CN114805109B - Efficient preparation method of fluoro [18F ] sand fenamide and PET imaging agent application - Google Patents
Efficient preparation method of fluoro [18F ] sand fenamide and PET imaging agent application Download PDFInfo
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- CN114805109B CN114805109B CN202210508969.4A CN202210508969A CN114805109B CN 114805109 B CN114805109 B CN 114805109B CN 202210508969 A CN202210508969 A CN 202210508969A CN 114805109 B CN114805109 B CN 114805109B
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- FXVQDNIIPGXEPX-UHFFFAOYSA-N spiro[1,3-dioxane-2,2'-adamantane]-4,6-dione Chemical compound O1C(=O)CC(=O)OC21C(C1)CC3CC1CC2C3 FXVQDNIIPGXEPX-UHFFFAOYSA-N 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 210000000626 ureter Anatomy 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
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Abstract
The invention discloses a high-efficiency preparation method of fluoro [18F ] salfenamide and an application of PET imaging agent, wherein the method comprises the steps of firstly designing and preparing a precursor of fluoro [18F ] salfenamide, and then realizing direct radioactive fluoro-labeling on an aromatic ring in the precursor through nucleophilic substitution reaction to obtain fluoro [18F ] salfenamide; the precursor is Mel-SF, SPIAd-SF, SPI5-SF, me 3 Sn-SF, or Bpin-SF; the prepared fluoro [18F ] sand fenamide can be used as PET imaging agent for imaging cell lines capable of secreting monoamine oxidase B, can be used for imaging living animals, can penetrate blood brain barrier and realize brain region imaging, can be combined with multiple targets in vivo in a reversible manner, can be used for imaging animal in-vitro tissues, and has potential application prospects in the medical fields of early diagnosis, auxiliary treatment, pathogenesis research and the like.
Description
Technical Field
The invention relates to the development of radiopharmaceuticals, belongs to the technical field of nuclear medicine, and in particular relates to a high-efficiency preparation method of fluoro [18F ] sand fenamide and application of the fluorous [18F ] sand fenamide serving as a PET probe with targeting monoamine oxidase B capability, and has potential application prospects in the medical fields of early diagnosis, auxiliary treatment, pathogenesis research and the like.
Background
The salfenamide is an auxiliary drug approved at present for treating parkinsonism, and can reduce the degradation of in-vivo levodopa and exogenously injected levodopa by inhibiting the activity of monoamine oxidase B (monoamine oxidase B), thereby maintaining the in-vivo levodopa at a higher level and achieving the effect of treating parkinsonism. However, as research progresses, the safinamide also shows potassium-sodium ion channel inhibition, glutamate receptor secretion inhibition and the like. These effects allow the treatment of diseases of the nervous system other than Parkinson's disease with the salfenamide or exhibit therapeutic potential such as depression, alzheimer's disease, palington's disease, ischemic cerebral infarction, etc. Unfortunately, the application research of the safinamide in the treatment of the parkinson's disease has been approved for the wide application in the market for more than ten years from the first clinical experiment, and the main reason for limiting the progress is that the current research means of the safinamide are mainly focused on anatomical plasma means or invasive in-vivo experiments, and the experimental period is long, the consumption is large, and the real metabolism, distribution, action and the like of the safinamide in the body are difficult to know. This results in even the case that the safinamide exhibits a wider application prospect and a greater application potential, and is difficult to be approved for application in other disease fields in a short period of time. Therefore, how to carry out noninvasive and real-time living study on the safinamide is important, so that the curative effect of the safinamide can be obtained rapidly, the mechanism in different diseases can be explored, the study period can be shortened, and the like.
Positron emission tomography (Positron Emission Tomography, PET) is the only novel imaging technology capable of displaying the activities of biomolecule metabolism, receptors and neuromediators on living bodies at present, and is widely used in the aspects of diagnosis and differential diagnosis of various diseases, efficacy evaluation, organ function research, new medicine development and the like. The PET imaging technology has high sensitivity, high specificity and strong penetrability, and can be used for whole-body imaging, and the image of each region of the whole body can be obtained by one-time whole-body imaging examination. PET is a display reflecting molecular metabolism, when the early stage of the disease is in the molecular level change stage and the morphological structure of a lesion area is not abnormal, and MRI and CT examination can not be clearly diagnosed, the PET examination can find the focus, can obtain a three-dimensional image, can perform quantitative analysis and realize early diagnosis, which is incomparable with other image examinations at present.
PET probes, also known as PET imaging agents, refer to radiopharmaceuticals that undergo imaging of organs, tissues or molecules after they are in vivo. After the radioactive drug is introduced into the body, the radioactive drug can be concentrated in target organs or tissues, and rays emitted by the radioactive drug are detected through an imaging instrument, so that a distribution image of the radioactive drug in the body is obtained and is used for diagnosing various diseases. Among PET probes, glucose mainly used in clinical studies, such as 18-fluoro-deoxyglucose ([ 18 F ] FDG), is the most common PET imaging agent, and has been intensively studied in tumor diagnosis and treatment.
Therefore, the use of the 18F nuclide labeled safinamide (fluorine [18F ] safinamide) can be used as a PET imaging agent to provide a noninvasive, rapid, sensitive and real-time living body research means for the safinamide research. Since the radionuclide half-life of the PET imaging agent is short (the half-life of 18F nuclide is 109.8 minutes), the radiosynthesis yield of the PET imaging agent can reach more than 10% to meet the experimental requirements, on the one hand, a sufficient radioactive dose is provided, and on the other hand, a sufficiently high radioactive concentration is provided, so that the use time range of the PET imaging agent and the number of experiments which can be arranged can be prolonged.
The invention determines five efficient synthetic routes of 18 F marked sandfenamide by adopting a radiolabeling strategy of directly carrying out radionucleophilic substitution on aromatic rings, and the radiosynthetic yield can reach more than 10%. The biological experiments on the cell level, the living animal level and the anatomical level prove that the application potential of the probe in the diseases of the nervous system, such as Alzheimer's disease, palington's chorea, depression, ischemic cerebral infarction and the like, is proved by the high affinity of the probe to monoamine oxidase B, the distribution condition of the probe in the whole body of the living animal, particularly in the brain region, the uptake dynamics of the probe and the like.
