CN102351941B - Method for labeling functional molecules with <18>F - Google Patents
Method for labeling functional molecules with <18>F Download PDFInfo
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- CN102351941B CN102351941B CN201110152843.XA CN201110152843A CN102351941B CN 102351941 B CN102351941 B CN 102351941B CN 201110152843 A CN201110152843 A CN 201110152843A CN 102351941 B CN102351941 B CN 102351941B
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- 0 Cc1c[n](**)nn1 Chemical compound Cc1c[n](**)nn1 0.000 description 5
- FZERHIULMFGESH-UHFFFAOYSA-N CC(Nc1ccccc1)=O Chemical compound CC(Nc1ccccc1)=O FZERHIULMFGESH-UHFFFAOYSA-N 0.000 description 1
- IGVQRLFQGDERCX-UHFFFAOYSA-N CNS(c(cc1)ccc1NC(SC)=O)(=O)=O Chemical compound CNS(c(cc1)ccc1NC(SC)=O)(=O)=O IGVQRLFQGDERCX-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention discloses a method for labeling functional molecules with <18>F. The method is any one of the followings: method one, in the solvent, in the presence of Cu(I) as catalyst, letting a compound B and a compound C be subject to 1,3-polar cycloaddition of nitrine and terminal acetylene; method 2, in the solvent, in the presence of Cu(I) as catalyst, letting a compound F and a compound G be subject to 1,3-polar cycloaddition of nitrine and terminal acetylene; wherein, R' represents various functional molecules which are suitable for being used as PET imaging probes, and R'' represents alkane. The method has the advantages of stable reaction system, mild conditions, simple operation, and versatility, and can be used for labeling compounds of similar construction. Excepting biological reagents, the other raw materials used in the method are cheap, and the structure of the precursor compound B or F is stable. The method is suitable for large scale preparation and production, and can be used for synthesizing PET imaging probes.
Description
Technical field
The present invention is specifically related to functional molecular to carry out
18the method of F mark.
Background technology
Current, as a kind of representative of molecular image technology, positron emission tomography (Positronemission tomography, PET) can be quantitatively and high resolution detection data are provided, be widely used in the diagnosis of tumour, mechanism research and clinical treatment (Simon M.Ametamey, Michael Honer, Pius August Schuiger. " Molecular Imaging with PET ", Chem.Rev.2008, 108, 1501-1516.PET molecular image, Paul J.Cassidy, George K.Radda, Molecular imagingperspectives, J.R.Soc.Interface, 2005, 2 (3): 133-144. molecular image prospect).
And in a large amount of positron radiation nucleic, fluoro-18[
18f] atomic radius is little, transformation period is 109.8min, maximum positron energy 0.64MeV, mean range 0.22mm, there is good nulcear properties and chemical property, be PET probe preferred nucleic (Okarvi, S.M. (2001). " Recent progress influorine-18labelled peptide radiopharmaceuticals. " Eur J Nucl Med 28 (7): 929-38.
18f labeling polypeptide radiopharmaceuticals progress).
Now clinical PET image probe mainly contains: organic molecule class, polypeptide class, antibody class, gene fragment class, oligonucleotides, wherein polypeptide class is with its good character and pharmacological characteristics, become in imaging medicament preferably.Along with
18f labeling polypeptide carries out Tumor receptor imaging and becomes one of major domain of current molecular nuclear medicine research, how to utilize
18the mark that F carries out polypeptide also becomes an important research topic.Totally it seems, polypeptide
18f mark mainly contains direct mark and two kinds of mark modes of indirect labelling, and in indirect labelling mode as main.Utilize the carboxyl, hydroxyl, the amino isoreactivity group that in polypeptide, exist, use difunctional aglucon to connect radionuclide
18f and peptide molecule, form peptide-linker-
18the typical structure of F is the most frequently used multistep marking method.(Shi Lingli. (2008). nuclear technique. (10). polypeptide
18f marking method)
Summary of the invention
Technical problem to be solved by this invention is functional molecular to be carried out with prior art is diverse in order to provide a kind of
18the method of F mark.In marking method of the present invention, reaction system is stable, mild condition, simple to operate, and the method has versatility, applicable to the series compound mark of similar.Except biological reagent, this marking method other raw materials cost used are cheap, and precursor compound B or F Stability Analysis of Structures are suitable for a large amount of preparations and produce, and the method can be used for the synthetic of PET Imaging probe.
The inventor for
18the compound of F and some type and invented a kind of brand-new
18f marking method.It is at present conventional that the method can be avoided
18f label probe some shortcomings in synthetic: as reaction yield is not high, labeling process does not have versatility etc.
