CN118344408A - Nitrosyl ruthenium complex with hydroxyproline and 5, 7-dichloro-8-hydroxyquinoline as ligands, and preparation method and application thereof - Google Patents
Nitrosyl ruthenium complex with hydroxyproline and 5, 7-dichloro-8-hydroxyquinoline as ligands, and preparation method and application thereof Download PDFInfo
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- PMMYEEVYMWASQN-UHFFFAOYSA-N dl-hydroxyproline Natural products OC1C[NH2+]C(C([O-])=O)C1 PMMYEEVYMWASQN-UHFFFAOYSA-N 0.000 title claims abstract description 66
- PMMYEEVYMWASQN-DMTCNVIQSA-N Hydroxyproline Chemical compound O[C@H]1CN[C@H](C(O)=O)C1 PMMYEEVYMWASQN-DMTCNVIQSA-N 0.000 title claims abstract description 62
- 229960002591 hydroxyproline Drugs 0.000 title claims abstract description 52
- FGMPLJWBKKVCDB-UHFFFAOYSA-N trans-L-hydroxy-proline Natural products ON1CCCC1C(O)=O FGMPLJWBKKVCDB-UHFFFAOYSA-N 0.000 title claims abstract description 50
- MEMIGISGUUDFSL-UHFFFAOYSA-N nitroxyl anion ruthenium(1+) Chemical compound [Ru]N=O MEMIGISGUUDFSL-UHFFFAOYSA-N 0.000 title claims abstract description 32
- WDFKMLRRRCGAKS-UHFFFAOYSA-N chloroxine Chemical compound C1=CN=C2C(O)=C(Cl)C=C(Cl)C2=C1 WDFKMLRRRCGAKS-UHFFFAOYSA-N 0.000 title claims abstract description 27
- 239000003446 ligand Substances 0.000 title claims abstract description 25
- 238000002360 preparation method Methods 0.000 title claims abstract description 25
- 239000012327 Ruthenium complex Substances 0.000 title claims abstract description 21
- PMMYEEVYMWASQN-IMJSIDKUSA-N cis-4-Hydroxy-L-proline Chemical compound O[C@@H]1CN[C@H](C(O)=O)C1 PMMYEEVYMWASQN-IMJSIDKUSA-N 0.000 claims abstract description 15
- PMMYEEVYMWASQN-IUYQGCFVSA-N trans-4-hydroxy-D-proline Chemical compound O[C@@H]1CN[C@@H](C(O)=O)C1 PMMYEEVYMWASQN-IUYQGCFVSA-N 0.000 claims abstract description 15
- 239000000126 substance Substances 0.000 claims abstract description 4
- 229910052707 ruthenium Inorganic materials 0.000 claims description 61
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 44
- 239000000243 solution Substances 0.000 claims description 32
- 238000006243 chemical reaction Methods 0.000 claims description 26
- 229910001868 water Inorganic materials 0.000 claims description 24
- 239000002243 precursor Substances 0.000 claims description 22
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 19
- 206010008342 Cervix carcinoma Diseases 0.000 claims description 12
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 claims description 12
- 201000010881 cervical cancer Diseases 0.000 claims description 12
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- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 6
- 239000002246 antineoplastic agent Substances 0.000 claims description 6
- 229940041181 antineoplastic drug Drugs 0.000 claims description 6
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- KJTLSVCANCCWHF-UHFFFAOYSA-N Ruthenium Chemical compound [Ru] KJTLSVCANCCWHF-UHFFFAOYSA-N 0.000 claims description 3
- MXLKRGIOUWUOIP-UHFFFAOYSA-M ethanol;tetramethylazanium;chloride Chemical compound [Cl-].CCO.C[N+](C)(C)C MXLKRGIOUWUOIP-UHFFFAOYSA-M 0.000 claims description 3
- 239000000047 product Substances 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- OKIZCWYLBDKLSU-UHFFFAOYSA-M N,N,N-Trimethylmethanaminium chloride Chemical compound [Cl-].C[N+](C)(C)C OKIZCWYLBDKLSU-UHFFFAOYSA-M 0.000 claims description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 2
- 238000013375 chromatographic separation Methods 0.000 claims description 2
- 239000012043 crude product Substances 0.000 claims description 2
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- 230000035755 proliferation Effects 0.000 claims 1
- MWUXSHHQAYIFBG-UHFFFAOYSA-N Nitric oxide Chemical compound O=[N] MWUXSHHQAYIFBG-UHFFFAOYSA-N 0.000 abstract description 256
- 230000027455 binding Effects 0.000 abstract description 18
- 230000000694 effects Effects 0.000 abstract description 17
- 102000007562 Serum Albumin Human genes 0.000 abstract description 8
- 108010071390 Serum Albumin Proteins 0.000 abstract description 8
- 239000002840 nitric oxide donor Substances 0.000 abstract description 3
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 24
- 102000008100 Human Serum Albumin Human genes 0.000 description 22
- 108091006905 Human Serum Albumin Proteins 0.000 description 22
- 210000004027 cell Anatomy 0.000 description 20
- 238000005286 illumination Methods 0.000 description 14
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 12
- 238000001228 spectrum Methods 0.000 description 10
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- 238000005160 1H NMR spectroscopy Methods 0.000 description 6
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 6
- 230000005764 inhibitory process Effects 0.000 description 6
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 6
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 5
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- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 description 4
- 229960004316 cisplatin Drugs 0.000 description 4
- 230000002401 inhibitory effect Effects 0.000 description 4
- 238000011160 research Methods 0.000 description 4
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- 150000004325 8-hydroxyquinolines Chemical class 0.000 description 3
- -1 N-methyl-D-glucosamine dithioformate Chemical compound 0.000 description 3
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- WHTVZRBIWZFKQO-AWEZNQCLSA-N (S)-chloroquine Chemical compound ClC1=CC=C2C(N[C@@H](C)CCCN(CC)CC)=CC=NC2=C1 WHTVZRBIWZFKQO-AWEZNQCLSA-N 0.000 description 1
- HNUFDBQIXGQAEX-UHFFFAOYSA-N 2-chloroquinolin-8-ol Chemical compound C1=C(Cl)N=C2C(O)=CC=CC2=C1 HNUFDBQIXGQAEX-UHFFFAOYSA-N 0.000 description 1
- PMMYEEVYMWASQN-SRBOSORUSA-N 4-hydroxy-D-proline Chemical compound OC1CN[C@@H](C(O)=O)C1 PMMYEEVYMWASQN-SRBOSORUSA-N 0.000 description 1
- 239000005725 8-Hydroxyquinoline Substances 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 102000004225 Cathepsin B Human genes 0.000 description 1
- 108090000712 Cathepsin B Proteins 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
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- 229960003677 chloroquine Drugs 0.000 description 1
- WHTVZRBIWZFKQO-UHFFFAOYSA-N chloroquine Natural products ClC1=CC=C2C(NC(C)CCCN(CC)CC)=CC=NC2=C1 WHTVZRBIWZFKQO-UHFFFAOYSA-N 0.000 description 1
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- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 description 1
- 229910052753 mercury Inorganic materials 0.000 description 1
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- 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
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F15/00—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table
- C07F15/0006—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table compounds of the platinum group
- C07F15/0046—Ruthenium compounds
- C07F15/0053—Ruthenium compounds without a metal-carbon linkage
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
Abstract
The invention belongs to the technical field of complexes and application, and provides a nitrosyl ruthenium complex taking hydroxyproline and 5, 7-dichloro-8-hydroxyquinoline as ligands, and a preparation method and application thereof. The chemical formula is [ Ru Ⅱ(NO)(HYP)(HqCl2) Cl ], wherein HYP is cis-4-hydroxy-L-proline or trans-4-hydroxy-D-proline, and HqCl 2 is 5, 7-dichloro-8-hydroxyquinoline; the serum albumin can be used as a carrier for transferring the serum albumin in organisms, has different recognition binding capacities with the serum albumin, and has a difference of 16.5 times of binding constants. Has the activity of photoinduction quantitative release of physiologically active molecule nitric oxide in solution and cell system, and can be used as nitric oxide donor.
