CN105669785B - 99mThe fan-shaped tree-shaped molecular complex of multivalence sugar of Tc marks and its purposes and the fan-shaped dendrimer ligands of multivalence sugar and its preparation method - Google Patents

99mThe fan-shaped tree-shaped molecular complex of multivalence sugar of Tc marks and its purposes and the fan-shaped dendrimer ligands of multivalence sugar and its preparation method Download PDF

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CN105669785B
CN105669785B CN201610151130.4A CN201610151130A CN105669785B CN 105669785 B CN105669785 B CN 105669785B CN 201610151130 A CN201610151130 A CN 201610151130A CN 105669785 B CN105669785 B CN 105669785B
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杨文江
刘宇
薛井泉
汪建军
张延华
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Institute of High Energy Physics of CAS
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Abstract

The invention provides formula to be99mTc (R HYNIC) (Tricine) m (L) n's99mThe fan-shaped tree-shaped molecular complex of multivalence sugar of Tc marks, additionally provide the purposes of the complex and the fan-shaped dendrimer ligands of multivalence sugar and its preparation method for preparing the complex.The fan-shaped dendrimer ligands of multivalence sugar provided by the invention part, optional synergy modes and radiopertechnetate can be formed together99mThe complex of Tc marks, the complex radiochemical purity is high, stability is good, biological property is good, specific activity is high, and liver uptake values and target to non-target ratio value are high, can be used as liver cell asialoglycoprotein receptor SPECT developer applications, and preparation process is easy, cost is low, there is extensive wide variety of value.

Description

99mThe fan-shaped tree-shaped molecular complex of multivalence sugar of Tc marks and its purposes and multivalence sugar Fan-shaped dendrimer ligands and its preparation method
Technical field
The present invention relates to radiopharmaceutical chemistry and clinical nuclear medicine technical field, and in particular to a kind of99mTc marks The fan-shaped tree-shaped molecular complex of multivalence sugar and its purposes, available for described in preparation99mThe multivalence sugar fan-shaped tree of the complex of Tc marks Shape molecule ligand and its preparation method.
Background technology
Liver diseases are always one of global important public hygiene problem.World Health Organization's statistics shows, entirely Ball HBV carrier about 3.5 hundred million, the 5% of world population is accounted for, HCV infection person about 1.7 hundred million.China is liver diseases state occurred frequently, hepatopathy Number accounts for global hepatopathy number 1/3, has 110,000 people to die from liver cancer every year, accounts for the 45% of whole world PLC mortality number.For The liver diseases such as chronic hepatitis, hepatic sclerosis and liver cancer are early diagnosed and carry out accurate evaluation to the functional status of liver, Be guiding treatment and evaluation prognosis effective foundation, also Hepatic Perioperative is significant (perioperative refer to from patient because From when needing the operative treatment to be in hospital, to time limit when leaving hospital only).
Molecular image (Molecular Imaging) can be studied under normal or pathological state by noninvasive imaging technique Molecular process.The wherein molecular image diagnostic techniques of nuclear medicine, including single photon emission computerized tomgraphy (SPECT) and positive electron Emission tomography images (PET), has been widely applied clinically, and it has high specific and sensitivity, is molecular image neck The important research direction in domain.The development of liver nuclear medicine image, particularly ASGPr (asialoglycoprotein receptor) developer Occur, a kind of noninvasive, sensitive, accurate method is provided for the assessment of liver function.
ASGPr (Asialoglycoprotein receptor) is asialoglycoprotein receptor, is a kind of cross-film egg In vain, it is predominantly located on mammalian liver parenchyma, participates in the liver metabolism process of haemocyanin.ASGPr can be certain Reflect effective hepatocyte function in degree, when the liver-injuring disease such as hepatitis, hepatic sclerosis or liver cancer occurs, its quantity and work Property suffers damage.Therefore the endocytic mechanism of ASGPr mediations can be utilized to prepare the molecular probe of ASGPr targetings, realizes liver The purpose of diagnosis and the evaluation of function.Vera in 1984 etc. is by chemical synthesis process by galacto configuration and human serum albumins Coupling obtains new lactose albumin (galactosyl-neoglycoalbumin, NGA), and passes through99mLiver imaging is carried out after Tc marks Research.Kubota in 1986 etc. have developed one kind99mTc mark GSA (99mTc-diethylenetriamine Pentaacetic acid-galactosyl-human serum albumin), by increasing bifunctional linking reagent DTPA (two Ethene pentaacetic acid) structure, flag condition is simplified, reduces non-specific binding.Japan utilizes ASGPr developers Three-dimensional hepatic model is established for nuclear medicine liver SPECT imagings, the imaging technique can truly, digitally reflect liver work( Can, so both the preoperative liver function of liver cirrhosis patient can correctly be assessed, aid forecasting operation risk, formulate treatment side Case, reduce the Peri operative mortality of operation on liver.