Therefore, the present invention relates to functional molecular to carry out
18the method of F mark, it is any one in following two kinds of methods:
Method one: in solvent, under the catalysis of Cu (I), compd B and Compound C are carried out to nitrine and 1 of end position alkynyl, 3-Dipolar Cycloaddition, can make Compound D;
Method two: in solvent, under the catalysis of Cu (I), compound F 17-hydroxy-corticosterone and compound G are carried out to nitrine and 1 of end position alkynyl, 3-Dipolar Cycloaddition, can make compound H;
Wherein, R ' is C
1～C
12straight chained alkyl or branched-chain alkyl, C
5～C
7cycloalkyl, C
6～C
10aryl, C
6～C
10aryl-C
1～C
4alkyl, or C
1～C
4alkyl-C
6～C
10aryl-C
1～C
4alkyl, R " be applicable to doing the polypeptide class, benzene sulfonamide of PET Imaging probe or other can be by the functional molecular substituting group of nitrine or Terminal Acetylenes base group modification for this area.
In R ', described C
1～C
12straight chained alkyl or branched-chain alkyl be preferably following arbitrary group:
N=0～8, preferably 2 or 3;
Described aryl-C
1～C
4alkyl is preferably following group:
Described C
1～C
4alkyl-C
6～C
10aryl-C
1～C
4alkyl is preferably following arbitrary group:
Wherein n1=1～4, n2=0～4;
Described cycloalkyl is preferably following group:
Wherein n=1～3;
R " in, the described applicable functional molecular substituting group that does PET Imaging probe is preferably following arbitrary group or polypeptide class functional molecular substituting group (as RGD peptide or Leu-Ala-Arg-Leu-Leu-Thr peptide, structure is as follows); Deng;
Described RGD peptide is following structure:
Described Leu-Ala-Arg-Leu-Leu-Thr peptide is following structure:
Preferably, the boiling point of above-claimed cpd B is lower than 200 DEG C.
In method one and method two, described 1, the method for 3-Dipolar Cycloaddition and condition can be method and condition used in this type of reaction of organic synthesis field, and described Cu (I) be monovalence copper, generally participates in reacting with the form of cupprous salt.
The inventor, through great many of experiments, particularly preferably goes out following method and condition:
In method one, described 1,3-Dipolar Cycloaddition comprises the following step: in solvent, pH is 3～12, under the catalysis of Cu (I), compd B and C is carried out to nitrine and 1 of end position alkynyl, 3-Dipolar Cycloaddition.
In method two, described 1,3-Dipolar Cycloaddition comprises the following step: in solvent, pH is 3～12, under the catalysis of Cu (I), compound F 17-hydroxy-corticosterone and G is carried out to nitrine and 1 of end position alkynyl, 3-Dipolar Cycloaddition.
In method one or two, described pH is preferably 6～8.Described preferred solvents be one or more in water, the trimethyl carbinol, acetonitrile and tetrahydrofuran (THF), as R " while being polypeptide compounds molecule substituting group, preferably water is solvent, compd B or F are (5.85 × 10 with the molecular volume of solvent than preferably
-14mol～5.85 × 10
-10mol)/(0.2～1mL); Or compd B or the F radioactive activity in solvent is preferably 1mCi～2Ci.When moisture in reaction solvent, the volume of other organic solvents is preferably no more than the volume of water.Described Compound C or the G concentration in reaction solution is preferably 0.2～20mmol/L, and that better is 3.8～7mmol/L.The amount of described Cu (I) is preferably 1 times～20 times of molar weight of Compound C or G, and better is 8 times～15 times.The concentration of described Cu (I) in reaction solution is preferably 5mmol/L～100mmol.
Described 1, the temperature of 3-Dipolar Cycloaddition can be according to participating in the Compound C of reaction or the stability of G, and the boiling point of the reaction solvent system adopting, and suitably regulates upper temperature limit, is preferably 10～100 DEG C, and better is 30～65 DEG C.
Described 1,3-Dipolar Cycloaddition can complete in a short period of time, and as 1～80 minute, better was 10～20 minutes.
Wherein, described pH value can regulate by this area ordinary method, as adds the phosphate buffered saline buffer of required pH scope.
Described Cu (I) can be the common form of the Cu (I) of this type of reaction in organic field, and the present invention particularly preferably Cu of following arbitrary form (I) participates in reaction:
1. cupric strong acid salt and xitix or its highly basic salt are carried out to reduction reaction, make Cu (I);
2. the solution of cuprous iodide or cuprous bromide and weakly alkaline copper part;
3. by Cu (O) (as copper wire, copper powder or nano copper particle etc.) oxidation, make Cu (I).
Above-mentioned Cu (I) effect in is 2. better.
1., in, described cupric strong acid salt can be one or more in copper sulfate, cupric nitrate and cupric chloride, preferably sulfuric acid copper.The highly basic salt of described xitix can be one or more in sodium ascorbate, potassium ascorbate and calcium ascorbate etc., because sodium ascorbate is more soluble in water than xitix, more common and cost is lower than other ascorbate salts, preferably sodium ascorbate.The mol ratio of described cupric strong acid salt and xitix or its highly basic salt is preferably 1: 1.1～1: 3, and better is 1: 1.5～1: 1.8.
2., in, described weakly alkaline copper part is to form the part that coordinates ion with Cu (I), is preferably ammoniacal liquor, triethylamine or diisopropylethylamine; The mol ratio of described cuprous iodide or cuprous bromide and weakly alkaline copper part is preferably 1: 5～1: 1000, and better is 1: 100～1: 300.Preferred solvents in described solution be water or conventional inert organic solvents.Cuprous iodide or the cuprous bromide concentration in solution is preferably 0.05mol/L～1.5mol/L, and that better is 0.4mol/L～1.2mol/L.