Description
Technical Field
The invention belongs to the technical field of complexes and application, and particularly relates to a nitrosylruthenium complex taking hydroxyproline and 5, 7-dichloro-8-hydroxyquinoline as ligands, and a preparation method and application thereof.
Background
The application of cisplatin antitumor drugs in clinical treatment greatly promotes the synthesis of ruthenium complexes and the development of cytotoxicity and mechanisms. The ruthenium nitrosyl complex has rich photophysical and photochemical properties, obvious cell activity and important application value in the biomedical field. The reactivity and the cell biological activity of the complex can be regulated by selecting and changing the ligand, thereby providing basis for the discovery of new antitumor drugs and physiological regulation preparations.
Cisplatin antitumor drugs have been successfully used in clinical treatment. However, because cisplatin has remarkable inhibition effect on tumor cells and also has inhibition effect on normal cells of human body, obvious toxic and side effects in vivo are caused. Ruthenium and platinum are in the VIIIB group of the periodic Table of elements, and ruthenium complexes and platinum complexes are similar to each other in inhibiting activity on tumor cell growth, but have different action mechanisms and action modes with biological macromolecules, so that the ruthenium complex and the platinum complex have relatively low cytotoxicity on normal cells. Meanwhile, nitric Oxide (NO) is a key biological signal molecule for regulating immune reaction and apoptosis process, so that synthesis of nitrosyl ruthenium complexes with different configurations has important significance for synthesis and screening of novel antitumor drugs and obtaining Nitric Oxide (NO) donors with physiological regulation activity.
The research shows that the 8-hydroxyquinoline derivative is a good metal chelating agent, chloroquine and metal complex formed by other halogenated 8-hydroxyquinoline derivatives, has an inhibiting effect on cathepsin B and has the anti-tumor, anti-leukemia, antibacterial and antiviral activity. Studies show that chloro-8-hydroxyquinoline complex may improve its antitumor activity. Hydroxyproline is a non-essential amino acid and plays a role in maintaining the cellular structure and function of plant, animal and human cells. The nitrosyl ruthenium complex which takes the halogenated 8-hydroxyquinoline derivative and the hydroxyproline as the ligand and is mixed and coordinated integrates the characteristics of the ligand, and the synthesis, separation, purification and property research of the nitrosyl ruthenium complex have application values. At present, no nitrosylruthenium complex using 5, 7-dichloro-8-hydroxyquinoline and hydroxyproline as ligands has been reported.
Disclosure of Invention
In view of the above, the present invention aims to provide a ruthenium nitrosylate complex using Hydroxyproline (HYP) and 5, 7-dichloro-8-hydroxyquinoline (HqCl 2) as ligands, and a preparation method and application thereof. Among them, hydroxyproline includes cis-4-hydroxy-L-proline (CHYP) and trans-4-hydroxy-D-proline (THYP).
The invention is realized by the following technical scheme: nitrosyl ruthenium complex with hydroxyproline HYP and 5, 7-dichloro-8-hydroxyquinoline HqCl 2 as ligands, wherein the chemical formula of the nitrosyl ruthenium complex is [ Ru Ⅱ(NO)(HYP)(HqCl2) Cl ], wherein HYP is cis-4-hydroxy-L-proline or trans-4-hydroxy-D-proline, and HqCl 2 is 5, 7-dichloro-8-hydroxyquinoline;
HYP is cis-4-hydroxy-L-proline, and the ruthenium nitrosyl complex is any complex shown in formula 1, formula 2 or formula 3; HYP is trans-4-hydroxy-D-proline, and the ruthenium nitrosyl complex is a complex shown in the following formula 4, formula 5 or formula 6:
。
The preparation method of the nitrosyl ruthenium complex taking hydroxyproline HYP and 5, 7-dichloro-8-hydroxyquinoline HqCl 2 as ligands comprises the following steps:
(1) Preparation of a crude ruthenium nitrosyl complex: carrying out coordination reaction on a precursor ruthenium nitrosyl complex [ (CH 3)4N][RuⅡ(NO)(HqCl2)Cl3 ] and hydroxyproline in a mixed solvent of ethanol and water, and carrying out reduced pressure distillation or vacuum drying on the obtained coordination reaction liquid until the solvent is evaporated to dryness to remove the solvent, thereby obtaining a crude ruthenium nitrosyl complex product;
Wherein: the precursor ruthenium nitrosyl complex [ (CH 3)4N][RuⅡ(NO)(HqCl2)Cl3 ] HqCl 2 is 5, 7-dichloro-8-hydroxyquinoline), the hydroxyproline is cis-4-hydroxy-L-proline or trans-4-hydroxy-D-proline, the molar ratio of [ (CH 3)4N][RuⅡ(NO)(HqCl2)Cl3 ] to hydroxyproline is 1:1-1:2 ], the volume ratio of ethanol to water in a mixed solvent of ethanol and water is 1:1-1:2, the temperature of the coordination reaction is 80-85 ℃ and the time is 4-6 h, and the coordination reaction is carried out in a reflux reaction under the conditions of light shielding and stirring;
(2) And (3) separating and purifying a final product: dissolving the crude product of the ruthenium nitrosyl complex obtained in the step (1) by using a mixed solution of ethanol and dichloromethane with the volume ratio of 1:10, and performing silica gel column chromatographic separation, wherein the specific separation method comprises the following steps: when the hydroxyproline is cis-4-hydroxy-L-proline, eluting agent for silica gel column chromatography is mixed solution of CH 2Cl2 and CH 3 OH with the volume ratio of 50:1 to obtain a complex 1,2 or 3; when the hydroxyproline is trans-4-hydroxy-D-proline, the eluent of silica gel column chromatography is a mixed solution of CH 2Cl2 and CH 3 OH with the volume ratio of 50:1, the complexes 4 and 5 are obtained by separation, and when the eluent is a mixed solution of CH 2Cl2 and CH 3 OH with the volume ratio of 30:1, the complex 6 is obtained by separation.