Disclosure of Invention
The invention aims at providing a high-efficiency preparation method of fluorine [18F ] sand-fenamide and application of PET imaging agent thereof, aiming at the defects of the prior art.
The technical scheme adopted by the invention is as follows:
fluoro [18F ] sand fenamide has the structure shown below:
The preparation method comprises the following steps: firstly, designing and preparing a precursor of the fluoro [18F ] sand-fenamide, and then realizing direct radioactive fluoro-marking on an aromatic ring in the precursor through nucleophilic substitution reaction to obtain the fluoro [18F ] sand-fenamide. The precursor is Mel-SF, SPIAd-SF, SPI5-SF, me 3 Sn-SF, or Bpin-SF; the structural general formula is as follows:
The corresponding R is respectively:
The preparation method of the precursor comprises the following steps:
1) For three types of precursors of Mel-SF, SPIAd-SF and SPI5-SF, the following method is adopted:
The compound 4A [ tert-butyl (S) - (1- ((tert-butoxycarbonyl) amino) -1-oxypropane-2-yl) (4- ((3-iodophenyl) oxy) benzyl) carbamate ] ] (1 equivalent) is dissolved with m-chloroperoxybenzoic acid (1 to 2 equivalents) using an anhydrous organic solution (such as tetrahydrofuran, dichloromethane, N-dimethylformamide, etc.) with stirring, and stirred at 40 ℃ to 80 ℃ for 1 hour to 6 hours. The reaction mixture is cooled to room temperature, potassium hydroxide or sodium hydroxide (5 to 10 equivalents) and 1 to 2 equivalents of mevalonic acid, spiro [ decane-2, 2'- [1,3] dioxane ] -4',6 '-dione (SPI 5), or spiro [ adamantane-2, 2' - [1,3] dioxane ] -4',6' -dione (SPIAd) are added, and the reaction is stirred for 30 minutes to 12 hours. The reaction is then diluted with a highly polar organic solution (e.g., dichloromethane, methanol, ethyl acetate, etc.), filtered and the filtrate concentrated until solid precipitation occurs, at which point 4 to 20 volumes of a low polar organic solution (e.g., n-hexane, petroleum ether, etc.) are added to promote solid precipitation, and the mixture is allowed to stand at-30 to 0 ℃ for 2 to 24 hours until all solids are precipitated. The solid is obtained after filtration and drying, and finally the product is further purified by column chromatography. The synthetic route is (wherein KOH can also be NaOH):
2) The synthesis method of the Me 3 Sn-SF precursor comprises the following steps:
The compound 4B [ tert-butyl (S) - (1- ((tert-butoxycarbonyl) amino) -1-oxypropane-2-yl) (4- ((3-bromophenyl) oxy) benzyl) carbamate ] ] (1 eq) was dissolved using an anhydrous organic solution (tetrahydrofuran, 1, 4-dioxane, etc.), and hexamethylditin (1 eq to 8 eq) was added under inert gas with stirring by displacing an inert gas (such as nitrogen, argon), followed by adding triphenylphosphine palladium dichloride (0.01 eq to 0.5 eq) for catalysis, and stirring under reflux with heating for 2 hours to 48 hours. The reaction was cooled and dried to remove the solvent, and the crude product was purified by column chromatography.
3) The method for synthesizing Bpin-SF precursor comprises the following steps:
Compound 4B (1 equivalent) was dissolved in an anhydrous organic solution (tetrahydrofuran, 1, 4-dioxane, etc.), the inert gas was replaced, potassium acetate (1 equivalent to 5 equivalents) and [1,1' -bis (diphenylphosphino) ferrocene ] dichloropalladium (II) (0.01 equivalent to 0.5 equivalent) were sequentially added under an inert gas atmosphere, and after mixing uniformly, 4, 5-tetramethyl-1, 3, 2-dioxaborane (1 equivalent to 10 equivalents) was added and stirred under reflux for 0.5 to 72 hours. Cooling and extracting after the reaction, drying the extracted organic phase by a drying agent (such as anhydrous magnesium sulfate, etc.), filtering, concentrating, and purifying by column chromatography.
The synthetic route for Bpin-SF precursor, me 3 Sn-SF precursor is as follows:
In the above technical scheme, the preparation method of the compound 4A, namely [ tert-butyl (S) - (1- ((tert-butoxycarbonyl) amino) -1-oxypropane-2-yl) (4- ((3-iodophenyl) oxy) benzyl) carbamate ] ], and the compound 4B, namely [ tert-butyl (S) - (1- ((tert-butoxycarbonyl) amino) -1-oxypropane-2-yl) (4- ((3-bromophenyl) oxy) benzyl) carbamate ] ], is as follows:
Compound 3A or 3B (1 equivalent) was dissolved in an anhydrous organic solution (e.g., tetrahydrofuran, methylene chloride, etc.), sodium cyanide material (2 to 4 equivalents) was slowly added at low temperature, and after stirring well, the temperature of the mixture was raised to 20 to 30 degrees celsius. Di-t-butyl dicarbonate (2 to 20 equivalents) was then slowly added and the mixture was stirred continuously. The reaction was cooled to room temperature at the end and the crude product was transferred to the organic solution by extraction and washing of the organic and aqueous solutions. The collected organic solution was dehydrated by adding a desiccant and concentrated by filtration. Finally, the crude product was purified by column chromatography. The structural formula of the compounds 3A and 3B is as follows:
the method for radiolabelling the precursor is as follows:
(1) Bombarding H 2 18 O by a cyclotron to obtain the radionuclide 18F, purifying by an ion exchange method under the condition of leaching solution containing alkali and a phase transfer catalyst, or directly transferring the radionuclide 18F into a reaction bottle containing the alkali and the phase transfer catalyst, and removing water at 80-120 ℃ (anhydrous acetonitrile can be added and water can be removed by an azeotropic mode).
(2) Then using anhydrous polar aprotic solution (such as dimethyl sulfoxide, acetonitrile, etc.) to dissolve the precursor (precursor concentration is 0.5mmol/L to 20 mmol/L) and adding the precursor into the reactant obtained in step (1), reacting at 80-160 ℃ for 5-40 minutes, diluting at the end of the reaction, and purifying by a semi-preparative high performance liquid chromatography system. For Me 3 Sn-SF and Bpin precursors, it is also necessary to add copper catalysts (such as copper triflate, cu (OTf) 2(py)4) and pyridine, respectively, for catalytic reactions.
