Side chain modified nitrosyl iron-sulfur complex as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of complexes and application thereof, and provides a side chain modified nitrosyl iron sulfide complex and a preparation method and application thereof. The side chain modified nitrosyl iron sulfur complex is a nitrosyl iron sulfur complex [Fe2 (-SL) 2 (NO) 4] taking [Fe2S2 (NO) 4] 2-as a raw material and halogenated hydrocarbon as a ligand, L is a ligand modified on an S atom, and the halogenated hydrocarbon is 3, 5-dimethoxybenzyl bromide, 3-bromomethylbenzonitrile, 4-bromomethylphenylacetate or 3-bromomethylphenylboronic acid. And the compound can be stably formed with the human serum albumin and can be used as a nitric oxide donor for preparation and can be applied as a nitric oxide donor. All the complexes can be recognized and combined with serum albumin, the crystal structure of the complex shows that the nitrosyl iron sulfur complex can be combined in the region of a serum albumin structural domain 1, and the formed complex also has the properties of light-induced nitric oxide release and obvious inhibition of human cervical cancer cell growth activity.

Description

Technical Field

[0001] The present invention belongs to the technical field of complexes and their applications, and particularly relates to a side-chain modified nitrosyl iron-sulfur complex, its preparation method and application, and more particularly to the synthesis of a nitrosyl iron-sulfur complex using [Fe 2 S 2 (NO) 4 2- and halogenated hydrocarbons as reactants, and its preparation method and application. Four different ligand-modified nitrosyl iron-sulfur complexes [Fe 2 (µ-SL) 2 (NO) 4 have been isolated and purified. Further, the complex can release NO by photoinduction, providing an application prospect for photodynamic therapy; this type of nitrosyl iron complex is applied in the screening and preparation of anti-tumor lead compounds. The prepared nitrosyl iron complex can recognize and bind to human serum albumin, and the formed complex still has the effect of inhibiting cancer cell growth. Serum albumin can be used as a carrier for this type of complex and is applied in biomedicine. Background Art

[0002] Metal ions play a key role in the life process. A variety of inorganic metal drugs based on metal ion complexes have been successfully applied clinically and shown good effects in the diagnosis and treatment of diseases. In recent years, nitrosyl metal compounds have been applied to the screening of anti-tumor activities, and NO, as a cell signal transduction messenger, plays a unique role synergistically in vivo. Nitrosylated Fe / S clusters produce polynuclear nitrosyl iron complexes, and the active centers of some metal enzymes in biological systems can exist in the form of this type of complex, which is also one of the important inorganic NO donors. It has been found that its photolysis can efficiently produce nitric oxide, which has important application value in the field of biomedicine [1-5].

[0003] By selecting and changing ligands, the reactivity and biological activity of nitrosyl iron complexes can be regulated, thus providing a basis for the discovery of new anti-tumor drugs and physiological regulators. After being modified by halide substitution, [Fe 2 S 2 (NO) 4 2- exhibits appropriate stability and photoinduced biological activity. This type of complex shows good effects in the treatment of diseases such as cardiovascular and cancer [6-14]. Human serum albumin (HSA) is one of the most abundant plasma proteins in the human body and is a natural carrier for endogenous and exogenous small molecules. The construction of a protein and drug molecule composite carrier system has important application value in clinical treatment [15-17]. Currently, the crystal structure of the complex of serum albumin and nitrosyl iron-sulfur complex has not been reported.

[0004] ​​References: [1] Wink D, Mitchell J. Chemical biology of nitric oxide: insights into regulatory, cytotoxic, and cytoprotective mechanisms of nitric oxide. Free Radical Biology and Medicine, 1998, 25(4-5): 434-456. [2] Murad F. Discovery of some of the biological effects of nitric oxide and its role in cell signaling. Bioscience Reports, 2004, 24(4-5): 452-474. [3] Ekanger L A, Oyala P H, Moradian A, et al .. Nitric oxide modulates endonuclease III redox activity by a 800 mV negative shift upon [Fe4S4] cluster nitrosylation. Journal of the American Chemical Society, 2018, 140(37): 11800-11810. [4] Crack J C, Green J, Thomson A J, et al .. Iron–sulfur clusters as biological sensors: the chemistry of reactions with molecular oxygen and nitric oxide[J]. Accounts Of Chemical Research, 2014, 47(10): 3196-3205. [5] Crack J C, Green J, Thomson A J, et al.. Iron–sulfur clusters as biological sensors: the chemistry of reactions with molecular oxygen and nitric oxide. Accounts Of Chemical Research, 2014, 47(10): 3196-3205. [6] Bourassa J, DeGraff W, Kudo S, et al .. Photochemistry of Roussin's Red Salt, Na2[Fe2S2(NO)4], and of Roussin's Black Salt, NH4[Fe4S3(NO)7]. In Situ Nitric Oxide Generation To Sensitize γ-Radiation Induced Cell Death[J]. Journal of the American Chemical Society, 1997, 119(12): 2853-2860. [7] Conrado C L, Wecksler S, Egler C, et al .. Synthesis and Photochemical Properties of a Novel Iron− Sulfur− Nitrosyl Cluster Derivatized with the Pendant Chromophore Protoporphyrin IX1. Inorganic Chemistry, 2004, 43(18): 5543-5549. [8] Tsai M L, Hsieh C H, Liaw W F. Dinitrosyl iron complexes (DNICs) containing S / N / O ligation: Transformation of Roussin's red ester into the neutral {Fe(NO)2}10 DNICs. Inorganic Chemistry, 2007, 46(12): 5110-5117. [9] hen Y J, Ku W C, Feng L T,et al .. Nitric oxide physiologicalresponses and delivery mechanisms probed by water-soluble Roussin’s red esterand {Fe(NO) 2 } 10 DNICs. Journal of the American Chemical Society, 2008, 130(33):10929-10938.

[10] Wu C R, Huang Y D, Hong Y H, et al .. Endogenous conjugation ofbiomimetic dinitrosyl iron complex with protein vehicles for oral delivery ofnitric oxide to brain and activation of hippocampal neurogenesis. JACS Au,2021, 1(7): 998-1013.

[11] Pulukkody R, Chupik R B, Montalvo S K, et al .. Towardbiocompatible dinitrosyl iron complexes: sugar-appended thiolates. ChemicalCommunications, 2017, 53(6): 1180-1183.

[12] Pectol D C, Khan S, Elsabahy M, et al .. Effects of glutathioneand histidine on NO release from a dimeric dinitrosyl iron complex (DNIC).Inorganic Chemistry, 2020, 59(23): 16998-17008.

[13] Szaciłowski K, Chmura A, Stasicka Z. Interplay between ironcomplexes, nitric oxide and sulfur ligands: Structure, (photo) reactivity andbiological importance. Coordination Chemistry Reviews, 2005, 249(21-22):2408-2436.

[14] Py B, Moreau P L, Barras F. Fe–S clusters, fragile sentinels ofthe cell. Current Opinion In Microbiology, 2011, 14(2): 218-223.

[15] Li X, Zhang Y, Sun J, et al .. Protein nanocage-based photo-controlled nitric oxide releasing platform. ACS Applied Materials&Interfaces,2017, 9(23): 19519-19524.

[16] Li L, Lin Z, Lu X, et al .. Photo-controlled and photo-calibratednanoparticle enabled nitric oxide release for anti-bacterial and anti-biofilmapplications. RSC Advances, 2022, 12(51): 33358-33364.

