CN105732723A - Ruthenium (II) polypyridine complex and preparation method and application thereof - Google Patents

Ruthenium (II) polypyridine complex and preparation method and application thereof Download PDF

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CN105732723A
CN105732723A CN201610157250.5A CN201610157250A CN105732723A CN 105732723 A CN105732723 A CN 105732723A CN 201610157250 A CN201610157250 A CN 201610157250A CN 105732723 A CN105732723 A CN 105732723A
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ruthenium
telomere
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serobila
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CN105732723B (en
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尹盼盼
尚英辉
姚天明
石硕
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Tongji University
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    • C07F15/0006Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table compounds of the platinum group
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    • C07F15/0053Ruthenium compounds without a metal-carbon linkage

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Abstract

The invention relates to ruthenium (II) polypyridine complex.The complex uses 2-(1, 10-phenanthroline monohydrate-5-base)-benzimidazole) as the main ligand and 2, 2'-bipyridine or 1, 10'-phenanthroline as the auxiliary ligand, and the chemical formula of the complex is [Ru(bpy)2pbi]2+ or [Ru(phen)2pbi]2+.A preparation method of the complex includes the steps of firstly, preparing pbi; secondly, synthesizing a ruthenium metal compound with the auxiliary ligand group; thirdly, synthesizing the target ruthenium (II) polypyridine complex; fourthly, using silica gel chromatography column separation to obtain the product.The ruthenium (II) polypyridine complex is used for preparing medicine combining with telomere G-quadruplex to increase the stability of the telomere G-quadruplex.Compared with the prior art, the ruthenium (II) polypyridine complex prepared by the method has the advantages that the ruthenium (II) polypyridine complex can recognize, combine and stabilize the telomere G-quadruplex, inhibit telomerase activity and regulate proto-oncogene expression, and a theoretical foundation is provided for anticancer medicine screening.

Description

A kind of ruthenium (II) multi-pyridine ligand and preparation and application thereof
Technical field
The present invention relates to metal complex, be specifically related to a kind of ruthenium (II) multi-pyridine ligand and preparation and application thereof.
Background technology
Nucleic acid is the material base of biological heredity.The research of nucleic acid is not only facilitated us and explores the origin of life, and be also particularly important when solving gene-associated diseases.In addition to traditional double-spiral structure, nucleic acid there is also a lot of non-classical configuration, the most noticeable surely belongs to G-tetra-serobila (G4) structure, and being primarily due to it is that people regulate the Effective target site of biological function by chemical interference.G4 is the nucleic acid structure of a kind of four chains, is to be piled up by the G-tetrad formed by Huo Shi (Hoogsteen) pairing between G base to form, and this assembling phenomenon is extremely widespread in rich in the nucleotide sequence of G.Due to rich G sequence rich content in a lot of functional gene regions (such as the telomere of eukaryotic cell, the promoter of some important proto-oncogene), therefore G4 is considered as suppression telomerase activation and the critical medication target spot of regulation and control expression of proto-oncogenes.Research display, the human body telomeric dna (Tel22, also known as 22AG) containing 22 bases can form four serobilas of various configuration under Different Alkali metal ion existence condition.At Na+In ion buffer, 22AG uses antiparallel G4 conformation, and at K+In ion buffer, but present parallel/antiparallel and mix type conformation.
Ruthenium outermost layer has 4d75s1Structure, its common valence state is Ru (I), Ru (II) and Ru (III), and is easily formed the coordination compound of hexa-coordinate.Transition metal ruthenium (II) multi-pyridine ligand has the DNA binding ability of uniqueness, good electrochemistry, photochemistry, Photophysics and abundant fluorescent property, and has hypotoxicity, the feature easily absorbing and easily draining.As Chinese patent CN 102464676 A discloses a kind of ruthenium (II) multi-pyridine ligand, described ruthenium (II) multi-pyridine ligand is with two pyridines [3,2-a, 2 ', 3 '-c] and azophenlyene-11,12-imidazoles is main part, and using bipyridyl or adjacent luxuriant and rich with fragrance quinoline as ruthenium (II) multi-pyridine ligand of assistant ligand, this ruthenium (II) multi-pyridine ligand is used for G-tetra-serobila DNA molecular photoswitch.But this compound is only used for the searching of G4, it is used for suppressing telomerase activation and regulation and control expression of proto-oncogenes not to have good effect for being combined with G-4 tetrad.
Summary of the invention
Defect that the purpose of the present invention is contemplated to overcome above-mentioned prior art to exist and a kind of ruthenium (II) multi-pyridine ligand having identification ability, binding ability and stabilizing power to telomeric dna and preparation and application thereof are provided.