The fluoro [18F ] sand fenamide prepared by the invention can be used as PET imaging agent for imaging cell lines capable of secreting monoamine oxidase B.
The fluoro [18F ] sand-fenamide can be used as PET imaging agent for living animal imaging. Furthermore, the PET imaging agent can penetrate through blood brain barrier and realize brain region imaging when in living animal imaging, can be combined with multiple targets in vivo, comprises monoamine oxidase B, potassium sodium ion channel receptor and glutamate receptor, is reversible, and can separate fluorine [18F ] sand fenamide which is combined with the targets from the combined targets under the competitive action by adding 19-fluoro-sand fenamide, monoamine oxidase B inhibitor and potassium sodium ion channel inhibitor.
Fluoro [18F ] sand-fenamide can also be used as PET imaging agent to image animal's isolated tissue by autoradiography.
The invention has the beneficial effects that:
① The invention provides routes for the radioactive preparation of fluoro [18F ] sand-fenamide and corresponding reaction steps and parameters, which can obtain more than 10% radioactive yield to provide sufficient radioactive dose and radioactive concentration to meet various experimental requirements.
② The invention provides 5 precursors for preparing fluorine [18F ] sand fenamide in radioactivity, and 3 synthetic routes are provided. This is advantageous to those in need of fluoro [18F ] sand-fenamide, and suitable precursors and synthetic routes are selected according to actual production conditions.
③ The invention discloses biological experimental result information of fluoro [18F ] sand fenamide on a cell level, a living animal level and an animal isolated tissue level for the first time, and can be used as an imaging research means of multi-level information of a user.
④ The fluorine [18F ] sand-fenamide disclosed by the invention is proved by experiments, and a plurality of characteristics of the 19-fluorine-sand-fenamide are maintained, so that the fluorine [18F ] sand-fenamide can be used as an imaging research means of the 19-fluorine-sand-fenamide.
⑤ The biological experimental result of the fluoro [18F ] sand fenamide disclosed by the invention shows that the fluoro [18F ] sand fenamide has the properties of combining monoamine oxidase B, potassium-sodium ion channel receptor, penetrating blood brain barrier, combining reversibility in vivo and the like, combines the current research status of some diseases, especially neurological diseases, and indicates that the fluoro [18F ] sand fenamide can be used as PET imaging agent in the aspects of drug screening, curative effect monitoring, early disease diagnosis, pathogenesis research and the like of diseases such as Alzheimer's disease, huntington chorea, parkinson's disease, depression, ischemic cerebral infarction and the like.
Drawings
In order that the contents of the present invention may be more clearly understood, the present invention will be further described in detail below with reference to specific embodiments thereof with reference to the accompanying drawings.
FIG. 1 radiolabeled precursor of fluoro [18F ] sand fenamide
FIG. 2 Synthesis of fluoro [18F ] salfenamide radiolabelled precursor (A) Mel-SF, SPIAd-SF, SPI5-SF; (B) Me 3 Sn-SF & Bpin-SF.
FIG. 3 1H-NMR spectra of molecules (A) 4A, (B) 4B, (C) Mel-SF, (D) SPIAd-SF, (E) SPI5-SF, (F) Me 3 Sn-SF, (G) Bpin-SF in the process of synthesis of a fluoro [18F ] saphenonamide radiolabelled precursor
FIG. 4 radiosynthesis of fluoro [18F ] salfenamide based on different precursors (A) Mel-SF, SPIAd-SF, SPI5-SF; (B) Me 3 Sn-SF; (C) Bpin-SF
FIG. 5 optimization of experimental conditions of the fluoro18F-saphenofenamide radiolabelling process based on Mel-SF, SPIAd-SF, SPI5-SF (A) alkali amount and species screening, (B) precursor species and amount screening, (C) reaction time and temperature screening, (D) reaction solvent screening
FIG. 6 radioactivity quality control of fluoro [18F ] sand fenamide (A) radiochemical purity and identity test (B) injection stability test
FIG. 7 uptake profile of fluoro [18F ] saphenolide in astrocytoma cells U87, breast cancer cells MCF-7, saphenolide pretreated astrocytoma cells
FIG. 8 systemic distribution of fluoro [18F ] sand fenamide in Sprague Dawley rats (PET dynamic scan) in FIG. 9 (A) fluoro [18F ] sand fenamide in Sprague Dawley rats brain area distribution (PET dynamic scan), (B) and corresponding activity/time curves
FIG. 10 brain area distribution (PET dynamic scan) of fluoro [18F ] saphenolamide in Sprague Dawley rats, (B) and corresponding activity/time curves under displacement experiments
FIG. 11 distribution of fluoro [18F ] sand fenamide in isolated tissue in C57 mice (autoradiography)
Detailed Description
The technical scheme of the invention is further described in detail below through the attached drawings and specific examples, and all reagents are available unless otherwise specified.
The structure of the fluoro [18F ] sand fenamide is as follows:
The invention designs and determines a high-efficiency preparation method thereof, wherein the structure of the related marking precursor is shown in a specific figure 1;
The synthetic routes for each precursor are shown in FIG. 2, and in order to better illustrate the synthesis of each precursor, the present invention is further described below with reference to some specific examples.
① Preparation method of molecule 4A [ tert-butyl (S) - (1- ((tert-butoxycarbonyl) amino) -1-oxypropane-2-yl) (4- ((3-iodophenyl) oxy) benzyl) carbamate ] ] and molecule 4B [ tert-butyl (S) - (1- ((tert-butoxycarbonyl) amino) -1-oxypropane-2-yl) (4- ((3-bromophenyl) oxy) benzyl) carbamate ] ]:
Molecules 3A or 3B (1 eq.) are dissolved in an anhydrous organic solution (e.g. tetrahydrofuran, dichloromethane, etc.), sodium cyanide material (2 to 4 eq.) is slowly added at low temperature and after stirring, the temperature of the mixture is raised to 20 to 30 ℃. Di-t-butyl dicarbonate (2 to 20 equivalents) was then slowly added and the mixture was stirred continuously. The reaction was cooled to room temperature at the end and the crude product was transferred to the organic solution by extraction and washing of the organic and aqueous solutions. The collected organic solution was dehydrated by adding a desiccant and concentrated by filtration. Finally, the crude product was purified by column chromatography.