[17] Rabbani G, Lee EJ, Ahmad K, et al.. Binding of TolperisoneHydrochloride with Human Serum Albumin: Effects on the Conformation,Thermodynamics, and Activity of HSA. Molecular Pharmaceutics, 2018,15(4):1445-1456。 Summary of the Invention

[0005] In view of this, the object of the present invention is to provide nitrosyl iron sulfur complexes modified with different side chain groups and their preparation methods and applications. The present invention also provides a preparation method and application of a complex of nitrosyl iron sulfur complex and human serum albumin HSA. The nitrosyl iron sulfur complex provided by the present invention is a novel nitrosyl iron sulfur complex [Fe 4 N) 2 [Fe 2 S 2 (NO) 4 synthesized with (Me 2 (µ-SL) 2 (NO) 4 and halogenated hydrocarbons as ligands, and the nitrosyl iron sulfur complex can significantly inhibit the growth activity of cancer cells. Its stable complex with human serum albumin can be prepared as a nitric oxide donor and applied as a nitric oxide donor in solution systems and cell systems, and can also be applied in the preparation and screening of anti-tumor drug lead compounds.

[0006] The present invention is achieved by the following technical solutions: A side chain-modified nitrosyl iron sulfur complex, wherein the side chain-modified nitrosyl iron sulfur complex is: [Fe 2 S 2 (NO) 4 2- as a raw material and a nitrosyl iron sulfur complex [Fe 2 (µ-SL) 2 (NO) 4 with halogenated hydrocarbons as ligands, where L is a ligand modified on the S atom. Among them: the halogenated hydrocarbon is 3,5-dimethoxybenzyl bromide, 3-bromomethylbenzonitrile, methyl 4-bromomethylphenylacetate or 3-bromomethylphenylboronic acid, and its structural schematic diagram is as follows: .

[0007] A method for preparing the side chain-modified nitrosyl iron sulfur complex, characterized in that it includes the following steps: (1) Prepare (Me 4 N) 2 [Fe 2 S​2 (NO) 4 Raw materials: 24.0 g of sodium sulfide nonahydrate Na 2 S·9H 2 O, 2.4 g of sodium nitrite NaNO 2 and 11.0 g of sodium hydroxide NaOH were mixed, 120 mL of distilled water was added and stirred to dissolve them. After complete dissolution, 22.4 g of ferrous sulfate heptahydrate FeSO 4 ·7H 2 O was slowly added to the solution. The solution immediately turned black. After adding a magnetic stirrer, it was placed in an oil bath, heated with stirring and boiled for 50 min, then filtered while it was hot. The filtrate was placed in an 80 °C water bath. 11 mL of saturated aqueous solution of tetramethylammonium chloride (CH 3 ) 4 NCl was slowly added to the filtrate with stirring. Subsequently, the solution was allowed to stand. After cooling to room temperature, a large amount of viscous solid was produced at the bottom. The supernatant was removed, the solid was dissolved in distilled water, filtered, recrystallized, and finally dark red-brown crystals with metallic luster were obtained, which was (Me 4 N) 2 [Fe 2 S 2 (NO) 4 ; (2) Preparation of the crude product of nitrosyl iron sulfur complex: The precursor nitrosyl iron sulfur complex (Me 4 N)[Fe 2 S 2 (NO) 4 obtained in step (1) and the halogenated hydrocarbon ligand were subjected to a ligand reaction in a solvent of methanol or water at room temperature in the dark for 5 - 6 h in a molar ratio of 1:2 - 1:2.5. The resulting ligand reaction solution was rotary evaporated to remove the solvent and dried to obtain the crude product of the nitrosyl iron sulfur complex; (3) Purification: The crude product of the nitrosyl iron sulfur complex obtained in step (2) was dissolved and separated by silica gel column chromatography; The selected halogenated hydrocarbon ligand was 3,5-dimethoxybenzyl bromide, 3-bromomethylbenzonitrile or methyl 4-bromomethylphenylacetate. The silica gel column chromatography separation was the first silica gel column chromatography separation, and the eluent was CH 2 Cl 2 ; The selected halogenated hydrocarbon was 3-bromomethylphenylboronic acid. The silica gel column chromatography separation was the second silica gel column chromatography separation. The eluent was a mixed reagent of CH 2 Cl 2 and CH 3 OH. The volume ratio of CH 2 Cl 2 to CH 3 OH in the eluent was 100:1.

[0008] Furthermore, the temperature of the coordination reaction in step (2) is 25 °C and the time is 5 h; the coordination reaction is carried out under light-shielded conditions.

[0009] The present invention also provides the use of the side-chain modified nitrosyl iron-sulfur complex in the preparation of a serum albumin complex; the serum albumin complex includes serum albumin and the side-chain modified nitrosyl iron-sulfur complex loaded on the serum albumin, and the molar ratio of the serum albumin to the side-chain modified nitrosyl iron-sulfur complex is 1:1 to 1:2.

[0010] Furthermore, the preparation method of the serum albumin complex is as follows: dissolving the side-chain modified nitrosyl iron-sulfur complex in an organic solvent DMSO, and diluting with deionized water to obtain a complex solution; mixing the complex solution with a serum albumin buffer solution for a binding reaction to obtain a serum albumin complex.

[0011] The present invention also provides the use of the side-chain modified nitrosyl iron-sulfur complex or the serum albumin complex in the preparation of an anti-tumor drug.

[0012] Furthermore, the anti-tumor drug is an anti-cervical cancer drug.

[0013] The present invention also provides the use of the side-chain modified nitrosyl iron-sulfur complex or the serum albumin complex in the preparation of a light-regulated nitric oxide donor reagent.

[0014] Compared with the prior art, the present invention synthesizes a novel class of nitrosyl iron-sulfur complexes that are relatively stable under light-shielded conditions and can regulate the release of NO by photoinduction. The structures of the four complexes of the present invention have not been reported. The release of nitric oxide in solution and cell systems was tested, and it was confirmed that the release amount of nitric oxide can be controlled by regulating the illumination time, and this complex can be used as a nitric oxide donor reagent. The inhibitory effects of the four iron-sulfur complexes on the growth activity of human cervical cancer HeLa cells were tested, and these complexes showed obvious inhibitory effects. The complex can also bind to human serum albumin and can be used in the preparation and screening of anti-tumor drug carriers. Description of the Drawings