3., in, described oxidation can be by airborne oxygen and is oxidized, or by adding oxygenant (as copper sulfate) to be oxidized.
Above-mentioned 1, after 3-Dipolar Cycloaddition finishes, can purify to product according to the aftertreatment of this area routine and method of purification, as used radioactivity HPLC separation and purification, before with radioactivity HPLC separation and purification, also can first carry out purifying with Sep-Pak C18 post to product.
In the present invention, described compd B can be made by following method: by compd A and
18f
-carry out nucleophilic substitution reaction;
Described compound F 17-hydroxy-corticosterone can be made by following method: by compd E and
18f
-carry out nucleophilic substitution reaction;
Wherein, R
1for leavings group conventional in nucleophilic substitution reaction, as-OTs ,-OMs or-OTf, described in the definition ditto of R '.
Wherein, the method for described nucleophilic substitution reaction and condition can be this area this type of
18the ordinary method of F labeled reactant and condition, the present invention is following method and condition particularly preferably: in organic solvent, under protection of inert gas, will contain K
222, K
2cO
3with
18f
-mixture and compd A or E carry out nucleophilic substitution reaction.
Wherein, described organic solvent is preferably one or more in anhydrous acetonitrile, anhydrous dimethyl formamide and anhydrous dimethyl sulfoxide, preferably acetonitrile.Described K
222and K
2cO
3mol ratio be preferably 1: 3～7: 1, better is 1.3: 1～3.3: 1.
18f
-activity be preferably 10 μ Ci～2Ci, that better is 5mCi～700mCi.Compd A or the E concentration in reaction solution is preferably 0.01～1mol/L, and that better is 0.05～0.2mol/L.K
222with the mass ratio of compd A or E be preferably 1: 1～7: 1, better is 2: 1-5: 1.Described rare gas element is preferably nitrogen and/or argon gas.The temperature of described nucleophilic substitution reaction is preferably 80～150 DEG C.The time of described nucleophilic substitution reaction is preferably 2～15min.
The described K that contains
222, K
2cO
3with
18f
-mixture can make by following method: use K
222(being Kryptofix 222) solution drip washing enrichment
18f
-qMA post, solvent evaporated.
Wherein, K
222solution can make by following method: by K
222, K
2cO
3, acetonitrile and water wiring solution-forming.Wherein, each component content scope is as follows: in every 1mL acetonitrile, have 30～150 μ L water, 1～7mg K
2cO
3, 5～30mg K
222.The method of configuration can be for to add 30～150 μ L water in 1mL acetonitrile, 1～7mg K
2cO
3, 5～30mg K
222.The most frequently used a kind of proportioning is: in every 960 μ L acetonitriles, have 14.4mg K
222, 3mgK
2cO
3, 40 μ L water, each composition wiring solution-forming.
After above-mentioned nucleophilic substitution reaction completes, aftertreatment and the method for purification of available this area routine are purified.The preferred following method of purification of the present invention and condition: when the boiling point of compd B or F is during lower than 200 DEG C, in reaction solution, add acetonitrile, using nitrogen as carrier gas, adopt distillating method separating impurity, collect the acetonitrile condensing soln of compd B or F.Described distillation temperature is preferably 85～150 DEG C, and distillation time is preferably 5～30 minutes.
Meeting on the basis of this area general knowledge, above-mentioned each optimum condition, can arbitrary combination, obtains the preferred embodiments of the invention.
Agents useful for same of the present invention and raw material be commercially available obtaining all.
Positive progressive effect of the present invention is:
1, in method of the present invention, the productive rate of two-step reaction is all very high, and the reaction times is very short.
2, of the present invention
18f labelled precursor, i.e. compd B and F Stability Analysis of Structures, separating and purifying method is simple.
3, the realization that the present invention can be rapidly and efficiently
18f mark is with organic molecule, polypeptide, other biological macromole, functional material or the nanoparticle compounds of end alkynyl radical group, and method has versatility.
4, the present invention can, by adjusting the structure of Compound C or G, prepare PET Imaging probe according to demand, and for PET video picture.
5, in the present invention, cuprous iodide used, copper sulfate, sodium ascorbate, weak base part, copper etc. are commercialization reagent, and raw material is cheap and easy to get.
Brief description of the drawings
Fig. 1 is the HPLC spectrogram (UV, 220nm) of the reaction system taking copper sulfate/sodium ascorbate as catalyst system in the example two of embodiment 2.
Fig. 2 is the HPLC spectrogram (UV, 220nm) of the reaction system taking cuprous iodide/ammoniacal liquor as catalyst system in the example two of embodiment 2.
Fig. 3 is the radioassay HPLC spectrogram in the reaction process taking copper sulfate/sodium ascorbate as catalyst system in the example two of embodiment 2 (reaction 5min).
Fig. 4 is the radioassay HPLC spectrogram in the reaction process taking cuprous iodide/ammoniacal liquor as catalyst system in the example two of embodiment 2 (reaction 5min).
