CN102643423B - A kind of 99mtc title complex, its preparation method, intermediate and application thereof - Google Patents

A kind of 99mtc title complex, its preparation method, intermediate and application thereof Download PDF

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CN102643423B
CN102643423B CN201110040441.0A CN201110040441A CN102643423B CN 102643423 B CN102643423 B CN 102643423B CN 201110040441 A CN201110040441 A CN 201110040441A CN 102643423 B CN102643423 B CN 102643423B
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chemical compounds
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沈玉梅
张元庆
许晓平
孙艳红
杨光
崔巍
朱华
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Shanghai Institute of Applied Physics of CAS
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Abstract

The invention discloses a kind of such as formula shown in II 99mtc title complex; Wherein m=1 ~ 10, n=60 ~ 85, p=10 ~ 50; Ball-like structure represent in Polyamidoamine Dendrimers except 128-NH 2structure in addition.The invention also discloses its preparation method, intermediate and application thereof.Of the present invention 99mtc title complex has excellent tumor-targeting, water-soluble and stability, is a kind of SPECT developer with better metabolic capacity.

Description

A kind of 99mtc title complex, its preparation method, intermediate and application thereof
Technical field
The present invention relates to a kind of radiometal complex, its preparation method, intermediate and application thereof, relate to one particularly 99mtc title complex, its preparation method, intermediate and application thereof.
Background technology
SPECT (single photon emission computed tomography) (Single-Photon Emission Computed Tomography, SPECT) is the gamma-ray nuclear medicine Computed tomography of a kind of use.
SPECT utilizes traditional core to cure contrast medium (radiotracer) and pacifies form γ-camera with the target organ of certain angle ring around patient, obtain a series of flat image, obtain another a series of image with computer image recombinant technology again, thus obtain the stereopsis of Transaxial, Sagittal and Coronal.
The basic image-forming principle of SPECT: general kernel doctor nuclear species is produced by producer, such as: Tc-99m, I-131 or I-125.It is excessively many types of that the nuclear species of this type belongs to neutron more, and in production process, with the female target of neutron bombardment, make it obtain superfluous neutron, and become unstable, after beta-decay, nucleus remains more energy.After getting back to ground state, unnecessary photon produces with single direction.Each sensitive spot of γ-camera probe detects the γ photon of coming in along a projection line (Ray), and its observed value represents the radioactivity sum of human body on this projection line.Radiopharmaceuticals on sensitive spot on same straight line detectable human body tomography, their output is called the One Dimensional Projection of this tomography (Projection).In figure, each bar projection line is all parallel to each other perpendicular to detector, therefore is referred to as collimated beam, and the normal of detector and the angle of cut θ of X-axis be called observing angle (View).γ-camera is two-dimensional detector, after having installed parallel aperture collimator, can obtain the parallel beam projection of multiple tomography simultaneously, Here it is plain film.Plain film can not reflect the context of each point on projection line.Wonder the structure of human body on depth direction, just need to observe from different perspectives.Can prove, be aware of the One Dimensional Projection of certain tomography in all observing angles, just can calculate the image of this tomography.The process solving faultage image from projection is called reconstruction (Reconstruction).It is similar to the planar imaging techniques of traditional γ-camera, but can provide 3-D view.
99mthe transformation period of Tc is 6 hours, and length is comparatively applicable to medical imaging. 99mtc is pure gamma-rays, and without β radiation, therefore the injury caused human body is very little.Its photon production rate reaches 86%, and energy is 140KeV, is suitable for video picture, is convenient to estimate absorption dose clinically and carry out pharmacokinetic.In addition, 99mtc chemically reactive is high, can be combined with chemical compound lot.
Traditional preparation zeroth order 99mthe method of Tc is: by five hydration potassium tetraborate (K 2b 4o 75H 2o), Rochelle salt (KNaC 4h 4o 64H 2and K O) 2(BH 3cO 2) mixing, more under nitrogen protection, add 1 ~ 2ml brand-new wherein 99mtcO 4 -solution, reacts 15min under room temperature, then carries out conventional aftertreatment.
The reduction ratio of the method is very high, can reach more than 90%.But the reduzate Fac-obtained [ 99mtc (CO) 3(H 2o) 3] +very low to the mark rate of diethylene triamine pentacetic acid (DTPA) class bifunctional chelating agent, only have about 20 ~ 30%.
99mthough Tc can be used for SPECT video picture, because it is non-targeted, distribution in vivo lacks selectivity, therefore needs the guiding of targeting agent directedly could arrive tumor locus.
Research shows, tumor cell surface exists some acceptors like this, and they are overexpression in tumor tissues, does not then express in the normal tissue or low expression.By these receptor mediated endocytosis, can be transported to the ligands specific of its combination or antibody target and specifically organize or cell, to strengthen the selectivity of medicine and to reduce toxic side effect, become the study hotspot of tumor biotherapy.