The purpose of the present invention can be achieved through the following technical solutions: a kind of ruthenium (II) multi-pyridine ligand, this coordination compound is with 2-(1,10-phenanthrene quinoline-5-base)-benzimidazole) it is main part, coordination compound is with 2,2 '-bipyridyl or 1,10 '-adjacent luxuriant and rich with fragrance quinoline is co-ligand, and the chemical formula of described coordination compound is [Ru (bpy)2pbi]2+Or [Ru (phen)2pbi]2+, the structural formula of described coordination compound is:
Wherein, bpy is 2,2 '-bipyridyl, and phen is 1,10 '-adjacent luxuriant and rich with fragrance quinoline, and pbi is 2-(1,10-phenanthrene quinoline-5-base)-benzimidazole).
Wherein Ru is bivalence in this coordination compound, and therefore this coordination compound is ruthenium (II) multi-pyridine ligand.
The synthesis step of the part that the present invention uses is simple, and productivity is high so that production cost is substantially reduced, it addition, main part uses the benzimidazole of synthesis, this material is exactly a lot of pharmaceutical intermediates, and for other parts, toxicity is less relatively, and medical potentiality is higher.
A kind of preparation method of ruthenium (II) multi-pyridine ligand as mentioned above, this preparation method includes following step:
(1) synthesis of pbi: weigh o-phenylenediamine by the mol ratio of 1:1:2,5-aldehyde radical Phen and sodium sulfite, add DMF dissolving and obtain solution, it is heated at reflux until solution starts precipitation occur, in solution, add water obtain yellow mercury oxide, by yellow mercury oxide sucking filtration, washing, i.e. obtain pbi after drying, stand-by;
(2) RuCl is weighed by the mol ratio of 1:1:23·3H2O, chloride hydrate lithium and a co-ligand, add DMF dissolving and obtain solution, be heated at reflux and obtain solidliquid mixture under the protection of protection gas; adding acetone after being cooled to room temperature, stirring makes solid dispersed, preserves 15~30h at-25~-4 DEG C; sucking filtration obtains purple black crystal, stand-by;
(3) purple to step (1) gained pbi and step (2) gained black crystal is dissolved in the mixed solution of ethylene glycol and water; in being heated at reflux under the protection of protection gas; it is then cooled to room temperature; add water dilution; sucking filtration obtains filtrate; adding hexafluoro in gained filtrate and close ammonium phosphate or sodium perchlorate, stirring is precipitated, and standing, sucking filtration, washs, is vacuum dried and obtains thick product;
(4) step (3) gained thick product silica gel chromatography is separated, obtain powdery product after drying and be ruthenium (II) multi-pyridine ligand.Ruthenium (II) multi-pyridine ligand prepared by above-mentioned reaction condition, productivity is high.
The synthesis step of the present invention is simple, only needs a step just can synthesize main part, and without purifying, productivity is high.Part earlier above synthesizes simple reduction production cost.
Described add water in solution volume is liquor capacity 5~10 times of step (1), the temperature being heated at reflux described in step (1) is 120~180 DEG C, and the time being heated at reflux is 5~12h.
Step (2) described co-ligand includes bipyridyl or adjacent luxuriant and rich with fragrance quinoline.
Amount is described solidliquid mixture 4~6 times of step (2) described addition acetone, the temperature being heated at reflux described in step (2) is 130~150 DEG C, and the time being heated at reflux is 5~10h.
Step (2), step (3) described protection gas are noble gas, including argon, nitrogen, helium.
Step (3) described ethylene glycol and H2Ethylene glycol and H in the mixed solvent of O2The mol ratio of O is 7:1, the hexafluoro added closes ammonium phosphate or the quality of sodium perchlorate is 1g:(15~20 with the ratio of the mole of ethylene glycol) mol, described washing is to wash at least 3 times with water and ether successively, being heated at reflux temperature described in step (3) is 110~130 DEG C, and the time of being heated at reflux is 5~8h.
The ratio that eluant is acetonitrile and toluene, acetonitrile and toluene used by step (4) described silica gel chromatography is acetonitrile: toluene=3:1.
The application of a kind of ruthenium (II) multi-pyridine ligand as mentioned above, described coordination compound is used for being combined with telomere G-tetra-serobila, improving the medicine of the stability of described telomere G-tetra-serobila for preparation.
The action principle of this coordination compound is as follows: states ruthenium (II) multi-pyridine ligand and uses the model of action of pi-pi accumulation to interact with telomere G-tetra-serobila; in normal somatic cell; telomere can shorten along with the division of cell, when its length reaches critical point, cell will become feeble and die.And in major part tumor cell and cancerous cell, telomere is lengthened by telomerase, compensate for its loss in normal cell, makes cancerous cell infinitely divide and is able to immortality.Research shows, the G4 that telomerase is formed can effectively suppress the activity of telomerase so that G4 becomes the important target spot of cancer therapy drug.It is possible to the molecule stablizing G4 structure becomes potential cancer therapy drug.