[0015] Figure 1 It is a structural diagram of the crystals of four different ligand-modified nitrosyl iron-sulfur complexes of the present invention; Figure 1 In it: (a) is [Fe 2 (µ-SC 9 H 11 O 2 ) 2 (NO) 4 ; (b) is [Fe 2 (µ-SC 8H 6 N) 2 (NO) 4 ; (c) is [Fe 2 (µ-SC 10 H 11 O 2 ) 2 (NO) 4 ;(d) is [Fe 2 (µ-SC 8 H 10 BO 2 ) 2 (NO) 4 ; Figure 2 This is the crystal structure diagram of the complex of the nitrosyl iron-sulfur complex [Fe 2 (µ-SC 7 H 8 BO 2 ) 2 (NO) 4 modified by the ligand in the present invention and serum albumin; Figure 3 This is the time-resolved infrared spectrum diagram of four nitrosyl iron-sulfur complexes modified by different ligands in the present invention under the irradiation conditions of a certain power and wavelength (420 nm); In the figure, (a)-(d) are [Fe 2 (µ-SC 9 H 11 O 2 ) 2 (NO) 4 , [Fe 2 (µ-SC 8 H 6 N) 2 (NO) 4 , [Fe 2 (µ-SC 10 H 11 O 2 ) 2 (NO) 4 , [Fe 2 (µ-SC 7 H 8 BO 2 ) 2 (NO) 4 ; Figure 4 This is the inhibitory activity of four nitrosyl iron-sulfur complexes modified by different ligands in the present invention on the growth of human cervical cancer cells HeLa; In the figure: (a)-(d) are [Fe 2 (µ-SC 9 H 11 O2 ) 2 (NO) 4 )、[Fe 2 (µ-SC 8 H 6 N) 2 (NO) 4 )、[Fe 2 (µ-SC 10 H 11 O 2 ) 2 (NO) 4 )、[Fe 2 (µ-SC 7 H 8 BO 2 ) 2 (NO) 4 ) at different concentrations in the range of 0 - 12.5 µM on the growth of HeLa cells under dark and light conditions; (e)-(h) are [Fe 2 (µ-SC 9 H 11 O 2 ) 2 (NO) 4 )、[Fe 2 (µ-SC 8 H 6 N) 2 (NO) 4 )、[Fe 2 (µ-SC 10 H 11 O 2 ) 2 (NO) 4 )、[Fe 2 (µ-SC 7 H 8 BO 2 ) 2 (NO) 4 ) at different concentrations in the range of 0 - 36 µM on the growth of human normal liver cells HL7702 under dark and light conditions; Figure 5 Effects of four nitrosyl iron sulfur complexes modified with different ligands of the present invention on the HeLa cell cycle under dark and light irradiation conditions; In the figure: Complexes 1, 2, 3, and 4 correspond to [Fe 2 (µ-SC 9 H 11 O 2 ) 2 (NO) 4 )、[Fe 2 (µ-SC 8 H6 N) 2 (NO) 4 ]、[Fe 2 (µ-SC 10 H 11 O 2 ) 2 (NO) 4 ] and [Fe 2 (µ-SC 7 H 8 BO 2 ) 2 (NO) 4 ]; Figure 6 The effects of four nitrosyl iron-sulfur complexes modified with different ligands on HeLa cell apoptosis under dark and light irradiation conditions are shown in the figure: complexes 1, 2, 3 and 4 correspond to [Fe 2 (µ-SC 9 H 11 O 2 ) 2 (NO) 4 ]、[Fe 2 (µ-SC 8 H 6 N) 2 (NO) 4 ]、[Fe 2 (µ-SC 10 H 11 O 2 ) 2 (NO) 4 ] and [Fe 2 (µ-SC 7 H 8 BO 2 ) 2 (NO) 4 ]; Figure 7 Figure 2 is a real-time cell fluorescence imaging diagram of NO of four nitrosyl iron-sulfur complexes modified with different ligands of the present invention under light irradiation conditions; in the figure: (a) to (d) correspond to the complexes [Fe 2 (µ-SC 9 H 11 O 2 ) 2 (NO) 4 ], complex [Fe 2 (µ-SC 8 H 6 N) 2 (NO) 4 ], complex [Fe 2 (µ-SC 10 H11 O 2 ) 2 (NO) 4 , complex [Fe 2 (µ-SC 7 H 8 BO 2 ) 2 (NO) 4 ; Figure 8 These are the fluorescence spectra and the fitting curves of the binding constants of four nitrosyl iron-sulfur complexes modified with different ligands of the present invention with human serum albumin (HSA); in the figure: (a) is the [Fe 2 (µ-SC 9 H 11 O 2 ) 2 (NO) 4 -HSA complex; (b) is the [Fe 2 (µ-SC 8 H 6 N) 2 (NO) 4 -HSA complex; (c) is the [Fe 2 (µ-SC 10 H 11 O 2 ) 2 (NO) 4 -HSA complex; (d) is the [Fe 2 (µ-SC 7 H 8 BO 2 ) 2 (NO) 4 -HSA complex; Figure 9 These are the inhibitory activities of four nitrosyl iron-sulfur complexes modified with different ligands of the present invention with the human cervical cancer cell line HeLa; in the figure: (a)-(d) are the [Fe 2 (µ-SC 9 H 11 O 2 ) 2 (NO) 4 -HSA complex, [Fe 2 (µ-SC 8 H 6 N) 2 (NO) 4 -HSA complex, [Fe 2 (µ-SC 10 H 11 O2 ) 2 (NO) 4 -HSA complex, [Fe 2 (µ-SC 7 H 8 BO 2 ) 2 (NO) 4 Cell viability graphs of the -HSA complex's effect on HeLa cell growth under non-irradiated and irradiated conditions within different concentration ranges; Figure 10 For the effects of four nitrosyl iron-sulfur complexes modified with different ligands of the present invention complexed with human serum albumin (HSA) on the HeLa cell cycle. Complexes 1, 2, 3, and 4 are respectively [Fe 2 (µ-SC 9 H 11 O 2 ) 2 (NO) 4 complexed with HSA, [Fe 2 (µ-SC 8 H 6 N) 2 (NO) 4 complexed with HSA, [Fe 2 (µ-SC 10 H 11 O 2 ) 2 (NO) 4 complexed with HSA, [Fe 2 (µ-SC 7 H 8 BO 2 ) 2 (NO) 4 complexed with HSA; Figure 11 For the effects of four nitrosyl iron-sulfur complexes modified with different ligands of the present invention complexed with human serum albumin (HSA) on HeLa cell apoptosis. Complexes 1, 2, 3, and 4 are respectively [Fe 2 (µ-SC 9 H 11 O 2 ) 2 (NO) 4 complexed with HSA, [Fe 2 (µ-SC 8 H 6 N) 2 (NO) 4 complexed with HSA, [Fe 2 (µ-SC10 H 11 O 2 ) 2 (NO) 4 complex with HSA, [Fe 2 (µ-SC 7 H 8 BO 2 ) 2 (NO) 4 complex with HSA; Figure 12 This is the real-time NO cellular fluorescence imaging diagram of the complexes of four nitrosyl iron-sulfur complexes modified with different ligands of the present invention and human serum albumin (HSA) under light irradiation; in the figure: (a) is the [Fe 2 (µ-SC 9 H 11 O 2 ) 2 (NO) 4 -HSA complex; (b) is the [Fe 2 (µ-SC 8 H 6 N) 2 (NO) 4 -HSA complex; (c) is the [Fe 2 (µ-SC 10 H 11 O 2 ) 2 (NO) 4 -HSA complex; (d) is the [Fe 2 (µ-SC 7 H 8 BO 2 ) 2 (NO) 4 -HSA complex. Detailed implementation manners

[0016] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the following embodiments are used to describe the preparation method and application of nitrosyl iron-sulfur complexes synthesized with (Me 4 N) 2 [Fe 2 S 2 (NO) 4 and halogenated hydrocarbons modified with different ligands as reactants. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all embodiments; all other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0017] Unless otherwise defined, 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. All references cited herein and the materials to which they refer are hereby incorporated by reference.

[0018] Equivalent techniques to the specific embodiments described that can be understood by those skilled in the art through routine experiments will be included in this application.

[0019] Unless otherwise specified, the experimental methods in the following examples are all conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples are all obtained from conventional biochemical reagent stores.

[0020] Example 1: A novel nitrosyl iron-sulfur complex was synthesized by modifying halohydrocarbons with (Me 4 N) 2 [Fe 2 S 2 (NO) 4 as the raw material. The chemical formula of the nitrosyl iron-sulfur complex is: [Fe 2 (µ-SL) 2 (NO) 4 , where L is the ligand modified on the S atom and is derived from 3,5-dimethoxybenzyl bromide, 3-bromomethylbenzonitrile, methyl 4-bromomethylphenylacetate, and 3-bromomethylphenylboronic acid respectively.

[0021] The specific preparation method is as follows: (1) Preparation of the (Me 4 N) 2 [Fe 2 S 2 (NO) 4 raw material. Weigh 24.0 g of sodium sulfide nonahydrate (Na 2 S·9H 2 O), 2.4 g of sodium nitrite (NaNO 2 ), and 11.0 g of sodium hydroxide (NaOH) respectively, place them in a 200 mL beaker, add 120 mL of distilled water and stir to dissolve them. After complete dissolution, slowly add 22.4 g of ferrous sulfate heptahydrate (FeSO 4 ·7H 2 O) to the solution. The solution immediately turns black. After adding a magnetic stir bar, place the beaker in an oil bath and heat it with stirring until it boils. After 50 minutes, filter while it is hot. Place the filtrate in a water bath at about 80 °C, and slowly add 11 mL of saturated tetramethylammonium chloride ((CH 3 ) 4An aqueous solution of NCl was prepared, and then the solution was allowed to stand. After cooling to room temperature, a large amount of viscous solid was found at the bottom of the beaker. The supernatant was removed, and the solid was just dissolved with distilled water, filtered, recrystallized, and finally dark red-brown crystals with metallic luster were obtained. (2)The precursor nitrosyl iron sulfur complex (Me 4 N)[Fe 2 S 2 (NO) 4 and the haloalkane ligand were subjected to a ligand coordination reaction in a solvent of methanol or water at room temperature in the dark for 5 hours in a molar ratio of 1:2. The resulting ligand coordination reaction solution was rotary evaporated to remove the solvent and evaporated to dryness to obtain a crude product of the nitrosyl iron sulfur complex.