But use the macromolecule ASGPr probes of albumen or polymer backbone that there is higher molecular weight and wider molecule Measure distribution.The number (carbohydrate density) for the galactosyl being coupled on each of which molecular skeleton can produce to ASGPr affinity Influence, and then influence its pharmacokinetic property in vivo；On the other hand due to the larger molecule body of macromolecule ASGPr probes Product so that it is because cytophagy often has higher non-specific uptake in the organs such as liver, spleen, and just Clearance rate often in tissue is slower.The foundation of model, the processing of data impact when these factors all can be to imaging, and then Influence the accurate evaluation to liver function.Therefore the ASGPr molecular probes of small molecule are found, overcomes and uses high polymer material as bone The shortcomings that ASGPr molecular probes of frame, non-specific uptake is reduced, be the important topic that current the art needs to solve.
The content of the invention
It is existing to overcome99mTc marks complex liver SPECT imaging arts are present the defects of, of the invention mesh Be to provide one kind99mThe fan-shaped tree-shaped molecular complex of multivalence sugar of Tc marks, its chemical stability is strong, biological property is good.
It is a further object to provide described99mThe purposes of the complex of Tc marks.
The present invention's a further object is that offer is described for preparing99mThe multivalence sugar of the complex of Tc marks is fan-shaped tree-shaped Molecule ligand and its preparation method.
It is provided by the invention99mThe fan-shaped tree-shaped molecular complex of multivalence sugar of Tc marks, its formula are：
99mTc(R-HYNIC)(Tricine)m(L)n
Wherein, m represents 1 or 2, n expression 0 or 1；L represents synergy modes；R-HYNIC represents 2LA-G1-HYNIC, 4LA- One kind in structure shown in G2-HYNIC, 8LA-G3-HYNIC,
The 2LA-G1-HYNIC represents following structure：
The 4LA-G2-HYNIC represents following structure：
The 8LA-G3-HYNIC represents following structure：
LA groups therein represent following structure：
It is above-mentioned99mIn the complex of Tc marks, part, synergy modes can use the common ligand species in this area altogether, its In, part can be N- tri- (methylol) methylglycine (Tricine) altogether, and its molecular structural formula is：
Synergy modes preferably use three (3- sulfonyl-phenyls) phosphine sodium salts (TPPTS) or triphenylphosphine list sulfonic acid monosodium salt (TPPMS) one kind in, its molecular structural formula are respectively：
It is provided by the invention99mThe complex of Tc marks has to be imaged as liver cell asialoglycoprotein receptor SPECT The purposes of agent.
Present invention also offers for of the present invention99mThe fan-shaped dendrimer ligands of multivalence sugar of Tc mark complexs, tool There is any shown structure in 2LA-G1-HYNIC-R ', 4LA-G2-HYNIC-R ', 8LA-G3-HYNIC-R ',
The 2LA-G1-HYNIC-R ' represents following structure：
The 4LA-G2-HYNIC-R ' represents following structure：
The 8LA-G3-HYNIC-R ' represents following structure：
Wherein, R ' group represents diazanyl blocking group.
In the fan-shaped dendrimer ligands of above-mentioned multivalence sugar, R ' group represents the diazanyl blocking group that aldehyde compound is formed, The stability of part can be increased.The species present invention of aldehyde compound is not specially limited, as long as blocking group and energy can be formed Conveniently sloughed when forming complex, as preferred embodiment, including but not limited to benzaldehyde, 4- Dimethylaminobenzenes Formaldehyde, benzaldehyde -2- sodium sulfonates, 4- carboxyl benzaldehydes etc..
The preparation method of the fan-shaped dendrimer ligands of multivalence sugar provided by the invention, using ethylenediamine as initiation material, including Following steps：
1) amido of ethylenediamine is protected using blocking group R "；
2) product obtained by step 1) obtains intermediate G1, the intermediate with methyl acrylate, reacting ethylenediamine successively G1 obtains intermediate G2 with methyl acrylate, reacting ethylenediamine successively, or the intermediate G2 successively with methyl acrylate, second two Amine reacts to obtain intermediate G3；
3)-the NH of intermediate obtained by step 2)2With lactobionolactone react, formed intermediate 2LA-G1,4LA-G2 or 8LA-G3；
4) the blocking group R " in intermediate 2LA-G1,4LA-G2 or 8LA-G3 obtained by step 3) is removed；
5) the 6- hydrazino pyridine -3- formic acid that step 4) products therefrom is protected with R ' group is reacted and produced；
Wherein, the reaction equation of step 2) is as follows：
In above-mentioned preparation method, R " represents tert-butyl carbonyl, is removed in step 4) using trifluoroacetic acid.
In above-mentioned preparation method, the preparation of lactobionolactone and with-NH2Reaction can use prior art.