Telomere G-tetra-serobila that described telomere G-tetra-serobila is behaved, this telomere G-tetra-serobila includes 22 bases.
The application of obtained ruthenium (II) coordination compound can pass through UV spectrum titration, and the spectroscopic data peak value display obtained reduces and with red shift, ruthenium (II) coordination compound described in explanation occurs combination with telomere G-tetra-serobila DNA.
Ruthenium (II) coordination compound passes through the raising to telomere G-tetra-serobila melting temperature of the DNA melting temperature determination experiment of PCR instrument, illustrates that coordination compound can stablize G-tetra-stranded structure.
The means such as double light path uv-vis spectra and PCR instrument are utilized to have detected ruthenium (II) coordination compound of designed synthesis to G-tetra-serobila binding ability and stabilizing power.
Coordination compound uses double beam system with the measurement of the uv-vis spectra of DNA effect, and instrument is Perkin Elmer lambda Bio 40 type ultraviolet-uisible spectrophotometer, and cuvette is 10mm x10mm quartz colorimetric utensil, sweep limits 230-600nm.Shown that ruthenium (II) coordination compound and telomere G-tetra-serobila have stronger binding ability by the red shift occurred in ultraviolet spectra and blue shift
PCR instrument (BIORAD icycler (iQ5)) is carried out apply FRET technology, test ruthenium (II) coordination compound to fluorescent label DNA F22T (melting temperature T of sequence (FAM-[(G3T2A) 3AG3]-TAMRA)mThe change of value.Thus reflect the Different Complex difference to G-tetra-serobila DNA Thermodynamically stable effect.
Compared with prior art, beneficial effects of the present invention is embodied in:
(1) present invention has significantly interaction by description of test G4 and DNA that the obvious red shift of ultraviolet chromatogram and hypochromic effect and PCR temperature raise, and the structure of G4 can be stablized, due to the more difficult destruction of the rigid structure of G4, so speculating that both model of action are the patterns of outside stacking binding;
(2) synthesis step of part is simple, and productivity is high, the benzimidazole of synthesis originally a lot of pharmaceutical intermediates, so toxicity may be less for other parts relatively, medical potentiality is higher.
Accompanying drawing explanation
Fig. 1 is embodiment 1 gained coordination compound and telomere G-tetra-serobila uv absorption spectra in potassium ion buffer solution;
Fig. 2 is embodiment 1 gained coordination compound and telomere G-tetra-serobila uv absorption spectra in sodium ion buffer solution;
Fig. 3 is embodiment 1 gained coordination compound and telomere G-tetra-serobila melting temperature experimental curve diagram in potassium ion buffer solution;
Fig. 4 is embodiment 2 gained coordination compound and telomere G-tetra-serobila uv absorption spectra in potassium ion buffer solution;
Fig. 5 is embodiment 2 gained coordination compound and telomere G-tetra-serobila uv absorption spectra in sodium ion buffer solution;
Fig. 6 is embodiment 2 gained coordination compound and telomere G-tetra-serobila melting temperature experimental curve diagram in sodium ion buffer solution.
Detailed description of the invention
Elaborating embodiments of the invention below, the present embodiment is implemented under premised on technical solution of the present invention, gives detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.
Embodiment 1
A kind of ruthenium (II) multi-pyridine ligand, this coordination compound is with 2-(1,10-phenanthrene quinoline-5-base)-benzimidazole) it is main part, coordination compound is with 2,2 '-bipyridyl is co-ligand, and the chemical formula of described coordination compound is [Ru (bpy)2pbi]2+, the structural formula of described coordination compound is:
It is denoted as a1
This coordination compound is through the following steps that prepare:
1. synthetic ligands pbi (pbi=2-(1,10-Phenanthrolin-5-yl)-benzo [d] imidazole) obtain yellow powder: in single neck flask, add 0.001mol o-phenylenediamine, 0.001mol 5-aldehyde radical Phen and 0.002mol sodium sulfite, and add 3mL DMF dissolving, it is heated to reflux 6 hours at 150 DEG C, obtain brown solution, and have and precipitate generation on a small quantity.Adding 30mL water in reactant liquor has a large amount of yellow mercury oxide to produce, and sucking filtration washes filter cake with water for several times, vacuum drying.Obtain yellow powder 0.255g, productivity 86%.