Fig. 5 be in the example two of embodiment 2 taking copper sulfate/sodium ascorbate as catalyst system the radioassay HPLC spectrogram (reaction 15min) of reaction after finishing.
Fig. 6 be in the example two of embodiment 2 taking cuprous iodide/ammoniacal liquor as catalyst system the radioassay HPLC spectrogram (reaction 15min) of reaction after finishing.
Embodiment
Mode below by embodiment further illustrates the present invention, but does not therefore limit the present invention among described scope of embodiments.The experimental technique of unreceipted actual conditions in the following example, according to ordinary method and condition, or selects according to catalogue.
P-[
18f] FSTC, [
18f] F-cRGDfK and [
18f] F-Clodinafop etc. synthetic:
Embodiment 12-nitrine-1-[
18f] Radio-synthesis of fluoroethane
20mCi
18f
-by quaternary ammonium type anion post QMA (Waters company of U.S. product,
18f
-provided by Shanghai City Tumor Hispital Attached to Fudan Univ and Ke Xing pharmaceutcal corporation, Ltd) catch after, get 1.2mLK
222(being Kryptofix 222) solution (17.3mgK
222, 3.6mgK
2cO
3, 1152 μ L acetonitriles, the solution that 48 μ L water are made into) and will
18f is flushed in reaction flask, and reaction flask immerses the oil bath of 95 DEG C, and nitrogen dries up, and then adds 500 μ L anhydrous acetonitriles to dry up, and repeats anhydrous acetonitrile and dries up twice; Then 5mg 2-azidoethyl p-toluenesulfonic esters is dissolved in to 400 μ L anhydrous acetonitriles, under nitrogen protection, adds rapidly reaction flask, confined reaction 5min at 95 DEG C, stopped reaction, ice-water bath is cooling.Mark rate can reach more than 97%.
In reaction solution, add acetonitrile 200 μ L, nitrogen is assisted current-carrying, and distill, and collect phlegma, distillation 10-20min, distillation efficiency can reach 79.5%.
Containing 2-nitrine-1-[
18f] the acetonitrile condensation of fluoroethane collects liquid (approximately 600 μ L) and can be used in the reaction of lower step mark.
The putting productive rate of this step can reach more than 70%.
Embodiment 2 employings contain azido group
18f compound is realized the mark with the functional molecular of Terminal Acetylenes group
[example one], realize organic micromolecule compound by 1,3-Dipolar Cycloaddition
18f mark
This example selects compound 4-aminobenzene sulfonamide as organic molecule model.The carbonic anhydrase 9 (CAIX) of abnormal expression when the weary oxygen of small molecules energy target tumor of this structure, therefore p-[
18f] FSTC (1-(2-[
18f] fluorine second)-N-(4-sulfoamido phenyl)-1H-1,2,3-triazole-4-acid amides) the potential PET Imaging probe that becomes tumor hypoxia.
Under nitrogen protection; in 100 μ L0.10mol/L pH6.0 phosphoric acid salt (PBS) damping fluids; add successively 50 μ L 0.45mol/L copper-baths and 100 μ L1.50mol/L sodium ascorbate solutions; 4-propiolyl amido benzsulfamide (5mg; 22.5 μ mol) 150 μ L DMF and 100 μ Lt-BuOH solution, and 2-nitrine-1-[of making of previous step
18f] the acetonitrile solution 300 μ L (also can use not the solution system before distillation) of fluoroethane.At 35 DEG C of oscillatory reaction 15min, the 10mL that adds water dilution, obtain crude product with Sep-Pak C18 column purification, again with partly preparing high performance liquid phase system purification of target product (the P680summit HPLC of Dionex company of U.S. analytical system, semipreparative column is LoChrosorb C18:10 μ m, 300mm × 7.8mm.Moving phase is water (A) and ethanol (B), and gradient separations condition is: 0-5-10-40-45min, 5% → 5% → 20% → 20% → 5%B.Flow velocity 1.5mL/min, detects and radioassay through UV (254nm).)
Radioactivity HPLC analyzes demonstration, the mark rate > 98% of this experiment, p-[
18f] the retention time t of FSTC
r=13.4min, radiochemical purity > 98%, yield > 96% (decaying, it is rear to proofread and correct).
[example two], realize polypeptide by 1,3-Dipolar Cycloaddition
18f mark:
This example selects cRGDfK as polypeptide model.Research shows that its prototype polypeptide cRGDfK has certain targeting to tumor neogenetic blood vessels, therefore [
18f] FcRGDfK is also a kind of potential PET Imaging probe.
By polypeptide
18f mark, checking 1,3-dipole-ring addition marking method versatility and validity.
And by the use of two kinds of catalyzer, prove the diversity of catalyst system.
