are not limited to, dichloromethane, methanol, dimethyl sulfoxide, ethanol, acetone, chloroform, etc. which can dissolve the tripterine.
The dissolution process is carried out at room temperature, and the dissolution can be accelerated by magnetic stirring, vortexing, ultrasound, and the like.
(2) Dissolving ferric salt in a solvent to obtain a solution B;
the iron-containing salts used include, but are not limited to: hydrochloric acid, sulfuric acid, nitrate salts of ferrous and ferric iron and crystalline hydrates thereof.
The solvent used includes distilled water, methanol, ethanol, dimethyl sulfoxide and other polar solvents capable of dissolving ferric salt.
The dissolution process is carried out at room temperature, and the dissolution can be accelerated by magnetic stirring, vortexing, ultrasound, and the like.
(3) Adding the solution B into the solution A, fully and uniformly mixing, and standing for 5min to obtain a CEL-Fe complex;
if the organic solvent used in the step (1) is not mutually soluble with the solvent in the step (2), separating an organic matter layer containing the CEL-Fe complex through liquid separation operation after layering, drying and rotary steaming to remove the organic solvent, and fully drying and collecting the CEL-Fe complex powder in a dark place;
if the organic solvent used in the step (1) is mutually soluble with the solvent in the step (2), adding double distilled water for layering, separating an organic matter layer containing the CEL-Fe complex through a liquid separation operation, drying, performing rotary evaporation to remove the organic solvent, and fully drying in a dark place to obtain CEL-Fe complex powder;
the molar ratio of iron ions to tripterine in the solution A and the solution B is more than 1:1, preferably 2:1, so that the excessive metal ions are ensured.
The dripping and mixing process is carried out at room temperature, and the process can be accelerated by magnetic stirring, vortex, ultrasonic and the like.
To increase the purity of the complex, the separation by layering can be repeated 2-3 times by adding double distilled water.
The water scavenger used for drying includes, but is not limited to: anhydrous sodium sulfate, anhydrous calcium chloride, and the like.
The CEL-Fe complex is used for preparing anticancer prodrugs and shielding agents; ATP is used as the trigger for the prodrug and the shielding agent.
The beneficial technical effects are as follows:
(1) The invention successfully synthesizes the celastrol iron complex (CEL-Fe) as a novel celastrol derivative, and has simple preparation method and strong operability.
(2) The invention successfully synthesizes the celastrol iron complex (CEL-Fe), is hopeful to combine the function of metal ions and increases the functionality of the celastrol iron complex in the in-vivo treatment process.
(3) The successfully synthesized celastrol iron complex (CEL-Fe) can recover the initial structure of CEL by using the competitive coordination effect at the tumor site of ATP over-expression, and enhance the selectivity of CEL to tumor cells, thereby becoming a promising CEL prodrug.
Drawings
Fig. 1: structural formulas of Celastrol (CEL) and celastrol iron complex (CEL-Fe) and transformation relation thereof.
Fig. 2: CEL-Fe related color (physical image)
Fig. 3: CEL and CEL-Fe ultraviolet-visible light absorption spectrum test result
Fig. 4: the ultraviolet-visible absorption spectrum of the CEL-Fe complex after ATP treatment was changed with time.
Fig. 5: and (3) analyzing results of the CEL, the CEL-Fe complex, the ATP treated CEL-Fe complex Fourier transform infrared spectrum and the Raman spectrum.
Fig. 6: cell viability statistics for CEL, CEL-Fe complex, and after ATP treatment, the CEL-Fe complex was applied to non-small cell lung cancer A549 cells for 24 h.
Detailed Description
The present invention will be further illustrated with reference to the following examples, but the present invention is not limited to the following examples.
Example 1
(1) Tripterine (90 mg) is added to a 50mL round bottom flask at room temperature, and 10mL of methylene chloride is added thereto and then dissolved by sonication for 10s to give solution A (CEL: 20 mM).