2. three-neck flask adds 6mmol ruthenium trichloride (RuCl3·3H2O), 6mmol mono-chloride hydrate lithium, and 12mmol bipyridyl, it is dissolved in 10ml DMF, under argon shield, is heated to reflux 8 hours in 140 DEG C.It is cooled to room temperature, adds 50mL acetone, dispersed to bottom solid with Glass rod stirring, put refrigerator freezing overnight.Sucking filtration obtains atropurpureus solid.All solids adding the alcohol heating reflux 0.5 hour of 150mL, is cooled to room temperature, sucking filtration obtains atropurpureus crystallite, is washed till the most colourless with frozen water, is dried, obtains cis-[Ru (bpy)2Cl2]·2H2O.Calculate with bipyridyl, productivity about 65%.
The most accurately weigh 0.4mmol cis-[Ru (bpy)2Cl2]·2H2The product of O, 0.229g (0.6mmol) step 1, is dissolved in 35mL ethylene glycol and 5mL H2The mixed solvent of O, under argon shield, refluxes 6 hours in 120 DEG C.It is atropurpureus that reaction starts solution, gradually becomes red along with reaction tends to perfect solution color.Reaction is cooled to room temperature after terminating, add the dilution of 50mL water, and sucking filtration removes unreacted part, obtains claret clear filtrate.In filtrate, add 2g hexafluoro close ammonium phosphate, stirring, obtain peony precipitation, after standing a night, sucking filtration, use water and ether washing filter cake respectively for several times, vacuum drying.Crude product is crossed 200~300 mesh neutral alumina columns separate, with acetonitrile/toluene (3:1, v/v) eluting, red zone eluent in the middle of collecting, being placed on fume hood with preservative film sealing makes solvent volatilize, and can obtain product about 0.2~0.3g, productivity about 50%.
Gained coordination compound has been carried out hydrogen spectrum (1H NMR) and Electrospray Mass Spectrometry (ES-MS) sign, result is as follows:
1H-NMR(DMSO-d6, 400Hz): 13.46 (1H, s), 10.08 (1H, d), 8.88 (6H, m), 8.23 (4H, dd), 8.13 (2H, t), 8.01 (1H, dd), 7.96 (1H, dd), 7.85 (3H, t), 7.65 (5H, m), 7.34 (4H, dd, TOF-MS for a1:m/z 709.1 (M-2PF6-H), 355.07 (M-2PF6/2)。
By gained ruthenium (II) multi-pyridine ligand for being combined with telomere G-tetra-serobila, the stability of described telomere G-tetra-serobila can be improved, thus suppress telomerase activation, regulate and control expression of proto-oncogenes.Telomere G-tetra-serobila that this telomere G-tetra-serobila is behaved, this telomere G-tetra-serobila includes 22 bases.
Ruthenium (II) multi-pyridine ligand is for comprising the following steps with telomere G-tetra-serobila:
(1) Tris-HCl buffer:
Buffer A: 10mM Tris, 100mM KCl, pH=7.0;
Buffer B: 10mM Tris, 100mM NaCl, pH=7.0;
General compound method: accurately weigh 0.303g Tris salt, 1.865g KCl or 1.460g NaCl, be completely dissolved with 60mL sterilizing triple distillation water, pH value is slowly regulated to 7.0 with dilute hydrochloric acid, proceed to 250ml volumetric flask, with triple distillation water constant volume, standby after mix homogeneously.
(2) phosphate buffer:
Buffer C:10mM KH2PO4-K2HPO4, 100mM KCl, pH=7.0.
Compound method: take 0.174g tri-hypophosphite monohydrate hydrogen dipotassium and 0.746g KCl with electronic balance scale, be configured to the KCl that concentration is 100mM, the K of 10mM with the volumetric flask that specification specification is 100mL and triple distillation water2HPO4Solution;Weigh 0.136g potassium dihydrogen phosphate and 0.746gKCl, be configured to the KCl that concentration is 100mM, the KH of 10mM by above-mentioned same compound method2PO4Solution.Take above-mentioned liquor capacity 61.5mL dipotassium hydrogen phosphate solution and 38.5mL potassium dihydrogen phosphate is mutually mixed, i.e. can obtain the potassium phosphate buffer of 10mM containing the KCl that concentration is 100mM.
(3) preparation of complex solution:
Accurately weigh 2~3mg coordination compounds (depending on complex molecule amount, the ruthenium complex expection concentration prepared herein is 200 μMs, volume is 10mL, preparation container is 15mL Corning centrifuge tube, so the theoretical value that needs weigh is: molecular weight/1000*2mg), first dissolve with 50-100 μ L DMSO, then be settled to 10mL with pure water, obtain the coordination compound storing solution of 200 μMs.
