CN103275188B - Radio-labeling T140 class polypeptide compound and its preparation method and application - Google Patents

Abstract

The invention discloses radio-labeling T140 class polypeptide compound and its preparation method and application.Radionuclide ¹⁸the T140 class polypeptide compound of F mark, contains the polypeptide (Ac-TC14012) of radionuclide and target CXCR4 acceptor in its structure, different according to Radiolabelling method, described compound is divided into compound 1 and compound 2 two kinds.The invention still further relates to described compound purposes as developer in the organ or tissue of human or animal, in particular as tumor developer, it has, and tumor uptake is high, non-specific uptake is low and the advantage of higher target/non-target ratio.

Description

Radio-labeling T140 class polypeptide compound and its preparation method and application

Technical field

The present invention relates to radio-labeled compound, its preparation method and described compound are as the application of tumor developer.

Background technology

According to the incidence trend of current cancer, the year two thousand twenty whole world cancer morbidity will than increasing by 50% now, and the annual newly-increased cancer patients's number in the whole world will reach 1,500 ten thousand people (World Health Organization (WHO) delivers in the recent period " report of world's cancer ").Discovery and early treatment reduce cancer mortality effective means the most early.Implement the generaI investigation system to intestinal cancer, cervical cancer, mammary cancer etc., global number of cancer deaths can be made to reduce 1/3rd.At present in developing country, the cancer patients of 80% was just found in ill late period.

Although the precise mechanism that cancer occurs is still unclear at present, if but can diagnose cancer in early days, and take operation as early as possible, radiation or chemotherapy (or combination of this several method), most of tumour patient is the possibility having survival.Therefore in order to cancer cells diffusion before to patient, we need the technology developing energy Sensitive Detection cancer, formation method at present for cancer diagnosis mainly contains: X-ray CT, ultrasonic (CS), Magnetic resonance imaging (MRI) and nuclear medicine SPECT/PET.US and MRI technology may be used for the dissection and analysis of noumenal tumour, but the shortcoming of these technology is: they can not detect the Biochemical changes of tumor tissues on a molecular scale, because MRI and CT technology need in tumor tissues containing a lot of contrast agents with and healthy tissues forms very strong contrast, another shortcoming of these technology is that they are for frequently-occurring tumour (such as mammary cancer, carcinoma of gallbladder, lung cancer and prostate cancer) specificity and susceptibility poor, therefore we need the radiotracer developing a kind of new tumour-specific badly, it not only can be used in the early diagnosis of tumour, and the reaction for pharmacological agent of the growth of tumour and tumour can be monitored.

Chemokine family is the small molecules secreted protein that a class is close with glycoprotein structure, and Main Function is the migration of chemotactic cell, and wherein stroma cell derivative factor SDF-1 α is considered to play an important role in revascularization process.SDF-1 α is combined with its receptor CXCR 4, and costimulatory receptor forms dimer, activates downstream signaling molecules, comprises contact adhesion kinase, extracellular signal-regulated kinase, protein kinase C, JAK/Stat and NF-KB transcriptional pathway.

Now there are some researches show that Chemokine receptor CXCR4 has overexpression on the surface of a variety of human tumor cell, such as CXCR4 acceptor has expression in lymphoma, neurospongioma, ovarian cancer and carcinoma of the pancreas in position, is also found in the mammary cancer of transfer, lung cancer, thyroid carcinoma, neuroblastoma, leukemia and diseases associated with inflammation.Also expression is had in healthy tissues is as spleen, pancreas and marrow.And research shows, CXCR4 acceptor and tumor prognosis is poor, chemotherapy resistance and transfer have substantial connection.One of CXCR4 acceptor two major co-receptors being also confirmed as HIV poisoning intrusion, therefore CXCR4 receptor antagonist is developed to the medicine of AIDS, therefore it can be used for blocking HIV cell entry host cell by capturing co-receptor to prevent virus attachment and incorporating cell.As can be seen here, the part of CXCR4 acceptor or antagonist can effectively for oncotherapy and video picture objects.In fact, a kind of small molecules CXCR4 receptor antagonist AMD3100(Plerixafor) clinically as immunostimulant for the protection of hematopoietic stem cell transplantation.

The radioactive tracer of development CXCR4 receptor-specific, the expression level being used for assessing acceptor by positron emission tomography (PET) video picture in vivo can provide guidance for the Diagnosis and Treat of tumour.

Because SDF-1 α/CXCR4 system especially has keying action at tumorigenesis in metastases; therefore the molecular probe for CXCR4 target spot studied at present is used for tumor imaging (ID Weiss; O Jacobson.Molecular Imaging of Chemokine Receptor CXCR4.Theranostics, 2013; 3 (1): 76-84).

By studying extensively and profoundly in a large number; Tamamura and co-worker thereof are developed and are optimized a kind of 14 amino acid whose CXCR4 acceptor inhibitors: T140 polypeptide and derivative (Tamamura H thereof; Omagari A; Oishi S; Kanamoto T; Yamamoto N; Peiper SC; et al.Pharmacophore identification of a specific CXCR4inhibitor; T140, leads to development of effective anti-HIV agents with very high selectivity indexes.Bioorg Med Chem Lett.2000; 10:2633-7).This seminar had previously reported TN14003 after radio-labeling, although the avidity of itself and CXCR4 is very high, was combined and had had a strong impact on imaging results, particularly greatly reduce the ratio of tumour and background with erythrocytic height.And peptide side chain the 7th and 8 Lys residues also need protection; add remove-insurance and protect step (Jacobson O, Weiss ID, Kiesewetter DO; Farber JM, Chen X.PET of Tumor CXCR4Expression with4-18F-T140.J Nucl Med.2010; 51:1796-804.).Utilize further the 7th and 8 Lys residues carry out ⁶⁴cu isotope labeling; still can keep very high receptor affinity; but the combination of erythrocyte does not reduce (Jacobson O WI; Szajek LP; Niu G, Ma Y, Kiesewetter DO; Farber JM, Chen X..PETimaging of CXCR4using copper-64labeled peptide antagonist.Theranostics.2011; 1:251-62).Utilize polypeptide N-to hold again and carry out 64Cu isotope labeling; although the marker ligand compound obtained reduces by 1 ~ 2 magnitude on receptor affinity; but the combination of itself and erythrocyte almost disappears; therefore the better tumor imaging result of contrast gradient (Jacobson O, Weiss ID, Szajek LP can be obtained; Niu G; Ma Y, Kiesewetter DO, et al.Improvement ofCXCR4tracer specificity for PET imaging.J Controlled Release.2012; 157:216-23).Except above-mentioned radioactive probe, the tracer agent of the target CXCR4 acceptor of the radioisotope labeling reported at present is multiple in addition, nucleic used comprise iodine-125 ( ¹²⁵i), technetium-99m ( ^99mtc), Value linear ( ¹⁸f), copper-64 ( ⁶⁴cu) and gallium-68 ( ⁶⁸ga) etc., part used comprises small molecules and polypeptide.But up to the present also do not have a kind of radiological imaging agent of satisfaction, the developer therefore developing a kind of excellent performance is still underway.