Example 1:
3A (616 mg,1.5 mmol) was dissolved in anhydrous tetrahydrofuran (10 mL) and then sodium hydride (60% dispersed in mineral oil, 132mg,3.3 mmol) was added dropwise to the solution at 0deg.C and stirred for 1h. The reaction mixture was heated to 25 ℃, di-tert-butyl dicarbonate (1.63 g,24 mmol) was slowly added and the mixture was stirred for an additional 2 hours. Finally, the reaction mixture was quenched with water and extracted with ethyl acetate. The organic solution was then washed sequentially with water, 1M HCl and brine and the organic fraction was collected, and the final organic solution was dried over Na2SO4, filtered and concentrated. Finally, the product was purified by silica gel column chromatography (5-50% ethyl acetate/petroleum ether) to give a colorless solid (4A, 170mg,0.28mmol,68% yield) ).1H NMR(400MHz,Chloroform-d)δ7.67(d,J＝1.8Hz,1H),7.54(dt,J＝7.8,1.5Hz,1H),7.26(dd,J＝7.8,1.5Hz,1H),7.13–6.96(m,3H),6.80(dd,J＝8.3,4.6Hz,2H),4.86(s,2H),4.69–3.70(m,3H),1.33(d,J＝4.1Hz,18H),1.21(d,J＝7.0Hz,3H)ppm.
Example 2:
similarly, 4B was prepared by replacing 3A with 3B in an equimolar amount in example 1, and the other steps were the same, and a colorless oily substance was finally obtained 4B.1H NMR(400MHz,Chloroform-d)δ7.59(t,J＝1.8Hz,1H),7.46(dt,J＝7.9,1.6Hz,1H),7.34(dt,J＝7.8,1.3Hz,1H),7.25(p,J＝4.8Hz,3H),6.99–6.88(m,2H),5.01(s,2H),4.90–3.89(m,3H),1.71(d,J＝2.8Hz,2H),1.46(d,J＝4.3Hz,18H),1.34(d,J＝7.0Hz,3H),1.31–1.22(m,1H).
Wherein the preparation of 3A/3B is based on the prior art, one method of preparation is shown in FIG. 2, starting with 1A/1B.
② Synthesis of precursors Mel-SF, SPIAd-SF, SPI5-SF
Compound 4A (1 equivalent) and m-chloroperoxybenzoic acid (1 to 2 equivalents) are dissolved with stirring using an anhydrous organic solution (such as tetrahydrofuran, dichloromethane, N-dimethylformamide, etc.), and stirred at 40 ℃ to 80 ℃ for 1 hour to 6 hours. The reaction mixture was cooled to room temperature, potassium hydroxide or sodium hydroxide (5 to 10 equivalents) and 1 to 2 equivalents of mevalonic acid/spiro [ decane-2, 2'- [1,3] dioxane ] -4',6 '-dione (SPI 5)/spiro [ adamantane-2, 2' - [1,3] dioxane ] -4',6' -dione (SPIAd) were added, and the reaction was stirred for 30 minutes to 12 hours. The reaction is then diluted with a highly polar organic solution (e.g., dichloromethane, methanol, ethyl acetate, etc.), filtered and the filtrate concentrated until solid precipitation occurs, at which time 4 to 20 volumes of a low polar organic solution (e.g., n-hexane, petroleum ether, etc.) are added to promote solid precipitation, and the mixture is allowed to stand at a low temperature of-30 to 0 ℃ for 2 to 24 hours until all solids are precipitated. The solid is obtained after filtration and drying, and finally the product is further purified by column chromatography.
Example 3:
In a closed reaction flask, compound 4A (1 eq) and 85% m-chloroperoxybenzoic acid (1.1 eq) were stirred in Dichloromethane (DCM) at 40 ℃ for 80 min. After cooling to room temperature, KOH (7 eq) and Makinic acid (1.3 eq) were added and stirred for a further 45 minutes. The reaction mixture was then diluted with DCM and filtered through filter paper. The solvent of the collected organic phase was removed under reduced pressure at 35 ℃ until the first solid precipitated. Then, hexane was slowly added, and the mixture was left to stand at-20℃for 12 hours to complete precipitation. The solid was collected by filtration, washed with n-hexane, and dried in air and vacuum. Finally, the crude product was purified by silica gel column chromatography (SiO 2, 40-100% ethyl acetate/petroleum ether) to give the Mel-SF precursor.
Mel-SF (41% yield, white solid) ):1H NMR(400MHz,Chloroform-d)δ7.86–7.76(m,1H),7.69–7.57(m,1H),7.47–7.36(m,1H),7.25–7.05(m,2H),6.92(t,J＝7.6Hz,2H),5.03(d,J＝37.9Hz,2H),4.90–3.80(m,3H),1.72(s,6H),1.49–1.41(m,18H),1.33(dd,J＝7.1,3.0Hz,3H)ppm.
The same method can prepare SPIAd-SF precursor and SPI5-SF precursor
SPIAd-SF (33% yield, yellowish solid) ):1H NMR(400MHz,Chloroform-d)δ7.83(s,1H),7.75–7.69(m,1H),7.52(d,J＝7.8Hz,1H),7.35(t,J＝7.9Hz,1H),7.18–6.95(m,3H),6.82(d,J＝8.6Hz,2H),5.00(s,2H),4.79–3.92(m,3H),2.36(s,2H),2.09(d,J＝12.6Hz,4H),1.78(s,2H),1.64(s,4H),1.39(d,J＝12.6Hz,18H),1.26(d,J＝7.0Hz,3H)ppm.
SPI5-SF (36% yield, yellow solid ):1H NMR(400MHz,Chloroform-d)δ7.81(d,J＝1.7Hz,1H),7.68(dt,J＝7.9,1.5Hz,1H),7.40(dt,J＝7.8,1.3Hz,1H),7.14(t,J＝7.8Hz,3H),6.94(d,J＝6.6Hz,2H),5.01(s,2H),4.95–3.78(m,3H),2.40–2.21(m,2H),2.18(d,J＝7.5Hz,2H),1.98(td,J＝4.5,2.2Hz,2H),1.95–1.88(m,2H),1.48(d,J＝4.3Hz,18H),1.35(d,J＝7.1Hz,3H).