Copper sulfate/sodium ascorbate catalyst system: the cRGDfK that 2mg propynoic acid is modified is dissolved in the mixing solutions of 200 μ LpH6.0 phosphoric acid buffers and the 200uL trimethyl carbinol, successively add 50 μ L0.4M copper sulfate and 100 μ L1.2M sodium ascorbate solutions, then the 2-nitrine-1-[that adds distillation condensation to obtain
18f] fluoroethane acetonitrile solution 200 μ L, 50 DEG C of reaction 5min, with radioactivity HPLC, (U.S. Agilent 1100HPLC system, semipreparative column is Waters C18column:7.3mm × 300mm.Moving phase is to have added water (A) and the acetonitrile (B) of 0.1% trifluoroacetic acid, and gradient separations condition is: 0-20min, 5% → 50%B.Flow velocity is 2.0mL/min.Detect and radioassay through UV (220nm)) detect, see Fig. 3, the radioactive product appearance time of paying close attention to is t
1=11.2min (90.1%), t
2=14.8min (9.9%).After reaction 15min, mark rate approaches 100%.With radioactivity HPLC separation and purification, (U.S. Agilent1100HPLC system, semipreparative column is Waters C18column:7.3mm × 300mm to product.Moving phase is to have added water (A) and the acetonitrile (B) of 0.1% trifluoroacetic acid, and gradient separations condition is: 0-20min, 5% → 50%B.Flow velocity is 2.0mL/min.Detect and radioassay through UV (220nm)).
(U.S. Agilent 1100HPLC system, semipreparative column is WatersC18column:7.3mm × 300mm in radioactivity HPLC analysis.Moving phase is to have added water (A) and the acetonitrile (B) of 0.1% trifluoroacetic acid, and gradient separations condition is: 0-20min, 5% → 50%B.Flow velocity is 2.0mL/min.Detect and radioassay through UV (220nm)) (seeing Fig. 5) demonstration, the mark rate > 98% of this experiment, [
18f] the retention time t of cRGDfK
r=14.8min, radiochemical purity > 97% (98.1%), yield > 95% (decaying, it is rear to proofread and correct).
Above-mentioned reaction system is carried out to HPLC detection, and HPLC condition is: 0-20min, 5% → 50%B (A: containing the water of 0.1% trifluoroacetic acid; B: containing 0.1% trifluoroacetic acid and acetonitrile; Flow velocity is 2.0mL/min.The results are shown in Figure 1.Can be seen in 7.5min-17.5min time range, there is the very large peak of the very high width of absorption value by Fig. 1, can affect paid close attention to radioactive product (t
1=11.2min, t
2=14.8min) separation.
Cuprous iodide/ammonia-catalyzed system: the cRGDfK that 2mg propynoic acid is modified is dissolved in 200 μ LpH6.0 phosphoric acid buffers, adds 40 μ L0.5M cuprous iodide ammonia solns, then the 2-nitrine-1-[that adds distillation condensation to obtain
18f] fluoroethane acetonitrile solution 200uL, 50 DEG C of reaction 5min, with radioactivity HPLC, (U.S. Agilent 1100HPLC system, semipreparative column is Waters C18column:7.3mm × 300mm.Moving phase is to have added water (A) and the acetonitrile (B) of 0.1% trifluoroacetic acid, and gradient separations condition is: 0-20min, 5% → 50%B.Flow velocity is 2.0mL/min.Detect and radioassay through UV (220nm)) detect, see Fig. 4, the radioactive product appearance time of paying close attention to is t
1=11.2min (68.4%), t
2=14.8min (31.6%).After reaction 15min, cooling rear use radioactivity HPLC separation and purification product [
18f] cRGDfK (separation condition is the same).
(U.S. Agilent 1100HPLC system, semipreparative column is WatersC18column:7.3mm × 300mm in radioactivity HPLC analysis.Moving phase is to have added water (A) and the acetonitrile (B) of 0.1% trifluoroacetic acid, and gradient separations condition is: 0-20min, 5% → 50%B.Flow velocity is 2.0mL/min.Detect and radioassay through UV (220nm)) (seeing Fig. 6) demonstration, [
18f] appearance time of cRGDfK is t
r=14.8min (92.2%), the mark rate > 92% of this experiment.
Above-mentioned reaction system is carried out to HPLC detection, and HPLC condition is: 0-20min, 5% → 50%B (A: containing the water of 0.1% trifluoroacetic acid; B: containing 0.1% trifluoroacetic acid and acetonitrile; Flow velocity is 2.0mL/min.The results are shown in Figure 2.Can be seen there is not the existence of disturbing broad peak by Fig. 2.This advantage can improve the sharpness of reaction process monitoring, can also ensure, in the time carrying out radioactivity separation, will can not introduce too much impurity (being other materials that have ultraviolet absorption peak), improves the specific activity of product.
[example three], realize and contain arbitrarily end alkynyl compounds by 1,3-Dipolar Cycloaddition
18f mark:
This example selection compound clodinafop-propargyl ((R)-2-[4-(the fluoro-2-pyridyloxy of the chloro-3-of 5-) phenoxy group] propionic acid propargyl ester, Clodinafop-propargyl) as the model that contains end alkynyl compounds.