(4) preparation of DNA solution and the mensuration of concentration:
G-tetra-serobila DNA (22AG)
Compound method: take the 22AG DNA of about 10OD, dissolves with the buffer A of corresponding volume and B, with heating in water bath to 90 DEG C and keep 5 minutes after sealing, puts into cold preservation more than 24 hours in 4 DEG C of refrigerators after being slowly cooled to room temperature, standby.
The application of obtained ruthenium (II) coordination compound can pass through UV spectrum titration, and step is as follows:
Take the coordination compound storing solution (200uM) of 70uL, in 2000uL buffer Tris-KCl pH 7.0 or Tris-NaCl pH 7.0 to 10mm × 10mm quartz colorimetric utensil, concussion mixing, it is placed in ultraviolet light absorption photometer sample cell.Reference cell puts the buffer display scanning recording curve containing 2000uL Tris-KCl pH 7.0 or Tris-NaCl pH 7.0.In sample cell and reference cell, add the 22AG DNA solution (100uM Tris salt buffer) of 10uL the most respectively, add mixing, scanning record curve obtained.Repetitive operation at least four times titration and no longer change, illustrate that titration reaches saturated.Preserving data, Origin maps, and result is as depicted in figs. 1 and 2.
Spectroscopic data peak value display has obvious hypochromic effect and red shift, illustrates that coordination compound is strong with G-tetra-serobila DNA effect.But, it is extremely difficult for being inserted into for the part of these four ruthenium complex between two G-tetrads of G-tetra-serobila, this is because four serobilas are a rigidity and stable structure, if to destroy its structural intergrity is to need to consume higher energy.Therefore speculate that coordination compound, by being stacked into G-tetra-serobila distal portion and G-tetra-serobila effect, is namely called outside accumulation mode.
Ruthenium (II) coordination compound is measured by the DNA melting temperature of PCR instrument, and concrete operation step is as follows:
Application FRET technology, tests the present embodiment gained coordination compound to fluorescent label DNA F22T (melting temperature T of sequence (FAM-[(G3T2A) 3AG3]-TAMRA)mThe change of value.Thus reflect the Different Complex difference to G-tetra-serobila DNA Thermodynamically stable effect.FRET experiment is carried out in PCR instrument, use the dipotassium hydrogen phosphate containing 100mM KCl or disodium hydrogen phosphate buffer solution (10mM, pH=7.0), add annealing and form the F22T (250nM) of G-tetra-serobila, and the coordination compound of finite concentration ratio, jointly hatch 1 hour, carry out TmValue is measured as shown in Figure 3.
Result shows, in the presence of having coordination compound (0.5uM G4,100mM NaCl), the Tm value of the F22T of G-tetra-serobila is less;Along with addition and the increase of concentration of coordination compound, the Tm value of F22T is gradually increased.Ultimately joining coordination compound concentration to be 7 μMs and make G-tetra-serobila Tm value increase 3 DEG C, therefore, under potassium buffer, coordination compound has concentration dependent to the Stabilization of G-tetra-serobila.
Embodiment 2
A kind of ruthenium (II) multi-pyridine ligand, this coordination compound is with 2-(1,10-phenanthrene quinoline-5-base)-benzimidazole) it is main part, coordination compound is with 1,10 '-adjacent luxuriant and rich with fragrance quinoline for co-ligand, and the chemical formula of coordination compound is [Ru (phen)2pbi]2+, the structural formula of described coordination compound is:
It is denoted as a2
This coordination compound is through the following steps that prepare:
1. synthetic ligands pbi (pbi=2-(1,10-Phenanthrolin-5-yl)-benzo [d] imidazole) obtain yellow powder: in single neck flask, add 0.001mol o-phenylenediamine, 0.001mol 5-aldehyde radical Phen and 0.002mol sodium sulfite, and add 3mL DMF dissolving, it is heated to reflux 6 hours at 150 DEG C, obtain brown solution, and have and precipitate generation on a small quantity.Adding 30mL water in reactant liquor has a large amount of yellow mercury oxide to produce, and sucking filtration washes filter cake with water for several times, vacuum drying.Obtain yellow powder 0.255g, productivity 86%.
2. three-neck flask adds 6mmol ruthenium trichloride (RuCl3·3H2O), 6mmol mono-chloride hydrate lithium, and 12mmol Phen, it is dissolved in 10ml DMF, under argon shield, is heated to reflux 8 hours in 140 DEG C.It is cooled to room temperature, adds 50mL acetone, dispersed to bottom solid with Glass rod stirring, put refrigerator freezing overnight.Sucking filtration obtains atropurpureus solid.All solids adding the alcohol heating reflux 0.5 hour of 150mL, is cooled to room temperature, sucking filtration obtains atropurpureus crystallite, is washed till the most colourless with frozen water, is dried.Calculate with bipyridyl, productivity about 65%.