As one of the specific receptors of mediated cell internalization, folic acid can be taken in eukaryotic endochylema by folacin receptor (folate receptor, FR), is a kind of high-affinity receptor.Due to folacin receptor at a part of human tumor as the cell surfaces such as ovarian cancer, mammary cancer, cervical cancer, colorectal carcinoma and nasopharyngeal carcinoma can both too be expressed, and expression high conservative in the normal tissue, therefore folacin receptor can be used for tumour-specific video picture and treatment.Reddy JA：Allagadda VM，Leamon CP(Targeting therapeutic and imaging agents to folate receptor positive tumors，Current Pharm Biotech，2005，6：131-150。) carry out the research of the imageable agents of folic acid active targeting.But cause the water-soluble also not good of this type of medicine due to the water-soluble of folic acid extreme difference, simultaneously due to the molecular properties of folic acid, directly mark the pharmacological property that metal may change it thereon.
Dendrimer (dendrimer) is a class new function polymer of Abroad in Recent Years exploitation, due to its distinctive advantage: the geometrical symmetry of height in structure, accurate molecular structure, a large amount of functional groups, molecular memory increase at cavity and molecular chain and have controllability, and the character of many uniquenesses and cause the extensive concern of association area.Polyamidoamine Dendrimers structure is as follows:
There is a large amount of active group in dendrimer surface, can react with multiple compounds, and it is inner containing a large amount of hydrophobic cavity, can be connected with hydrophobic drug molecule with non-covalent fashion.In addition, because dendrimer has ultrahigh molecular weight, can not be fallen by kidney metabolism very soon as small molecules, thus can stay longer in blood, thus increase organ to the absorption of medicine.
Summary of the invention
Technical problem to be solved by this invention be in order to overcome existing Fac-[ 99mtc (CO) 3(H 2o) 3] +the defect lower to the mark rate of diethylene triamine pentacetic acid (DTPA) class bifunctional chelating agent, provides one 99mtc title complex, its preparation method, intermediate and application thereof.Of the present invention 99mtc title complex has excellent tumor-targeting, water-soluble and stability, is a kind of SPECT developer with better metabolic capacity.
Contriver finds through further investigation, for overcoming the defect of folic acid poorly water-soluble, and ensureing that the pharmacokinetics of folic acid is unaffected as far as possible, polyamide-amide class dendrimer can be selected as carrying platform, carries folate molecule and bifunctional chelating agent; And in order to reduce the toxicity of polyamide-amide class dendrimer, first contriver uses diacetyl oxide (Ac 2o) part on acetylize dendrimer surface is amino, then introduces folate molecule by the amino on dendrimer surface, to realize the cancer target of imageable agents, finally reconnects bifunctional chelating agent, obtain labelled precursor chemical compounds I.
Traditional Fac-[ 99mtc (CO) 3(H 2o) 3] +very low to the mark rate of diethylene triamine pentacetic acid (DTPA) class bifunctional chelating agent, and the present inventor has found Na 99mtcO 4the single stage method marked diethylene triamine pentacetic acid (DTPA) class bifunctional chelating agent while reduction, namely utilizes SnCl 2reduction 99mtcO 4 -, simultaneously with chemical compounds I coordination.This method mark rate can reach 93%.
The present invention relates to the derivative of Polyamidoamine Dendrimers and the preparation method based on it, and Polyamidoamine Dendrimers complex structure, therefore its part-structure elliptical technique of painting is replaced, the structure ball-like structure namely beyond 128 amino represent.
Therefore the present invention relates to a kind of such as formula shown in II 99mtc title complex;
Wherein m=1 ~ 10, n=60 ~ 85, p=10 ~ 50, preferred m=4 ~ 8, n=76 ~ 78, p=25 ~ 40; Best, m=7, n=77, p=35.Compound ii can be abbreviated as PAMAM-Ac-pegFa-DTPA- 99mtc.
The invention further relates to the preparation method of above-mentioned title complex II, it comprises the following step: in aqueous hydrochloric acid, at SnCl 2effect under, by chemical compounds I and 99mtcO 4 -an alkali metal salt and/or alkaline earth salt carry out labeled reactant;
Wherein, m=1 ~ 10, n=60 ~ 85, p=10 ~ 50, preferred m=4 ~ 8, n=76 ~ 78, p=25 ~ 40; Best, m=7, n=77, p=35.