Select from nucleic, be applicable to the video picture nucleic Value linear of PET, copper-64 and gallium-68 and there is advantage. ¹⁸f is positron radionuclide the most conventional in the world at present, its core excellent property, atomic radius is little affects scalar nature hardly, and transmitting positron energy is low can better spatial resolution, when transformation period is applicable to mark and dispensing, and accelerator is convenient for production to be easy to get. ¹⁸f is radionuclide the most frequently used during PET video picture checks, in the PET imaging medicament of current whole world application, ¹⁸f tagged compound accounts for more than 90%, is widely used in the inspection of the multiple internal organs illness such as the heart, brain, bone, tumour.

In T140 series derivates; the Ac-TC14012 of latest report shows very high avidity (29nM) and HIV (human immunodeficiency virus)-resistant activity (Tamamura H; Hiramatsu K; Kusano S; Terakubo S; Yamamoto N, Trent JO, et al.Synthesis of potent CXCR4inhibitors possessing low cytotoxicity and improved biostabilitybased on T140derivatives.Org Biomol Chem.2003; 1:3656-62).And this peptide side chain only has the Lys residue of the 7th to can be used for radio-labeling, can ensure the marker obtaining one-component, improve radiochemical purity.And research shows that the Lys of this position and ligand activity have nothing to do, can carry out transforming or marking and not affect the activity of polypeptide and receptors bind.

For this reason, the present invention adopts ¹⁸f is radionuclide, is connected with Ac-TC14012 polypeptide by certain way, obtains ¹⁸the developer for CXCR4 acceptor of F mark, the diagnosing tumor of highly expressing for CXCR4 in conjunction with PET imaging technique or therapeutic evaluation.

Summary of the invention

Primary and foremost purpose of the present invention is to provide the new radiolabeled T140 compound with premium properties;

Another object of the present invention is to provide the preparation method of new radiolabeled T140 compound;

Another object of the present invention is to provide the application of described compound as tumor developer.

The invention provides two kinds of radioactivity containing T140 structure ¹⁸the compound (compound 1 and 2) of F mark.

formula 1

Object of the present invention can be achieved through the following technical solutions:

The radiopharmaceuticals containing T140 polypeptide derivative of radioisotope labeling, described radionuclide is ¹⁸f, described T140 polypeptide is Ac-TC14012.The T140 polypeptide radiopharmaceutical of described radioisotope labeling is that described radionuclide is combined with described T140 polypeptide and obtains, and is water white liquid injection.The chemical structural formula of compound 1 and 2 is see formula 1.

The preparation method of compound 1, comprises the following steps:

1) 4-nitrophenyl-2-[ ¹⁸f] fluorine propionic ester ([ ¹⁸f] FPA) preparation

General appropriate (20 ~ 300mCi) [ ¹⁸f] F ^-from separator column (CHROMAFIX), drip washing is got off, and is placed in reaction flask, is removed by solvent by vacuum drying method, obtain anhydrous [ ¹⁸f] F ^-, add appropriate (1 ~ 5mg) ethyl 2 bromopropionic acid ester (0.1 ~ 1mL acetonitrile) after cooling, be heated to 70 ~ 150 ° of C and react 5 ~ 20min, obtain ethyl 2-[ ¹⁸f] fluorine propionic ester.At the end of reaction, add appropriate (50 ~ 200 μ L) 0.2mol/L potassium hydroxide solution and acetonitrile solution (200 ~ 500 μ L), be again heated to 70 ~ 150 ° of C and react 5 ~ 20min, obtain ethyl 2-[ ¹⁸f] fluorine propionic acid sylvite, then with appropriate (0.2 ~ 2mL) dilution in acetonitrile and vacuum-drying except desolventizing, add appropriate (2 ~ 40mg) two-(4-nitrophenyl) acetonitrile solution of carbonic ether afterwards, under 70 ~ 150 ° of C conditions, react 5 ~ 20min, generate 4-nitrophenyl-2-[ ¹⁸f] fluorine propionic ester ([ ¹⁸f] FPA), after cooling, reaction mixture is with half preparative HPLC separation and purification and concentrate volume with C18 post.The whole reaction times is about 100min, without the productive rate about 10 ~ 15% of decay correction.

2) [ ¹⁸f] FP-Ac-TC14012(compound 1) preparation

By above-mentioned gained and be adsorbed on C18 post [ ¹⁸f] FPA gets off with appropriate (0.2 ~ 2mL) eluent methylene chloride, manual removing upper strata aqueous phase, at room temperature applying argon gas evaporate dichloromethane solvent, then the DIPEA that appropriate (20 ~ 600 μ g) is dissolved in Ac-TC14012 polypeptide (customized in polypeptide company of the U.S.) in DMSO and (1 ~ 10 μ L) is in right amount added, be heated to react 5 ~ 20min under 50 ~ 120 ° of C conditions, at the end of reaction, cooling is also diluted with the aqueous solution containing TFA.After HPLC purifying, obtain compound 1, this step reaction times is about 50min, and the productive rate after decay correction about 30 ~ 40%, radiochemical purity is greater than 95%.