[0022] (3)Separation and purification of the complexes modified with four different ligands: The crude product of the nitrosyl iron sulfur complex was dissolved and then separated by silica gel column chromatography. When the selected haloalkane is 3,5-dimethoxybenzyl bromide, the silica gel column chromatography is the first silica gel column chromatography, and the eluent is CH 2 Cl 2 ; When the selected haloalkane is 3-bromomethylbenzonitrile, the silica gel column chromatography is the first silica gel column chromatography, and the eluent is CH 2 Cl 2 ; When the selected haloalkane is methyl 4-bromomethylphenylacetate, the silica gel column chromatography is the first silica gel column chromatography, and the eluent is CH 2 Cl 2 ; When the selected haloalkane is 3-bromomethylphenylboronic acid, the silica gel column chromatography is the second silica gel column chromatography, and the eluent is a mixed reagent of CH 2 Cl 2 and CH 3 OH. The volume ratio of CH 2 Cl 2 to CH 3 OH in the eluent is 100:1.

[0023] The four different ligand-modified nitrosyl iron sulfur complexes obtained by separation and purification were analyzed and identified by structural characterization methods such as nuclear magnetic resonance hydrogen spectrum and infrared spectrum. For the sake of convenience of expression, the nitrosyl iron sulfur complexes modified with different ligands shown in Formulas 1-4 were respectively denoted as Complexes 1-4.

[0024] Complex 1: Nuclear magnetic resonance HNMR spectrum 600 M ( d 6-DMSO): δ 6.52 (d, J = 69.2 Hz, 3H), 4.33 (d, J = 45.4 Hz, 2H), 3.74 (s, 6H). v NO : 1745.68(cm -1 )、1603.31 (cm -1 ).

[0025] Complex 2: Nuclear magnetic resonance HNMR spectrum 600 M ( d 6 -DMSO): δ 7.86 - 7.83 (m, 2H), 7.78 (s, 1H), 7.67 (s, 1H), 4.54 (s, 1H), 4.47 (s, 1H). Infrared IR spectrum: v NO : 1747.34 (cm -1 )、1731.84 (cm -1 ).

[0026] Complex 3: Nuclear magnetic resonance HNMR spectrum 600 M ( d 6 -DMSO): δ 7.37 (d, J = 6.4 Hz, 1H), 7.31 (d, J = 6.9 Hz, 1H), 4.43 (s, 0.61H), 4.36 (s, 0.4H), 3.71 (s, 1H), 3.62 (d, J = 2.5 Hz, 1.5H). Infrared IR spectrum: v NO : 1766.65 (cm -1 )、1724.72 (cm -1 ).

[0027] Complex 4: Nuclear magnetic resonance HNMR spectrum 600 M ( d 6 -DMSO): δ 8.08 (d, J = 5.2 Hz, 2H), 7.85 (d, J = 9.1 Hz, 1H), 7.76 (d, J = 7.1 Hz, 1H), 7.41 (d, J = 7.3 Hz, 1H), 7.37 (t, J = 7.4 Hz, 1H), 4.43 (s, 1H), 4.34 (s, 1H). Infrared IR spectrum: v NO : 1765.94(cm -1 ).-1 )。

[0028] Example 2: The four different ligand-modified nitrosyl iron-sulfur complexes synthesized in Example 1 were prepared into single crystals by the following method: The nitrosyl iron-sulfur complexes with different ligands prepared in Example 1 were dissolved in a methanol and dichloromethane solution with a volume ratio of 1:1 - 2:1, and the evaporation rate of the solvent was adjusted. Four single crystals suitable for X-ray crystallography determination, namely regular-shaped reddish-brown crystals a, b, c, and d, were prepared by the slow evaporation method. The preferred crystallization condition was a solvent volume ratio of 2:1 (methanol / dichloromethane).

[0029] The atomic-resolution structures were determined using an X-ray single-crystal diffractometer, and their accurate spatial structures were obtained by analysis. The crystal structures of Complexes 1 - 3 were exactly the same as the expected structures. It is worth noting that one hydroxyl group on the B atom was methylated to a methoxy group after crystallization of Complex 4 in a methanol solution, which was confirmed by NMR and mass spectrometry methods. The nuclear magnetic resonance 1 H NMR spectrum 600M ( d 6 -DMSO): δ 8.09 (s, 1H), 7.86 (s, 1H), 7.77 (s, 1H), 7.41 (s, 1H), 7.39 (s, 1H), 4.43 (s, 1H), 4.36 (s, 1H), 1.25 (d, J = 14.3 Hz, 3H). ESI-MS: m / z calcd. for 592.9521 [M - H + . Found: 592.9174。

[0030] Data collection was carried out at room temperature of 298 K using a Bruker D8 Venture diffractometer, and its radiation source was Mo-Kα radiation monochromatized by a graphite monochromator with a wavelength of 0.71073 Å. The unit cell parameters were determined and the data were reduced by running the SAINT program of the SMART software; the structure analysis was completed using the SHELXTL-97 program package.

[0031] The crystal structure of the complex is as Figure 1 shown. Combining the analysis of the nuclear magnetic resonance spectra of the complexes modified with different ligands, the structures of the four nitrosyl iron-sulfur complexes are as follows: ; Among them, (a) is [Fe 2 (µ-SC 9 H 11 O 2 ) 2 (NO)4 ; (b) is [Fe 2 (µ-SC 8 H 6 N) 2 (NO) 4 ; (c) is [Fe 2 (µ-SC 10 H 11 O 2 ) 2 (NO) 4 ; (d) is [Fe 2 (µ-SC 8 H 10 BO 2 ) 2 (NO) 4 ; The optimized obtained crystal is a single crystal. The ORTEP diagram of the complex crystal is as shown in Figure 1 . The crystal structure and the 1H NMR spectrum confirm the successful synthesis of four nitrosyl iron-sulfur complexes.

[0032] Example 3: Preparation of a modified nitrosyl iron-sulfur complex-human serum albumin complex, and successfully resolving the crystal structure of the complex of complex 4 and HSA, specifically as follows: By centrifuging the mixed solution of HSA and palmitic acid through an ultrafiltration tube with a molecular weight cut-off of 30 kDa at 3000 rpm in a high-speed refrigerated centrifuge and concentrating to a protein concentration of 40 mg / mL; preparing the crystallization reservoir solution with 50% PEG3350, DMSO, glycerol and 0.2 M phosphate buffer (pH = 7.2) in different volume ratios; mixing 1.5 μL of the concentrated protein solution with an equal volume of the crystallization reservoir solution on a cover slip, and carrying out crystal cultivation in a constant temperature cabinet at 18 °C using the hanging drop vapor diffusion method. After two days, regular-shaped transparent crystals were obtained. Adding 5 mM of the nitrosyl iron-sulfur complex to the crystal droplet for cocrystallization cultivation, and after two days, light yellow complex crystals were obtained. Diffraction data at 2.6 Å were collected using the Shanghai Synchrotron Radiation Facility, and its atomic resolution structure was resolved, as shown in Figure 2 . The crystal structure of the complex of the nitrosyl iron-sulfur complex and serum albumin consists of three domains: domain I (blue-green), domain II (yellow), and domain III (red). It can be seen from the figure that a nitrosyl iron-sulfur complex molecule binds to region I of domain IB1 of the serum albumin molecule.