Wherein, described reaction method and condition all can be ordinary method and the condition of this type of labeled reactant of this area.The preferred following condition of the present invention: the volume mass ratio of described aqueous hydrochloric acid and chemical compounds I is preferably 10 ~ 200ml/g; The concentration of described aqueous hydrochloric acid is preferably 0.05 ~ 0.15M; The mol ratio of hydrochloric acid and chemical compounds I is preferably 0.1: 1 ~ 20: 1, and better is 5: 1 ~ 10: 1; Described SnCl 2consumption can in very large range select, consumption is more, and effect is better, is preferably 3 ~ 20 times of chemical compounds I molar weight, and better is 5 ~ 10 times; Described 99mtcO 4 -an alkali metal salt and/or the preferred Na of alkaline earth salt 99mtcO 4; 99mtcO 4 -an alkali metal salt and/or the radioactive activity of alkaline earth salt be preferably 1mci: 10 with the ratio of the molar weight of chemical compounds I -6mmol ~ 1mci: 1mmol, better is 1mci: 10 -4mmol ~ 1mci: 10 -2mmol; Described temperature of reaction is preferably 60 ~ 100 DEG C, and better is 75 ~ 85 DEG C; The described reaction times preferably with detection reaction completely till, as by radioactivity TLC detection reaction completely till, be generally 0.2 ~ 1 hour.
In the present invention, described chemical compounds I can be obtained by following method: in water, and under the effect of alkali, compound III and compounds Ⅳ carry out reaction that is amino and isothiocyano;
Wherein, m=1 ~ 10, n=60 ~ 85, p=10 ~ 50, preferred m=4 ~ 8, n=76 ~ 78, p=25 ~ 40; Best, m=7, n=77, p=35.Chemical compounds I also can be abbreviated as PAMAM-Ac-pegFa-DTPA.
Wherein, described reaction method and condition can be ordinary method and the condition of this type of reaction of this area, and the present invention is following condition particularly preferably: the consumption of described water and the volume mass of compound III are 10 ~ 200ml/g than preferably; The consumption of described compounds Ⅳ is preferably 5 ~ 40 times of compound III molar weight, and better is 10 ~ 20 times; Described alkali is preferably inorganic strong alkali, and as sodium hydroxide, the consumption of alkali is preferably for the pH scope controlling reaction soln is the amount of 8 ~ 10, and preferable ph is 9; Described temperature of reaction is preferably 30 ~ 50 DEG C, and better is 35 ~ 45 DEG C; The described reaction times preferably with detection reaction completely till, be generally 24 ~ 72 hours.
After having reacted, the post-treating method that this type of reaction is conventional through this area, as deionized water dissolving, filtration and PBS solution dialysis, can obtain pure chemical compounds I.
In the present invention, described compound III can be obtained by following method: compound V and compound VII are carried out condensation reaction;
Wherein, m=1 ~ 10, n=60 ~ 85, p=10 ~ 50, preferred m=4 ~ 8, n=76 ~ 78, p=25 ~ 40; Best, m=7, n=77, p=35.Compound III also can be abbreviated as PAMAM-Ac-pegFa.
Wherein, described reaction method and condition can be ordinary method and the condition of this type of condensation reaction of this area, and the consumption of described compound VII is preferably 1 ~ 12 times of compound V molar weight, and better is 5 ~ 10 times; Described temperature of reaction is preferably 15 ~ 37 DEG C, and better is 20 ~ 30 DEG C; The described reaction times preferably with detection reaction completely till, be generally 24 ~ 120 hours.
After having reacted, the post-treating method that this type of reaction is conventional through this area, as deionized water dissolving, filtration and PBS solution dialysis, can obtain pure compound III.
In preparation method of the present invention, the optimum condition of each processing step can arbitrary combination, i.e. obtained each preferred embodiments.
In the present invention, described compound V can be obtained by following method: acetylization reaction compound VI being carried out part amino;
Wherein, n=60 ~ 85, preferred n=76 ~ 78; Best, n=77.
Wherein, described reaction method and condition all can be ordinary method and the condition of this area acetylization reaction; concrete steps can reference Istva ' n J.Majoros; Acetylation of Poly (amidoamine) Dendrimers; Macromolecules 2003; 36,5526-5529.
The invention still further relates to a kind of such as formula the preparation shown in I 99mthe midbody compound of Tc title complex II, namely 99mthe labelled precursor compound of Tc, it is polyamide-amide class dendrimer derivative;
Be abbreviated as PAMAM-Ac-pegFa-DTPA; Wherein m=1 ~ 10, n=60 ~ 85, p=10 ~ 50, preferred m=4 ~ 8, n=76 ~ 78, p=25 ~ 40; Best, m=7, n=77, p=35.
The invention further relates to 99mthe application of Tc title complex II in preparation SPECT developer.
Except specified otherwise, the raw material that the present invention relates to and reagent are all commercially.
Positive progressive effect of the present invention is: in the present invention 99mtc title complex II utilizes polyamide-amide class dendrimer as pharmaceutical carrier, overcome the shortcoming of folic acid poorly water-soluble, improve its water-soluble and stability, increase drug loading thus strengthen imaging results, it has tumor-targeting simultaneously, be a kind of SPECT developer with better metabolic capacity, there is good video picture prospect.In addition, in the present invention, preparation method's step of title complex II is simple, can a step realize 99mtcO 4 -reduction and mark to diethylene triamine pentacetic acid (DTPA) class bifunctional chelating agent, mark rate can reach 93%.