The preparation method of compound 2, comprises the following steps:

1) N-succinimide-4- ¹⁸f-fluorobenzoate ([ ¹⁸f] SFB) preparation

General appropriate (20 ~ 300mCi) [ ¹⁸f] F ^-from separator column (CHROMAFIX), drip washing is got off, and is placed in reaction flask, is removed by solvent by vacuum drying method, obtain anhydrous [ ¹⁸f] F ^-appropriate (1 ~ 5mg) 4-Trimethylamine ethyl benzoate trifluoromethyl sulfonic acid (0.1 ~ 2mLDMSO) is added after cooling, be heated to 90 ~ 150 ° of C and react 5 ~ 20min, at the end of reaction, add appropriate (200 ~ 1000 μ L) 0.2mol/L sodium hydroxide solution, be again heated to 90 ~ 150 ° of C and react 5 ~ 20min, cooling reaction solution, add appropriate (0.1 ~ 1mL) 1mol/L HCl and carry out acidifying, obtain 4- ¹⁸f-fluorobenzoic acid ([ ¹⁸f] FB), then by reaction mixture by C18 column purification, and with acetonitrile drip washing obtain after purifying [ ¹⁸f] FB, add appropriate (2 ~ 20 μ L) 40% tetrabutylphosphoniuhydroxide hydroxide amine afterwards, after drying up acetonitrile solvent, again add appropriate (8 ~ 20mg) TSTU(N, N, N ', N '-tetramethyl-urea a tetrafluoro borate) solution, 5 ~ 20min is reacted under 70 ~ 100 ° of C conditions, generate [ ¹⁸f] SFB, after cooling, reaction mixture is with half preparative HPLC separation and purification and concentrate volume with C18 post.The whole reaction times is about 120min, without the productive rate about 20% of decay correction.

2) [ ¹⁸f] FB-Ac-TC14012(compound 2) preparation

By above-mentioned gained and be adsorbed on C18 post [ ¹⁸f] SFB gets off with appropriate (0.2 ~ 2mL) eluent methylene chloride, and manually removing upper strata aqueous phase, at room temperature applying argon gas evaporate dichloromethane solvent, then adds appropriate (20 ~ 600 μ g) and is dissolved in 0.1mL Na ₂hPO ₄ac-TC14012 polypeptide (customized in polypeptide company of the U.S.) in (pH8.5,100mM), is heated to react 5 ~ 30min under 20 ~ 60 ° of C conditions, and at the end of reaction, cooling is also diluted with the aqueous solution containing TFA.After HPLC purifying, obtain compound 2, this step reaction times is about 60min, and the productive rate after decay correction about 30 ~ 40%, radiochemical purity is greater than 98%.

The stable reference compou nd synthesis (FP-Ac-TC14012) of compound 1

Appropriate (1 ~ 20mg) Ac-TC14012 polypeptide is dissolved in right amount in (100 ~ 1000 μ L) DMSO, adds suitably excessive (1.1eq) 4-nitrophenyl 2-fluorine propionic ester and appropriate (1 ~ 10 μ L) DIPEA, room temperature reaction 8 ~ 40min.With appropriate (1 ~ 20 μ L) TFA termination reaction.Product, through half preparative HPLC separation and purification, collects target compound cut and freeze-drying.Obtain the stable reference compound of compound 1: FP-Ac-TC14012.Result confirms through mass spectrum.

The stable reference compou nd synthesis (FB-Ac-TC14012) of compound 2

The Na of (100 ~ 1000 μ L) 100mM will be dissolved in right amount by (1 ~ 20mg) Ac-TC14012 polypeptide in right amount ₂hPO ₄(pH8.5), in, suitably excessive (1.5eq) N-succinimide-4-fluorobenzoate (10 ~ 1000 μ L acetonitrile) is added, room temperature reaction 10 ~ 40min.With appropriate (1 ~ 60 μ L) TFA termination reaction.Product, through half preparative HPLC separation and purification, collects target compound cut and freeze-drying.Obtain the stable reference compound of compound 2: FB-Ac-TC14012.Result confirms through mass spectrum.

Other chemical substance used in above-mentioned synthesis step is commercial goods.

Beneficial effect of the present invention:

1. the present invention is in T140 series derivates, selects the Ac-TC14012 the highest with CXCR4 receptor affinity to be part, introduces radioactivity ¹⁸f is as visualization portion.Effectively can obtain the high tumor developer of avidity, increase tumor uptake value, be conducive to the early diagnosis of tumour.

2. the marking method of two kinds of markers of the present invention easily is automated synthesis, and this is for extremely important transformation period shorter radionuclide, advantageously in commercial applications and the clinical expansion of radioactively labelled substance.

3. the compound structure that formed of T140 part of the present invention and radionuclide is more single, is conducive to the chemical purity and the radiochemical purity that improve tagged compound, and avoids the impact of radioactivity mixture on video picture quality.

4. the marker site of part Ac-TC14012 of the present invention is the Lys of side chain the 7th, and the activity relationship of this site and part is little, can not affect its biological activity through radio-labeling or after modifying.

5. the combination of radio-labeled compound of the present invention and erythrocyte is very low, accelerates tracer agent removing speed in blood, increases target/non-target ratio, makes video picture more clear, reaches better diagnosis effect by improving video picture quality.

Accompanying drawing explanation

Fig. 1 is A: the expression of flow cytometry analysis CXCR4 acceptor in CHO-CXCR4 and Chinese hamster ovary celI; B: the stable reference compound (FP-Ac-TC14012) of prototype compound (Ac-TC14012), compound 1 and the stable reference compound (FB-Ac-TC14012) of compound 2 in CHO-CXCR4 cell with [ ¹²⁵i] competitive binding assay of CXCL12.

Fig. 2 is compound 1(A: picked-up; B: be detained) and compound 2(C: picked-up; D: be detained) picked-up respectively in CHO-CXCR4 and Chinese hamster ovary celI and delay.