(2) Ferric trichloride hexahydrate (FeCl) at room temperature 3 ·6H 2 O,108 mg) was dissolved in 10mL double distilled water to obtain a solution B (Fe (III): 40 mM).
(3) Adding the solution B into the solution A, and fully and uniformly mixing by ultrasonic for 10 s. Subsequently, the mixture was transferred to a separating funnel, and after mixing again, the mixture was allowed to stand for 5 minutes.
(4) The lower methylene chloride layer containing the CEL-Fe complex was separated, 20mL of double distilled water was added, the upper aqueous phase was discarded, and the above procedure was repeated 3 times. The lower methylene chloride layer containing the CEL-Fe complex at the last wash was collected in a beaker containing 20g of anhydrous sodium sulfate, after stirring thoroughly, the excess water and metal ions were removed by suction filtration, the filtrate was collected in a 50mL round bottom flask, and the complex attached to the surface of the anhydrous sodium sulfate crystal was washed twice with 10mL of methylene chloride, and all the filtrates collected were combined.
(5) Removing the organic solvent by rotary evaporation, drying in a vacuum drying oven at room temperature for 24 hours, and collecting the CEL-Fe complex powder.
The CEL-Fe powder was reconstituted with methanol at a concentration of 1mM and diluted 10-fold, and a photograph was taken as shown in FIG. 2b, and the CEL-Fe color was changed to dark green by comparison. Ultraviolet-visible absorption spectra were measured using Nanodrop (Thermo Fisher, USA), and the experimental results after treatment as shown in fig. 3, the ligand-to-metal electron transfer bands (LMCT) present in the spectra provide evidence for complex formation compared to the initial state.
To confirm the ATP responsiveness of the complex, we incubated CEL-Fe with ATP solution after dissolution in dimethyl sulfoxide and tested the uv-vis absorbance spectrum over time as shown in figure 4. As a result, it was found that in a short time, the ligand-to-metal electron transfer band disappeared, and the solution color was restored to the original level (fig. 2 c), confirming the ligand dissociation.
The sample powder after ATP treatment was obtained by adding an excess amount of ATP aqueous solution (100 mM,30 mL) to CEL-Fe, mixing thoroughly, repeating the above steps of separating liquid, washing with water, suction filtration, spin-steaming, and drying after color change. The complex formation and its mechanism of reversion after ATP competition is further verified by fourier transform infrared spectroscopy and raman spectroscopy. The results of the Fourier transform infrared spectrum show (FIG. 5 a), 601cm in the CEL-Fe complex -1 A new strong peak appears at the position, which is attributed to the stretching vibration of Fe-O. The results of the Raman spectrum are consistent with the presence of infrared (b in FIG. 5), 3008cm in the CEL spectrum -1 The stretching vibration peak of some hydroxyl near the center disappears, and at the same time, 250-750cm of CEL-Fe complex -1 The obvious characteristic peak after complexation appears in the wave band, and the change trend is recovered after ATP treatment.
Cytotoxicity of CEL, CEL-Fe complexes and ATP-treated complexes was performed in human non-small lung cancer cell line a 549. CEL, CEL-Fe complex and ATP treated complex were reconstituted in dimethyl sulfoxide to prepare a mother solution of 5 mM. A549 was plated at 6000/well in 96-well plates (Corning, USA) and after 12h the medium was removed. Cells were incubated with CEL, CEL-Fe complex and ATP treated complex medium (phenol red free RPMI 1640,3% FBS) solutions at final concentrations of 2,4,6,8, 12 (. Mu.M), respectively, for 24 hours. The medium was removed and then 10% cck-8 reagent diluted with RPMI 1640 was added in air and after incubation for 2h at 37 ℃ the absorbance of the wells was measured with a microplate reader (spectromax) at 450nm (peak absorbance). Cell viability was expressed as the ratio of absorbance of the test wells and control wells. At the same concentration, CEL-Fe shows the best cell activity after incubation, and the drug effect shielding and attenuation effects of the complex are proved; whereas cell activity was significantly reduced and restored to a level similar to that of CEL after ATP treatment after co-incubation of CEL-Fe with cells, confirming reactivation of the drug activity by ATP. Above, the change in cell viability demonstrates the feasibility of CEL-Fe as a CEL prodrug that can be reactivated at the tumor site.