Further, the preparation method of the precursor ruthenium nitrosyl complex [ (CH 3)4N][RuⅡ(NO)(HqCl2)Cl3 ] comprises the steps of carrying out reflux coordination substitution reaction on [ Ru Ⅱ(NO)(H2O)2Cl3 ] and 5, 7-dichloro-8-hydroxyquinoline in an ethanol solvent according to a molar ratio of 1:1, controlling the reaction temperature to be 85 ℃, reacting 3 h to obtain a coordination substitution reaction solution, controlling the molar ratio of [ Ru Ⅱ(NO)(H2O)2Cl3 ] to tetramethyl ammonium chloride to be 1:4, adding tetramethyl ammonium chloride ethanol solution into the coordination substitution reaction solution, controlling the reaction temperature to be 4 ℃ for equilibrium precipitation reaction 24 h to obtain a precipitate, sequentially carrying out suction filtration on the precipitate, and carrying out vacuum drying and suction filtration on the precipitate to obtain the precursor ruthenium nitrosyl complex [ (CH 3)4N][RuⅡ(NO)(HqCl2)Cl3 ].
Further, the molar ratio of the precursor ruthenium nitrosyl complex [ (CH 3)4N][RuⅡ(NO)(HqCl2)Cl3 ] to hydroxyproline is 1:2, the volume ratio of ethanol to water in the mixed solvent of ethanol and water is 1:1, and the temperature of the coordination reaction is 85 ℃ and the time is 5 h.
The specific method of the coordination reaction is as follows: dissolving a precursor ruthenium nitrosyl complex [ (CH 3)4N][RuⅡ(NO)(HqCl2)Cl3 ] in ethanol to obtain a precursor ruthenium nitrosyl complex ethanol solution, dissolving hydroxyproline in water to obtain a hydroxyproline water solution, mixing the precursor ruthenium nitrosyl complex ethanol solution and the hydroxyproline water solution, and carrying out coordination reaction at the temperature of 85 ℃.
The invention also provides application of the nitrosyl ruthenium complex taking the hydroxyproline HYP and the 5, 7-dichloro-8-hydroxyquinoline HqCl 2 as ligands in preparing antitumor drugs.
Furthermore, the application of the ruthenium nitrosyl complex in preparing the medicine for resisting cervical cancer cell proliferation.
The IC 50 values of the complexes of formulas 1 to 6 are respectively: 6.63 mu.M, 5.24. Mu.M, 2.08. Mu.M, 1.29. Mu.M, 10.76. Mu.M, 4.43. Mu.M.
Furthermore, the complex shown in the formula 3 and the formula 4 is applied to the preparation of anti-cervical cancer drugs.
The invention also provides application of the nitrosyl ruthenium complex taking the hydroxyproline HYP and the 5, 7-dichloro-8-hydroxyquinoline HqCl 2 as ligands in preparation of NO molecular donor reagents in a solution system and a cell system which are regulated and controlled by light.
The source of the precursor ruthenium nitrosyl complex [ (CH 3)4N][RuⅡ(NO)(HqCl2)Cl3 ] is not particularly limited, and the precursor ruthenium nitrosyl complex can be prepared by adopting commercial products or by adopting a preparation method well known to a person skilled in the art, and specifically can be prepared by referring to the literature "Xie L., et al Inorg. Chem. 2021, 60, 8826-8837".
Compared with platinum medicines, the ruthenium complex has the advantages of low toxicity, small drug resistance and the like, has remarkable anti-tumor activity, has stronger cell growth inhibition activity than cisplatin on certain cancer cells, and is expected to become a metal-based clinical anti-tumor medicine of a new generation medicine. Therefore, the invention designs and synthesizes a novel nitrosyl ruthenium complex containing 5, 7-dichloro-8-hydroxyquinoline and different chiral hydroxyproline coordination configurations. It has the activity of inhibiting tumor cell growth, can be combined with serum albumin in blood, and has the activity of light-induced release of Nitric Oxide (NO).
The ruthenium nitrosyl complex provided by the invention is a new-configuration ruthenium nitrosyl complex taking hydroxyproline and 5, 7-dichloro-8-hydroxyquinoline as ligands, and the ruthenium nitrosyl complex can obviously inhibit the activity of cancer cell growth. The ruthenium nitrosyl complex prepared by the invention can also have a combination effect with serum albumin (HSA) in blood, and the serum albumin can be used as a carrier for transporting the complex in organisms. The nitrosyl ruthenium complex with different configurations prepared by the invention has different recognition binding capacities with serum albumin, and the binding constants are different by 16.5 times. Has the activity of photoinduction quantitative release of physiologically active molecule Nitric Oxide (NO) in solution and cell system, and can be used as nitric oxide donor.