Fig. 3 is on compound 1() and compound 2(under) bio distribution in lotus knurl (CHO-CXCR4 and CHO) nude mice after administration 120min and inject Ac-TC14012 polypeptide (50 μ g) altogether and suppress result.The mean value of 4-6 mouse.

Fig. 4 is that compound 1 is at tumor bearing nude mice (right shoulder: CHO-CXCR4; Left shoulder: CHO) in PET video picture coronal-plane tomography (on, the representative picture of 1 nude mice) and the quantitative uptake values (under, the average result of 4-6 mouse) that calculates thus.A figure is the result of 30min after administration, and B figure is the result of 60min after administration.

Fig. 5 is that compound 2 is at tumor bearing nude mice (right shoulder: CHO-CXCR4; Left shoulder: CHO) in PET video picture coronal-plane tomography (on, the representative picture of 1 nude mice) and the quantitative uptake values (under, the average result of 4-6 mouse) that calculates thus.A figure is the result of 30min after administration, and B figure is the result of 60min after administration.

Embodiment

The present invention can be made to be illustrated more clearly in below by way of concrete preparation example and embodiment:

One, preparation example:

1. the preparation of compound 1:

1) 4-nitrophenyl-2-[ ¹⁸f] fluorine propionic ester ([ ¹⁸f] FPA) preparation

15mg K222(amino-polyether is contained with 1mL) and 3.5mg K ₂cO ₃90% acetonitrile solution by 100mCi [ ¹⁸f] F ^-from separator column (CHROMAFIX), drip washing is got off, and is placed in glass reaction bottle, is removed by solvent by vacuum pumping method, obtain anhydrous [ ¹⁸f] F ^-.Add the 5mg ethyl 2 bromopropionic acid ester being dissolved in 0.5mL acetonitrile after cooling, be heated to 100 ° of C and react 10min, obtain ethyl 2-[ ¹⁸f] fluorine propionic ester.At the end of reaction, add 125 μ L0.2mol/L potassium hydroxide solutions and 375 μ L acetonitrile solutions, be again heated to 100 ° of C and react 10min, obtain ethyl 2-[ ¹⁸f] fluorine propionic acid sylvite, then with 1mL dilution in acetonitrile and vacuum-drying except desolventizing, add the acetonitrile solution (20mg bis--(4-nitrophenyl) carbonic ether) of appropriate 1mL bis--(4-nitrophenyl) carbonic ether afterwards, 10min is reacted under 100 ° of C conditions, generation 4-nitrophenyl-2-[ ¹⁸f] fluorine propionic ester ([ ¹⁸f] FPA).After cooling, reaction mixture is with 1mL acetonitrile and 5% acetum (25%:75%) dilution, then with half preparative HPLC separation and purification, collecting retention time is the peak of 21min, dilute with the acetum of 30mL0.1%, then by C18 post (Waters Sep-Pak Plus), radioactive product is adsorbed on pillar, with 1mL water wash removing water-soluble impurity.The whole reaction times is about 100min, without the average yield about 12.4 ± 3.4% (n=4) of decay correction.

2) [ ¹⁸f] FP-Ac-TC14012(compound 1) preparation

By above-mentioned gained and be adsorbed on C18 post [ ¹⁸f] FPA gets off with 1mL eluent methylene chloride, and be placed in 1.5mL polypropylene reaction tubes, the upper strata aqueous phase of methylene dichloride is manually removed with syringe, then at room temperature applying argon gas evaporate dichloromethane solvent, add 200 μ g and be dissolved in 0.1mL DMSO(dimethyl sulfoxide (DMSO)) in Ac-TC14012 polypeptide and 5 μ LDIPEA(N, N-diisopropylethylamine), react 10min under reaction tubes being heated to 80 ° of C conditions.At the end of reaction, cooling reaction solution also with 0.5mL containing 0.1%TFA(trifluoroacetic acid) aqueous solution dilution.Compound 1 is obtained after half preparative HPLC purifying, collecting retention time is the peak of 18.9min, to be loaded to C18 post (VarianBond Elute after the dilution of 10mL water, radioactive product 100mg) is made to be stranded in post, then with the drip washing of 0.15mL10mM HCl ethanolic soln, blow away ethanol with argon gas, last product is dissolved in PBS.This step reaction times is about 50min, the productive rate after decay correction about 38.1 ± 10.5% (n=4), substantially from 9.4mCi [ ¹⁸f] FPA starts to obtain 2.4mCi product (compound 1).Be greater than 95% with analysis mode HPLC analytical radiochemistry purity, relative retention time is 17.5min.

2. the preparation of compound 2:

1) N-succinimide-4- ¹⁸f-fluorobenzoate ([ ¹⁸f] SFB) preparation

15mg K222 and 3.5mg K is contained with 1mL ₂cO ₃90% acetonitrile solution by 160mCi [ ¹⁸f] F ^-from separator column (CHROMAFIX), drip washing is got off, and is placed in glass reaction bottle, by being heated to 110 ° of C and solvent removes by the method for inflated with nitrogen, obtain anhydrous [ ¹⁸f] F ^-0.5mL4-Trimethylamine ethyl benzoate trifluoromethyl sulfonic acid (3mg is dissolved in 0.5mL DMSO) is added after cooling, be heated to 120 ° of C and react 10min, at the end of reaction, add 500 μ L0.2mol/L sodium hydroxide solutions, be again heated to 120 ° of C and react 10min, cooling reaction solution, add 0.5mL1mol/L HCl and carry out acidifying, obtain 4- ¹⁸f-fluorobenzoic acid ([ ¹⁸f] FB), then by reaction mixture by C18 column purification, and with acetonitrile drip washing obtain after purifying [ ¹⁸f] FB, add 5 μ L40% tetrabutylphosphoniuhydroxide hydroxide amine afterwards, after inflated with nitrogen dries up acetonitrile solvent, add 0.5mL TSTU(N, N, N ', N '-tetramethyl-urea a tetrafluoro borate) and acetonitrile solution (15mg), under 90 ° of C conditions, react 5min, generate [ ¹⁸f] SFB.Cooling reaction solution after, reaction mixture with half preparative HPLC separation and purification, obtain about 20mCi [ ¹⁸f] SFB, and adsorb this product with C18 post.Reaction times is about 100min, without the average yield about 17.4 ± 5.7% (n=4) of decay correction.