Accompanying drawing explanation
Fig. 1 is 99mtcO 4 -radioactivity HPLC spectrogram, retention time t=6.15min.
The radioactivity HPLC spectrogram of the title complex II of Fig. 2 obtained by embodiment 4, retention time t=26.54min.
The SPECT image of the title complex II pair tumor mouse of Fig. 3 obtained by embodiment 4.
Embodiment
Further illustrate the present invention by embodiment below, but the present invention is not limited.
Embodiment 1: the synthesis (partial acetylations of five PAMAM dendrimers) of compound V (n=77)
According to document (Istva ' n J.Majoros; Acetylation of Poly (amidoamine) Dendrimers; Macromolecules 2003; 36,5526-5529) acetylize dendrimer, concrete steps are as follows: 100mg five PAMAM dendrimer (compound VI; G5PAMAM; molecular weight 28824g/mol, is stored in methyl alcohol, wt:5%) (be about 3.47 × 10 -6mol), 24.9 μ l acetic anhydride (Ac 2o) (2.43 × 10 are about -4mol) and 43.4 μ l triethylamine (Et 3n) (3.04 × 10 are about -4mol) mix, under nitrogen protection room temperature, (25 DEG C) stirring reaction 24h, obtains colourless transparent liquid.Be spin-dried for solvent, use deionized water lysate, filter by aqueous phase syringe filter (specification 25mm × 0.20 μm), filtrate is dialysed in PBS solution, and (PBS fills a prescription: NaCl 8.0g, KCl 0.2g, Na 2hPO 41.44g, KH 2pO 40.27g is dissolved in 1000ml deionized water, measures pH=7.31).Dialyse one day, change three PBS solution.Above-mentioned product is continued dialyse two days in deionized water, change three deionized waters every day.Dialysis terminates, and by the product lyophilize obtained, productive rate is about 94.7%.
Its appraising datum is as follows:
1H-NMR(D 2O，TMS，500MHz)，δ1.88(230H，s)，2.33(504H，s)
Embodiment 2: the synthesis (synthesis of PAMAM-Ac-pegFa) of compound III (m=7, n=77, p=10)
0.090g compound VII (is about 3.27 × 10 -5mol) be dissolved in 3ml DMSO, at 15 DEG C, add wherein and be dissolved with 3.27 × 10 -6the 3ml DMSO solution of mol PAMAM-Ac, reaction 3d.Obtain yellow transparent solution.Be spin-dried for solvent, use deionized water lysate, aqueous phase syringe filter (specification 25mm × 0.20 μm) is filtered, and filtrate is dialysed one day in PBS solution, changes three PBS solution.Above-mentioned product is continued dialyse two days in deionized water, change three deionized waters every day.Dialysis terminates, and by the product lyophilize obtained, productive rate is about 93.7%.
Its appraising datum is as follows:
1H-NMR(D 2O，TMS，500MHz)，δ1.89(230H，s)，2.28(504H，s)，8.64(7H)，7.62(14H)，6.68(14H)
Embodiment 3: the synthesis (synthesis of PAMAM-Ac-pegFa-DTPA) of chemical compounds I (m=7, n=77, p=10)
Containing having an appointment 2.94 × 10 -6in the aqueous solution of mol PAMAM-Ac-pegFa (compound III) (5mL), add 0.0572g compounds Ⅳ and (be about 2.94 × 10 -5mol), by NaOH solution (1M) adjust ph to about 9.At 40 DEG C, reaction 24h, obtains yellow transparent solution.Be spin-dried for solvent, use deionized water lysate, aqueous phase syringe filter (specification 25mm × 0.20 μm) is filtered, and filtrate is dialysed one day in PBS solution, changes three PBS solution.Above-mentioned product is continued dialyse two days in deionized water, change three deionized waters every day.Dialysis terminates, and by the product lyophilize obtained, productive rate is about 94.4%.
Nuclear magnetic data is as follows:
1H-NMR(D 2O，TMS，500MHz)，δ1.88(230H，s)，2.33(504H，s)，6.78(7H，d，J＝5.0)，7.56(31H，d，J＝8.0)，6.84(30H，d，J＝8.2)
Embodiment 4: the synthesis (PAMAM-Ac-pegFa-DTPA-of compound ii (m=7, n=77, p=15) 99mthe synthesis of Tc)
By type I compound (50mg, 1.2 × 10 -6mol) SnCl of 5ml is dissolved in 2(SnCl 2concentration 5.27 × 10 -3m) hydrochloric acid soln (concentration of hydrochloric acid: 1 × 10 -3m) in, then 1mCiNa is added wherein 99mtcO 4solution, reacts 30min, obtains marked product in the water-bath of 80 DEG C.Mark rate is about 93%, with radioactivity HPLC qualification, the results are shown in accompanying drawing 2, t=26.54min.