[0033] From the electron density map of the complex of the nitrosyl iron-sulfur complex and serum albumin measured and the structure of the nitrosyl iron-sulfur complex resolved by measurement and analysis, [Fe 2 (µ-SC 7 H 8 BO 2 )2 (NO) 4 When the complex binds to serum albumin, it binds in the form of Fe(SL)(NO) 2 in the cavity formed by amino acids Ile142, His146, Phe149, Arg186, Gly189, Lys190, and Ser193 in HSA domain 1. From the structure of the nitrosyl iron-sulfur complex in the crystal structure of the complex, it can be seen that human serum albumin may play a role in the form of a mononuclear nitrosyl iron-sulfur complex binding when delivering the complex as a carrier.

[0034] Example 4: The infrared spectra of four ligand-modified nitrosyl iron-sulfur complexes (Complex 1, 2, 3, and 4) were measured using a Nicolet iS50R FT-IR Fourier transform infrared spectrometer. The method is as follows: The complexes were separately dissolved in DMSO to prepare a 1×10 -3 M stock solution. Using a pipette, 50 µL of DMSO and the sample solution to be measured were slowly loaded into an infrared sample cell composed of two calcium fluoride window plates with a diameter of 25 mm and a thickness of 2 mm and a 100 µm thick "O"-shaped polytetrafluoroethylene gasket, and then fixed through an infrared sample holder. When there were no bubbles in the middle, the background and sample data were collected in sequence; the latter mainly observed the release of NO from the sample under 420 nm light illumination for 30 min. Before measurement, the DMSO solution spectrum was measured and the background was subtracted. The number of scans was set to 64 times, and the scan was performed in the wavenumber range of 2000~1500 cm -1 . A strong infrared vibration peak near 1700~1800 cm -1 is the stretching vibration peak of the NO group in the complex.

[0035] The test results are as Figure 3 shown, Figure 3 in (a) is Complex 1, i.e., [Fe 2 (µ-SC 9 H 11 O 2 ) 2 (NO) 4 , (b) is Complex 2, i.e., [Fe 2 (µ-SC 8 H 6 N) 2 (NO) 4 , (c) is Complex 3, i.e., [Fe 2 (µ-SC 10 H 11 O 2 ) 2 (NO) 4 , (d) is Complex 4, i.e., [Fe2 (µ-SC 7 H 8 BO 2 ) 2 (NO) 4 . As can be seen from the figure, as the illumination time increases, the complex dissociates and releases nitric oxide, and the NO vibration peak decreases significantly. The NO release rates of complexes 1-4 are slightly different. At the same time, as the illumination time increases, the vibration peak of NO in the complex gradually weakens. By changing the illumination time, the release amount of NO can be regulated.

[0036] Example 5: Under dark and light conditions, the inhibitory activities of four different ligand-modified iron-sulfur complexes prepared in Example 1 on the growth of human cervical cancer HeLa cells and human normal liver cells HL7702 were tested. The method is as follows: Dissolve the complex sample with DMSO as the mother liquor, dilute two samples with 5 identical concentration gradients each to incubate human cervical cancer HeLa cells and human normal liver cells HL7702. Set 4 replicates for each concentration. Finally, control the DMSO concentration to be 1% in 200 µL of culture medium. The operation of the blank group and the control group is the same. Take two cell culture plates and add 0-10 µM of the complex respectively. Place the cell culture plates in a 37 °C, 5% CO 2 After incubating in the cell culture incubator for 2 h, take out one cell plate as the light group, place it under a 420 nm 96-well LED special light source with a voltage of 21 V for 20 min of illumination, and then put it back into the cell culture incubator for incubation for 24 h; after the incubation time ends, take out the culture plate, add 10 µL of CCK-8 solvent to each well, and then take it out after incubating for 4 h. Measure the absorbance at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader, calculate the inhibition rate respectively, and then make a statistical graph.

[0037] The results are as Figure 4 shown, Figure 4 In (a)-(d) are HeLa cells: (a) is complex 1, namely [Fe 2 (µ-SC 9 H 11 O 2 ) 2 (NO) 4 , (b) is complex 2, namely [Fe 2 (µ-SC 8 H 6 N) 2 (NO) 4 , (c) is complex 3, namely [Fe 2 (µ-SC 10 H 11 O 2 ) 2 (NO) 4, (d) is complex 4, i.e., [Fe 2 (µ-SC 7 H 8 BO 2 ) 2 (NO) 4 . (e)-(h) are HL7702 cells: (e) is complex 1, i.e., [Fe 2 (µ-SC 9 H 11 O 2 ) 2 (NO) 4 , (f) is complex 2, i.e., [Fe 2 (µ-SC 8 H 6 N) 2 (NO) 4 , (g) is complex 3, i.e., [Fe 2 (µ-SC 10 H 11 O 2 ) 2 (NO) 4 , (h) is complex 4, i.e., [Fe 2 (µ-SC 7 H 8 BO 2 ) 2 (NO) 4 .

[0038] Calculated by Figure 4 through the SPSS software program, it is known that the IC 50 values of complexes 1-4 for HeLa cells are 6.31, 6.29, 7.30, 6.03 µM respectively. Among them, the toxicity levels are: complex 4 > complex 2 > complex 1 > complex 3. Under light illumination conditions, the IC 50 values of complexes 1-4 are 4.34, 5.03, 5.27, 4.77 µM respectively. The results show that light illumination can enhance the inhibitory effect on tumor cell growth. The IC 50 values of complexes 1-4 for human normal liver cells HL7702 are 24.55, 25.42, 23.21, 18.66 µM respectively. Under light illumination conditions, the IC 50The values were 26.80, 26.73, 25.40, and 19.99 μM, respectively. The Selective Indexes for HeLa cells were 3.89, 4.04, 3.18, and 3.09, respectively, indicating that complexes 1-4 showed relatively low cytotoxicity to human normal liver cells HL7702, while showing relatively high cytotoxicity to human cervical cancer HeLa cells, and having good selectivity for tumor HeLa cells.

[0039] Example 6: The effects of the four modified nitrosyl iron sulfur complexes on the cell cycle and apoptosis of HeLa cells were analyzed by a CytoFLEX flow cytometer.

[0040] HeLa cells in the logarithmic growth phase were seeded in 6-well plates at a cell density of 5×10 5 per well and incubated in a constant temperature cell incubator at 37°C with 5% CO 2 for 24 h. When the cell density in each well reached about 80%, the drug addition group was added with DMSO solutions of different concentrations of nitrosyl iron sulfur complexes, and the control group was added with an equal volume of DMSO, with the volume ratio of DMSO in each well being 1%. After continuing to incubate for 2 h, the light irradiation group was treated with a 420 nm LED high-power monochromatic light source for 20 min and then continued to be cultured, while the non-irradiated group was not treated. After 24 h, the cells were collected, and the residual culture medium and trypsin were washed away with PBS. After centrifugation at 1000 rpm for 5 min, the supernatant was removed, and 500 μL of pre-cooled ethanol at 70% was added to fix the cells at 4°C for 12 h. The fixing solution was washed away by centrifugation with PBS, and 100 μL of RNase A solution, a DNA content detection reagent, was added to the cell pellet and incubated in a water bath at 37°C for 30 min. Then, 400 μL of PI staining solution was added and incubated in the dark at 4°C for 30 min for staining. Before detecting on the machine, the cells were first filtered through a 200-mesh nylon mesh, and then the red fluorescence at 488 nm was detected with a Beckman Coulter flow cytometer. The measured results were processed and analyzed with FlowJo software.