2) [ ¹⁸f] FB-Ac-TC14012(compound 2) preparation

By above-mentioned gained and be adsorbed on C18 post [ ¹⁸f] SFB gets off with 1mL eluent methylene chloride, and is placed in the polypropylene tube of 1.5mL, manually remove upper strata aqueous phase, at room temperature applying argon gas evaporate dichloromethane solvent, then add 200 μ g and be dissolved in 0.1mL100mM Na with syringe ₂hPO ₄(pH8.5) the Ac-TC14012 polypeptide in solution, and react 20min at ambient temperature, at the end of reaction, cooling is also diluted containing the aqueous solution of 0.1%TFA with 0.5mL.Compound 2 is obtained after half preparative HPLC purifying, collecting retention time is the peak of 20.5min, to be loaded to C18 post (Varian Bond Elute after the dilution of 10mL water, 100mg), then with the HCl ethanolic soln drip washing of 0.15mL10mM, after blowing away alcohol solvent with argon gas, the finished product are dissolved in PBS.This step reaction times is about 60min, and the average yield after decay correction is 31.6 ± 7.0% (n=4), in general, from 10.7mCi [ ¹⁸f] SFB starts to obtain 2.0mCi product.Finally detect radiochemical purity with analysis mode HPLC and be greater than 98%, relative retention time is 19.5min.

3. the stable reference compou nd synthesis (FP-Ac-TC14012) of compound 1

3mg Ac-TC14012 polypeptide is dissolved in 400 μ L DMSO, adds 4-nitrophenyl 2-fluorine propionic ester and 5 μ L DIPEA, the room temperature reaction 20min of 1.1 times of equivalents.With appropriate 10 μ L TFA termination reactions.Product, through half preparative HPLC separation and purification, collects target compound cut (retention time is 27min) and freeze-drying.Obtain the stable reference compound of white powder compound 1: FP-Ac-TC14012.Productive rate is about 56%, and result confirms through mass spectrum, high resolution mass spectrum (C ₉₅h ₁₄₆fN ₃₄o ₂₁s ₂): [M+H] ⁺: 2182.4392, calculated value: 2181.0827 (m/z).

4. the stable reference compou nd synthesis (FB-Ac-TC14012) of compound 2

3mg Ac-TC14012 polypeptide is dissolved in the Na of 500 μ L100mM ₂hPO ₄(pH8.5), in damping fluid, 100 μ L N-succinimide-4-fluorobenzoate acetonitrile solutions of 1.5 times of equivalents are added, room temperature reaction 20min.With 20 μ L TFA termination reactions.Product, through half preparative HPLC separation and purification, collects target compound cut (retention time 31min) and freeze-drying.Obtain the stable reference compound of white powder compound 2: FB-Ac-TC14012.Productive rate about 42%, result confirms (C through high resolution mass spectrum ₉₉h ₁₄₆fN ₃₄o ₂₁s ₂), [M+H] ⁺: 2230.1590, calculated value: 2230.0827 (m/z).

5.HPLC gradient condition

Preparation HPLC: C18 preparative column (Phenomenex Luna, 5 μm, 250 × 10mm).Linear drip washing gradient: 0 ~ 5 minute: 5%B(0.1%TFA acetonitrile solution) and the 95%A(0.1%TFA aqueous solution), 5 ~ 35 minutes: be increased to 65%B and 35%A; Flow velocity: 5mL/min; Compound 1 retention time is 21.6min.

Analysis mode HPLC:C18 analytical column (Phenomenex Luna, 5 μm, 150 × 4.6mm).Linear drip washing gradient: 0 ~ 30 minute: 5%A(0.1%TFA acetonitrile solution) and the 95%A(0.1%TFA aqueous solution) be increased to 50%A and 50%B; Flow velocity: 1mL/min.

Two, embodiment:

Compound 1 and 2 is prepared by above-mentioned preparation example, and reference compound stable accordingly (nonradioactive labeling) FP-Ac-TC14012 and FB-Ac-TC14012.Below that their performance measurement is described:

1. cell experiment.Choose wild-type CHO(Chinese hamster ovarian) cell and determine the Chinese hamster ovary celI (CHO-CXCR4) of transfection CXCR4 acceptor, Chinese hamster ovary celI growth is in F-12K nutrient solution, containing 10% foetal calf serum, 1mM Sodium.alpha.-ketopropionate, 2mM Pidolidone and some non-essential amino acid, at 37 ° of C and 5%CO ₂cultivate under environment.CXCR4 acceptor quantitative expression in CHO-CXCR4 cell and Chinese hamster ovary celI is measured respectively by flow-cytometry method, the Chinese hamster ovary celI (CHO-CXCR4) of result display transfection CXCR4 acceptor exceeds 30 times than the fluorescence intensity of the wild-type CHO cells of untransfected, show that the transfection of CXCR4 acceptor is successful, detailed results is see Figure 1A.

2.Ac-TC14012 the avidity of FP-Ac-TC14012 and FB-Ac-TC14012 and CXCR4 acceptor measures

Adopt cell competition Binding experiment method, measure the Ac-TC14012 polypeptide (prototype) of unmodified, the stable reference compound F 17-hydroxy-corticosterone P-Ac-TC14012 polypeptide of the compounds of this invention 1 and the stable reference compound F 17-hydroxy-corticosterone B-Ac-TC14012 polypeptide of compound 2 and the CXCR4 receptor binding capacity of CHO-CXCR4 cell expressing respectively, test with ¹²⁵the radioactive ligand that I-CXCL12 combines as receptor CXCR 4 protein-specific.Experimental result shows: all compounds containing T140 polypeptide structure unit and CXCR4 receptor protein all have higher avidity, their 503nhibiting concentration (IC ₅₀) be respectively 2.47nM(Ac-TC14012), 7.52nM(FP-Ac-TC14012) and 25.1nM(FB-Ac-TC14012).The present invention transforms the 503nhibiting concentration of rear T140 polypeptide and former Compound Phase is worked as can be seen here, wherein transforms the larger and fat-soluble stronger FB group of volume and introduces and slightly lower avidity.The results detailed in accompanying drawing 1B.