Drawings
FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of six nitrosyl ruthenium complexes of different configurations of the invention; in the figure, (a) is complex 1, [ Ru Ⅱ(NO)(CHYP1)(HqCl2 ] Cl ]; (b) Is complex 2, [ Ru Ⅱ(NO)(CHYP2)(HqCl2) Cl ]; (c) Is complex 3, [ Ru Ⅱ(NO)(CHYP3)(HqCl2) Cl ];
FIG. 2 is a nuclear magnetic resonance hydrogen spectrum of six nitrosyl ruthenium complexes of different configurations of the invention; in the figure, (d) is complex 4, [ Ru Ⅱ(NO)(THYP1)(HqCl2 ] Cl ]; (e) Is complex 5, [ Ru Ⅱ(NO)(THYP2)(HqCl2) Cl ]; (f) Is complex 6, [ Ru Ⅱ(NO)(THYP3)(HqCl2) Cl ];
FIG. 3 is a nuclear magnetic resonance carbon spectrum of six nitrosyl ruthenium complexes of different configurations of the invention; in the figure, (a) is complex 1, [ Ru Ⅱ(NO)(CHYP1)(HqCl2 ] Cl ]; (b) Is complex 2, [ Ru Ⅱ(NO)(CHYP2)(HqCl2) Cl ]; (c) Is complex 3, [ Ru Ⅱ(NO)(CHYP3)(HqCl2) Cl ];
FIG. 4 is a nuclear magnetic resonance carbon spectrum of six nitrosyl ruthenium complexes of different configurations of the invention; in the figure, (d) is complex 4, [ Ru Ⅱ(NO)(THYP1)(HqCl2 ] Cl ]; (e) Is complex 5, [ Ru Ⅱ(NO)(THYP2)(HqCl2) Cl ]; (f) Is complex 6, [ Ru Ⅱ(NO)(THYP3)(HqCl2) Cl ];
FIG. 5 is a block diagram of four ruthenium nitrosyl complex crystals of different configurations according to the invention; in the figure: (a) Is complex 1, namely [ Ru Ⅱ(NO)(CHYP1)(HqCl2) Cl ]; (b) Is complex 2, [ Ru Ⅱ(NO)(CHYP2)(HqCl2) Cl ]; (c) Is complex 3, [ Ru Ⅱ(NO)(CHYP3)(HqCl2) Cl ]; (d) Is complex 4, [ Ru Ⅱ(NO)(THYP1)(HqCl2) Cl ];
FIG. 6 shows the inhibitory activity of six ruthenium nitrosyl complexes of different configurations of the invention on cervical cancer cell growth; in the figure, (a) is complex 1, namely [ Ru Ⅱ(NO)(CHYP1)(HqCl2 ] Cl ]; (b) Is complex 2, [ Ru Ⅱ(NO)(CHYP2)(HqCl2) Cl ]; (c) Is complex 3, [ Ru Ⅱ(NO)(CHYP3)(HqCl2) Cl ]; (d) Is complex 4, [ Ru Ⅱ(NO)(THYP1)(HqCl2) Cl ]; (e) Is complex 5, [ Ru Ⅱ(NO)(THYP2)(HqCl2) Cl ]; (f) Is complex 6, [ Ru Ⅱ(NO)(THYP3)(HqCl2) Cl ]; cell activity patterns affecting cervical cancer cell growth over different concentration ranges;
FIG. 7 is a fluorescence spectrum of binding of six ruthenium nitrosyl complexes of various configurations of the invention to Human Serum Albumin (HSA); in the figure: (a) Is complex 1, namely [ Ru Ⅱ(NO)(CHYP1)(HqCl2) Cl ]; (b) Is complex 2, [ Ru Ⅱ(NO)(CHYP2)(HqCl2) Cl ]; (c) Is complex 3, [ Ru Ⅱ(NO)(CHYP3)(HqCl2) Cl ]; (d) Is complex 4, [ Ru Ⅱ(NO)(THYP1)(HqCl2) Cl ]; (e) Is complex 5, [ Ru Ⅱ(NO)(THYP2)(HqCl2) Cl ]; (f) Is complex 6, [ Ru Ⅱ(NO)(THYP3)(HqCl2) Cl ];
FIG. 8 is a graph showing the fit of the binding constants of six nitrosyl ruthenium complexes of various configurations of the invention to Human Serum Albumin (HSA); in the figure: (a) Is complex 1, namely [ Ru Ⅱ(NO)(CHYP1)(HqCl2) Cl ]; (b) Is complex 2, [ Ru Ⅱ(NO)(CHYP2)(HqCl2) Cl ]; (c) Is complex 3, [ Ru Ⅱ(NO)(CHYP3)(HqCl2) Cl ]; (d) Is complex 4, [ Ru Ⅱ(NO)(THYP1)(HqCl2) Cl ]; (e) Is complex 5, [ Ru Ⅱ(NO)(THYP2)(HqCl2) Cl ]; (f) Is complex 6, [ Ru Ⅱ(NO)(THYP3)(HqCl2) Cl ];
FIG. 9 is an electron spin resonance (EPR) spectrum of six ruthenium nitrosyl complexes of different configurations of the invention; in the figure, (a) is complex 1, [ Ru Ⅱ(NO)(CHYP1)(HqCl2 ] Cl ]; (b) Is complex 2, [ Ru Ⅱ(NO)(CHYP2)(HqCl2) Cl ]; (c) Is complex 3, [ Ru Ⅱ(NO)(CHYP3)(HqCl2) Cl ]; (d) Is complex 4, [ Ru Ⅱ(NO)(THYP1)(HqCl2) Cl ]; (e) Is complex 5, [ Ru Ⅱ(NO)(THYP2)(HqCl2) Cl ]; (f) Is complex 6, [ Ru Ⅱ(NO)(THYP3)(HqCl2) Cl ];
FIG. 10 is a graph of real-time fluorescence imaging of NO under light irradiation conditions for six ruthenium nitrosyl complexes of various configurations in accordance with the invention; in the figure, (a) is complex 1, [ Ru Ⅱ(NO)(CHYP1)(HqCl2 ] Cl ]; (b) Is complex 2, [ Ru Ⅱ(NO)(CHYP2)(HqCl2) Cl ]; (c) Is complex 3, [ Ru Ⅱ(NO)(CHYP3)(HqCl2) Cl ]; (d) Is complex 4, [ Ru Ⅱ(NO)(THYP1)(HqCl2) Cl ]; (e) Is complex 5, [ Ru Ⅱ(NO)(THYP2)(HqCl2) Cl ]; (f) Is complex 6, namely [ Ru Ⅱ(NO)(THYP3)(HqCl2) Cl ].
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments; all other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, the disclosure of which is incorporated herein by reference as is commonly understood by reference.
Those skilled in the art will recognize that equivalents of the specific embodiments described, as well as those known by routine experimentation, are intended to be encompassed within the present application.
The experimental methods in the following examples are conventional methods unless otherwise specified. The instruments used in the following examples are laboratory conventional instruments unless otherwise specified; the experimental materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores.
Example 1: nitrosyl ruthenium complex (1-6) with Hydroxyproline (HYP) and 5, 7-dichloro-8-hydroxyquinoline (HqCl 2) as ligands and with different configurations, wherein the chemical formula of the nitrosyl ruthenium complex is as follows: [ Ru Ⅱ(NO)(HYP)(HqCl2) Cl ], wherein HYP is cis-4-hydroxy-L-proline (CHYP) or trans-4-hydroxy-D-proline (THYP). The specific structural formula of the ruthenium nitrosyl complex 1-6 is as follows:
。
The preparation method comprises the following steps:
(1) The preparation of the precursor ruthenium nitrosyl complex [ (CH 3)4N][RuⅡ(NO)(HqCl2)Cl3 ] comprises the steps of weighing 2 mmol [ Ru Ⅱ(NO)(H2O)2Cl3 ] reactant and an equimolar 5, 7-dichloro-8-hydroxyquinoline ligand, respectively dissolving in 15 mL ethanol solvent, mixing the two, heating and refluxing in water bath at 85 ℃ to react for 3 h, adding 5 mL tetramethyl ammonium chloride ethanol solution (8 mmol) after the reaction is finished, standing in a refrigerator at 4 ℃ for 24 hours, filtering, and vacuum drying to obtain a reddish brown solid product, namely the precursor ruthenium nitrosyl complex [ (CH 3)4N][RuⅡ(NO)(HqCl2)Cl3 ].