[0041] The results of the cell cycle are as Figure 5 shown, Figure 5 in which complex 1, namely [Fe 2 (µ-SC 9 H 11 O 2 ) 2 (NO) 4 , complex 2, namely [Fe 2 (µ-SC 8 H 6 N) 2 (NO) 4 , complex 3, namely [Fe 2 (µ-SC 10H 11 O 2 ) 2 (NO) 4 (NO) 2 (µ-SC 7 H 8 BO 2 ) 2 (NO) 4 (NO) 。In the absence of light, compared with the control group, the addition of the four nitrosyl iron sulfur complexes reduced the G1 phase by 14.6%, 16.8%, 8.4% and 18.2% respectively; the S phase increased by 8.1%, 10.4%, 6.2% and 11.0% respectively, and the proportion of cells in the G2 phase was not significantly different from that in the G2 phase of the control group, indicating that the complexes arrested the cell cycle in the S phase. After light irradiation, the G1 phase decreased by 24.9%, 16.8%, 16.5% and 24.9% respectively, the S phase increased by 21.9%, 22.1%, 26.8% and 27.4% respectively, and the cells in the G2 phase decreased by 4.4%, 4.7%, 9.35% and 3.5% respectively, indicating that after light irradiation, the complexes still arrested the cell cycle in the S phase and enhanced this arrest effect.

[0042] HeLa cells in the logarithmic growth phase were seeded in 6-well plates at a cell density of 5×10 5 per well and incubated in a constant temperature cell incubator for 24 h. The drug-treated groups were added with DMSO solutions of different concentrations of nitrosyl iron sulfur complexes, and the control group was added with an equal volume of DMSO. After continuing to incubate for 2 h, the light-irradiated group was treated with a 420 nm LED high-power monochromatic light source for 20 min and then continued to be cultured, while the non-light-irradiated group was not treated. After 24 h, the cells were digested with trypsin without EDTA (0.25% Typsin - no EDTA), the medium was added, and the cells were collected by centrifugation at 1000 rpm for 5 min, washed twice with PBS, the cell pellet was resuspended in 1× binding buffer, 5 μL of Annexin V-FITC and 10 μL of PI were added to 100 μL of the cell suspension, gently mixed, incubated in the dark at room temperature for 15 min, and then 400 μL of 1× binding buffer working solution was added. The cells were filtered through a 200-mesh nylon mesh and detected by flow cytometry. The measured results were processed and analyzed by FlowJo software.

[0043] The results of cell apoptosis are as Figure 6 shown, Figure 6 Complex 1, namely [Fe 2 (µ-SC 9 H 11 O 2 ) 2(NO) 4 , Complex 2, i.e., [Fe 2 (µ-SC 8 H 6 N) 2 (NO) 4 , Complex 3, i.e., [Fe 2 (µ-SC 10 H 11 O 2 ) 2 (NO) 4 , Complex 4, i.e., [Fe 2 (µ-SC 7 H 8 BO 2 ) 2 (NO) 4 . In the absence of light, compared with the control group, the addition of the four nitrosyl iron-sulfur complexes decreased the percentage of viable cells (Annexin V-FITC- / PI-), while the percentage of early apoptotic cells (Annexin V-FITC+ / PI-) increased from 6.47% to 13.1%, 13.5%, 6.77% and 8.26% respectively, and the percentage of late apoptotic and necrotic cells (Annexin V-FITC+ / PI+) increased from 5.81% to 8.04%, 7.71%, 6.49% and 3.81% respectively. It indicates that the [Fe 2 (µ-SL) 2 (NO) 4 complex can still promote the apoptosis of HeLa cells in the absence of light.

[0044] Under the 420 nm light treatment, compared with the control group, the complexes can also promote the apoptosis of HeLa cells, that is, the phototoxicity of the modified nitrosyl iron-sulfur complexes is not obvious. However, compared with the non-irradiated group, the proportions of early apoptotic and late apoptotic cells both increased to varying degrees, indicating that light can promote the effect of nitrosyl complexes on the apoptosis of HeLa cells.

[0045] Example 7: Using a ZEISS LSM-880 confocal laser scanning microscope and a selective NO fluorescence probe DAX-J2 Red, the nitric oxide release performance of Complexes 1-4 in the cell system under light irradiation and non-irradiation conditions was detected in real time. The method is as follows: Add 10 mM NO probe DAX-J2 Red to an EP tube containing PBS buffer solution and dilute it to 5 µM. Take 1 mL and add it to a confocal dish containing cultured HeLa cells and incubate for 20 min. Take 10 µL of the pre-prepared 2 mM complex sample and add it to the confocal dish and continue to incubate for 20 min. Finally, wash it 3 times with PBS, add 1 mL of PBS buffer solution and place it under a confocal microscope for observation; then irradiate it with a 420 nm LED monochromatic light source for 0, 5, 10, and 15 min respectively, and observe the imaging of NO in HeLa cells after irradiation. The control group was only added with 5 µM NO probe DAX-J2 Red and 20 µM complex in the cell culture medium, but without light irradiation; in the experimental group, DAX-J2 Red and 20 µM complex were added respectively, and then irradiated for different times.

[0046] The results of real-time fluorescence imaging of NO of four different ligand-modified nitrosyl iron-sulfur complexes at different light irradiation times are as Figure 7 shown, Figure 7 in which (a) is complex 1, namely [Fe 2 (µ-SC 9 H 11 O 2 ) 2 (NO) 4 , (b) is complex 2, namely [Fe 2 (µ-SC 8 H 6 N) 2 (NO) 4 , (c) is complex 3, namely [Fe 2 (µ-SC 10 H 11 O 2 ) 2 (NO) 4 , (d) is complex 4, namely [Fe 2 (µ-SC 7 H 8 BO 2 ) 2 (NO) 4 .

[0047] DAX-J2 Red is a specific NO fluorescent probe. When it reacts with NO, it emits red fluorescence and can selectively identify the production of NO. Figure 7Among (a), (b), (c), and (d), 5 μM DAX-J2 Red and 20 μM of Complex 1, Complex 2, Complex 3, and Complex 4 were added respectively. Using a 420 nm light source, HeLa cells were irradiated for 0, 5, 10, and 15 min respectively, and a laser confocal imaging analyzer was used to measure the production of NO in HeLa cells in real time. From Figure 7 It can be seen that when there is no light or the light irradiation time is 0 min, no or only very weak NO fluorescence can be observed; however, when there is a nitrosyl iron sulfur complex in the culture medium, obvious NO fluorescence can be observed, and with the increase of the light irradiation time, the generated NO fluorescence gradually increases. Figure 7 The results confirmed the controllable release of NO by the photoinduced complex in the cell system.

[0048] Example 8: Prepare a 1×10 -5 M human serum albumin (HSA) solution with ultrapure water for standby. Dissolve and prepare a 1×10 -3 M mother liquor of nitrosyl iron sulfur complexes 1-4 with dimethyl sulfoxide (DMSO), and store it in the dark for standby; take 1.5 mL of 1 × 10 -5 M HSA into a fluorescence absorption cup. The instrument parameters of the fluorescence spectrometer were set as: excitation wavelength 280 nm, emission wavelength 300-500 nm, and a fluorescence titration experiment was carried out. Take 1×10 -3 M mother liquor of nitrosyl iron sulfur complexes 1-4 and add it to the HSA solution successively at intervals of 2.5 μL, mix well, let it stand for 2 min and then measure the fluorescence spectrum until the fluorescence spectrum diagram tends to be stable. After the test, heat it up to 30 and 35 °C and continue to measure. Then, use Origin software to draw the fluorescence spectrum diagram with wavelength and fluorescence intensity as coordinates respectively, and calculate the binding constant and the number of binding sites with lg[Q] and lg[(F 0 -F) / F] as coordinates, where Q is the actual concentration of the quencher (nitrosyl iron sulfur complex), F 0 is the initial maximum fluorescence intensity of HSA without adding the quencher, and F is the maximum fluorescence intensity of HSA at the same wavelength after adding the quencher. The fluorescence spectra of HSA titrated by nitrosyl iron sulfur complexes 1-4 modified with different ligands and the linear fitting curves of lg[F 0 / F - 1] changing with lg[Q] are as Figure 8 shown.