The most accurately weigh 0.4mmol cis-[Ru (bpy)2Cl2]·2H2The product of O, 0.229g (0.6mmol) step 1, is dissolved in 35mL ethylene glycol and 5mL H2The mixed solvent of O, under argon shield, refluxes 6 hours in 120 DEG C.It is atropurpureus that reaction starts solution, gradually becomes red along with reaction tends to perfect solution color.Reaction is cooled to room temperature after terminating, add the dilution of 50mL water, and sucking filtration removes unreacted part, obtains claret clear filtrate.In filtrate, add 2g hexafluoro close ammonium phosphate, stirring, obtain peony precipitation, after standing a night, sucking filtration, use water and ether washing filter cake respectively for several times, vacuum drying.Crude product is crossed 200~300 mesh neutral alumina columns separate, with acetonitrile/toluene (5:1, v/v) eluting, red zone eluent in the middle of collecting, being placed on fume hood with preservative film sealing makes solvent volatilize, and can obtain product about 0.2~0.3g, productivity about 50%.
Gained coordination compound has been carried out hydrogen spectrum (1H NMR) and Electrospray Mass Spectrometry (ES-MS) sign, result is as follows:
1H-NMR(DMSO-d6, TMS, 400Hz): 13.46 (1H, s), 10.07 (1H, d), 8.95 (2H, s), 8.79 (5H, s), 8.41 (4H, s), 8.18 (3H, s), 8.09 (2H, s), 7.79 (8H, m), 7.35 (2H, d), TOF-MS for a1:m/z 757.1 (M-2PF6-H), 379.06 (M-2PF6/2)
By gained ruthenium (II) multi-pyridine ligand for being combined with telomere G-tetra-serobila, the stability of described telomere G-tetra-serobila can be improved, thus suppress telomerase activation, regulate and control expression of proto-oncogenes.Telomere G-tetra-serobila that this telomere G-tetra-serobila is behaved, this telomere G-tetra-serobila includes 22 bases.
The application of obtained ruthenium (II) coordination compound uses method same as in Example 1 to be measured, and result is as shown in Figure 4 and Figure 5.
Spectroscopic data peak value display has obvious hypochromic effect and red shift, illustrates that coordination compound is strong with G-tetra-serobila DNA effect.But, it is extremely difficult for being inserted into for the part of these four ruthenium complex between two G-tetrads of G-tetra-serobila, this is because four serobilas are a rigidity and stable structure, if to destroy its structural intergrity is to need to consume higher energy.Therefore coordination compound a is speculated2By being stacked into G-tetra-serobila distal portion and G-tetra-serobila effect, namely it is called outside accumulation mode.And compared by spectrogram, either in potassium ion or sodium ion buffer, the effect power of four kinds of coordination compounds and telomere G-tetra-serobila is all a2> a1
Ruthenium (II) coordination compound a obtained by the present embodiment2For the stabilizing power research of telomere G-tetra-serobila DNA, specifically comprise the following steps that
Application FRET technology, tests coordination compound a2 to fluorescent label DNA F22T (melting temperature T of sequence (FAM-[(G3T2A) 3AG3]-TAMRA)mThe change of value.Thus reflect the Different Complex difference to G-tetra-serobila DNA Thermodynamically stable effect.FRET experiment is carried out in PCR instrument, use the dipotassium hydrogen phosphate containing 100mM NaCl or disodium hydrogen phosphate buffer solution (10mM, pH=7.0), add annealing and form the F22T (250nM) of G-tetra-serobila, and the coordination compound of finite concentration ratio, jointly hatch 1 hour, carry out TmValue is measured as shown in Figure 6.
Result shows, (0.5uM G4,100mM NaCl), the T of the F22T of G-tetra-serobila in the presence of having coordination compoundmIt is worth less;Along with coordination compound a2Addition and the increase of concentration, the T of F22TmValue is gradually increased, as coordination compound a2When concentration increases to 7 μMs, G-tetra-serobila TmValue improves 5 DEG C.Therefore, under potassium buffer, coordination compound has concentration dependent, and coordination compound a to the Stabilization of G-tetra-serobila2The effect of stable G-tetra-serobila more than coordination compound a1
Embodiment 3
Use preparation method similar to Example 1, specifically include following step:
1. synthetic ligands pbi (pbi=2-(1,10-Phenanthrolin-5-yl)-benzo [d] imidazole) obtain yellow powder: in single neck flask, add 0.001mol o-phenylenediamine, 0.001mol 5-aldehyde radical Phen and 0.002mol sodium sulfite, and add 3mL DMF dissolving, it is heated to reflux 12 hours at 120 DEG C, obtain brown solution, and have and precipitate generation on a small quantity.In reactant liquor, add the water of 5 times of liquor capacities, have a large amount of yellow mercury oxide to produce, sucking filtration, wash filter cake with water for several times, vacuum drying.Obtain yellow powder 0.255g, productivity 86%.