③ Synthesis of precursor Me 3 Sn-SF
Compound 4B (1 equivalent) was dissolved with an anhydrous organic solution (tetrahydrofuran, 1, 4-dioxane, etc.), and by substituting an inert gas (such as nitrogen, argon), hexamethylditin (1 equivalent to 8 equivalents) was added under the protection of the inert gas and stirred, followed by adding triphenylphosphine palladium dichloride (0.01 equivalent to 0.5 equivalent) and stirring under reflux for 2 hours to 48 hours for complete reaction. The reaction was cooled and dried to remove the solvent, and the crude product was purified by column chromatography.
Example 4:
In a two-port reaction flask, compound 4B (282 mg,0.500 mmol) was added, and 5mL of 1, 4-dioxane was dissolved and subjected to degassing and nitrogen-charging treatment. Hexamethyl-ditin (193. Mu.l, 0.930 mmol) and triphenylphosphine palladium dichloride (3.9 mg) were added. The mixture was heated to 65 ℃ and stirred for 12 hours. The mixture was distilled to dryness by rotary evaporation, and purified by silica gel column chromatography (0-80% ethyl acetate/n-hexane, containing 1% by volume of triethylamine) to give a yellow oily product (Me 3 Sn-SF,21% yield ).1H NMR(400MHz,Chloroform-d)δ8.80(s,1H),7.35–7.18(m,6H),6.88–6.72(m,2H),5.14–5.02(m,2H),4.68(q,J＝6.8Hz,1H),4.35(dt,J＝12.5,1.0Hz,1H),4.28(dt,J＝12.4,0.9Hz,1H),1.46(d,J＝2.8Hz,18H),1.38(d,J＝6.8Hz,3H),0.78(s,9H).
④ Synthesis of precursor Bpin-SF
Compound 4B (1 equivalent) was dissolved in an anhydrous organic solution (tetrahydrofuran, 1, 4-dioxane, etc.), the inert gas was replaced, potassium acetate (1 equivalent to 5 equivalents) and [1,1' -bis (diphenylphosphino) ferrocene ] dichloropalladium (II) (0.01 equivalent to 0.5 equivalent) were sequentially added under an inert gas atmosphere, and after mixing uniformly, 4, 5-tetramethyl-1, 3, 2-dioxaborane (1 equivalent to 10 equivalents) was added and stirred under reflux for 0.5 to 72 hours. Cooling and extracting after the reaction, drying the extracted organic phase by a drying agent (such as anhydrous magnesium sulfate, etc.), filtering, concentrating, and purifying by column chromatography.
Example 5:
Compound 4B (4.8 g,8.7 mmol), potassium acetate (2.55 g,26 mmol), [1,1' -bis (diphenylphosphino) ferrocene ] dichloropalladium (II) (0.63 g,0.87 mmol) and 4, 5-tetramethyl-1, 3, 2-dioxaborane (4.39 g) were dissolved in 1, 4-dioxane (40 ml) under nitrogen, stirred at 80℃under nitrogen for 2h, and then cooled to room temperature. Water (30 ml) was added to the reaction solution, and the mixture was extracted with ethyl acetate (3X 20 ml). The combined organic phases were concentrated by rotary evaporation. The crude product was purified by silica gel column chromatography (0-40% ethyl acetate/n-hexane) to give a transparent gum-like substance (62% yield) ).1H NMR(300MHz,Chloroform-d)δ7.59–7.47(m,2H),7.40–7.21(m,4H),6.89–6.80(m,2H),5.19–5.03(m,2H),4.68(q,J＝6.8Hz,1H),4.41–4.30(m,1H),4.30–4.22(m,1H),1.46(d,J＝2.2Hz,18H),1.41(d,J＝6.8Hz,3H),1.24(d,J＝15.1Hz,12H).
⑤ Nuclear magnetic resonance hydrogen spectrogram spectrum: the 1H-NMR chart of each of the above molecules is shown in FIG. 3. FIG. 3 1H-NMR spectra of molecules (A) 4A, (B) 4B, (C) Mel-SF, (D) SPIAd-SF, (E) SPI5-SF, (F) Me 3 Sn-SF, (G) Bpin-SF in the process of synthesis of a fluoro [18F ] saphenonamide radiolabelled precursor
Depending on the precursors, different processes for the radiosynthesis of fluoro [18F ] sand fenamide were designed, as shown in FIG. 4. The method comprises the following steps: bombarding H 2 18 O by a cyclotron to obtain the radionuclide 18F, purifying by an ion exchange method (by using an eluent containing alkali and a phase transfer catalyst), or directly transferring the radionuclide to a reaction bottle containing the alkali and the phase transfer catalyst; the water is removed at 80 to 120℃ (anhydrous acetonitrile can be added in the volatilizing process, and water is removed by utilizing an azeotropic mode). Then using anhydrous polar aprotic solution (such as dimethyl sulfoxide, acetonitrile, DMF, DMA, etc.) to dissolve the precursor (0.5 mmol/L to 20 mmol/L) and adding into a reaction bottle, and for Me 3 Sn-SF and Bpin precursor, copper catalyst (such as copper triflate, cu (OTf) 2(py)4) and pyridine are respectively added for catalytic reaction, reacting for 5 minutes to 40 minutes at 80-160 ℃, ending the reaction, diluting and purifying by a semi-preparation high performance liquid chromatography system.
Example 6:
the radioactive synthesis process based on Mel-SF, SPIAd-SF and SPI5-SF comprises the following steps:
Oxygen-enriched water H 2 18 O was bombarded by a cyclotron to give 18F ions, 18 F ions were adsorbed by an anion exchange column (QMA), 18 F ions were eluted from the QMA column with 1mL of an eluent containing 0.8mL of acetonitrile, 0.2mL of H 2 O, 10mg of TEAB, and transferred to a reaction flask, azeotropically dehydrated using 1mL of anhydrous acetonitrile, and the azeotropic procedure was repeated three times until drying was complete. 3mg of the labeled precursor compound Mel-SF, SPIAd-SF or SPI5-SF prepared in example 1 was added, dissolved using 1mL of anhydrous acetonitrile, and reacted at 120℃for 15 minutes with heating. And adding 1mL of 6M hydrochloric acid aqueous solution into the obtained crude product solution, and carrying out hydrolysis reaction for 10min at 110 ℃ to obtain the crude product solution of the target compound fluorine [18F ] sand fenamide.