Embodiment 5: the synthesis (synthesis of PAMAM-Ac-pegFa) of compound III (m=1, n=77, p=50)
0.0090g compound VII (is about 3.27 × 10 -6mol) be dissolved in 3ml DMSO, under room temperature (25 DEG C), add wherein and be dissolved with 3.27 × 10 -6the 3mlDMSO solution of mol PAMAM-Ac (compound V), reacts 3d under room temperature.Obtain yellow transparent solution.Be spin-dried for solvent, use deionized water lysate, aqueous phase syringe filter (specification 25mm × 0.20 μm) is filtered, and filtrate is dialysed one day in PBS solution, changes three PBS solution.Above-mentioned product is continued dialyse two days in deionized water, change three deionized waters every day.Dialysis terminates, and by the product lyophilize obtained, productive rate is about 91.7%.
Its appraising datum is as follows:
1H-NMR(D 2O，TMS，500MHz)，δ1.89(230H，s)，2.28(504H，s)，8.64(1H)，7.62(2H)，6.68(2H)
Embodiment 6: the synthesis (synthesis of PAMAM-Ac-pegFa-DTPA) of chemical compounds I (m=1, n=77, p=50)
Containing having an appointment 2.94 × 10 -6add compounds Ⅳ in the aqueous solution (5ml) of mol PAMAM-Ac-pegFa (compound III) and (be about 14.7 × 10 -6mol), by NaOH solution (1M) adjust ph to about 8, at 30 DEG C, react 24h, obtain yellow transparent solution.Be spin-dried for solvent, use deionized water lysate, aqueous phase syringe filter (specification 25mm × 0.20 μm) is filtered, and filtrate is dialysed one day in PBS solution, changes three PBS solution.Above-mentioned product is continued dialyse two days in deionized water, change three water every day.Dialysis terminates, and by the product lyophilize obtained, productive rate is about 91%.HPLC analysis is carried out to it, retention time t=10.825min.
Nuclear magnetic data is as follows:
1H-NMR(D 2O，TMS，500MHz)，δ1.88(230H，s)，2.33(504H，s)，6.78(1H，d，J＝5.0)，7.56(18H，d，J＝8.0)，6.84(17H，d，J＝8.2)
Embodiment 7: the synthesis (PAMAM-Ac-Fa-DTPA-of compound ii (m=1, n=77, p=50) 99mthe synthesis of Tc)
Type I compound (1mmol) is dissolved in SnCl 2(3mmol), in aqueous hydrochloric acid (concentration is 0.15M, and hydrochloric acid is 0.1mmol), 1mCi Na is then added 99mtcO 4solution, reacts 30min, obtains marked product in the water-bath of 60 DEG C.Mark rate is about 92%, with radioactivity HPLC qualification, and retention time t=11.10min.
Embodiment 8: the synthesis (synthesis of PAMAM-Ac-pegFa) of compound III (m=10, n=77, p=35)
By 0.108g (about 3.93 × 10 -5mol) compound VII is dissolved in 3ml DMSO, adds wherein and is dissolved with 3.27 × 10 -6the 3ml DMSO solution of mol PAMAM-Ac (compound V), reacts 3d at 37 DEG C, obtains yellow transparent solution.Be spin-dried for solvent, use deionized water lysate, aqueous phase syringe filter (specification 25mm × 0.20 μm) is filtered, and filtrate is dialysed one day in PBS solution, changes three PBS solution.Above-mentioned product is continued dialyse two days in deionized water, change three deionized waters every day.Dialysis terminates, and by the product lyophilize obtained, productive rate is about 90.1%.
Its appraising datum is as follows:
1H-NMR(D 2O，TMS，500MHz)，δ1.89(230H，s)，2.28(504H，s)，8.64(10H)，7.62(20H)，6.68(21H)
Embodiment 9: the synthesis (synthesis of PAMAM-Ac-pegFa-DTPA) of chemical compounds I (m=10, n=77, p=35)
Containing having an appointment 3.01 × 10 -6add compounds Ⅳ in the aqueous solution (5ml) of mol PAMAM-Ac-pegFa (compound III) and (be about 12.1 × 10 -5mol), by NaOH solution (1M) adjust ph to about 10, at 50 DEG C, react 24h, obtain yellow transparent solution.Be spin-dried for solvent, use deionized water lysate, aqueous phase syringe filter (specification 25mm × 0.20 μm) is filtered, and filtrate is dialysed one day in PBS solution, changes three PBS solution.Above-mentioned product is continued dialyse two days in deionized water, change three deionized waters every day.Dialysis terminates, and by the product lyophilize obtained, productive rate is about 91%.HPLC analysis is carried out to it.