2. three-neck flask adds 6mmol ruthenium trichloride (RuCl3·3H2O); 6mmol mono-chloride hydrate lithium; and 12mmol bipyridyl; it is dissolved in 10ml DMF, is heated to reflux 10 hours in 130 DEG C under argon shield, obtains solidliquid mixture; it is cooled to room temperature; add 4 times of volumes in the acetone of solidliquid mixture, dispersed to bottom solid with Glass rod stirring, preserve 15h at being placed in-25 DEG C.Sucking filtration obtains atropurpureus solid.All solids adding the alcohol heating reflux 0.5 hour of 150mL, is cooled to room temperature, sucking filtration obtains atropurpureus crystallite, is washed till the most colourless with frozen water, is dried, obtains cis-[Ru (bpy)2Cl2]·2H2O.Calculate with bipyridyl, productivity about 65%.
The most accurately weigh 0.4mmol cis-[Ru (bpy)2Cl2]·2H2The product of O, 0.229g (0.6mmol) step 1, is dissolved in 35mol ethylene glycol and 5mol H2The mixed solvent of O, under argon shield, refluxes 8 hours in 110 DEG C.It is atropurpureus that reaction starts solution, gradually becomes red along with reaction tends to perfect solution color.Reaction is cooled to room temperature after terminating, add the dilution of 50mL water, and sucking filtration removes unreacted part, obtains claret clear filtrate.In filtrate, add 2.33g hexafluoro close ammonium phosphate, stirring, obtain peony precipitation, after standing a night, sucking filtration, use water and ether washing filter cake respectively for several times, vacuum drying.Crude product is crossed 200~300 mesh neutral alumina columns separate, with acetonitrile/toluene (3:1, v/v) eluting, red zone eluent in the middle of collecting, being placed on fume hood with preservative film sealing makes solvent volatilize, and can obtain product about 0.2~0.3g, productivity about 50%.
Embodiment 4
Use preparation method similar to Example 1, specifically include following step:
1. synthetic ligands pbi (pbi=2-(1,10-Phenanthrolin-5-yl)-benzo [d] imidazole) obtain yellow powder: in single neck flask, add 0.001mol o-phenylenediamine, 0.001mol 5-aldehyde radical Phen and 0.002mol sodium sulfite, and add 3mL DMF dissolving, it is heated to reflux 5 hours at 180 DEG C, obtain brown solution, and have and precipitate generation on a small quantity.In reactant liquor, add the water of 10 times of liquor capacities, have a large amount of yellow mercury oxide to produce, sucking filtration, wash filter cake with water for several times, vacuum drying.Obtain yellow powder 0.255g, productivity 86%.
2. three-neck flask adds 6mmol ruthenium trichloride (RuCl3·3H2O); 6mmol mono-chloride hydrate lithium; and 12mmol bipyridyl; it is dissolved in 10ml DMF, is heated to reflux 5 hours in 150 DEG C under argon shield, obtains solidliquid mixture; it is cooled to room temperature; add 6 times of volumes in the acetone of solidliquid mixture, dispersed to bottom solid with Glass rod stirring, preserve 30h at being placed in-4 DEG C.Sucking filtration obtains atropurpureus solid.All solids adding the alcohol heating reflux 0.5 hour of 150mL, is cooled to room temperature, sucking filtration obtains atropurpureus crystallite, is washed till the most colourless with frozen water, is dried, obtains cis-[Ru (bpy)2Cl2]·2H2O.Calculate with bipyridyl, productivity about 65%.
The most accurately weigh 0.4mmol cis-[Ru (bpy)2Cl2]·2H2The product of O, 0.229g (0.6mmol) step 1, is dissolved in 35mol ethylene glycol and 5mol H2The mixed solvent of O, under argon shield, refluxes 5 hours in 130 DEG C.It is atropurpureus that reaction starts solution, gradually becomes red along with reaction tends to perfect solution color.Reaction is cooled to room temperature after terminating, add the dilution of 50mL water, and sucking filtration removes unreacted part, obtains claret clear filtrate.In filtrate, add 1.75g sodium perchlorate, stirring, obtain peony precipitation, after standing a night, sucking filtration, wash filter cake for several times with water and ether respectively, vacuum drying.Crude product is crossed 200~300 mesh neutral alumina columns separate, with acetonitrile/toluene (3:1, v/v) eluting, red zone eluent in the middle of collecting, being placed on fume hood with preservative film sealing makes solvent volatilize, and can obtain product about 0.2~0.3g, productivity about 50%.