Example 7:
the radiosynthesis process based on Me 3 Sn-SF comprises the following steps:
Oxygen-enriched water H 2 18 O is bombarded by a cyclotron to obtain 18F ions, 18 F ions are adsorbed by an anion exchange column (Chromafix-PS-HCO 3), and the exchange column is sequentially washed and activated by 5mL of ethanol, 5mL of 90mg/mL of potassium triflate solution and 5mL of deionized water before use. The adsorbed fluoride ions were then eluted into the reaction flask using 1mL of eluent (10 mg/mL of potassium triflate 0.45mL,1mg/mL of aqueous potassium carbonate 0.05mL and anhydrous acetonitrile 0.5 mL). Azeotropic removal of water was performed using 1mL of anhydrous acetonitrile and the azeotropic step was repeated three times until drying was complete. 3mg of the labeling precursor compound Me 3 Sn-SF prepared in example 1, 0.4mL of anhydrous Dimethylacetamide (DMA), 0.1mL of 1M pyridine DMA solution and 0.067mL of 0.2M copper triflate DMA solution were added, and after thorough mixing, heated at 110℃for 20min. Then cooling to room temperature, adding 1mL of 6M hydrochloric acid aqueous solution, and hydrolyzing at 110 ℃ for 10min to obtain the crude product solution of the target compound fluorine [18F ] sand fenamide.
Example 8:
the process for radiosynthesis based on Bpin-SF comprises the following steps:
oxygen-enriched water H 2 18 O was bombarded by a cyclotron to give 18F ions, 18 F ions were adsorbed by an anion exchange column (QMA), 18 F ions were eluted from the QMA column with 1mL of an eluent containing 0.8mL of acetonitrile, 0.2mL of H 2 O, 10mg of TEAB, and transferred to a reaction flask, azeotropically dehydrated using 1mL of anhydrous acetonitrile, and the azeotropic procedure was repeated three times until drying was complete. 1-3mg of the labeled precursor compound Bpin-SF prepared in example 1, 0.15mL of 1M Cu (OTf) 2(py)4, and 1mL of anhydrous DMA solution were added, and the reaction was heated at 140℃for 20min. And adding 1mL of 6M hydrochloric acid aqueous solution into the obtained crude product solution, and carrying out hydrolysis reaction for 10min at 110 ℃ to obtain the crude product solution of the target compound fluorine [18F ] sand fenamide.
Semi-preparative liquid chromatography purification process is carried out on the obtained crude product solution:
To the crude reaction solutions obtained in examples 6, 7, 8 were added 2.5mL of solution (30% acetonitrile, 70% water) for dilution, followed by purification using a semi-preparative high performance liquid chromatography system (column C18, 250mm x 10mm,4.6um particle size) under chromatographic conditions (5 mL/min,30% -80% acetonitrile/0.1% tfa in water), and the purified products were transferred to a sterile vial, diluted with 40mL of deionized water and adsorbed by WATERS SEP-Pak C18 column. The product was then eluted from the reagent loading module using 0.5 ml ethanol and 4.5 ml saline. The final solution of fluoro [18F ] sand fenamide was passed through a sterile membrane filter (Millex LG,0.20 um) and stored in another sterile vial as an injection for subsequent experiments.
The radioactive yield (Radiochemical yield, RCY) of the synthesis process can reach 10 percent basically; furthermore, we have done extensive optimization of the synthesis conditions for the various radiosynthesis processes described above to maximize the radioproductivity to better meet the dose requirements of biological experiments. Wherein the radioactive yield of the method can reach more than 25% by optimizing the process of radiolabeling the fluorine [18F ] sand fenamide based on SPIAd-SF, and the specific synthesis conditions are as follows:
10mgTEAB is used in the eluent; the precursor is 15mgSPIAd-SF, the polar aprotic solution is DMSO solution, and the radioactive reaction condition is 120 ℃ for 15min; the highest radioactive yield was obtained under the conditions of 25.6.+ -. 4.2% (decay correction) and molar activity was 286.2.+ -. 31.5GBq/umol, as in example 6. All procedures, including radiosynthesis and purification, take 88.+ -. 4 minutes in total. Correspondingly, when 7mgTEAB is used as the leaching solution, 2mgSPIAd-SF is used as the precursor, N-dimethylacetamide is used as the polar aprotic solution, and the radioactive reaction condition is 120 ℃ for 10min, the radioactive yield is only 4-9%.
The precursor is Me 3 Sn-SF, and the radiosynthesis optimization process comprises the following steps: 3 to 5mg of potassium triflate, 0.05mg of potassium carbonate, 3 to 8mg of precursor, DMA solution as polar aprotic solution, and the radioactive reaction temperature of 110 to 125 ℃ for 10 to 30min are used in the leacheate, and the other conditions are the same as those in example 7, the radioactive yield can be more than 10%, and the molar activity is 166GBq/umol. All procedures, including radiosynthesis and purification, take 94 to 106 minutes.
When the precursor is Bpin-SF, the radiosynthesis optimization process is as follows: 6mgTEAB,10mg precursor and 0.25mL Cu (OTf) 2(py)4 solution are used in the eluent, and the radioactive reaction is carried out for 10min at 155 ℃ under the condition that the polar aprotic solution is a DMA solution; the highest radioactivity yields were obtained at 11-17% (n=5, decay correction) with a molar activity of 182GBq/umol. All procedures, including radiosynthesis and purification, took 101 minutes.
Aiming at the prepared fluoro [18F ] sand fenamide injection, a high performance liquid chromatography system is utilized to control the quality of the injection to ensure that the quality meets the experimental requirements. The results are shown in FIG. 6.
① By co-injection of HPLC analysis after mixing the injection with non-radioactive safinamide (red peak line), the difference in retention time between the product fluoro [18F ] safinamide (black peak line of a in fig. 6) and 19-fluoro-safinamide (red line in fig. 6A) was confirmed to be within 0.1 minutes. Meanwhile, the radiochemical purity of the fluoro [18F ] sand fenamide purified in the radioHPLC exceeds 99%. Proved that the fluorine [18F ] sand fenamide is successfully prepared, and the purity meets the requirement
② The newly prepared fluoro [18F ] sand fenamide injection is stirred (550 rpm) for 0, 30, 90 and 180 minutes at 37 ℃, 1/10 volume of solution is taken out at each time point, 10 percent of physiological saline solution is added for dilution, and the radioactive chemical purity analysis is carried out in a high performance liquid chromatography system provided with a radioactive NaI detector, finally, the fluoro [18F ] sand fenamide injection is proved to have extremely high stability (the radioactive chemical purity is higher than 98 percent) all the time within 180 minutes.