3. cellular uptake and retardation assay

CHO-CXCR4 cell and Chinese hamster ovary celI are seeded in 24 orifice plates respectively, every hole kind 10 ⁵individual cell, every hole adds about 25 μ Ci the compounds of this invention 1([ ¹⁸f] FP-Ac-TC14012) or compound 2([ ¹⁸f] FB-Ac-TC14012), under 37 ° of C conditions, cultivate 5,15,30,60 and 120min.Then with cold PBS washed cell twice, then 1min is hatched to remove the radioreagent of cell surface absorption with pickling damping fluid (50mM glycine, 0.1M NaCl, pH=2.8).Then cell washes twice with the PBS of cooling and every hole adds 250 μ L0.1M NaOH peptic cells and collects again, measures radiocounting.Each time point is averaged in triplicate.Result is shown in Fig. 2 A.As can be seen here, the compounds of this invention 1 reaches 29.94 ± 1.16% in the intracellular picked-up of CHO-CXCR4 after hatching 2h, and the uptake values under similarity condition in Chinese hamster ovary celI is only 13.17 ± 1.04%, illustrate that the picked-up of this compound 1 is CXCR4 receptor-specific.For compound 2, cellular uptake and compound 1 similar, hatch 2h in CHO-CXCR4 cell after, picked-up reaches 46.18 ± 1.05%, and the uptake values under similarity condition in Chinese hamster ovary celI is only 27.23 ± 0.46%, illustrates that the picked-up of this compound 2 is also CXCR4 receptor-specific.The results are shown in Fig. 2 C.

For radioreagent in the intracellular outflow of CHO-CXCR4 (Efflux) experiment, in 24 orifice plates, every hole adds 25 μ Ci the compounds of this invention 1([ ¹⁸f] FP-Ac-TC14012) or 2([ ¹⁸f] FB-Ac-TC14012), 60min is cultivated under 37 ° of C conditions, then with cold PBS washed cell twice, cell is hatched 0 in F-12K nutrient solution, 30 and 60min, then cell washes twice with the PBS of cooling and every hole adds 250 μ L0.1M NaOH peptic cells and collects again, measures radiocounting.Each time point is averaged in triplicate.Result is shown in Fig. 2 B, as can be seen here, the compounds of this invention 1 will apparently higher than Chinese hamster ovary celI in the intracellular delay of CHO-CXCR4, be respectively 21.97 ± 0.73% and 6.47 ± 0.47% in intracellular picked-up after hatching 60min, therefore the retention rate of compound 1 in CHO-CXCR4 cell and Chinese hamster ovary celI is respectively 73% and 49%.Same visible, the compounds of this invention 2 will apparently higher than Chinese hamster ovary celI in the intracellular delay of CHO-CXCR4, after hatching 60min, the picked-up of compound 2 in CHO-CXCR4 cell and Chinese hamster ovary celI is respectively 37.38 ± 0.77% and 18.76 ± 0.26%, and therefore the retention rate of compound 2 in CHO-CXCR4 cell and Chinese hamster ovary celI is respectively 81% and 68%.Detailed results is see Fig. 2 D.

Above-mentioned cell experiment result is visible, and fat-soluble stronger compound 2 is higher in intracellular picked-up, is probably caused by non-specific uptake increases.

4. ¹⁸the bio distribution of F tagged compound in tumor bearing nude mice body

Inoculate Chinese hamster ovary celI and CHO-CXCR4 cell (every side joint kind 10 respectively the upper limbs both sides of nude mice in 4-6 age in week are subcutaneous ⁷individual cell), test for bio distribution and PET video picture after growing general 2 time-of-weeks.

Tail vein injection 0.1mL(about 50 μ Ci from tumor bearing nude mice) prepare ¹⁸f tagged compound (compound 1 or 2) injection liquid (PBS), then after administration 120 minutes by sacrifice, get the tissues such as its blood, the heart, liver, lung, kidney, muscle, marrow, spleen and knurl carry out weighing and measure radiocounting, calculate the percentage injection dose rate (%ID/g) of every gram of tissue.Marrow obtains by rinsing in bone.In order to verify the specificity of tumor uptake, the unlabelled Ac-TC14012 of co-injection, carries out biodistribution experiments again.Often organize 4-6 mouse.Bio distribution the results are shown in accompanying drawing 3.As can be seen here, upon administration during 120min, the compounds of this invention 1 and 2 all absorbs higher than CHO tumour (compound 1:4.30 ± 0.86vs.0.93 ± 0.06%ID/g, p<0.01 in CHO-CXCR4 tumour; Compound 2:1.86 ± 0.19vs.0.44 ± 0.02%ID/g, p<0.01)).About excessive 50 times of use 50 μ g() Ac-TC14012 is when suppressing, and CHO-CXCR4 tumor uptake obviously reduces, now the picked-up difference of CHO-CXCR4 and CHO two kinds of tumours little (p>0.05).Although the CHO tumor uptake of the low expression of CXCR4 raises to some extent after suppressing, difference is not significantly (p>0.05).

5. tumor model mouse MicroPET video picture

Prepare ¹⁸f tagged compound (compound 1 or 2) solution (PBS), gets 0.1mL(about 100 μ Ci) be injected in tumor bearing nude mice tail vein, 30min and 60min carries out static MicroPET tomography surface sweeping (Siemens Inveon) upon administration respectively.In order to verify the specificity of radioactive uptake, while injection radioactive drug, the unmarked polypeptide A c-TC14012 injecting various dose (10,50 and 200 μ g) carries out acceptor suppression.Carry out three-dimensional reconstruction after IMAQ, gained whole body decay correction coronal image delineates region of interest (ROI).From multiple ROI average pixel value, obtain the radioactive activity in the organs such as tumour, muscle, liver, kidney and urine and be converted into MBq/mL, income value obtains %ID/g(divided by injected dose and supposes that tissue density is 1g/mL).Often organize 4-6 mouse.