Example 2
The iron trichloride hexahydrate of example 1 was changed to the corresponding iron dichloride, and the divalent iron ions were converted into 3-valent iron ions during the preparation by the operation under the air condition line, so that the corresponding complex could also be finally obtained.
The present invention has been described in detail above. While the principles and embodiments of the present invention have been described herein with reference to specific examples, the foregoing examples are provided to assist in understanding the core concept of the invention and are intended to provide numerous modifications and adaptations of the invention without departing from the principles of the invention.
Claims (10)
1. The preparation method of the celastrol iron complex CEL-Fe is characterized by comprising the following steps:
(1) Adding tripterine (2R, 4aS,6aS,12bR,14aS,14 bR) -10-hydroxy-2,4a,6a,9,12b,14 a-hexa-methyl-11-oxo-1, 2,3, 4a,5, 6a,11,12b,13,14 a,14 b-tetradecahydro-2-carboxic acid into a reactor, and dissolving with an organic solvent to obtain a solution A;
(2) Dissolving ferric salt in a solvent to obtain a solution B;
(3) Adding the solution B into the solution A, fully and uniformly mixing, and standing for 5min to obtain a CEL-Fe complex;
in the step (3), if the organic solvent used in the step (1) is not mutually soluble with the solvent in the step (2), separating an organic matter layer containing the CEL-Fe complex through liquid separation operation after layering, drying and rotary steaming to remove the organic solvent, and fully drying in a dark place to obtain CEL-Fe complex powder;
if the organic solvent used in the step (1) is mutually soluble with the solvent in the step (2), adding double distilled water for layering, separating an organic substance layer containing the CEL-Fe complex through a liquid separation operation, drying, performing rotary evaporation to remove the organic solvent, and fully drying in a dark place to obtain CEL-Fe complex powder.
2. The method according to claim 1, wherein the organic solvent used in the step (1) is selected from the group consisting of dichloromethane, methanol, dimethyl sulfoxide, ethanol, acetone, chloroform-soluble organic solvents of tripterine;
the dissolution process is carried out at room temperature and the dissolution is accelerated by magnetic stirring, vortexing or sonication.
3. The method of claim 1, wherein the iron-containing salt used in step (2) is selected from the group consisting of: hydrochloric acid, sulfuric acid, nitrate of ferrous and ferric iron, and one or more of its crystalline hydrates.
4. The process according to claim 1, wherein the solvent used in step (2) is selected from the group consisting of double distilled water, methanol, ethanol, a polar solvent in which dimethyl sulfoxide is capable of dissolving iron-containing salts;
the dissolution process is carried out at room temperature, and the dissolution is accelerated by magnetic stirring, vortexing and ultrasound.
5. The method according to claim 1, wherein the molar ratio of iron ions to tripterine in the solution a and the solution B in the step (3) is 1:1 or more, thereby ensuring the excessive metal.
6. The method of claim 1, wherein the step (3) is repeated 2 to 3 times by adding double distilled water for the separation of the layers to increase the purity of the complex.
7. The method of claim 1, wherein the water scavenger used in the drying of step (3) is selected from the group consisting of anhydrous sodium sulfate, anhydrous calcium chloride; the dropping and mixing process in the step (3) is carried out at room temperature, and the process is accelerated through magnetic stirring, vortex and ultrasonic.
8. The process according to any one of claims 1-7, to obtain celastrol iron complex CEL-Fe.
9. Use of celastrol iron complex CEL-Fe prepared according to the method of any one of claims 1-7 for the preparation of anticancer prodrugs, screening agents.
10. The use according to claim 9, wherein ATP is used as the trigger for the prodrug, the shielding agent.
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