In above-mentioned preparation method, the reaction of step 5) can deposit in succinimide and dicyclohexylcarbodiimide (DCC) Reacted under.
It is provided by the invention99mThe fan-shaped tree-shaped molecular complex of multivalence sugar of Tc marks can be fan-shaped tree-shaped by multivalence sugar Molecule ligand prepares to be formed, and preparation process can be that existing method replaces to obtain by part.
Specifically, when without using synergy modes, its preparation process can be：
A. the fan-shaped dendrimer ligands of multivalence sugar are dissolved in buffer solution；According to part than common part be 1:5~2000, Preferably 1:5~1000 weight ratio, add (methylol) methylglycines (Tricine) of part N- tri- altogether.
B. it is 1 then to press reducing agent than part:0.5~800, it is preferably 1:1~800 weight ratio weighs reducing agent and molten In hydrochloric acid solution；It is added in step a resulting solutions, is well mixed, controls its pH between 5.5~6.5.
C. will be from medical99Mo-99mThe pertechnetate of acquisition is eluted in Tc generators99mTcO4 -Leacheate adds step b systems In standby solution, reacted 10~100 minutes under the conditions of boiling water bath after sealing.
D. labeled compound obtained by step c is purified by gel chromatographic columnses.
Buffer solution in above-mentioned steps a can be phosphate buffer, pH value 6.0.
Reducing agent in above-mentioned steps b can be by high price technetium (99mTcVIIO4 -) the conventional reduction agent of low price technetium is reduced to, Such as two hydrated stannous chloride (SnCl2·2H2O)。
In above-mentioned steps c, the blocking group of diazanyl is hydrolyzed and left away in part, is exposed diazanyl and is come, so as to be marked.
Gel chromatographic columnses in above-mentioned steps d can coagulating for sephadex G 25 (Sephadex G25) from filler Glue post.
When using synergy modes, its preparation process can be：
A. the fan-shaped dendrimer ligands of multivalence sugar are dissolved in buffer solution；According to part than common part be 1:5~2000, Preferably 1:5~1000 weight ratio, add (methylol) methylglycines (Tricine) of part N- tri- altogether.
B. it is 1 than common part according to synergy modes:2~10 weight ratio, add synergy modes.
C. it is 1 then to press reducing agent than part:0.5~800, it is preferably 1:1~800 weight ratio weighs reducing agent and molten In hydrochloric acid solution；It is added in step B resulting solutions, is well mixed, controls its pH between 5.5~6.5.
D. will be from medical99Mo-99mElution obtains in Tc generators99mTcO4 -Leacheate adds solution prepared by step C In, reacted 10~100 minutes under the conditions of boiling water bath after sealing.
E. labeled compound obtained by step d is purified by gel chromatographic columnses.
Buffer solution in above-mentioned steps A can be phosphate buffer, pH value 6.0.
Reducing agent in above-mentioned steps C can be by high price technetium (99mTcVIIO4 -) the conventional reduction agent of low price technetium is reduced to, Such as two hydrated stannous chloride (SnCl2·2H2O)。
In above-mentioned steps D, the blocking group of diazanyl is hydrolyzed and left away in part, is exposed diazanyl and is come, so as to be marked.
Gel chromatographic columnses in above-mentioned steps E can coagulating for sephadex G 25 (Sephadex G25) from filler Glue post.
The fan-shaped dendrimer ligands of multivalence provided by the invention sugar can part, optional synergy modes and radioactivity together Pertechnetate (99mTcO4 -) formed99mThe fan-shaped tree-shaped molecular complex of multivalence sugar of Tc marks, the complex radiochemical purity High (being more than 95%), stability is good, and biological property is good, and specific activity is high, and liver uptake values and target to non-target ratio value are high, can conduct Liver cell asialoglycoprotein receptor SPECT developer applications.And preparation process is easy, cost is low, have extensive wide The value of general application.
Brief description of the drawings
Fig. 1 is the fan-shaped dendrimer ligands 2LA-G1-HYNIC-1 of multivalence of the present invention sugar synthetic route chart.
Fig. 2 is the when m- activity curve figure of test case 2 of the present invention.
Embodiment
Below by embodiment, the present invention is described in detail, so that the features and advantages of the present invention become apparent from.But should This points out that embodiment is used for the design for understanding the present invention, and the scope of the present invention is not limited only to reality listed herein Apply example.
Such as be not particularly illustrated, raw material used in embodiment be commercially available prod, it is used operation be this area Routine operation.
The preparation of the part of embodiment 1
With reference to figure 1 (by taking 2LA-G1-HYNIC-1 as an example).