[0049] It can be seen from the fluorescence spectrum diagram that with the increase of the concentration of Complexes 1-4, obvious fluorescence quenching of HSA occurs. From the lg[F 0 / F - 1]-lg[Q] diagram, the binding constants of Complexes 1-4 with HSA at 25 °C are 4.90×10 4 M -1, 2.68×10 4 M -1 , 6.79×10 5 M -1 and 8.07×10 4 M -1 , the number of binding sites are 1.01, 2.68, 1.24, and 1.14 respectively. There are certain differences in the binding strength of nitrosyl iron-sulfur complexes modified with different ligands to HSA, and the magnitudes of their binding constants are in the order of Complex 3 > Complex 4 > Complex 1 > Complex 2. At the same time, through variable-temperature titration experiments, it can be calculated that both ΔH and ΔS during the binding of the four complexes to HSA are negative values, indicating that their binding mainly depends on hydrogen bonds and van der Waals forces, and ΔG < 0 indicates that the binding between the two can occur spontaneously.

[0050] Example 9: Under dark, light-free, and light conditions, test the inhibitory activities of the complexes formed by incubating four different ligand-modified complexes 1 - 4 with HSA on the growth of human cervical cancer HeLa cells. The method is as follows: Prepare a 1 mM human serum albumin HSA solution with PBS buffer (10 mM, pH = 7.2). Mix complexes 1 - 4 with HSA at a molar concentration ratio of 1:10, and control the DMSO content in the system to be less than 5%. Heat the mixed solution at 50 °C for 20 min, cool it to room temperature, and then transfer it to the refrigerator at 4 °C and let it stand overnight. Use an ultrafiltration centrifugal tube with a cut-off molecular weight of 30 kDa and centrifuge it at 3000 rpm in a high-speed refrigerated centrifuge for 10 h. Continuously supplement PBS solution during the process to wash away the unbound complex small molecules in the system, and store the obtained complex in the dark for later use. Dilute two samples with five identical concentration gradients each to incubate human cervical cancer HeLa cells. Set 4 replicates for each concentration. Finally, control the DMSO concentration in the 200 µL culture medium solution not to exceed 2%. The operation of the blank group and the control group is the same. Take two cell culture plates and add complexes HSA complexes with different concentrations from 0 - 10 µM. 2 After incubating the cell culture plates in a 37 °C, 5% CO 2 cell culture incubator for 2 h, take out one cell plate as the light group, place it under a 420 nm 96-well LED special light source with a voltage of 21 V for 20 min, and then put it back into the cell culture incubator for 24 h; after the incubation time ends, take out the culture plate, add 20 µL of CCK-8 solvent to each well, incubate for another 4 h and then take it out. Measure the absorbance at 450 nm with an enzyme-linked immunosorbent assay reader, calculate the inhibition rate respectively, and then make a statistical graph.

[0051] The results are as Figure 9 shown, Figure 9 in (a), (b), (c), and (d) respectively correspond to Complex 1, i.e., [Fe 2 (µ-SC 9H 11 O 2 ) 2 (NO) 4 Complex formed with HSA, Complex 2, namely [Fe 2 (µ-SC 8 H 6 N) 2 (NO) 4 Complex formed with HSA, Complex 3, namely [Fe 2 (µ-SC 10 H 11 O 2 ) 2 (NO) 4 Complex formed with HSA, and Complex 4, namely [Fe 2 (µ-SC 7 H 8 BO 2 ) 2 (NO) 4 Complex formed with HSA.

[0052] From Figure 9 Calculated by SPSS software program, it is known that the IC 50 values of the complexes formed by Complexes 1-4 with HSA acting on HeLa cells are 5.64, 5.13, 6.14, 5.45 µM respectively. Under light illumination conditions, the IC 50 values of the complexes formed by Complexes 1-4 with HSA are 3.40, 4.01, 4.73, 4.27 µM respectively. These results indicate that light illumination can enhance the inhibitory effect of the complexes on tumor cell growth, and loading drugs through serum albumin can promote the inhibitory effect of the drugs on tumor cell growth and exhibit certain photodynamic activity.

[0053] Example 10: The effects of the complexes formed by four different ligand-modified nitrosyl iron sulfur complexes with serum albumin after modification on the cell cycle and apoptosis of HeLa cells were analyzed by CytoFLEX flow cytometer. The specific experimental methods were the same as those in the implementation, and the results are as Figure 10 shown. Figure 10 Among them, Complex 1, namely [Fe 2 (µ-SC 9 H 11 O 2 ) 2 (NO) 4 Complex formed with HSA, Complex 2, namely [Fe 2 (µ-SC 8 H 6 N) 2 (NO) 4The complex formed with HSA, Complex 3, i.e., [Fe 2 (µ-SC 10 H 11 O 2 ) 2 (NO) 4 The complex formed with HSA, Complex 4, i.e., [Fe 2 (µ-SC 7 H 8 BO 2 ) 2 (NO) 4 The complex formed with HSA.

[0054] In the absence of light, compared with the control group, the addition of the complexes reduced the proportion of cells in the G1 phase by 1.0%, 18.5%, 15.6% and 11.1% respectively, and increased the proportion of cells in the S phase by 6.5%, 7.6%, 3.9% and 4.2% respectively, indicating that the complexes arrested the HeLa cell cycle in the S phase, which was consistent with the mechanism of the effect of the individual complexes on the HeLa cell cycle, suggesting that the binding of HSA to the [Fe 2 (µ-SR) 2 (NO) 4 complex had little effect on its pathway of arresting the proliferation of HeLa cells. Under 420 nm light treatment, compared with the control group, the addition of the complexes reduced the proportion of cells in the S and G2 phases, while the proportion of cells in the G1 phase increased by 24.0%, 19.6%, 16.3% and 20.3% respectively, indicating that under the action of light, the complexes arrested the HeLa cell cycle in the G1 phase, and the light treatment after binding to serum albumin changed the pathway of the [Fe 2 (µ-SL) 2 (NO) 4 complex in arresting cell proliferation.

[0055] The results of cell apoptosis are as Figure 11 shown. In the absence of light, compared with the control group, the addition of the four complexes decreased the percentage of live cells (Annexin V-FITC- / PI-), the percentage of early apoptotic cells (Annexin V-FITC+ / PI-) changed little compared with the control group, while the percentage of late apoptotic cells (Annexin V-FITC+ / PI+) increased from 7.19% to 22.1%, 20.1%, 16.4% and 30.1% respectively, indicating that under the condition of no light, the [Fe 2 (µ-SL) 2 (NO) 4 complex still promoted the apoptosis of HeLa cells after being complexed with serum albumin.

[0056] Under the treatment of 420 nm light irradiation, compared with the control group, the complex still promoted the apoptosis of HeLa cells. The percentage of early apoptotic cells (Annexin V-FITC+ / PI-) increased from 4.09% to 5.57%, 7.23%, 6.95% and 10.80% respectively, and the percentage of late apoptotic cells (Annexin V-FITC+ / PI+) increased from 8.32% to 37.80%, 47.70%, 39.00% and 31.90% respectively. The proportions of early apoptotic and late apoptotic cells both increased to varying degrees, indicating that the modified nitrosyl iron sulfur complex promoted the apoptosis of HeLa cells after binding to HSA under light conditions.