The preparation of the precursor ruthenium nitrosyl complex [ (CH 3)4N][RuⅡ(NO)(HqCl2)Cl3 ] involves the reaction formula shown in the formula A:。
(2) Synthesizing and compounding a ruthenium nitrosyl complex: dissolving 1 mmol of a precursor ruthenium nitrosyl complex [ (CH 3)4N][RuⅡ(NO)(HqCl2)Cl3 ] in a 10mL ethanol solvent, dissolving 2 mmol cis-4-hydroxy-L-proline or trans-4-hydroxy-D-proline ligand in 10mL water, mixing the two solutions, keeping the reaction solution away from light, heating and refluxing for 5 hours at 85 ℃ under the stirring effect, and distilling under reduced pressure after the reaction is finished to remove the solvent.
The reaction involved in the preparation of the compound ruthenium nitrosyl complex is shown as the formula B and the formula C:
、。
(3) Separation and purification of six different configurations of complexes: purifying and separating by silica gel column chromatography, wherein when the ligand is cis-4-hydroxy-L-proline and 5, 7-dichloro-8-hydroxyquinoline, the used developing agent is CH 2Cl2:CH3 OH (volume ratio 50:1), and three complexes 1-3 with different configurations are respectively obtained. When the ligand is trans-4-hydroxy-D-proline and 5, 7-dichloro-8-hydroxyquinoline, the developing agent is CH 2Cl2:CH3 OH (volume ratio 50:1) to obtain complexes 4 and 5; the developing agent used was CH 2Cl2:CH3 OH (volume ratio 30:1) to give complex 6.
The six nitrosyl ruthenium complexes with different configurations obtained by separation and purification are respectively subjected to structural analysis and identification by using structural characterization methods such as nuclear magnetic resonance spectrum and the like. The invention obtains nuclear magnetic resonance hydrogen spectrograms of six nitrosyl ruthenium complexes with different configurations, as shown in figures 1 and 2. The invention obtains nuclear magnetic resonance carbon spectrograms of six nitrosyl ruthenium complexes with different configurations, as shown in fig. 3 and 4.
Complex compound 1：1H NMR (600 MHz, DMSO-d6) δ 9.12 (dd, J = 5.1, 1.3 Hz, 1H), δ 8.83 (dd, J = 8.7, 1.3 Hz, 1H), δ 7.99 (dd, J = 8.6, 5.0 Hz, 1H), δ 7.96 (s, 1H), δ 7.77 (q, J = 7.7 Hz, 1H), δ 5.24 (s, 1H), δ 4.33 – 4.27 (m, 1H), δ 4.24 (dd, J = 7.7, 2.1 Hz, 1H), δ 3.29 (t, J = 7.3 Hz, 2H), δ 2.42 (ddd, J = 12.8, 9.5, 6.8 Hz, 1H), δ 1.82 (dt, J = 12.8, 7.1 Hz, 1H).
13C NMR (151 MHz, DMSO-d6) δ 179.91, 161.15, 150.37, 144.36, 137.55, 129.81, 126.36, 124.03, 117.24, 115.24, 69.20, 63.15, 55.89, 37.39.
Complex compound 2：1H NMR (600 MHz, DMSO-d6) δ 9.27 (d, J = 4.9 Hz, 1H), δ 8.84 (d, J = 8.6 Hz, 1H), δ 8.09 (q, J = 8.1 Hz, 1H), δ 8.00 (dd, J = 8.6, 5.1 Hz, 1H), δ 7.97 (s, 1H), δ 5.32 (s, 1H), δ 4.22 (dq, J = 16.9, 8.6, 7.7 Hz, 2H), δ 3.39 (t, J = 9.8 Hz, 1H), δ 3.13 (dt, J = 11.7, 6.1 Hz, 1H), δ 2.43 (dt, J = 12.7, 8.1 Hz, 1H), δ 1.84 (dt, J = 12.4, 8.8 Hz, 1H).
13C NMR (151 MHz, DMSO-d6) δ 179.09, 161.86, 150.86, 143.88, 137.57, 129.80, 126.43, 123.99, 117.41, 115.53, 69.14, 62.55, 56.14, 36.61.
Complex compound 3：1H NMR (600 MHz, DMSO-d6) δ 9.08 (d, J = 5.0 Hz, 1H), δ 8.74 (d, J = 8.6 Hz, 1H), δ 8.00 (s, 1H), δ 7.98 (t, J = 8.3 Hz, 1H), δ 7.92 (dd, J = 8.4, 5.4 Hz, 1H), δ 5.48 (s, 1H), δ 4.42 (s, 1H), δ 4.06 (q, J = 7.1 Hz, 1H), δ 3.46 – 3.42 (m, 1H), δ 3.15 (d, J = 12.4 Hz, 1H) , δ 2.25 (d, J = 6.2 Hz, 2H).
13C NMR (151 MHz, DMSO-d6) δ 182.97, 158.86, 150.56, 142.14, 136.15, 130.22, 126.07, 124.61, 117.11, 116.22, 69.11, 63.05, 62.22, 40.06.
Complex compound 4：1H NMR (600 MHz, DMSO-d6) δ 9.12 (dd, J = 5.1, 1.3 Hz, 1H), δ 8.83 (dd, J = 8.6, 1.4 Hz, 1H), δ 7.99 (dd, J = 8.6, 5.1 Hz, 1H), δ 7.96 (s, 1H), δ 5.17 (s, 1H), δ 4.46 (s, 1H), δ 4.35 (q, J = 8.1 Hz, 1H), δ 3.52 (ddd, J = 12.1, 8.5, 3.9 Hz, 1H), δ 3.17 (d, J = 10.6 Hz, 2H), δ 2.18 (dd, J = 12.3, 10.1 Hz, 1H), δ 2.13 (dd, J = 7.7, 5.0 Hz, 1H).
13C NMR (151 MHz, DMSO-d6) δ 180.13, 161.29, 150.39, 144.37, 137.50, 129.79, 126.36, 124.02, 117.18, 115.10, 69.60, 63.67, 57.98, 38.87.
Complex compound 5：1H NMR (600 MHz, DMSO-d6) δ 9.10 (d, J = 7.7 Hz, 1H), δ 8.75 (d, J = 8.6 Hz, 1H), δ 8.02 (s, 1H), δ 7.97 – 7.91 (m, 2H), δ 5.15 (s, 1H), δ 4.39 (s, 1H), δ 4.26 (q, J = 8.6 Hz, 1H), δ 3.46 (dt, J = 16.4, 7.5 Hz, 2H), δ 2.24 (t, J = 10.3 Hz, 2H).
13C NMR (151 MHz, DMSO-d6) δ 181.49, 158.64, 150.80, 142.01, 136.38, 130.30, 126.09, 124.70, 117.18, 116.54, 69.52, 63.46, 63.32, 38.79.