1. fan-shaped dendrimer G1, G2, G3 of core synthesis centered on ethylenediamine
1.5g ethylenediamine is dissolved in 100mL dichloromethane, and the 2.18g for being dissolved in 20mL dichloromethane is added dropwise under ice-water bath Di-tert-butyl dicarbonate.Room temperature reaction is overnight.After removing precipitation, revolving removes solvent, is re-dissolved in 50mL ethyl acetate, with full Washed 3 times with sodium chloride solution.Organic phase is collected, solvent is spin-dried for after drying, obtains N- tertbutyloxycarbonyl ethylenediamine 1.1g, yield 69%.
1H NMR(400MHz,D2O)δ:1.45(s,9H,tBu CH3),2.80(t,2H,CH2), 3.17(m,2H,CH2), 5.0(s,1H,NH2)。
N- tertbutyloxycarbonyl ethylenediamine 0.6g, are dissolved in 5mL methanol, and 10mL methyl acrylates are added dropwise under ice-water bath.Drop After adding, reacted 24 hours under 37 degree of warm bath.After reaction terminates, revolving removes solvent repeatedly, obtains yellow oil 1.1g. Product is dissolved in 6mL methanol, is added dropwise under ice-water bath in 40mL ethylenediamines and 10mL methanol solution.After being added dropwise, room Temperature reaction 3 days.Revolving removes solvent repeatedly, after drying, obtains yellow oil G1 1.37g.
1H NMR(400MHz,D2O)δ:1.42(s,9H,tBu CH3),2.42(m,4H, CO-CH2),2.59(m,2H,N- CH2), 2.72~2.82 (m, 8H, N-CH2),3.16(m,2H, N-CH2),3.25(m,4H,N-CH2)；ESI-MS:calcd for C17H36N6O4 388.28,found m/z 389.29[M+H]。
G1 0.5g, are dissolved in 5mL methanol, and 10mL methyl acrylates are added dropwise under ice-water bath.After being added dropwise, 37 degree of temperature The lower reaction of bath 24 hours.After reaction terminates, revolving removes solvent repeatedly, obtains yellow oil 0.8g.Product is dissolved in 6mL first It is added dropwise in alcohol, under ice-water bath in 40mL ethylenediamines and 10mL methanol solution.After being added dropwise, react at room temperature 3 days.Revolve repeatedly Solvent is evaporated off, after drying, obtains yellow oil G2 0.9g.
1H NMR(400MHz,D2O)δ:1.41(s,9H,tBu CH3),2.41(m,12H, CO-CH2),2.59(m,6H, N-CH2),2.70(m,10H,N-CH2), 2.78~2.81 (m, 12H, N-CH2),3.23(t,10H,N-CH2),3.29(m,2H, N-CH2)；ESI-MS:calcd for C37H76N14O8 844.60,found m/z 845.60[M+H]。
G2 0.5g, are dissolved in 5mL methanol, and 10mL methyl acrylates are added dropwise under ice-water bath.After being added dropwise, 37 degree of temperature The lower reaction of bath 24 hours.After reaction terminates, revolving removes solvent repeatedly, obtains yellow oil 0.9g.Product is dissolved in 6mL first It is added dropwise in alcohol, under ice-water bath in 40mL ethylenediamines and 10mL methanol solution.After being added dropwise, react at room temperature 3 days.Revolve repeatedly Solvent is evaporated off, after drying, obtains yellow oil G3 1g.
1H NMR(400MHz,D2O)δ:1.41(s,9H,tBu CH3),2.43(m,28H, CO-CH2),2.59(m,12H, N-CH2),2.71(m,26H,N-CH2),2.80(m,28H, N-CH2),3.23(t,22H,N-CH2)；ESI-MS:calcd for C77H156N30O16 1757.23, found m/z 1757.51[M]。
2. multivalence sugar fan-shaped dendrimer 2LA-G1,4LA-G2,8LA-G3 synthesis
1.27g lactobionic acids are weighed, add 25mL methanol, revolving removes solvent after dispersing and dissolving.25mL methanol is added, point Revolving removes solvent after dissipating dissolving, is repeated 20 times, obtains lactobionolactone.
Lactose lactone 0.68g is dissolved in 7mL methanol and flowed back, G1 0.39g are dissolved in 3mL methanol and add reaction bulb.Return Stream reaction 120min, is cooled to room temperature, produces white crystal.After filtering, solid is washed with a small amount of cold methanol, is dried in vacuo, Obtain buff powder.Add after 3mL TFA all dissolve and 1h is stirred at room temperature.Solvent is spin-dried for, after precipitation is dissolved in into water, passes through Portugal Polysaccharide gel post (Sephadex G25) is purified, and 2LA-G1 is obtained after freeze-drying.
ESI-MS:calcd for C41H76N6O26 1068.48,found m/z 1069.47[M+H]。
Lactose lactone 0.68g is dissolved in 7mL methanol and flowed back, G2 0.42g are dissolved in 3mL methanol and add reaction bulb.Return Stream reaction 120min, is cooled to room temperature, produces white crystal.After filtering, solid is washed with a small amount of cold methanol, is dried in vacuo, Obtain buff powder.Add after 3mL TFA all dissolve and 1h is stirred at room temperature.Solvent is spin-dried for, after precipitation is dissolved in into water, passes through Portugal Polysaccharide gel post (Sephadex G25) is purified, and 4LA-G2 is obtained after freeze-drying.