Image results figure is shown in attached Figure 4 and 5.As can be seen here, tagged compound 1 and 2 of the present invention upon administration 30min all has obviously picked-up and good tumor background ratio in CHO-CXCR4 tumour, and picked-up in the CHO tumour of the low expression of CXCR4 acceptor is obviously much lower.Compared with the allied compound reported in the past, much less is wanted in the compounds of this invention delay in blood, may come from lower erythrocyte and combine.Except tumor tissues, other metabolic organ, such as liver and kidney also have higher picked-up.The inhibition test result that the unlabelled Ac-TC14012 polypeptide of co-injection carries out shows; for compound 1; injecting about excessive 10 times of 10 μ g(altogether) Ac-TC14012 can significantly improve the picked-up of CHO-CXCR4 tumour (30min after administration, absorbs after suppressing with before suppressing: 4.81 ± 0.33vs.2.43 ± 0.13%ID/g; 60min after administration, absorb after suppressing with before suppressing: 4.52 ± 0.24vs.2.37 ± 0.16%ID/g, P<0.01), possible reason is under 10 μ g inhibitor conditions, liver picked-up is suppressed cause (30min after administration, absorbs before suppressing with after suppressing: 14.27 ± 0.66vs.20.36 ± 1.15%ID/g; 60min after administration, absorbs before suppressing with after suppressing: 11.66 ± 0.46vs.19.37 ± 1.56%ID/g, P<0.01).When common injection suppresses dosage higher (50 or 200 μ g, ~ 50 or ~ 200 times excessive), the radioactive uptake of CHO-CXCR4 tumour significantly reduces (30min:1.53 ± 0.09 and 2.20 ± 0.17%ID/g; 60min:1.45 ± 0.17 and 1.65 ± 0.14%ID/g; Inject 10 μ g inhibitor together and compare P<0.01).Simultaneously visible, for the CHO tumour of CXCR4 expression of receptor feminine gender, before and after suppressing, radioactive uptake difference is not significantly (P>0.05).Injecting unlabelled polypeptide altogether also causes liver to absorb reduction and kidney picked-up increase, and display liver likely has the picked-up of CXCR4 receptor-specific, and kidney is main metabolism and Excretory organ.

For the compounds of this invention 2, its uptake pattern in CHO-CXCR4 and CHO tumour and compound 1 similar, but its picked-up level when suppressing dosage to be respectively 0 and 10 μ g is very low, and this may be cause because the avidity of compound is lower.Further, when high dosage suppresses (200 μ g), the liver picked-up of compound 2 is higher than corresponding compound 1(P<0.05).In a word, the higher experiment with cellular uptake of the non-specific uptake of compound 2 is consistent (cell experiment result is see Fig. 2).

Two kinds of compounds all demonstrate higher splenic uptake (compound 1:8.00 ± 1.07; Compound 2:7.53 ± 0.58%ID/g), mainly because spleen is the organ of CXCR4 expression of receptor.And the picked-up of this specificity also can be suppressed by common injection inhibitor, inhibition is about 50%, suitable with the tumor inhibitory effect of CXCR4 positive expression.In addition; the compounds of this invention in liver and kidney height picked-up with before based on similar (the Jacobson O WI of the developer of T140 polypeptide; Szajek LP; Niu G; Ma Y; Kiesewetter DO, Farber JM, Chen X.PET imagingof CXCR4using copper-64labeled peptide antagonist.Theranostics.2011; 1:251-62; JacobsonO, Weiss ID, Szajek LP, Niu G, Ma Y, Kiesewetter DO, et al.Improvement of CXCR4tracerspecificity for PET imaging.J Controlled Release.2012; 157:216-23; Demmer O; Dijkgraaf I; Schumacher U; Marinelli L; Cosconati S, Gourni E, et al.Design; Synthesis, andFunctionalization of Dimeric Peptides Targeting Chemokine Receptor CXCR4.J Med Chem.2011; 54:7648-62; Kuil J, Buckle T, Yuan H, van den Berg NS, Oishi S, Fujii N, et al.Synthesisand Evaluation of a Bimodal CXCR4Antagonistic Peptide.Bioconjugate Chem.2011; 22:859-64).

By common injection inhibitor, significantly can reduce the liver picked-up of two kinds of compounds (1 and 2), show that liver is specificity picked-up to a certain extent, this (Moepps B consistent with relevant mouse liver high expression level CXCR4 messenger RNA(mRNA) before, Frodl R, Rodewald H-R, Baggiolini M, Gierschik P.Two murine homologues of the humanchemokine receptor CXCR4mediating stromal cell-derived factor1 α activation of Gi2aredifferentially expressed in vivo.Eur J Immunol.1997, 27:2102-12).Find, along with the increase of common injection inhibitor, kidney picked-up also increases, and this point is consistent in bio distribution with PET image results simultaneously.Other picked-up as blood, muscle and other major organs is low-down, and the impact of the not suppressed preparation of intake.Good several CXCR4 receptor developer based on T140 polypeptide for SPECT and PET video picture is in the news.Each derivative respectively has relative merits, as by [ ¹¹¹in] DTPA mark Ac-TZ14011 polypeptide; picked-up in carcinoma of the pancreas AsPC-1 knurl is only 0.51%ID/g(Hanaoka H; Mukai T; Tamamura H, Mori T, Ishino S; Ogawa K; et al.Development of a111In-labeled peptide derivative targeting a chemokine receptor, CXCR4, forimaging tumors.Nucl Med Biol.2006; 33:489-94).The avidity of N-FB-TN14003 polypeptide and acceptor is very high; but the combination of erythrocyte is also very high in itself and body; have a strong impact on imaging results (Jacobson O; Weiss ID; Kiesewetter DO; Farber JM, Chen X.PET of Tumor CXCR4Expression with4-18F-T140.JNucl Med.2010; 51:1796-804; Jacobson O WI, Szajek LP, Niu G, Ma Y, Kiesewetter DO, Farber JM, Chen X.PET imaging of CXCR4using copper-64labeled peptide antagonist.Theranostics.2011; 1:251-62).