Nuclear magnetic data is as follows:
1H-NMR(D 2O，TMS，500MHz)，δ1.88(230H，s)，2.33(504H，s)，6.78(10H，d，J＝5.0)，7.56(36H，d，J＝8.0)，6.84(37H，d，J＝8.2)
Embodiment 10: synthesis (the PAMAM-Ac-Fa-DTPA-of compound ii (m=10, n=77, p=35) 99mthe synthesis of Tc)
By type I compound (10 -6mmol) SnCl is dissolved in 2(10 × 10 -6mmol) in aqueous hydrochloric acid, (concentration is 0.05M, and hydrochloric acid is 20 × 10 -6mmol), 1mCi Na is then added 99mtcO 4solution, reacts 30min, obtains marked product in the water-bath of 100 DEG C.Mark rate is about 93%, with radioactivity HPLC qualification, and retention time t=11.10min.
Embodiment 11: the synthesis (partial acetylations of five PAMAM dendrimers) of compound V (n=65)
Concrete steps are as follows: 100mg five PAMAM dendrimer (compound VI, G5PAMAM, molecular weight 28824g/mol are stored in methyl alcohol, wt:5%) (is about 3.47 × 10 -6mol), 21.1 μ l acetic anhydride (Ac 2o) (2.05 × 10 are about -4mol) and 36.7 μ l triethylamine (Et 3n) (2.57 × 10 are about -4mol) mix, under nitrogen protection room temperature, (25 DEG C) stirring reaction 24h, obtains colourless transparent liquid.Be spin-dried for solvent, use deionized water lysate, filter by aqueous phase syringe filter (specification 25mm × 0.20 μm), filtrate is dialysed in PBS solution, and (PBS fills a prescription: NaCl 8.0g, KCl 0.2g, Na 2hPO 41.44g, KH 2pO 40.27g is dissolved in 1000ml deionized water, measures pH=7.31).Dialyse one day, change three PBS solution.Above-mentioned product is continued dialyse two days in deionized water, change three deionized waters every day.Dialysis terminates, and by the product lyophilize obtained, productive rate is about 94.7%.
Its appraising datum is as follows:
1H-NMR(D 2O，TMS，500MHz)，δ1.88(194H，s)，2.33(504H，s)
Embodiment 12: the synthesis (partial acetylations of five PAMAM dendrimers) of compound V (n=85)
Concrete steps are as follows: 100mg five PAMAM dendrimer (compound VI, G5PAMAM, molecular weight 28824g/mol are stored in methyl alcohol, wt:5%) (is about 3.47 × 10 -6mol), 27.6 μ l acetic anhydride (Ac 2o) (2.68 × 10 are about -4mol) and 47.9 μ l triethylamine (Et 3n) (3.36 × 10 are about -4mol) mix, under nitrogen protection room temperature, (25 DEG C) stirring reaction 24h, obtains colourless transparent liquid.Be spin-dried for solvent, use deionized water lysate, filter by aqueous phase syringe filter (specification 25mm × 0.20 μm), filtrate is dialysed in PBS solution, and (PBS fills a prescription: NaCl 8.0g, KCl 0.2 g, Na 2hPO 41.44g, KH 2pO 40.27g is dissolved in 1000ml deionized water, measures pH=7.31).Dialyse one day, change three PBS solution.Above-mentioned product is continued dialyse two days in deionized water, change three deionized waters every day.Dialysis terminates, and by the product lyophilize obtained, productive rate is about 94.7%.
Its appraising datum is as follows:
1H-NMR(D 2O，TMS，500MHz)，δ1.88(254H，s)，2.33(504H，s)
Embodiment 13: the synthesis (partial acetylations of five PAMAM dendrimers) of compound V (n=60)
Concrete steps are as follows: 100mg five PAMAM dendrimer (compound VI, G5PAMAM, molecular weight 28824g/mol are stored in methyl alcohol, wt:5%) (is about 3.47 × 10 -6mol), 21.1 μ l acetic anhydride (Ac 2o) (2.05 × 10 are about -4mol) and 36.7 μ l triethylamine (Et 3n) (2.57 × 10 are about -4mol) mix, under nitrogen protection room temperature, (25 DEG C) stirring reaction 24h, obtains colourless transparent liquid.Be spin-dried for solvent, use deionized water lysate, filter by aqueous phase syringe filter (specification 25mm × 0.20 μm), filtrate is dialysed in PBS solution, and (PBS fills a prescription: NaCl 8.0g, KCl 0.2g, Na 2hPO 41.44g, KH 2pO 40.27g is dissolved in 1000ml deionized water, measures pH=7.31).Dialyse one day, change three PBS solution.Above-mentioned product is continued dialyse two days in deionized water, change three deionized waters every day.Dialysis terminates, and by the product lyophilize obtained, productive rate is about 93.9%.
1H-NMR(D 2O，TMS，500MHz)，δ1.88(179H，s)，2.33(504H，s)。
Embodiment 14: the synthesis (synthesis of PAMAM-Ac-pegFa) of compound III (m=7, n=60, p=10)
0.087g compound VII (is about 3.27 × 10 -5mol) be dissolved in 3ml DMSO, at 15 DEG C, add wherein and be dissolved with 3.27 × 10 -6the 3ml DMSO solution of mol PAMAM-Ac, reaction 3d.Obtain yellow transparent solution.Be spin-dried for solvent, use deionized water lysate, aqueous phase syringe filter (specification 25mm × 0.20 μm) is filtered, and filtrate is dialysed one day in PBS solution, changes three PBS solution.Above-mentioned product is continued dialyse two days in deionized water, change three deionized waters every day.Dialysis terminates, and by the product lyophilize obtained, productive rate is about 93.7%.