ESI-MS:calcd for C80H148N14O50 2104.95,found m/z 2109.02[M+4H]。
Lactose lactone 0.68g is dissolved in 7mL methanol and flowed back, G3 0.44g are dissolved in 3mL methanol and add reaction bulb.Return Stream reaction 120min, is cooled to room temperature, produces white crystal.After filtering, solid is washed with a small amount of cold methanol, is dried in vacuo, Obtain buff powder.Add after 3mL TFA all dissolve and 1h is stirred at room temperature.Solvent is spin-dried for, after precipitation is dissolved in into water, passes through Portugal Polysaccharide gel post (Sephadex G25) is purified, and 8LA-G3 is obtained after freeze-drying.
ESI-MS:calcd for C168H308N30O102 4377.98,found m/z 4378.98[M+H]。
3. multivalence sugar fan-shaped dendrimer ligands 2LA-G1-HYNIC-1,4LA-G2-HYNIC-2,8LA-G3-HYNIC-3 Synthesis
6- hydrazino pyridine -3- formic acid (HYNIC) 1g (6.5mmol) are dissolved in 40mL DMF, the 4- of the amount of substance such as addition Dimethylaminobenzaldehyde carries out diazanyl protection, and 3h is stirred at room temperature.Add succinimide 748mg (6.5mmol) and dicyclohexyl Carbodiimide DCC 2.76g (13.4mmol), react at room temperature 18h.Filtering, filtrate are spin-dried for, and add 50mL ethyl acetate, heat back 1h is flowed, heat filtering, obtains yellow powder.
The yellow powder of gained is pressed 1.2 with 2LA-G1,4LA-G2,8LA-G3 respectively:1 feeds intake, and room temperature is anti-in DMSO Answer 24h.After being dissolved in water, purified by sephadex column (Sephadex G25), yellow powder is obtained after freeze-drying End.As 2LA-G1-HYNIC-1 (ESI-MS: calcd for C51H82N10O25 1234.55,found m/z 1235.55[M +H])、 4LA-G2-HYNIC-2(ESI-MS:calcd for C95H162N18O51 2371.06,found m/z 2372.22[M+ H])、8LA-G3-HYNIC-3(ESI-MS:calcd for C183H322N34O103 4644.10,found m/z 4645.24[M+ H])。
Embodiment 299mTc(2LA-G1-HYNIC)(Tricine)2Preparation
Take 2LA-G1-HYNIC-1 0.05mg to be dissolved in 0.5mL phosphate buffers (0.2mol/L, pH6.0, to be total to containing 30mg Part Tricine) in, 2mg/mL SnCl are added after all dissolving2·2H2The μ L of O solution 10, fully shake up.It is eventually adding new It is fresh99mTcO4 -Eluent 0.5mL (37MBq), fully vibration, are sealed after reacting 20min in boiling water bath.Obtain99mTc(2LA- G1-HYNIC)(Tricine)2。
By HiTrap desalinations gel column (Sephadex G25), with 25mL leacheates, (0.05mol/L, pH 7.5 phosphoric acid delays Fliud flushing) balance, coutroi velocity is between 1~10mL/min.0.25mL has been marked99mTc(2LA-G1-HYNIC) (Tricine)2With 1mL syringe loadings, after first being eluted with 1.25mL leacheates, 1mL leacheates are collected, the impurity that is removed, The rate of recovery>95%99mTc(2LA-G1-HYNIC)(Tricine)2Solution.
By chromatography and radio-HPLC identifications, its retention time is 7.2min, and radiochemical purity is more than 99%.HPLC Condition is：High performance liquid chromatograph HITACHI (D-2000)；300 × 7.8mm of TSK-GEL G2500PWxL posts；Mobile phase is Water；Flow velocity 1mL/min.
Radiochemicsl purity is unchanged after placing 6 hours at room temperature, possesses preferable stability.
Embodiment 39mTc(4LA-G2-HYNIC)(Tricine)2Preparation
Take 4LA-G2-HYNIC-2 0.1mg to be dissolved in 0.5mL phosphate buffers (0.2mol/L, pH6.0, containing 25mg altogether to match somebody with somebody Body Tricine) in, 2mg/mL SnCl are added after all dissolving2·2H2The μ L of O solution 10, fully shake up.It is eventually adding new It is fresh99mTcO4 -Eluent 0.5mL (37MBq), fully vibration, are sealed after reacting 20min in boiling water bath.Obtain99mTc(4LA- G2-HYNIC)(Tricine)2。
By HiTrap desalinations gel column (Sephadex G25), with 25mL leacheates, (0.05mol/L, pH 7.5 phosphoric acid delays Fliud flushing) balance, coutroi velocity is between 1~10mL/min.0.25mL has been marked99mTc(4LA-G2-HYNIC) (Tricine)2With 1mL syringe loadings, after first being eluted with 1.25mL leacheates, 1mL leacheates are collected, the impurity that is removed, The rate of recovery>95%99mTc(4LA-G2-HYNIC)(Tricine)2Solution.