Under nitrogen protection; in 100 μ L0.10mol/L pH6.0 phosphoric acid salt (PBS) damping fluids; add successively 50 μ L0.45mol/L copper-baths and 100 μ L1.50mol/L sodium ascorbate solutions; clodinafop-propargyl (5mg; 14.3 μ mol) 150 μ LDMF and 100 μ L t-BuOH solution, and 2-nitrine-1-[of making of previous step
18f] the acetonitrile solution 300 μ L (also can use not the solution system before distillation) of fluoroethane.At 35 DEG C of oscillatory reaction 15min, the 10mL that adds water dilution, obtains crude product with Sep-Pak C18 post (Waters company of the U.S.) purifying, then with partly prepare high performance liquid phase system purification of target product [
18f] (U.S. Agilent 1100HPLC system, adopts semipreparative column to analyze to F-Clodinafop, and column type is Waters C18column:7.3mm × 300mm.Moving phase is to have added water (A) and the acetonitrile (B) of 0.1% trifluoroacetic acid, and gradient separations condition is: 0-20min, 5% → 90%B.Flow velocity is 2.0mL/min.Detect and radioassay through UV (220nm)).
(U.S. Agilent 1100HPLC system, adopts semipreparative column to analyze, and column type is Waters C18column:7.3mm × 300mm in radioactivity HPLC analysis.Moving phase is to have added water (A) and the acetonitrile (B) of 0.1% trifluoroacetic acid, and gradient separations condition is: 0-20min, 5% → 90%B.Flow velocity is 2.0mL/min.Detect and radioassay through UV (220nm)) show, the mark rate > 96% of this experiment, [
18f] the retention time t of F-Clodinafop
r=17.9min, radiochemical purity > 99%, yield > 95% (decaying, it is rear to proofread and correct).
Embodiment 3 employings contain Terminal Acetylenes group
18f compound is realized the mark with the functional molecular of azido group
[example one], realize by 1,3-Dipolar Cycloaddition the polypeptide that nitrine is modified
18f mark:
This example selects the Leu-Ala-Arg-Leu-Leu-Thr peptide of nitrine modification as the model that contains nitrine modified polypeptide.Leu-Ala-Arg-Leu-Leu-Thr peptide is the new peptides part of energy feature associative list skin growth factor EGFR, can be used for cancer target.
The Leu-Ala-Arg-Leu-Leu-Thr peptide (8.4 μ mol) that 2mg nitrine is modified is dissolved in the mixing solutions of 200 μ LpH6.0 phosphoric acid buffers and the 200uL trimethyl carbinol, successively add 50 μ L0.4M copper sulfate and 100 μ L 1.2M sodium ascorbate solutions, then the 5-[that adds distillation condensation to obtain
18f] the acetonitrile solution 200 μ L of fluorine pentyne, 50 DEG C of reaction 25min.With HPLC detection, (U.S. Agilent 1100HPLC system, analytical column is Zorbax C18column (4.6mm × 250mm) to product.Moving phase is to have added water (A) and the acetonitrile (B) of 0.1% trifluoroacetic acid, and gradient separations condition is: 0-20min, 5% → 90%B.Flow velocity is 1.0mL/min.Detect and radioassay through UV (220nm).)
Radioactivity HPLC analyzes demonstration, the mark rate > 93% of this experiment,
[
18f] the retention time t of F-Leu-Ala-Arg-Leu-Leu-Thr
r=13.1min.
[example two], realize with the micromolecular compound of azido group by 1,3-Dipolar Cycloaddition
18f mark:
This example selects p-chlorobenzyl nitrine as the micromolecular compound model that contains azido group.
10mg p-chlorobenzyl nitrine (59.3 μ mol) is dissolved in to 50 μ L acetonitrile solutions, successively adds 50 μ L0.4M copper sulfate and 100 μ L1.2M sodium ascorbate solutions, then the 5-[that adds distillation condensation to obtain
18f] the acetonitrile solution 200 μ L of fluorine pentyne, 50 DEG C of reaction 15min.With HPLC detection, (U.S. Agilent 1100HPLC system, analytical column is Zorbax C18column (4.6mm × 250mm) to product.Moving phase is to have added water (A) and the acetonitrile (B) of 0.1% trifluoroacetic acid, and gradient separations condition is: 0-20min, 5% → 90%B.Flow velocity is 1.0mL/min.Detect and radioassay through UV (220nm).)
Radioactivity HPLC analyzes demonstration, the mark rate > 63% of this experiment, [
18f] the retention time t of F-p-chlorobenzyl fluorine
r=15.7.
Embodiment 45-[
18f] Radio-synthesis of fluorine pentyne
20mCi
18f
-by quaternary ammonium type anion post QMA (Waters company of U.S. product,
18f
-provided by Shanghai City Tumor Hispital Attached to Fudan Univ and Ke Xing pharmaceutcal corporation, Ltd) catch after, get 1.2mLK
222(being Kryptofix 222) solution (17.3mgK
222, 3.6mgK
2cO
3, 1152 μ L acetonitriles, the solution that 48 μ L water are made into) and will
18f is flushed in reaction flask, and reaction flask immerses the oil bath of 95 DEG C, and nitrogen dries up, and then adds 500 μ L anhydrous acetonitriles to dry up, and repeats anhydrous acetonitrile and dries up twice; Then 5mg 5-tolysulfonyl pentyne is dissolved in to 400 μ L anhydrous acetonitriles, under nitrogen protection, adds rapidly reaction flask, confined reaction 5min at 95 DEG C, stopped reaction, ice-water bath is cooling.Mark rate can reach more than 96%.