Claims (10)

1. ruthenium (II) multi-pyridine ligand, it is characterised in that this coordination compound is with 2-(1,10-phenanthrene quinoline-5- Base)-benzimidazole) it is main part, coordination compound is with 2,2 '-bipyridyl or 1, and 10 '-adjacent luxuriant and rich with fragrance quinoline is co-ligand, described The chemical formula of coordination compound be [Ru (bpy)2pbi]2+Or [Ru (phen)2pbi]2+, the structural formula of described coordination compound is:
Wherein, bpy is 2,2 '-bipyridyl, and phen is 1,10 '-adjacent luxuriant and rich with fragrance quinoline, and pbi is 2-(1,10-phenanthrene quinoline-5- Base)-benzimidazole).
2. the preparation method of ruthenium as claimed in claim 1 (II) multi-pyridine ligand, it is characterised in that This preparation method includes following step:
(1) synthesis of pbi: weigh o-phenylenediamine, 5-aldehyde radical Phen and sulfurous acid by the mol ratio of 1:1:2 Hydrogen sodium, adds DMF dissolving and obtains solution, being heated at reflux until starting precipitation occur in solution, adding in solution Enter water and obtain yellow mercury oxide, by yellow mercury oxide sucking filtration, washing, i.e. obtain pbi after drying, stand-by;
(2) RuCl is weighed by the mol ratio of 1:1:23·3H2O, chloride hydrate lithium and a co-ligand, add DMF Dissolving obtains solution, is heated at reflux and obtains solidliquid mixture under the protection of protection gas, adds third after being cooled to room temperature Ketone, stirring makes solid dispersed, preserves 15~30h at-25~-4 DEG C, and sucking filtration obtains purple black crystal, stand-by;
(3) purple to step (1) gained pbi and step (2) gained black crystal is dissolved in the mixing of ethylene glycol and water In solution, in being heated at reflux under the protection of protection gas, being then cooled to room temperature, add water dilution, sucking filtration obtains Filtrate, adds hexafluoro in gained filtrate and closes ammonium phosphate or sodium perchlorate, and stirring is precipitated, standing, sucking filtration, Washing, vacuum drying obtain thick product;
(4) step (3) gained thick product silica gel chromatography is separated, obtain powdery product after drying and be Ruthenium (II) multi-pyridine ligand.
The preparation method of a kind of ruthenium (II) multi-pyridine ligand the most according to claim 2, its feature exists In, described add water in solution volume is liquor capacity 5~10 times of step (1), institute in step (1) Stating the temperature being heated at reflux is 120~180 DEG C, and the time being heated at reflux is 5~12h.
The preparation method of a kind of ruthenium (II) multi-pyridine ligand the most according to claim 2, its feature exists In, step (2) described co-ligand includes bipyridyl or adjacent luxuriant and rich with fragrance quinoline.
The preparation method of a kind of ruthenium (II) multi-pyridine ligand the most according to claim 2, its feature exists In, amount is described solidliquid mixture 4~6 times of step (2) described addition acetone, return described in step (2) The temperature of stream heating is 130~150 DEG C, and the time being heated at reflux is 5~10h.
The preparation method of a kind of ruthenium (II) multi-pyridine ligand the most according to claim 2, its feature exists In, step (2), step (3) described protection gas are noble gas, including argon, nitrogen, helium.
The preparation method of a kind of ruthenium (II) multi-pyridine ligand the most according to claim 2, its feature exists In, step (3) described ethylene glycol and H2Ethylene glycol and H in the mixed solvent of O2The mol ratio of O is 7:1, The hexafluoro added closes ammonium phosphate or the quality of sodium perchlorate is 1g:(15~20 with the ratio of the mole of ethylene glycol) mol, Described washing is to wash at least 3 times with water and ether successively, and being heated at reflux temperature described in step (3) is 110~130 DEG C, the time of being heated at reflux is 5~8h.
The preparation method of a kind of ruthenium (II) multi-pyridine ligand the most according to claim 2, its feature exists In, the ratio that eluant is acetonitrile and toluene, acetonitrile and toluene used by step (4) described silica gel chromatography For acetonitrile: toluene=3:1.
9. the application of ruthenium as claimed in claim 1 (II) multi-pyridine ligand, it is characterised in that described Coordination compound is used for being combined with telomere G-tetra-serobila, improving the medicine of the stability of described telomere G-tetra-serobila for preparation Thing.
The application of a kind of ruthenium (II) multi-pyridine ligand the most according to claim 8, it is characterised in that Telomere G-tetra-serobila that described telomere G-tetra-serobila is behaved, this telomere G-tetra-serobila includes 22 bases.
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