By chromatography and radio-HPLC identifications, its retention time is 7.3min, and radiochemical purity is more than 99%.HPLC Condition is：High performance liquid chromatograph HITACHI (D-2000)；300 × 7.8mm of TSK-GEL G2500PWxL posts；Mobile phase is Water；Flow velocity 1mL/min.
Radiochemicsl purity is unchanged after placing 6 hours at room temperature, possesses preferable stability.
Embodiment 499mTc(8LA-G3-HYNIC)(Tricine)2Preparation
Take 8LA-G3-HYNIC-3 0.2mg to be dissolved in 0.5mL phosphate buffers (0.2mol/L, pH6.0, containing 40mg altogether to match somebody with somebody Body Tricine) in, 2mg/mL SnCl are added after all dissolving2·2H2The μ L of O solution 10, fully shake up.It is eventually adding new It is fresh99mTcO4 -Eluent 0.5mL (37MBq), fully vibration, are sealed after reacting 20min in boiling water bath.Obtain99mTc(8LA- G3-HYNIC)(Tricine)2。
By HiTrap desalinations gel column (Sephadex G25), with 25mL leacheates, (0.05mol/L, pH 7.5 phosphoric acid delays Fliud flushing) balance, coutroi velocity is between 1~10mL/min.0.25mL has been marked99mTc(8LA-G3-HYNIC) (Tricine)2With 1mL syringe loadings, after first being eluted with 1.25mL leacheates, 1mL leacheates are collected, the impurity that is removed, The rate of recovery>95%99mTc(8LA-G3-HYNIC)(Tricine)2Solution.
By chromatography and radio-HPLC identifications, its retention time is 7.2min, and radiochemical purity is more than 99%.HPLC Condition is：High performance liquid chromatograph HITACHI (D-2000)；300 × 7.8mm of TSK-GEL G2500PWxL posts；Mobile phase is Water；Flow velocity 1mL/min.
Radiochemicsl purity is unchanged after placing 6 hours at room temperature, possesses preferable stability.
Embodiment 599mTc (4LA-G2-HYNIC) (Tricine) (TPPTS) preparation
Take 4LA-G2-HYNIC-2 0.1mg to be dissolved in 0.5mL phosphate buffers (0.2mol/L, pH6.0, containing 25mg altogether to match somebody with somebody Body Tricine, 5mg synergy modes TPPTS) in, add 2 mg/mL SnCl after all dissolving2·2H2The μ L of O solution 5, fully Shake up.It is eventually adding fresh99mTcO4 -Eluent 0.5mL (37MBq), fully vibration, are sealed after being reacted in boiling water bath 20min.Obtain99mTc(4LA-G2-HYNIC)(Tricine)(TPPTS)。
Purification process is the same as embodiment 3.Radio-HPLC retention times are 6.0min, and radiochemical purity is more than 99%.Room Radiochemicsl purity is unchanged after being placed 6 hours under temperature, possesses preferable stability.
Embodiment 699mTc (8LA-G3-HYNIC) (Tricine) (TPPMS) preparation
Take 8LA-G3-HYNIC-3 0.5mg to be dissolved in 0.5mL phosphate buffers (0.2mol/L, pH6.0, containing 40mg altogether to match somebody with somebody Body Tricine, 3mg synergy modes TPPMS) in, 2mg/mL SnCl are added after all dissolving2·2H2The μ L of O solution 10, fully Shake up.It is eventually adding fresh99mTcO4 -Eluent 0.5mL (37MBq), fully vibration, are sealed after being reacted in boiling water bath 20min.Obtain99mTc(8LA-G3-HYNIC)(Tricine)(TPPMS)。
Purification process is the same as embodiment 4.Radio-HPLC retention times are 6.1min, and radiochemical purity is more than 99%.Room Radiochemicsl purity is unchanged after being placed 6 hours under temperature, possesses preferable stability.
The Biodistribution in mice of test case 1 and Inhibition test
Using the complex obtained in embodiment 3 using Tricine as common part99mTc(4LA-G2-HYNIC) (Tricine)2Exemplified by.
15 normal Kunming small white mouses are taken, in tail vein injection 0.1mL99mTc(4LA-G2-HYNIC)(Tricine)2 (about 0.185MBq).The sacrificed by decapitation after 10,30 and 120min after injection.Take out the groups such as the heart, liver, lung, kidney, muscle, bone, blood Knit, weigh and its radiocounting is surveyed in gamma counter.