In reaction solution, add acetonitrile 200 μ L, nitrogen is assisted current-carrying, with the self-control water distilling apparatus shown in Fig. 1 (building according to routine distillation knowledge) distillation, and collects phlegma, distillation 10-20min, and distillation efficiency can reach 79.5%.
Containing 5-[
18f] the acetonitrile condensation of fluorine pentyne collects liquid (approximately 600 μ L) and can be used in the reaction of lower step mark.
The putting productive rate of this step can reach more than 70%.
In above-described embodiment, relate to three kinds
18f tagged compound 2-nitrine-1-[
18f] fluoroethane, p-[
18f] FSTC and [
18f] structure of cRGDfK is by the reference compound 2-nitrine-1-[corresponding with it
19f] fluoroethane p-[
19f] FSTC and [
19f] cRGDfK contrasts and determined.Concrete confirmation method is: will
18f tagged compound carries out HPLC detection simultaneously after mixing with its reference compound, contrast (due to after radioactive detector is connected to UV-detector by the ultraviolet peak appearance time to HPLC and radioactivity peak appearance time, its signal exists the regular time poor), appearance time difference is fixed, and thinks that their structures are consistent.Three kinds of reference compounds are that laboratory is synthetic, structure via
1the detections such as HNMR, IR, MS.
The reference compound structure using is as follows:
2-nitrine-1-[
18f] fluoroethane
1H?NMR(300MHz，CDCl
3)δ：4.685～4.498(dt，2H，CH
2-F，
2J
FH＝47Hz，?
2J
HH＝4.5Hz)；3.579～3.456(dt，2H，CH
2-CH
2-F，
3J
FH＝27Hz，
2J
HH＝4.5Hz).Analytical?HPLC，t＝11.1min，and?t
(DMF)＝
5.4min.
1H?NMR(400MHz，CDCl
3)δ：2.450(3H，CH
3-)；3.470-3.496(2H，NCH
2)；?4.148-4.175(2H，SCH
2)；7.357-7.829(4H).IR(KBr)υ：5556667128139151013?1095?1176?1299?1364?1458?1579?2112?2925?2962cm
-1.Analytical?HPLC，t＝13.2min.
ESI-MS(M+H＝745.2)。HPLC(t＝14.6min).
1HNMR(300MHz，CDCl
3)δ：1.594～1.618(3H，CH
3-CH)；4.592-4.661，(5H，CH-O，-CH
2-CH
2-)；5.296-5.326(2H，-CH
2-O-)；6.844-7.855(7H).Analytical?HPLC，t＝17.7min.
Claims (16)
1. pair functional molecular carries out
18the method of F mark, it is any one in following two kinds of methods:
Method one: in solvent, under the catalysis of Cu (I), compd B and Compound C are carried out to nitrine and 1 of end position alkynyl, 3-Dipolar Cycloaddition, can make Compound D;
Method two: in solvent, under the catalysis of Cu (I), compound F 17-hydroxy-corticosterone and compound G are carried out to nitrine and 1 of end position alkynyl, 3-Dipolar Cycloaddition, can make compound H;
Wherein, R ' is C
1～C
12straight chained alkyl;
In R ', described C
1～C
12straight chained alkyl be following group:
n=0～8；
R ' ' is following group;
2. the method for claim 1, is characterized in that: the boiling point of described compd B is lower than 200 DEG C.
3. method as claimed in claim 1 or 2, it is characterized in that: in method one, described 1,3-Dipolar Cycloaddition comprises the following step: in solvent, pH is 3～12, under the catalysis of Cu (I), compd B and C is carried out to nitrine and 1 of end position alkynyl, 3-Dipolar Cycloaddition;
In method two, described 1,3-Dipolar Cycloaddition comprises the following step: in solvent, pH is 3～12, under the catalysis of Cu (I), compound F 17-hydroxy-corticosterone and G is carried out to nitrine and 1 of end position alkynyl, 3-Dipolar Cycloaddition.
4. method as claimed in claim 3, is characterized in that: in method one or two:
Described pH is 6～8; Described solvent is one or more in water, the trimethyl carbinol, acetonitrile and tetrahydrofuran (THF); In the time that R ' ' is polypeptide compounds molecule substituting group, taking water as solvent; Compd B or F are (5.85 × 10 with the molecular volume ratio of solvent
-14mol～5.85 × 10
-10mol)/(0.2-1mL), or compd B or the radioactive activity of F in solvent are 1mCi～2Ci; When moisture in reaction solvent, the volume of other organic solvents is no more than the volume of water; Described Compound C or the G concentration in reaction solution is 0.2～20mmol/L; The amount of described Cu (I) is 1 times～20 times of molar weight of Compound C or G; The concentration of described Cu (I) in reaction solution is 5mmol/L～100mmol; Described 1, the temperature of 3-Dipolar Cycloaddition is 10～100 DEG C; Described 1, the time of 3-Dipolar Cycloaddition is 1～80 minute.