Example 9:
The prepared fluoro [18F ] sand fenamide can be used for imaging a cell line of monoamine oxidase B secreting monoamine oxidase B through cell experiments. In the experiment, astrocytoma cell U87 capable of producing monoamine oxidase B and breast cancer cell MCF-7 incapable of producing monoamine oxidase B were used as a comparison. The experimental results are shown in FIG. 7.
U87 astrocytes (uptake rate 2.01%) which produce monoamine oxidase B had about four times the uptake of fluoro [18F ] saphenonamide as MCF-7 breast cancer cells (uptake rate 0.53%) which did not produce monoamine oxidase B. This result shows that fluoro [18F ] salfenamide has a high affinity for monoamine oxidase B, which is consistent with the properties of salfenamide. Meanwhile, the radioisotope probe uptake rate (uptake rate 0.59%) of U87 cells pretreated with 100ug/mL of safinamide was significantly reduced as compared to U87 cells, and showed similar levels to MCF-7 cells. This suggests that the binding site of fluoro [18F ] safinamide is identical to that of safinamide and that there is a limit in binding capacity.
Example 10:
We performed the first living experiments of fluoro [18F ] sand-fenamide to obtain the information of the distribution, aggregation, migration, metabolism, etc. of fluoro [18F ] sand-fenamide in living organism. The uptake of the major organs is shown in figure 8.
An in vivo PET study of fluoro [18F ] salfenamide was performed for the first time on Sprague-Dawley (SD) rats to examine the biodistribution of fluoro [18F ] salfenamide and to further understand the in vivo mechanism and metabolic information of salfenamide. SD rats were intravenously injected with fluoro [18F ] saphenolamide followed by 3 minutes of CT scan to locate the rats followed by 90 minutes of dynamic PET scan. The obtained PET image is fused with the matched CT image as shown in fig. 8. We delineate the region of interest (ROI) and quantify these PET imaging data by normalizing the uptake values (suv=the radioconcentration of ROI/systemic radioconcentration). Of particular note, the bone uptake detected during PET scan was negligible, and this region also showed the lowest SUV of 0.0573 throughout the body, confirming that fluoro [18F ] sand fenamide is not or rarely defluorinated in the body. We then calculated the ratio of ROI to SUV of bone (labeled SUVROI/bone) using SUV of bone as baseline.
The highest uptake of fluoro [18F ] salfenamide was observed in the bladder (SUVROI/bone=11.99), which is consistent with the metabolic pathway of salfenamide, since elimination of salfenamide occurs primarily through bladder metabolism. Similarly, some fluoro [18F ] sand fenamides can also be found in the small intestine region (SUVROI/bone=1.69) 33. In addition, high uptake of fluoro [18F ] sand fenamide was observed in the liver, kidney and pancreas, corresponding SUVROI/bone values of 6.56, 6.43 and 5.00, respectively. An amount of fluoro [18F ] sand fenamide (SUVROI/bone=2.35) was also found in the lungs. The heart showed similar levels of uptake to bone (SUVROI/bone=1.15). According to literature reports, a large amount of monoamine oxidase B is found in the liver and pancreas in the peripheral tissues of rats; the renal cortex and ureter of the kidneys and lungs also produce some monoamine oxidase B, which can be observed in small or negligible amounts in the heart, spleen and muscles. Thus, these uptake showed that the distribution and abundance of fluoro [18F ] salfenamide in rat peripheral tissues was closely related to monoamine oxidase B, indicating that fluoro [18F ] salfenamide and monoamine oxidase B have high affinity in these regions.
Example 11:
monoamine oxidase B information of brain regions is the most difficult to obtain by conventional means, and is also the region most prominent in the living body detection advantage of PET. In addition, monoamine oxidase B changes in brain regions are closely related to a number of important neurological diseases. We explored the distribution of the tracer in the brain region and measured the time-activity curve (TAC), disclosing its kinetic characteristics in the brain region.
As shown in B in fig. 9, the radioactivity level in the rat brain was relatively high early in the PET scan (10 minutes ago) and then effectively cleared. The SUV then slowly declined and remained stable after 30 minutes. This TAC means that fluoro [18F ] salfenamide enters the rat brain, but most fluoro [18F ] salfenamide does not accumulate well and is rapidly cleared from the brain with metabolism; the remaining tracer accumulates in the brain and is believed to reflect the actual binding site of fluoro [18F ] sand fenamide in the rat brain.
Several representative slices of the dynamic PET image are shown as a in fig. 9. According to SUV of rat brain ROI, the radioactivity of cerebellum grey matter is minimal. To more conveniently compare SUVs, the SUV ratio between the ROI and cerebellum was calculated, labeled SUV ROI/ Cerebellum of cerebellum . The results indicate a high accumulation of fluoro [18F ] sand fenamide (SUV ROI/ Cerebellum of cerebellum exceeding 1.5) was found in hippocampus, septum, thalamus, striatum, cingulate cortex, nucleus accumbens and medial prefrontal cortex. There are also some radiotracers in the midbrain, the bridgebrain, the hypothalamus, medulla, pituitary and parietal cortex, the outer Zhou Eshe cortex, the auditory cortex, the island cortex and the somatosensory cortex (SUV ROI/ Cerebellum of cerebellum is between 1.3 and 1.5).
Furthermore, the distribution and abundance pattern of fluoro [18F ] safinamide in rat brain is not single targeted, with binding capacity to multiple targets within the brain. In one aspect, large amounts of fluoro [18F ] salfenamide are seen in monoamine oxidase B rich regions such as the hippocampus, amygdala, striatum, septum, vomica, medial prefrontal cortex and thalamus. On the other hand, radiotracers were also observed in areas of the cingulate cortex, the orbital cortex, the auditory cortex, etc. Monoamine oxidase B is relatively rare in these regions, with a large number of potassium/sodium channel receptors and glutamate distributed therein. The above-described profile of fluoro [18F ] salfenamide is consistent with the multi-target mode of action of salfenamide reported in recent years of research. This is also the first time verification achieved by non-invasive in vivo real-time monitoring means.