Complex compound 6：1H NMR (600 MHz, DMSO-d6) δ 9.07 (d, J = 5.0 Hz, 1H), δ 8.73 (d, J = 8.6 Hz, 1H), δ 8.06 – 8.01 (m, 1H), δ 8.00 (s, 1H), δ 7.91 (dd, J = 8.4, 5.2 Hz, 1H), δ 5.30 (s, 1H), δ 4.50 (s, 1H), δ 4.18 (q, J = 9.0 Hz, 1H), δ 3.82 (td, J = 8.3, 3.7 Hz, 1H), δ 3.21 (dd, J = 12.7, 6.6 Hz, 1H), δ 2.24 (d, J = 10.0 Hz, 2H).
13C NMR (151 MHz, DMSO-d6) δ 182.23, 158.27, 150.71, 142.43, 136.26, 130.00, 126.13, 124.56, 117.04, 116.25, 69.33, 62.60, 58.86, 38.72.
Example 2: the ruthenium nitrosyl complex with different configurations prepared in the example 1 is placed in CH 3 OH and CH 2Cl2 solution with the volume ratio of 1:1-3:1, the solvent volatilization speed is regulated, and four single crystals suitable for X-ray crystallography measurement are prepared through a slow evaporation method, wherein: (a) Is complex 1, namely [ Ru Ⅱ(NO)(CHYP1)(HqCl2) Cl ]; (b) Is complex 2, [ Ru Ⅱ(NO)(CHYP2)(HqCl2) Cl ]; (c) Is complex 3, [ Ru Ⅱ(NO)(CHYP3)(HqCl2) Cl ]; (d) Is complex 4, namely [ Ru Ⅱ(NO)(THYP1)(HqCl2 ] Cl ]. The single crystal structure of the complex 5 [ Ru Ⅱ(NO)(THYP2)(HqCl2) Cl ] and the complex 6 [ Ru Ⅱ(NO)(THYP3)(HqCl2) Cl ] was not obtained, but the nuclear magnetic resonance hydrogen spectrum and the carbon spectrum were measured respectively. At 298K, the collection of crystal diffraction data was performed using a Bruker D8 Venture diffractometer with a radiation source of Mo-K alpha radiation, wavelength 0.71073A, monochromatized by a graphite monochromator. The cell parameters and data were determined and restored by running the SMART software SAINT program, respectively, and the structural analysis was done by the SHELXTL package.
The three-dimensional structures of four different configurations of ruthenium nitrosyl complexes 1 to 4 can be obtained by single crystal diffraction, and ORTEP diagrams (ellipsoid rate is 30%) of the four complexes are shown in FIG. 5. In complex 1,2, 4-hydroxy-proline is coordinately bound to the para-position of the NO group, whereas in complex 35, 7-dichloro-8-hydroxyquinoline is coordinately bound to the para-position of the NO group. Complex 1 and complexes 2 and 4 exhibit approximately mirror-symmetrical spatial configurations centered on the metal ion Ru, with the configuration of the spatial coordination of complexes 2 and 4 being similar. However, in complex 2 the ligand is 4-hydroxy-L-proline, whereas in complex 2 the ligand is 4-hydroxy-D-proline. Thus, complexes 1,2,3 and 4 are four differently configured complex molecules. In combination with analysis of the nuclear magnetic resonance hydrogen spectra and carbon spectra of the six ruthenium nitrosyl complexes shown in FIGS. 1-4, complexes 5 and 6 also differ from complexes 1-4 in coordination configuration, and they are the ruthenium nitrosyl complex molecules of six different configurations.
Example 3: six different-structure complexes prepared in example 1 were tested for inhibition activity on cervical cancer cell growth under dark, no-light and light conditions. The method comprises the following steps:
And (3) taking a DMSO dissolved complex sample as a mother solution, diluting two samples with the same concentration gradient to incubate cervical cancer cells, and finally controlling the DMSO concentration to be 1% in a 200 mu L culture medium, wherein the blank group and the control group operate identically. Adding 0-1.2 mM concentration gradient complex mother liquor into the cell culture plate. Placing the cell culture plate in a cell culture box, and incubating at 37 ℃ for 24 h; and taking out the culture plate after the incubation time is over, adding 20 mu L of CCK-8 solvent into each hole, incubating for 3h, taking out, measuring the absorbance at the position of 450 nm by using an enzyme label instrument, calculating the inhibition rate respectively, and then carrying out statistical mapping.
As a result, FIG. 6 shows that (a) in FIG. 6 is complex 1, [ Ru Ⅱ(NO)(CHYP1)(HqCl2 ] Cl ]; (b) Is complex 2, [ Ru Ⅱ(NO)(CHYP2)(HqCl2) Cl ]; (c) Is complex 3, [ Ru Ⅱ(NO)(CHYP3)(HqCl2) Cl ]; (d) Is complex 4, [ Ru Ⅱ(NO)(THYP1)(HqCl2) Cl ]; (e) Is complex 5, [ Ru Ⅱ(NO)(THYP2)(HqCl2) Cl ]; (f) Is complex 6, namely [ Ru Ⅱ(NO)(THYP3)(HqCl2) Cl ].
As can be seen from FIG. 6, the IC 50 values of complexes 1-6 were 6.63. Mu.M, 5.24. Mu.M, 2.08. Mu.M, 1.29. Mu.M, 10.76. Mu.M, 4.43. Mu.M, respectively. The anti-cervical cancer cell proliferation activity is as follows: complex 4 > complex 3 > complex 6 > complex 2 > complex 1 > complex 5. Experimental results show that the complexes 1-6 can be applied to screening and preparing antitumor lead compound medicaments. Moreover, the complex 3 and the complex 4 have strong inhibition effect on proliferation of cervical cancer cells.
Example 4: preparing 10 -5 M Human Serum Albumin (HSA) solution for later use by using ultrapure water, dissolving and preparing mother liquor of 4mM ruthenium nitrosyl complex 1-6 by using dimethyl sulfoxide (DMSO), and preserving in a dark place for later use; 2mL of HSA solution was placed in a fluorescence absorbing cup. The fluorescence spectrometer parameters were set as: fluorescence titration experiments were performed with excitation wavelengths of 280 nm and emission wavelengths of 290-520: 520 nm. Mother solutions of the nitrosyl ruthenium complexes 1 to 6 of 4mM are respectively taken and added into the HSA solution successively at intervals of 1 mu L, evenly mixed, and stood for reaction 3 min until the fluorescence spectrogram tends to be stable, and then the measurement is carried out. Visual description of fluorescence spectrograms is carried out by Origin software by taking wavelength and fluorescence intensity as coordinates, and the binding constant and the number of binding sites are calculated by taking lg [ Q ] and lg [ (F 0 -F)/F ] as coordinates, wherein Q is the actual concentration of a quencher (nitrosyl ruthenium complex), F 0 is the initial maximum fluorescence intensity of HSA when the quencher is not added, and F is the maximum fluorescence intensity of HSA under the same wavelength after the quencher is added.