5. method as claimed in claim 4, is characterized in that: the amount of described Cu (I) is 8 times～15 times of molar weight of Compound C or G; Described Compound C or the G concentration in reaction solution is 3.8～7mmol/L; Described 1, the temperature of 3-Dipolar Cycloaddition is 30～65 DEG C; Described 1, the time of 3-Dipolar Cycloaddition is 10～20 minutes.
6. method as claimed in claim 3, is characterized in that:
Described Cu (I) participates in reaction by the Cu (I) of following arbitrary form:
1. cupric strong acid salt and xitix or its highly basic salt are carried out to reduction reaction, make Cu (I);
2. the solution of cuprous iodide or cuprous bromide and weakly alkaline copper part;
3. by Cu (O) oxidation, make Cu (I).
7. method as claimed in claim 6, is characterized in that:
2., in, described weakly alkaline copper part is ammoniacal liquor, triethylamine or diisopropylethylamine; The mol ratio of described cuprous iodide or cuprous bromide and weakly alkaline copper part is 1:5～1:1000.
8. method as claimed in claim 6, is characterized in that: 2., the mol ratio of described cuprous iodide or cuprous bromide and weakly alkaline copper part is 1:100～1:300.
9. method as claimed in claim 6, is characterized in that:
1., in, described cupric strong acid salt is one or more in copper sulfate, cupric nitrate and cupric chloride; The highly basic salt of described xitix is one or more in sodium ascorbate, potassium ascorbate and calcium ascorbate; The mol ratio of described cupric strong acid salt and xitix or its highly basic salt is 1:1.1～1:3;
3., in, described oxidation is oxidized by airborne oxygen, or by adding oxygenant to be oxidized.
10. method as claimed in claim 9, is characterized in that:
1., in, described cupric strong acid salt is one or more in copper sulfate, cupric nitrate and cupric chloride; The highly basic salt of described xitix is one or more in sodium ascorbate, potassium ascorbate and calcium ascorbate; The mol ratio of described cupric strong acid salt and xitix or its highly basic salt is 1:1.5～1:1.8;
3., in, described oxygenant is copper sulfate.
11. methods as claimed in claim 1 or 2, is characterized in that: described compd B is made by following method: by compd A and
18f
-carry out nucleophilic substitution reaction;
Described compound F 17-hydroxy-corticosterone is made by following method: by compd E and
18f
-carry out nucleophilic substitution reaction;
Wherein, R is leavings group conventional in nucleophilic substitution reaction, and the definition of R ' is with described in claim 1 or 2.
12. methods as claimed in claim 11, is characterized in that: described R for-OTs ,-OMs or-OTf.
13. methods as claimed in claim 11, is characterized in that: described nucleophilic substitution reaction comprises the following step: in organic solvent, under protection of inert gas, will contain K
222, K
2cO
3with
18f
-mixture and compd A or E carry out nucleophilic substitution reaction;
Wherein, described organic solvent is one or more in anhydrous acetonitrile, anhydrous dimethyl formamide and anhydrous dimethyl sulfoxide; Described K
222and K
2cO
3mol ratio be 1:3～7:1;
18f
-activity be 10 μ Ci～2Ci; Compd A or the E concentration in reaction solution is 0.01～1mol/L, and its liquor capacity consumption is 200～500 μ L; K
222with the mass ratio of compd A or E be 1:1～7:1; Described rare gas element is nitrogen and/or argon gas; The temperature of described nucleophilic substitution reaction is 80-150 DEG C; The time of described nucleophilic substitution reaction is 2-15min.
14. methods as claimed in claim 13, is characterized in that: the described K that contains
222, K
2cO
3with
18f
-mixture make by following method: use K
222solution drip washing enrichment
18f
-qMA post, solvent evaporated.
15. methods as claimed in claim 14, is characterized in that: described K
222solution makes by following method: by K
222, K
2cO
3, acetonitrile and water wiring solution-forming,, wherein, each component content scope is as follows: in every 1mL acetonitrile, have 30～150 μ L water, 1～7mg K
2cO
3, 5～30mg K
222.
16. methods as claimed in claim 11, it is characterized in that: after nucleophilic substitution reaction completes, purify by following method of purification: when the boiling point of compd B or F is during lower than 200 DEG C, in reaction solution, add acetonitrile, using nitrogen as carrier gas, adopt distillating method separating impurity, collect the acetonitrile condensing soln of compd B or F; Described distillation temperature is 85～150 DEG C, and distillation time is 5～30 minutes.
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| In Vivo Positron Emission Tomography (PET) Imaging with an αvβ6 Specific Peptide Radiolabeled using 18F-"Click" Chemistry:Evaluation and Comparison with the Corresponding 4-[18F]Fluorobenzoyl- and 2-[18F]Fluoropropionyl-Peptides;Sven H. Hausner et al.;《J. Med. Chem.》;20081231(第51期);第5901-5904页 * |
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