Compound 1 and 2 is new radio-labeling T140 compounds that the present inventor develops, compared with existing conventional tumor developer, itself and tumour CXCR4 receptor affinity are high, high specificity, there is very high tumor uptake and the delay of lower blood, can obtain tumour and background ratio better, we also notice that very high kidney metabolism may affect the video picture of abdominal tumor simultaneously.

The compounds of this invention also possesses the advantage that Radiochemical yield is higher and radiochemical purity is high simultaneously.In these two kinds of compounds, we think that compound 1 demonstrates higher tumor uptake, lower non-specific uptake, and more excellent tumour and background ratio, mark mark auxiliary group volume less as seen, lower be fat-solublely conducive to the effect improving tumor imaging.The compounds of this invention 1 can carry out live body without wound tumor imaging, the expression of qualitative assessment CXCR4 acceptor.

Claims (4)

1. the preparation method of the radiolabeled compound containing T140 polypeptide structure unit, this structural formula of compound is:

Method comprises the steps:

1) 4-nitrophenyl-2-[ ¹⁸f] preparation of fluorine propionic ester

By 20 ~ 300mCi [ ¹⁸f] F ^-from separator column CHROMAFIX, drip washing is got off, and is placed in reaction flask, is removed by solvent by vacuum drying method, obtain anhydrous [ ¹⁸f] F ^-, add the 1 ~ 5mg ethyl 2 bromopropionic acid ester being dissolved in 0.1 ~ 1mL acetonitrile after cooling, be heated to 70 ~ 150 DEG C reaction 5 ~ 20min, obtain ethyl 2-[ ¹⁸f] fluorine propionic ester;

At the end of reaction, add 50 ~ 200 μ L 0.2mol/L potassium hydroxide solutions and 200 ~ 500 μ L acetonitrile solutions, be again heated to 70 ~ 150 DEG C reaction 5 ~ 20min, obtain ethyl 2-[ ¹⁸f] fluorine propionic acid sylvite, then with 0.2 ~ 2mL dilution in acetonitrile and vacuum-drying except desolventizing, add the acetonitrile solution containing 2 ~ 40mg bis--(4-nitrophenyl) carbonic ether afterwards, under 70 ~ 150 DEG C of conditions, react 5 ~ 20min, generation 4-nitrophenyl-2-[ ¹⁸f] fluorine propionic ester, namely [ ¹⁸f] FPA, after cooling, reaction mixture is with half preparative HPLC separation and purification and concentrate volume with C18 post;

2) compound 1, namely [ ¹⁸f] preparation of FP-Ac-TC14012

By above-mentioned be adsorbed on C18 post [ ¹⁸f] FPA gets off with 0.2 ~ 2mL eluent methylene chloride, removing upper strata aqueous phase, at room temperature applying argon gas evaporate dichloromethane solvent, then the DIPEA that 20 ~ 600 μ g are dissolved in Ac-TC14012 polypeptide in DMSO and 1 ~ 10 μ L is added, heating also reacts 5 ~ 20min under 50 ~ 120 DEG C of conditions, at the end of reaction, cooling is also diluted with the aqueous solution containing TFA, obtains compound 1 after purified.

2. the method for claim 1, is characterized in that, step 2) purifying be through HPLC purifying.

3. the preparation method of the radiolabeled compound containing T140 polypeptide structure unit, this structural formula of compound is:

Method comprises the steps:

1) N-succinimide-4- ¹⁸the preparation of F-fluorobenzoate

By 20 ~ 300mCi [ ¹⁸f] F ^-from separator column CHROMAFIX, drip washing is got off, and is placed in reaction flask, is removed by solvent by vacuum drying method, obtain anhydrous [ ¹⁸f] F ^-1 ~ 5mg 4-Trimethylamine ethyl benzoate the trifluoromethyl sulfonic acid being dissolved in 0.1 ~ 2mLDMSO is added after cooling, be heated to 90 ~ 150 DEG C of reaction 5 ~ 20min, at the end of reaction, add 200 ~ 1000 μ L 0.2mol/L sodium hydroxide solutions, be again heated to 90 ~ 150 DEG C of reaction 5 ~ 20min, cooling reaction solution, add 0.1 ~ 1mL 1mol/L HCl and carry out acidifying, obtain 4- ¹⁸f-fluorobenzoic acid;

Then reaction mixture is passed through C18 column purification, and obtain the 4-after purifying with acetonitrile drip washing ¹⁸f-fluorobenzoic acid, adds 2 ~ 20 μ L 40% tetrabutylphosphoniuhydroxide hydroxide amine afterwards, again adds 8 ~ 20mg TSTU solution after drying up acetonitrile solvent, under 70 ~ 100 DEG C of conditions, react 5 ~ 20min, generate [ ¹⁸f] SFB, after cooling, reaction mixture is with half preparative HPLC separation and purification and concentrate volume with C18 post;

2) compound 2, namely [ ¹⁸f] preparation of FB-Ac-TC14012

By above-mentioned be adsorbed on C18 post [ ¹⁸f] SFB gets off with 0.2 ~ 2mL eluent methylene chloride, and removing upper strata aqueous phase, at room temperature applying argon gas evaporate dichloromethane solvent, then adds 20 ~ 600 μ g and is dissolved in 0.1mL Na ₂hPO ₄in Ac-TC14012 polypeptide, be heated to react 5 ~ 30min under 20 ~ 60 DEG C of conditions, at the end of reaction, cooling is also diluted with the aqueous solution containing TFA, obtains compound 2 after purified.

4. method as claimed in claim 3, is characterized in that, step 2) purifying be through HPLC purifying.