Example 12:
In the field of PET imaging agents, whether the binding of the tracer to the target is reversible or not, and whether the tracer has a higher signal-to-noise ratio, excellent image accuracy and a rapid kinetic curve is concerned. For this we disclose a reversible binding assay (i.e. displacement assay) of fluoro [18F ] sand fenamide in vivo experiments. The results are shown in FIG. 10.
To further verify the reversibility of fluoro [18F ] safinamide binding and the consistency of fluoro [18F ] safinamide and safinamide associated with the binding site, substitution experiments were performed in which SD rats were injected with a solution of safinamide mesylate as a competitor (in order to increase the solubility of safinamide) during PET scan. As shown in B in fig. 10, after injection of safinamide mesylate, whole brain SUV decreased by more than 50% and remained slightly declining in subsequent PET scans. This result indicates that the binding of fluoro [18F ] salfenamide in the rat brain is reversible, while indicating that the binding site of fluoro [18F ] salfenamide is highly consistent with that of salfenamide. This can be more intuitively demonstrated by a in fig. 10, where the degree of probe aggregation in the high uptake region is significantly reduced and continuously decreased after injection of safinamide mesylate.
Example 13:
To verify the accuracy of fluoro [18F ] sand fenamide in PET living body detection, partially experimental mice were subjected to ex vivo dissection and autoradiography, the accuracy was demonstrated by comparing PET experimental results with autoradiography results, and an autoradiography image is shown in FIG. 11.
The results show that the in-vitro whole body distribution condition of the fluorine [18F ] sand fenamide in animal body weight is basically consistent with the condition obtained by PET, and the experiment that the fluorine [18F ] sand fenamide replaces part of the separation body or other traditional laboratories is feasible, thereby being beneficial to greatly reducing the consumption of manpower and material resources and accelerating the development process of sand fenamide as a medicine for treating other diseases. Also proves the application value of the fluoro [18F ] sand fenamide in the fields of disease diagnosis, pathological research, curative effect evaluation and the like.
Claims (2)
1. The preparation method of the fluoro [18F ] salfenamide is characterized in that a precursor of the fluoro [18F ] salfenamide is firstly designed and prepared, and then the aromatic ring in the precursor is directly and radially fluorinated and labeled through nucleophilic substitution reaction to obtain the fluoro [18F ] salfenamide; the structure of the fluoro [18F ] sand-fenamide is as follows:
The precursor is Mel-SF, or SPIAd-SF; the structural general formula is as follows:
The corresponding R is respectively:
the preparation method comprises the following steps:
1 equivalent of compound 4A [ tert-butyl (S) - (1- ((tert-butoxycarbonyl) amino) -1-oxypropane-2-yl) (4- ((3-iodophenyl) oxy) benzyl) carbamate ] ] and 1 to 2 equivalents of m-chloroperoxybenzoic acid are stirred and dissolved by using an anhydrous organic solution, and stirred for 1 to 6 hours at a temperature of between 40 and 80 ℃; the reaction mixture is cooled to room temperature, 5 to 10 equivalents of potassium hydroxide or sodium hydroxide and 1 to 2 equivalents of McOtstool acid or spiro [ adamantane-2, 2' - [1,3] dioxane ] -4',6' -diketone (SPIAd) are added, and stirred for reaction for 30 minutes to 12 hours; then diluting the reactant by using a high-polarity organic solution, filtering, concentrating the filtrate until solid precipitation occurs, adding a low-polarity organic solution with the volume of 4-20 times to promote the solid precipitation, standing the mixture at the temperature of minus 30 ℃ to 0 ℃ for 2-24 hours until all the solid is precipitated, obtaining the solid after filtering and drying, and finally further purifying the product by column chromatography;
The anhydrous organic solution is tetrahydrofuran, dichloromethane or N, N-dimethylformamide; the high-polarity organic solution is dichloromethane, methanol or ethyl acetate; the low-polarity organic solution is n-hexane or petroleum ether;
the preparation method of the compound 4A comprises the following steps:
Dissolving 1 equivalent of compound 3A in anhydrous organic solution, adding 2 to 4 equivalents of sodium hydride substance at 0 ℃, uniformly stirring, heating the mixture to 20 to 30 ℃, then adding 2 to 20 equivalents of di-tert-butyl dicarbonate, continuously stirring the mixture for reaction, cooling the reaction to room temperature, extracting and cleaning the organic solution and aqueous phase solution, transferring the crude product into the organic solution, adding a drying agent into the collected organic solution for removing water, filtering and concentrating, and finally purifying the crude product by column chromatography; the structural formula of compound 3A is as follows:
The method for radiolabeling the precursor comprises:
(1) Bombarding H 2 18 O by a cyclotron to obtain radionuclide 18F, purifying by an ion exchange method under the condition of leacheate containing alkali and a phase transfer catalyst TEAB, or directly transferring the radionuclide 18F into a reaction bottle containing the alkali and the phase transfer catalyst; then removing water at 80-120 ℃;
(2) Then using anhydrous polar aprotic solution to dissolve the precursor, wherein the concentration of the precursor is 0.5mmol/L to 20mmol/L, adding the precursor into the reactant obtained in the step (1), reacting for 5 minutes to 40 minutes at the temperature of 80 ℃ to 160 ℃, ending the reaction, diluting and purifying by a semi-preparation high performance liquid chromatography system.
2. The method for preparing fluoro [18F ] sand fenamide according to claim 1, wherein when the precursor is SPIAd-SF, the method for radiolabelling comprises: bombarding oxygen-enriched water H 2 18 O by a cyclotron to obtain 18F ions, adsorbing 18 F ions by an anion exchange column, eluting 18 F ions from a QMA column by using 1mL of eluent containing 0.8mL of acetonitrile, 0.2mL of H 2 O and 10mg of TEAB, transferring the eluent into a reaction bottle, performing azeotropic dehydration by using 1mL of anhydrous acetonitrile for three times, repeating the azeotropic step until the azeotropic step is dried completely, adding 15mg of a marked precursor compound SPIAd-SF, dissolving by using a 1mL of a MSO solution, heating and reacting at 120 ℃ for 15min, adding 1mL of a 6M hydrochloric acid aqueous solution into the obtained crude product solution, and performing hydrolysis reaction at 110 ℃ for 10min to obtain the crude product solution of the target compound fluorine [18F ] sand-fenamide.
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