Its appraising datum is as follows:
1H-NMR(D 2O，TMS，500MHz)，δ1.89(179H，s)，2.28(504H，s)，8.64(7H)，7.62(14H)，6.68(14H)
Embodiment 15: the synthesis (synthesis of PAMAM-Ac-pegFa-DTPA) of chemical compounds I (m=7, n=60, p=10)
Containing having an appointment 2.76 × 10 -6in the aqueous solution of mol PAMAM-Ac-pegFa (compound III) (5mL), add 0.0572g compounds Ⅳ and (be about 2.94 × 10 -5mol), by NaOH solution (1M) adjust ph to about 9.At 40 DEG C, reaction 24h, obtains yellow transparent solution.Be spin-dried for solvent, use deionized water lysate, aqueous phase syringe filter (specification 25mm × 0.20 μm) is filtered, and filtrate is dialysed one day in PBS solution, changes three PBS solution.Above-mentioned product is continued dialyse two days in deionized water, change three deionized waters every day.Dialysis terminates, and by the product lyophilize obtained, productive rate is about 92.4%.
Nuclear magnetic data is as follows:
1H-NMR(D 2O，TMS，500MHz)，δ1.88(179H，s)，2.33(504H，s)，6.78(7H，d，J＝5.0)，7.56(31H，d，J＝8.0)，6.84(30H，d，J＝8.2)
Embodiment 16: synthesis (the PAMAM-Ac-pegFa-DTPA-of compound ii (m=7, n=60, p=15) 99mthe synthesis of Tc)
By type I compound (48mg, 1.2 × 10 -6mol) SnCl of 5ml is dissolved in 2(SnCl 2concentration 5.27 × 10 -3m) hydrochloric acid soln (concentration of hydrochloric acid: 1 × 10 -3m) in, then 1mCiNa is added wherein 99mtcO 4solution, reacts 30min, obtains marked product in the water-bath of 80 DEG C.Mark rate is about 94%.
Embodiment 17: the synthesis (synthesis of PAMAM-Ac-pegFa) of compound III (m=7, n=85, p=10)
0.089g compound VII (is about 3.27 × 10 -5mol) be dissolved in 3ml DMSO, at 15 DEG C, add wherein and be dissolved with 3.27 × 10 -6the 3ml DMSO solution of mol PAMAM-Ac, reaction 3d.Obtain yellow transparent solution.Be spin-dried for solvent, use deionized water lysate, aqueous phase syringe filter (specification 25mm × 0.20 μm) is filtered, and filtrate is dialysed one day in PBS solution, changes three PBS solution.Above-mentioned product is continued dialyse two days in deionized water, change three deionized waters every day.Dialysis terminates, and by the product lyophilize obtained, productive rate is about 94.1%.
Its appraising datum is as follows:
1H-NMR(D 2O，TMS，500MHz)，δ1.89(254H，s)，2.28(504H，s)，8.64(7H)，7.62(14H)，6.68(14H)
Embodiment 18: the synthesis (synthesis of PAMAM-Ac-pegFa-DTPA) of chemical compounds I (m=7, n=85, p=10)
Containing having an appointment 2.80 × 10 -6in the aqueous solution of mol PAMAM-Ac-pegFa (compound III) (5mL), add 0.0572g compounds Ⅳ and (be about 2.94 × 10 -5mol), by NaOH solution (1M) adjust ph to about 9.At 40 DEG C, reaction 24h, obtains yellow transparent solution.Be spin-dried for solvent, use deionized water lysate, aqueous phase syringe filter (specification 25mm × 0.20 μm) is filtered, and filtrate is dialysed one day in PBS solution, changes three PBS solution.Above-mentioned product is continued dialyse two days in deionized water, change three deionized waters every day.Dialysis terminates, and by the product lyophilize obtained, productive rate is about 92.4%.
Nuclear magnetic data is as follows:
1H-NMR(D 2O，TMS，500MHz)，δ1.88(254H，s)，2.33(504H，s)，6.78(7H，d，J＝5.0)，7.56(31H，d，J＝8.0)，6.84(30H，d，J＝8.2)
Embodiment 19: synthesis (the PAMAM-Ac-pegFa-DTPA-of compound ii (m=7, n=85, p=15) 99mthe synthesis of Tc)
By type I compound (51mg, 1.2 × 10 -6mol) SnCl of 5ml is dissolved in 2(SnCl 2concentration 5.27 × 10 -3m) hydrochloric acid soln (concentration of hydrochloric acid: 1 × 10 -3m) in, then 1mCiNa is added wherein 99mtcO 4solution, reacts 30min, obtains marked product in the water-bath of 80 DEG C.Mark rate is about 95.1%.