99mTc(4LA-G2-HYNIC)(Tricine)2Bio distribution in normal mouse is shown in Table 1.
The normal mouse vivo biodistribution distributed data (%ID/g ± sd, n=5) of table 1.
Bio distribution result in normal mouse shows,99mTc(4LA-G2-HYNIC)(Tricine)2Have in liver Higher initial intake, after injecting 10min, there are (32.20 ± 4.24) %ID/g uptake values in liver, and in liver There is preferable delay.It is relatively low in the radioactivity concentration of the non-target organ such as the heart, lung, spleen.The radioactivity of liver is introduced into blood Mainly quickly excreted by kidney.With Clinical practice99mTc-GSA is compared,99mTc(4LA-G2-HYNIC) (Tricine)2It is relatively low in the absolute uptake values of liver.But the radioactivity concentration in the non-target organ such as blood, kidney is obvious It is less than99mTc-GSA, and remove faster.Under same experimental conditions,99mTc-GSA putting in 30min kidneys and blood after injection Penetrating property is absorbed：(12.92 ± 0.50) %ID/g, (2.32 ± 0.15) %ID/g (n=3).
Inhibition test takes same 5 normal Kunming small white mouses of batch, and in tail vein preform injection 0.1mL inhibitor, (GSA is dissolved in Physiological saline, injected by the dosage of 10mg/kg body weight), after 5min, in tail vein injection 0.1mL99mTc(4LA-G2-HYNIC) (Tricine)2(about 0.185MBq).Sacrificed by decapitation after 10min after injection.Take out the groups such as the heart, liver, lung, kidney, muscle, bone, blood Knit, weigh and its radiocounting is surveyed in technetium analyzer.10min result compares after its result is injected with normal mouse.
99mTc(4LA-G2-HYNIC)(Tricine)2Inhibition assay result is shown in Table 2 in normal mouse body.
Inhibition test data (%ID/g ± sd, n=5) in the normal mouse body of table 2.
The result of Inhibition test shows, by preform injection GSA, can significantly suppress in 10min99mTc(4LA-G2- HYNIC)(Tricine)2Intake (P in mouse liver<0.001), the radioactivity in blood, kidney etc. in non-target organ Concentration increased.
The result explanation of Inhibition test passes through preform injection GSA, can effectively suppress99mTc(4LA-G2-HYNIC) (Tricine)2Intake in liver, it was demonstrated that it has compatibility to ASGPr acceptors.
Based on above mouse biodistribution experiments result,99mTc(4LA-G2-HYNIC)(Tricine)2Show excellent Biological property, be advantageous to enough in the imaging in future obtain clearly image and accurate data.Similarly, other embodiment 2nd, 4-6 also possesses similar excellent properties.
The rat SPECT dynamic scan results of test case 2
With the complex obtained in embodiment 299mTc(2LA-G1-HYNIC)(Tricine)2, obtain in embodiment 3 Complex99mTc(4LA-G2-HYNIC)(Tricine)2, the complex that obtains in embodiment 499mTc(8LA-G3-HYNIC) (Tricine)2Exemplified by.
Normal SD rats (200g) are taken, after isoflurane anesthesia, respectively at tri- kinds of tail vein injection 0.5mL about 37MBq The technetium-99 m labeled fan-shaped tree-shaped molecular complex of multivalence sugar and the MBq of 0.5mL about 3799mTc-GSA, injection dosage are 1.3 ×10-8mol/kg.Immediately using the dynamic scan of SPECT scanners progress 1 hour after injection.After collection terminates, liver is delineated The region of interest ROI of dirty, kidney, heart, the when m- activity curve TAC in liver, kidney, heart (blood) is drawn respectively.
Inhibition test takes same batch normal SD rats (200g), after isoflurane anesthesia, prior to tail vein preform injection 0.1mL inhibitor (GSA is dissolved in physiological saline, is injected by the dosage of 10mg/kg body weight), after 5min, respectively at tail vein injection The fan-shaped tree-shaped molecular complex of multivalence sugar and 0.5mL about 37MBq technetium-99 m labeled tri- kinds of 0.5mL about 37MBq99mTc-GSA, Injection dosage is 1.3 × 10-8mol/kg.Immediately using the dynamic scan of SPECT scanners progress 1 hour after injection.Adopt After collection terminates, delineate the region of interest ROI of liver, kidney, heart, draw respectively in liver, kidney, heart (blood) when M- activity curve TAC.