The fluorescence spectra of the different configurations of ruthenium nitrosyl complexes 1 to 6 were titrated for HSA respectively as shown in FIG. 7. A linear fit curve as a function of lg [ F 0/F-1 ] as a function of lg [ Q ] is shown in FIG. 8. As can be seen from the fluorescence spectrum, with increasing concentrations of complexes 1 to 6, the HSA exhibited significant fluorescence quenching. As is clear from the lg [ F 0/F ] -lg [ Q ] pattern, the binding constants of the complexes 1 to 6 and HSA were 5.81×104M-1、2.36×105M-1、1.25×104M-1、1.30×105M-1、2.08×104M-1、5.31×104M-1, binding sites number of 1.06, 1.15, 0.95, 1.14, 0.99 and 1.04, respectively, and R values were 0.9968, 0.9953, 0.9912, 0.9849, 0.9941 and 0.9928, respectively. The binding constants of the different-structure complex molecules and HSA show obvious difference, and the binding constant of the complex 2 and HSA is 16.5 times of the binding constant of the complex 3 and HSA, so that the different-structure complex molecules show space selective binding to the biological macromolecule HSA. The number of binding sites was close to 1, indicating that there was one binding site in complexes 1-6 and HSA.
Example 5: and detecting the NO release condition of the complexes 1-6 in the solution system under the condition of illumination and NO illumination by using a Bruker E300 electron paramagnetic resonance spectrometer and utilizing a NO molecule capturing technology. The method comprises the following steps:
dissolving complex samples to 5×10 -3 M with DMSO and water (volume ratio 1:1), respectively; feSO 4·7H2 O and N-methyl-D-glucosamine dithioformate (MGD) were prepared in 1X 10 -2 M in DMSO, respectively, in a volume ratio of 1:1 to obtain an NO trapping agent Fe (MGD) 2; and respectively taking 25 mu L of sample solution to be measured and Fe (MGD) 2 solution to be mixed, sucking a certain amount of mixed solution (30 mu L) by using a capillary tube, and placing the mixed solution in a resonant cavity of an electron paramagnetic resonance spectrometer for data collection. The spectrum width is 3400-3500G, after the EPR spectrogram without illumination is collected in a dark place, a light source is turned on, the EPR spectrogram with a certain interval time under the illumination condition is collected, and the illumination time of four curves from bottom to top is sequentially increased. The light source is a 100W mercury lamp.
The test results are shown in FIG. 9, (a) is complex 1, [ Ru Ⅱ(NO)(CHYP1)(HqCl2 ] Cl ]; (b) Is complex 2, [ Ru Ⅱ(NO)(CHYP2)(HqCl2) Cl ]; (c) Is complex 3, [ Ru Ⅱ(NO)(CHYP3)(HqCl2) Cl ]; (d) Is complex 4, [ Ru Ⅱ(NO)(THYP1)(HqCl2) Cl ]; (e) Is complex 5, [ Ru Ⅱ(NO)(THYP2)(HqCl2) Cl ]; (f) Is complex 6, namely [ Ru Ⅱ(NO)(THYP3)(HqCl2) Cl ].
The results show that the EPR spectra of the six complexes are almost horizontal lines in the non-illuminated condition, indicating NO molecular signaling. Immediately after illumination, EPR profile produces a characteristic triplet signal (g= 2.039) of NO molecules and signal intensity increases with increasing illumination time. Experiments show that the light condition can induce the complex to generate NO molecules, and the release amount can be controlled by adjusting the light time. The release of the complex NO can be quantitatively regulated through light excitation, the rates of the complex with different configurations for releasing NO show a certain difference, and the complex can be applied to the preparation of NO donors in a light regulated solution system and a cell system.
Example 6: the production of NO can be selectively identified using a ZEISS LSM-880 cell laser confocal microscope using a selective NO fluorescent probe DAX-J2 Red, which emits Red fluorescence when reacted with NO. Detecting the release condition of the complex 1-6 NO molecules in the cell system under the conditions of illumination and no illumination in real time. The method comprises the following steps:
Taking 10 mM NO probes DAX-J2 Red, adding an EP tube filled with PBS buffer solution, diluting to 5 mu M, taking 5 mu M DAX-J2 Red solution 1mL and 2mM complex sample 10 mu L, adding the complex sample to a focusing dish purged with PBS for 2-3 times, and incubating for 10 min. And then using the LED monochromatic light sources of 420 nm to illuminate 0, 5, 10, 15 and 20min respectively, and observing the imaging condition of NO in cervical cancer cells after illumination. The real-time fluorescent imaging results of NO of the complex formed by combining the nitrosyl ruthenium complex with the serum albumin with six different configurations at different illumination times are shown in FIG. 10, wherein (a) in FIG. 10 is complex 1, namely [ Ru Ⅱ(NO)(CHYP1)(HqCl2 ] Cl ]; (b) Is complex 2, [ Ru Ⅱ(NO)(CHYP2)(HqCl2) Cl ]; (c) Is complex 3, [ Ru Ⅱ(NO)(CHYP3)(HqCl2) Cl ]; (d) Is complex 4, [ Ru Ⅱ(NO)(THYP1)(HqCl2) Cl ]; (e) Is complex 5, [ Ru Ⅱ(NO)(THYP2)(HqCl2) Cl ]; (f) Is complex 6, namely [ Ru Ⅱ(NO)(THYP3)(HqCl2) Cl ].
As can be seen from fig. 10, the addition of the ruthenium nitrosyl complex to the solution, but no or only very weak red fluorescence was observed in the absence of light or t=0; however, when the light is irradiated for a certain time, red fluorescence can be obviously observed, and the generated red fluorescence is gradually enhanced along with the increase of the irradiation time, which indicates that the irradiation condition can induce the ruthenium nitrosyl complex to release NO molecules in cells. As the illumination time increases, the amount of NO molecules released increases gradually.
Human serum albumin is the most abundant protein in blood, and is also a natural transport protein for many small biological molecules and drug molecules. Drug delivery systems based on human serum albumin have received increasing attention in recent years due to their biodegradability, non-immunogenicity, biocompatibility, and longer half-life. The invention determines and researches the binding action mode of six nitrosyl ruthenium complexes and HSA, and preferably adopts fluorescence spectrum at room temperature for research. The fluorescence spectrum can be used for measuring fluorescence intensity, emission spectrum and the like, and further information such as characteristics of emission peaks of fluorescence, binding constant, quenching constant type and the like can be obtained.
The invention dissolves the complexes 1-6 in a solution system, and NO signal of released NO free radical can be detected by utilizing a NO molecule capturing technology under the condition of NO illumination. Whereas under light conditions a significant signal of NO free radical release can be detected. This indicates that complexes 1-6 can induce NO release under light conditions. Can be used as a photo-induced NO donor.