HPLC characterizes embodiment:
The chemical compounds I more than synthesized characterizes correct through nuclear-magnetism, for determining the exactness of compound ii structure, We conducted the experiment of reverse high resolution liquid chromatography.
Reverse high resolution liquid chromatography (Reverse Phase High Performance Liquid Chromatography, HPLC) condition:
HPLC column: Phenomenex (Torrance, CA) Jupiter C5 silica based HPLC column (250mm_4.6mm, 300 ).
Drip washing condition: eluent is water/acetonitrile.Drip washing gradient is 50: 50 (water/acetonitriles) when linearly dropping to 30min from 90: 10 during 0min (water/acetonitrile).
Under these conditions, we obtain radioactivity HPLC and compose Fig. 1 and 2.
99mtcO 4 -radioactivity HPLC spectrogram see Fig. 1, its retention time t=6.15min.
The radioactivity HPLC spectrogram of the title complex II that embodiment 4 is obtained is shown in Fig. 3, its retention time t=26.54min.
Can think that the title complex II that embodiment 4 obtains is correct from above-mentioned data.
Effect example: the SPECT imaging example of tumor mouse
BALB/c nude mice, male, body weight 18 ~ 20g, is provided by Shanghai Slac Experimental Animal Co., Ltd., raises at Shanghai Univ. of Traditional Chinese Medicine SPF level Animal Lab..Raise two days later in Animal House adaptability, in nude mice oxter injection human oral cancer KB cells, injection system is subcutaneous injection, and injection volume is 0.2ml (1 × 10 7cells/ml).Latter three weeks of injection, tumor size is 1cm × 1cm, carries out SPECT imaging experiment.
Tumor mouse, by tail vein injection, injects the PAMAM-Ac-pegFa-DTPA-that embodiment 4 is obtained 99mtc, radioactivity amount is 500 μ Ci.SPECT image after 6 hours is shown in accompanying drawing 3.

Claims (11)

1. one kind such as formula shown in II 99mtc title complex;
Wherein, m=1 ~ 10, n=60 ~ 85, p=10 ~ 50; Ball-like structure represent in Polyamidoamine Dendrimers except 128-NH 2structure in addition.
2. as claimed in claim 1 99mtc title complex, is characterized in that: described m=4 ~ 8, n=76 ~ 78, p=25 ~ 40.
3. as claimed in claim 2 99mtc title complex, is characterized in that: described m=7, n=77, p=35.
4. the preparation method of the title complex II as described in any one of claims 1 to 3, is characterized in that: the method comprises the following step: in aqueous hydrochloric acid, at SnCl 2effect under, by chemical compounds I and 99mtcO 4 -an alkali metal salt and/or alkaline earth salt carry out labeled reactant;
5. the preparation method of title complex II as claimed in claim 4, is characterized in that: the concentration of described aqueous hydrochloric acid is 0.05 ~ 0.15M; The mol ratio of hydrochloric acid and chemical compounds I is 0.1:1 ~ 20:1; Described SnCl 2consumption be 3 ~ 20 times of chemical compounds I molar weight; Described 99mtcO 4 -an alkali metal salt and/or alkaline earth salt be Na 99mtcO 4; 99mtcO 4 -an alkali metal salt and/or ratio between the radioactive activity of alkaline earth salt and chemical compounds I molar weight be 1mci:10 -6mmol ~ 1mci:1mmol; Described temperature of reaction is 60 ~ 100 DEG C; Till the described reaction times is complete with detection reaction.
6. the preparation method of title complex II as claimed in claim 4, is characterized in that: described chemical compounds I is obtained by following method: in water, and under the effect of alkali, compound III and compounds Ⅳ carry out reaction that is amino and isothiocyano;
7. the preparation method of title complex II as claimed in claim 6, is characterized in that: the consumption of described compounds Ⅳ is 5 ~ 40 times of compound III molar weight; Described alkali is inorganic strong alkali; The consumption of alkali is the pH scope controlling reaction soln is the amount of 8 ~ 10; The temperature of described reaction is 30 ~ 50 DEG C; Time of described reaction with detection reaction completely till.
8. the preparation method of title complex II as claimed in claim 6, is characterized in that: described compound III is obtained by following method: compound V and compound VII are carried out condensation reaction;
9. the preparation method of title complex II as claimed in claim 8, is characterized in that: the consumption of described compound VII is 1 ~ 12 times of compound V molar weight; The temperature of described reaction is 15 ~ 37 DEG C; Time of described reaction with detection reaction completely till.
10. one kind such as formula the preparation shown in I as described in any one of claims 1 to 3 99mthe midbody compound of Tc title complex II;
11. as described in any one of claims 1 to 3 99mthe application of Tc title complex II in preparation SPECT developer.
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