The result of SPECT dynamic acquisitions is shown in Fig. 2.The result of dynamic scan is shown in SD rat bodies.Three kinds of technetium -99m marks The fan-shaped tree-shaped molecular complex of multivalence sugar of note99mTc(2LA-G1-HYNIC)(Tricine)2、99mTc(4LA-G2-HYNIC) (Tricine)2、99mTc(8LA-G3-HYNIC)(Tricine)2There is specific intake in liver, in kidney and blood Remove very fast.The result of Inhibition test displays that, by preform injection GSA, can effectively suppress the intake of liver, illustrates that technetium -99m is marked The fan-shaped tree-shaped molecular complex of multivalence sugar of note is receptor-mediated into liver by ASGPr.With Clinical practice99mTc- GSA is compared, and it is relatively low in the absolute uptake values of liver, but is detained preferable.Simultaneously by the contrast of inhibition assay result, by It can be metabolized in the technetium-99 m labeled fan-shaped tree-shaped molecular complex of multivalence sugar by kidney, with99mTc-GSA is compared and is trapped in Label in blood can be removed rapidly, so as to be effectively increased the clearance rate of internal non-target tissue, reduce non-specificity and take the photograph Take.
Above-mentioned every description of test, the technetium-99 m labeled fan-shaped tree-shaped molecular complex of multivalence sugar of the present invention, puts Chemical purity height is penetrated, chemical stability is good, and has excellent biological property, there is higher initial intake in liver, and It can quickly be removed by kidney, reduce non-specific uptake, with99mTc-GSA, which is compared, relatively low non-target internal organs intake, can make For new liver cell asialoglycoprotein receptor SPECT developers clinically popularization and application.
Unless limited otherwise, term used herein is the implication that those skilled in the art are generally understood that.
Embodiment described in the invention is not used to limit the scope of the invention merely for exemplary purpose, Those skilled in the art can be made within the scope of the invention various other replacements, changes and improvements, thus, the invention is not restricted to Above-mentioned embodiment, and be only defined by the claims.

Claims (7)

  1. It is 1. a kind of99mThe fan-shaped tree-shaped molecular complex of multivalence sugar of Tc marks, its formula are：
    99mTc(R-HYNIC)(Tricine)m(L)n
    Wherein, m represents 1 or 2, n expression 0 or 1；Tricine represents N- tri- (methylol) methylglycine；L represents synergy modes； R-HYNIC represents one kind in structure shown in 2LA-G1-HYNIC, 4LA-G2-HYNIC, 8LA-G3-HYNIC,
    The 2LA-G1-HYNIC represents following structure：
    The 4LA-G2-HYNIC represents following structure：
    The 8LA-G3-HYNIC represents following structure：
    LA groups therein represent following structure：
  2. 2. complex according to claim 1, wherein, the synergy modes are selected from three (3- sulfonyl-phenyls) phosphine sodium salts or three Phenylphosphine list sulfonic acid monosodium salt.
  3. 3. purposes of the complex of claim 1 or 2 as liver cell asialoglycoprotein receptor SPECT developers.
  4. 4. a kind of fan-shaped dendrimer ligands of multivalence sugar for being used to prepare the complex of claim 1 or 2, have 2LA-G1- Any shown structure in HYNIC-R ', 4LA-G2-HYNIC-R ', 8LA-G3-HYNIC-R ',
    The 2LA-G1-HYNIC-R ' represents following structure：
    The 4LA-G2-HYNIC-R ' represents following structure：
    The 8LA-G3-HYNIC-R ' represents following structure：
    Wherein, R ' represents diazanyl blocking group.
  5. 5. the fan-shaped dendrimer ligands of multivalence sugar according to claim 4, wherein, R ' represents phenyl, 4- Dimethylaminobenzenes Base, 2- sodium sulfonate-phenyls or 4- carboxyl phenyls.
  6. 6. the preparation method of the fan-shaped dendrimer ligands of the multivalence sugar of claim 4 or 5, using ethylenediamine as initiation material, bag Include following steps：
    1) amido of ethylenediamine is protected using blocking group R "；
    2) product obtained by step 1) obtained successively with methyl acrylate, reacting ethylenediamine intermediate G1, the intermediate G1 according to It is secondary to obtain intermediate G2 with methyl acrylate, reacting ethylenediamine, or the intermediate G2 is anti-with methyl acrylate, ethylenediamine successively Intermediate G3 should be obtained；
    3)-the NH of intermediate obtained by step 2)2Reacted with lactobionolactone, form intermediate 2LA-G1,4LA-G2 or 8LA-G3；
    4) the blocking group R " in intermediate 2LA-G1,4LA-G2 or 8LA-G3 obtained by step 3) is removed；
    5) the 6- hydrazino pyridine -3- formic acid that step 4) products therefrom is protected with R ' group is reacted and produced；
    Wherein, the reaction equation of step 2) is as follows：
  7. 7. preparation method according to claim 6, wherein, R " represents tert-butyl carbonyl, and trifluoroacetic acid is used in step 4) Removed.
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CN101486764A (en) * 2009-03-09 2009-07-22 北京师范大学 Technetium-99m marked 6-hydrazino pyridine-3-formamido novel lactose albumin complexes, preparation and use thereof
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