The invention incubates the complex 1-6 with cervical cancer cells and NO probe DAX-J2 Red. Under illumination conditions, red fluorescence in cancer cells can be observed to different degrees by adjusting the excitation wavelength to be suitable under a confocal fluorescence microscope. This indicates that complexes 1-6 release NO molecules in living cells under light conditions.
The nitrosyl ruthenium complex 1-6 with different configurations has photosensitive activity and NO molecule release function, and can effectively release NO under the condition of light irradiation, so that the nitrosyl ruthenium complex can be applied to preparation of NO molecule donor reagents, and is suitable for a solution system and a cell system.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (10)
1. Nitrosyl ruthenium complex taking hydroxyproline HYP and 5, 7-dichloro-8-hydroxyquinoline HqCl 2 as ligands, and is characterized in that: the chemical formula of the ruthenium nitrosyl complex is [ Ru Ⅱ(NO)(HYP)(HqCl2) Cl ], wherein HYP is cis-4-hydroxy-L-proline or trans-4-hydroxy-D-proline, and HqCl 2 is 5, 7-dichloro-8-hydroxyquinoline;
HYP is cis-4-hydroxy-L-proline, and the ruthenium nitrosyl complex is any complex shown in formula 1, formula 2 or formula 3; HYP is trans-4-hydroxy-D-proline, and the ruthenium nitrosyl complex is a complex shown in the following formula 4, formula 5 or formula 6:
。
2. A process for the preparation of a ruthenium nitrosylate complex according to claim 1, comprising hydroxyproline HYP and 5, 7-dichloro-8-hydroxyquinoline HqCl 2 as ligands, characterized in that: the method comprises the following steps:
(1) Preparation of a crude ruthenium nitrosyl complex: carrying out coordination reaction on a precursor ruthenium nitrosyl complex [ (CH 3)4N][RuⅡ(NO)(HqCl2)Cl3 ] and hydroxyproline in a mixed solvent of ethanol and water, and carrying out reduced pressure distillation or vacuum drying on the obtained coordination reaction liquid until the solvent is evaporated to dryness to remove the solvent, thereby obtaining a crude ruthenium nitrosyl complex product;
Wherein: the precursor ruthenium nitrosyl complex [ (CH 3)4N][RuⅡ(NO)(HqCl2)Cl3 ] HqCl 2 is 5, 7-dichloro-8-hydroxyquinoline), the hydroxyproline is cis-4-hydroxy-L-proline or trans-4-hydroxy-D-proline, the molar ratio of [ (CH 3)4N][RuⅡ(NO)(HqCl2)Cl3 ] to hydroxyproline is 1:1-1:2 ], the volume ratio of ethanol to water in a mixed solvent of ethanol and water is 1:1-1:2, the temperature of the coordination reaction is 80-85 ℃ and the time is 4-6 h, and the coordination reaction is carried out in a reflux reaction under the conditions of light shielding and stirring;
(2) And (3) separating and purifying a final product: dissolving the crude product of the ruthenium nitrosyl complex obtained in the step (1) by using a mixed solution of ethanol and dichloromethane with the volume ratio of 1:10, and performing silica gel column chromatographic separation, wherein the specific separation method comprises the following steps: when the hydroxyproline is cis-4-hydroxy-L-proline, eluting agent for silica gel column chromatography is mixed solution of CH 2Cl2 and CH 3 OH with the volume ratio of 50:1 to obtain a complex 1,2 or 3; when the hydroxyproline is trans-4-hydroxy-D-proline, the eluent of silica gel column chromatography is a mixed solution of CH 2Cl2 and CH 3 OH with the volume ratio of 50:1, the complexes 4 and 5 are obtained by separation, and when the eluent is a mixed solution of CH 2Cl2 and CH 3 OH with the volume ratio of 30:1, the complex 6 is obtained by separation.
3. The preparation method according to claim 2, characterized in that: the preparation method of the precursor ruthenium nitrosyl complex [ (CH 3)4N][RuⅡ(NO)(HqCl2)Cl3 ] comprises the steps of carrying out reflux coordination substitution reaction on [ Ru Ⅱ(NO)(H2O)2Cl3 ] and 5, 7-dichloro-8-hydroxyquinoline in an ethanol solvent according to a molar ratio of 1:1, controlling the reaction temperature to be 85 ℃, reacting for 3 hours to obtain a coordination substitution reaction liquid, controlling the molar ratio of [ Ru Ⅱ(NO)(H2O)2Cl3 ] to tetramethyl ammonium chloride to be 1:4, adding tetramethyl ammonium chloride ethanol solution into the coordination substitution reaction liquid, controlling the reaction temperature to be 4 ℃ for equilibrium precipitation reaction for 24 hours to obtain a precipitate, sequentially carrying out suction filtration on the precipitate, and carrying out vacuum drying on the suction filtration to obtain the precursor ruthenium nitrosyl complex [ (CH 3)4N][RuⅡ(NO)(HqCl2)Cl3 ].
4. The preparation method according to claim 2, characterized in that: the molar ratio of the precursor ruthenium nitrosyl complex [ (CH 3)4N][RuⅡ(NO)(HqCl2)Cl3 ] to hydroxyproline is 1:2, the volume ratio of ethanol to water in the mixed solvent of ethanol and water is 1:1, and the temperature of the coordination reaction is 85 ℃ and the time is 5 h.
5. The method of manufacturing according to claim 4, wherein: the specific method of the coordination reaction is as follows: dissolving a precursor ruthenium nitrosyl complex [ (CH 3)4N][RuⅡ(NO)(HqCl2)Cl3 ] in ethanol to obtain a precursor ruthenium nitrosyl complex ethanol solution, dissolving hydroxyproline in water to obtain a hydroxyproline water solution, mixing the precursor ruthenium nitrosyl complex ethanol solution and the hydroxyproline water solution, and carrying out coordination reaction at the temperature of 85 ℃.
6. The use of a ruthenium nitrosyl complex with hydroxyproline HYP and 5, 7-dichloro-8-hydroxyquinoline HqCl 2 as ligands according to claim 1 in the preparation of antitumor drugs.
7. The use according to claim 6, characterized in that: the application of the ruthenium nitrosyl complex in preparing the medicine for resisting the proliferation of cervical cancer cells.
8. The use according to claim 7, characterized in that: the IC 50 values of the complexes of formulas 1 to 6 are respectively: 6.63 mu.M, 5.24. Mu.M, 2.08. Mu.M, 1.29. Mu.M, 10.76. Mu.M, 4.43. Mu.M.
9. The use according to claim 7, characterized in that: the application of the complex shown in the formula 3 and the formula 4 in preparing the anti-cervical cancer medicine.
10. The use of a ruthenium nitrosyl complex with hydroxyproline HYP and 5, 7-dichloro-8-hydroxyquinoline HqCl 2 as ligands according to claim 1 for the preparation of NO molecule donor reagents in light regulated solution systems and cell systems.
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