[0057] Example 11: Using a ZEISS LSM-880 confocal laser scanning microscope and a selective NO fluorescence probe DAX-J2 Red, the release of nitric oxide from the complexes 1-4 and serum albumin complexes in the cell system was detected in real time under light and dark conditions. The method is as follows: Add 10 mM NO probe DAX-J2 Red to an EP tube containing PBS buffer solution and dilute it to 5 μM. Take 1 mL and add it to a confocal dish containing cultured HeLa cells and incubate for 20 min. Take 10 μL of the pre-prepared 2 mM complex sample and add it to the confocal dish and continue to incubate for 20 min. Finally, wash it 3 times with PBS, add 1 mL of PBS buffer solution and observe it under a confocal microscope; then irradiate it with a 420 nm LED monochromatic light source for 0, 5, 10, 15 min respectively, and observe the imaging of NO in HeLa cells after irradiation. In the experimental group, DAX-J2 Red and 20 μM of the complex were added respectively. The real-time fluorescence imaging results of nitric oxide of the complexes formed by the combination of four different ligand-modified nitrosyl iron sulfur complexes and serum albumin at different light irradiation times are as Figure 12 shown, Figure 12 in (a) is the complex 1, namely [Fe 2 (µ-SC 9 H 11 O 2 ) 2 (NO) 4 and the serum albumin complex; (b) is the complex 2, namely [Fe 2 (µ-SC 8 H 6 N) 2 (NO) 4 and the serum albumin complex; (c) is the complex 3, namely [Fe 2 (µ-SC 10 H 11 O 2 ) 2 (NO)4 complex with serum albumin; (d) is complex 4, namely [Fe 2 (µ-SC 7 H 8 BO 2 ) 2 (NO) 4 complex with serum albumin.

[0058] DAX-J2 Red is a specific NO fluorescent probe. When it reacts with NO, it emits red fluorescence, which can selectively identify the production of NO. Figure 12 In (a), (b), (c) and (d), 5 µM DAX-J2 Red and 20 µM of complex 1, complex 2, complex 3 and complex 4 were added respectively. Using a 420 nm light source, HeLa cells were irradiated for 0, 5, 10, 15 min respectively, and the laser confocal imaging analyzer was used to measure the production of NO in HeLa cells in real time.

[0059] It can be seen from Figure 7 that when the complex is added to the solution, but there is no light irradiation or at t = 0, no or only very weak NO fluorescence can be observed; but when irradiated with light for a certain time, obvious fluorescence for capturing NO can be observed. As the light irradiation time increases, the generated NO fluorescence gradually increases, and its intensity is stronger than that of the complex alone. Figure 12 The results confirmed the release of NO by the photoinduced complex with serum albumin in the cell system.

[0060] In summary, the above experiments showed that the iron-sulfur complexes modified with four ligands can achieve photo-regulated NO release and can be used as NO donors in biomedical treatment. Complexes 1-4 modified with different ligands have obvious activities in inhibiting the growth of human cervical cancer HeLa cells. The IC 50 value of complex 1 is 6.31 µM, the IC 50 value of complex 2 is 6.29 µM, the IC 50 value of complex 3 is 7.30 µM, and the IC 50 value of complex 4 is 6.03 µM. Complexes 1-4 can be applied in the screening and preparation of anti-tumor lead compound drugs.

[0061] Through photoexcitation, the release of nitric oxide by the complex can be quantitatively regulated, and there are certain differences in the rates of nitric oxide release by complexes modified with different ligands, which can be applied in the preparation of nitric oxide donors in photo-regulated solution systems and cell systems. In addition, complexes 1-4 can all recognize and bind to serum albumin, and the complex still has obvious activities in inhibiting the growth of human cervical cancer cells. The IC 50They were 5.64, 5.13, 6.13, and 5.45 μM in sequence. By binding with serum albumin, the level of the complex releasing nitric oxide donors was further increased. Therefore, serum albumin can be used as a carrier of the complex in biomedicine.

[0062] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A side chain modified nitrosyl iron-sulfur complex, characterized in that: The side chain modified nitrosyl iron sulfur complex is: [Fe2S2(NO)4] 2- As raw material, nitrosyl iron sulfur complex [Fe2(µ-SL)2(NO)4] with halogenated hydrocarbon as ligand, wherein L is a ligand modified on the S atom, wherein the halogenated hydrocarbon is 3,5-dimethoxybenzyl bromide, 3-bromomethylbenzonitrile, 4-bromomethylphenylacetic acid methyl ester or 3-bromomethylphenylboronic acid, and its structural schematic diagram is as follows: 。 2. A method for preparing the side chain modified nitrosyl iron-sulfur complex according to claim 1, characterized in that: The following steps are involved: (1) Preparation of (Me4N)2[Fe2S2(NO)4] Raw materials: 24.0 g sodium sulfide nonahydrate Na2S·9H2O, 2.4 g sodium nitrite NaNO2 and 11.0 g sodium hydroxide NaOH were mixed, and 120 mL distilled water was added and stirred to dissolve. After all the solution was dissolved, 22.4 g ferrous sulfate heptahydrate FeSO4·7H2O was slowly added to the solution. The solution immediately turned black. After adding a magnet, it was heated and stirred in an oil bath and boiled for 50 min. It was then filtered while hot. The filtrate was placed in an 80°C water bath. 11 mL of saturated tetramethylammonium chloride (CH3)4NCl aqueous solution was slowly added to the filtrate while stirring. The solution was then allowed to stand. After cooling to room temperature, a large amount of sticky solid was produced at the bottom. The supernatant was removed, and the solid was dissolved with distilled water. The solid was filtered and recrystallized to obtain dark red-brown crystals with a metallic luster, namely (Me4N)2[Fe2S2(NO)4]; (2) Preparation of crude nitrosyl iron-sulfur complex: The precursor nitrosyl iron-sulfur complex (Me4N)[Fe2S2(NO)4] obtained in step (1) and a halogenated hydrocarbon ligand are subjected to a coordination reaction in a methanol or water solvent at a molar ratio of 1:2 at room temperature in the dark for 5 hours. The resulting coordination reaction solution is subjected to rotary evaporation to remove the solvent and dried to obtain a crude nitrosyl iron-sulfur complex; (3) Purification: the crude product of the nitrosyl iron-sulfur complex in step (2) is dissolved and then separated by silica gel column chromatography; The selected halogenated hydrocarbon ligand is 3,5-dimethoxybenzyl bromide, 3-bromomethylbenzonitrile or 4-bromomethylphenylacetic acid methyl ester, the silica gel column chromatography separation is the first silica gel column chromatography separation, and the eluent is CH2Cl2; The selected halogenated hydrocarbon is 3-bromomethylphenylboronic acid, the silica gel column chromatography separation is the second silica gel column chromatography separation, the eluent is a mixed reagent of CH2Cl2 and CH3OH, and the volume ratio of CH2Cl2 and CH3OH in the eluent is 100:

1.

3. The preparation method according to claim 2, characterized in that: The temperature of the coordination reaction in step (2) is 25° C. and the time is 5 h; the coordination reaction is carried out in the dark.

4. Use of the side chain modified nitrosyl iron-sulfur complex according to claim 1 in the preparation of a serum albumin complex; characterized in that: The serum albumin complex comprises serum albumin and a side-chain modified nitrosyl iron-sulfur complex loaded on the serum albumin, and the molar ratio of the serum albumin to the side-chain modified nitrosyl iron-sulfur complex is 1:1-1:

2.

5. The use according to claim 4, characterized in that: The preparation method of the serum albumin complex is as follows: dissolving the side chain modified nitrosyl iron-sulfur complex in an organic solvent DMSO, and diluting with deionized water to obtain a complex solution; mixing the complex solution with a serum albumin buffer solution for a binding reaction, thereby obtaining the serum albumin complex.

6. Use of the side-chain modified nitrosyl iron-sulfur complex of claim 1 or the serum albumin complex of claim 4 in screening or preparing anti-tumor lead compounds or drugs.

7. The use according to claim 6, characterized in that: The anti-tumor drug is an anti-cervical cancer drug.

8. Use of the side-chain modified nitrosyl iron-sulfur complex of claim 1 or the serum albumin complex of claim 4 in the preparation of a light-regulated nitric oxide donor reagent.

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